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Research Article Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through the AKT-PTEN-FOXO4 Pathway An-Ting Liou, 1 Mei-Fang Chen, 2 and Chu-Wen Yang 1 1 Department of Microbiology, Soochow University, Shihlin, Taipei 111, Taiwan 2 Department of Medical Research, Taipei Veterans General Hospital, Taipei 112, Taiwan Correspondence should be addressed to Chu-Wen Yang; [email protected] Received 25 February 2017; Accepted 6 June 2017; Published 9 July 2017 Academic Editor: Jae Youl Cho Copyright © 2017 An-Ting Liou et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. Curcumin (diferuloylmethane) is a yellow-colored polyphenol with antiproliferative and proapoptotic activities to various types of cancer cells. is study explored the mechanism by which curcumin induces p53-null hepatoma cell apoptosis. Results. AKT, FOXO1, and FOXO3 proteins were downregulated aſter curcumin treatment. Conversely, PTEN was upregulated. Subcellular fractionations revealed that the FOXO4 protein translocated from cytosol into the nucleus aſter curcumin treatment. Overexpression of FOXO4 increases the sensitivity of Hep3B cells to curcumin. Knockdown of the FOXO4 gene by siRNA inhibits the proapoptotic effects of curcumin on Hep3B cell. Conclusions. is study revealed the AKT/PTEN/FOXO4 pathway as a potential candidate of target for treatment of p53-null liver cancers. 1. Introduction Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and is the third most common cause of cancer-related death in the Asia-Pacific region [1]. HCC is a cancer with a high mortality rate [2]. e prognosis of liver cancer, even in developed countries, is very unfavorable. In the US, the five-year survival rates are 5% and 18% before 1989 and aſter 2005, respectively [3]. It has been reported that the absence of p53 promotes HCC metastasis in a mouse model [4]. Moreover, mutations in the TP53 tumor suppressor gene have been reported in 23–67% of HCC patients worldwide and in 50% of HCC patients in China and South Africa [5]. Curcumin is a natural polyphenol found in the rhizome of Curcuma longa (turmeric) [6]. Curcuma longa has been traditionally used in Asian countries as a medical herb for several pathologies due to its antioxidant, anti-inflammatory [7], cancer chemoprevention [8, 9], and anticancer properties [10–12]. Several studies have reported that curcumin could induce cancer cell apoptosis through p53-dependent and p53-independent pathways [13]. It has been reported that curcumin and related analogous compounds could induce apoptosis in hepatoma cells that express p53 protein normally (e.g., HepG2 cell) through p53/p21 pathway [14–16]. However, little is known regarding the effects of curcumin on p53-null liver cancer cells. In this study, the PI3K/AKT/PTEN/FOXO pathway was shown to mediate curcumin induced apoptosis in p53-null Hep3B hepatoma cells. 2. Materials and Methods 2.1. Chemicals and Antibodies. Chemicals and PI3K inhibitor LY294002 were purchased from Sigma-Aldrich (Sigma- Aldrich Co. LLC). Antibodies of the signal transduction path- way, apoptosis pathway, and FOXO family were purchased from Cell Signaling (Cell Signaling Technology, Inc.). 2.2. Cell Culture. HepG2 (ATCC HB-8065) and Hep3B (ATCC HB 8064), human hepatocellular carcinoma cell lines, were cultured in DMEM (Gibco) containing 10% FBS, 1% NEAA, and 1% glutamine and incubated in an incubator at 37 ∘ C with 5% CO 2 . Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2017, Article ID 4063865, 12 pages https://doi.org/10.1155/2017/4063865

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Page 1: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Research ArticleCurcumin Induces p53-Null Hepatoma Cell Line Hep3BApoptosis through the AKT-PTEN-FOXO4 Pathway

An-Ting Liou1 Mei-Fang Chen2 and Chu-Wen Yang1

1Department of Microbiology Soochow University Shihlin Taipei 111 Taiwan2Department of Medical Research Taipei Veterans General Hospital Taipei 112 Taiwan

Correspondence should be addressed to Chu-Wen Yang ycw6861scuedutw

Received 25 February 2017 Accepted 6 June 2017 Published 9 July 2017

Academic Editor Jae Youl Cho

Copyright copy 2017 An-Ting Liou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective Curcumin (diferuloylmethane) is a yellow-colored polyphenol with antiproliferative and proapoptotic activities tovarious types of cancer cells This study explored the mechanism by which curcumin induces p53-null hepatoma cell apoptosisResults AKT FOXO1 and FOXO3 proteins were downregulated after curcumin treatment Conversely PTEN was upregulatedSubcellular fractionations revealed that the FOXO4 protein translocated from cytosol into the nucleus after curcumin treatmentOverexpression of FOXO4 increases the sensitivity of Hep3B cells to curcumin Knockdown of the FOXO4 gene by siRNA inhibitsthe proapoptotic effects of curcumin onHep3B cellConclusionsThis study revealed theAKTPTENFOXO4pathway as a potentialcandidate of target for treatment of p53-null liver cancers

1 Introduction

Hepatocellular carcinoma (HCC) is the sixth most commoncancer worldwide and is the third most common cause ofcancer-related death in the Asia-Pacific region [1] HCC is acancer with a high mortality rate [2] The prognosis of livercancer even in developed countries is very unfavorable IntheUS the five-year survival rates are 5 and 18 before 1989and after 2005 respectively [3] It has been reported that theabsence of p53 promotes HCC metastasis in a mouse model[4] Moreover mutations in the TP53 tumor suppressor genehave been reported in 23ndash67 of HCC patients worldwideand in 50 of HCC patients in China and South Africa [5]

Curcumin is a natural polyphenol found in the rhizomeof Curcuma longa (turmeric) [6] Curcuma longa has beentraditionally used in Asian countries as a medical herb forseveral pathologies due to its antioxidant anti-inflammatory[7] cancer chemoprevention [8 9] and anticancer properties[10ndash12]

Several studies have reported that curcumin couldinduce cancer cell apoptosis through p53-dependent andp53-independent pathways [13] It has been reported that

curcumin and related analogous compounds could induceapoptosis in hepatoma cells that express p53 protein normally(eg HepG2 cell) through p53p21 pathway [14ndash16]Howeverlittle is known regarding the effects of curcumin on p53-nullliver cancer cells In this study the PI3KAKTPTENFOXOpathway was shown to mediate curcumin induced apoptosisin p53-null Hep3B hepatoma cells

2 Materials and Methods

21 Chemicals and Antibodies Chemicals and PI3K inhibitorLY294002 were purchased from Sigma-Aldrich (Sigma-AldrichCo LLC) Antibodies of the signal transduction path-way apoptosis pathway and FOXO family were purchasedfrom Cell Signaling (Cell Signaling Technology Inc)

22 Cell Culture HepG2 (ATCC HB-8065) and Hep3B(ATCCHB8064) human hepatocellular carcinoma cell lineswere cultured in DMEM (Gibco) containing 10 FBS 1NEAA and 1 glutamine and incubated in an incubator at37∘C with 5 CO

2

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 4063865 12 pageshttpsdoiorg10115520174063865

2 Evidence-Based Complementary and Alternative Medicine

23 Flow Cytometry For cell-cycle analysis cells were har-vested and fixed dropwise with 70 ethanol After incubationovernight at 4∘C ethanol was removed by centrifuge andRNase A was added followed by a propidium iodide (PI)solution The cell-cycle stages of stained cells were ana-lyzed by flow cytometry (Cytomics FC500 Beckman) Forcaspase analysis 25 120583L of FITC labeled caspase substrates(OncoImmuno Inc) was incubated with cells for 45minsPI solution was added and incubated for another 15minsCaspase substrate and propidium iodide were removed bycentrifuge and cells were washed by PBS Caspase intensitywas detected by flow cytometry

24 Immunoblotting Each well of polyacrylamide gel wasloaded with 55 120583g cell lysate After SDS-PAGE the stackinggel was discarded and separating gel was taken to thesemi-dryer blotting system Once blotting was finished thenitrocellulose (NC) membrane was washed with TTBS andwas blocked by 5 milk in TTBS solution Washed NCmembrane was incubated with specific primary antibodieswith shaking at 4∘C overnight Secondary antibody solutionwas added and incubated with shaking at room temperaturefor 1 hour NC membrane with secondary antibody waswashed with TTBS 3 times to remove nonspecific bindingEnhanced chemiluminescence (ECL) substrate solution wasadded to determine the results using luminescence readers

25 Cloning and Sequencing PCR primers of FOXO4 genewere designed by the Primer 3 software (httpbioinfouteeprimer3-040primer3) The forward primer sequence isFOXO4F 51015840-GAGCAGGAAGCTGAGTGAGAG-31015840 Thereverse primer sequence is FOXO4R 51015840-CCTAGTCCC-AGGACGCTAGTC-31015840 PCR products were subcloned intopcDNA 31 TOPO TA Expression Vector (Invitrogen) andvalidated by Sanger sequencing in the Genome Center ofNational Yang-Ming University

26 RNA Interference siRNAs of PTEN FOXO1 FOXO3aand FOXO4 genes were purchased from Invitrogen siR-NAs were transfected using Lipofectamine 2000 (Invitrogen)according to the instructions of the manufacturer The effec-tiveness of the siRNAs on protein expression was validated byWestern blotting

27 Statistics Nonparametric two sample tests were per-formed using the Mann-Whitney 119880 test by the wilcoxtestfunction of R 119901 values less than 005 were considered to bestatistically significant

3 Results

31 HepG2 and Hep3B Cells Exhibit Different Sensitivity inCurcumin Induced Cell Death The p53 protein expression inHepG2 and Hep3B cells was first checked by Western blotanalysis (Figure 1) As expected p53 protein is expressedin HepG2 cells but not expressed in Hep3B cells Flowcytometrywas used to analyze curcumin inducedHepG2 and

Hep3B cell death (Figure 1) The subG1 portions of HepG2cells were increased from 188 plusmn 15 245 plusmn 36 and339 plusmn 29 after 50 120583M curcumin treatment at 12 18 and24 hours respectively In contrast the subG1 portions ofHep3B cells were 173 plusmn 13 and 247 plusmn 31 after 50120583Mcurcumin treatment at 18 to 24 hours respectively Theseresults suggested that HepG2 cells (which express wild-typep53 protein) were more sensitive to curcumin treatment thanp53-null Hep3B cells

32 Curcumin Induces Apoptosis in Hep3B Cells To confirmthat the subG1 portion observed in Hep3B cells shown inFigure 1 was due to apoptosis caspase-3 caspase-8 andcaspase-9 activities were analyzed by flow cytometry Asshown in Figure 2 caspase-3 caspase-8 and caspase-9 wereactivated after 12 hours of curcumin treatments Westernblotting further confirmed the results of flow cytometryassays (Figure 3) The observations that both caspase-8 andcaspase-9 were activated indicate that curcumin inducedapoptosis may be mediated by both of the extrinsic andintrinsic pathways simultaneously

33 Treatments of Curcumin Activate AKTPTENFOXOPathway in Hep3B Cells Since the Hep3B cells are p53-nullthe p53p21 pathway for apoptosis is defective the responseof AKTPTEN pathway (for cell survival signal) to curcumintreatment was analyzed by Western blot analysis As shownin Figure 4(a) PTEN and phospho-PTEN proteins increasedaccompanied by the decreased native and phosphorylatedforms (p-AKT 308T and 473S) of the AKT1 protein aftertreatments of 50 120583M curcumin The FOXO1 and FOXO3aproteins were downregulated after 50120583M curcumin treat-ments (Figure 4(a)) In contrast the FOXO4 protein wasupregulated after 50 120583M curcumin treatments Subcellularfractionation revealed that the FOXO1 and FOXO3a proteinswere downregulated in both cytosol and the nucleus after50 120583M curcumin treatments (Figure 4(b)) On the otherhand the FOXO4 protein was translocated from cytosol tothe nucleus

34 PI3K Inhibitor Enhances Curcumin Induced Caspase 3Activation in Hep3B Cells Knockdown of PTEN by siRNAin Hep3B cells led to an increase in the native and phos-phorylation forms (p-AKT 308T and 473S) of AKT1 proteins(Figure 5(a)) and an increase in cell growth (Figure 5(b))To elucidate whether the downregulation of phospho-AKT1proteins and upregulation of PTEN proteins are associ-ated with PI3K activity the PI3K inhibitor LY294002 wasused to treat Hep3B cells with curcumin As shown inFigure 5(c) the effects of curcumin on AKT1 and PTENwere enhanced by addition of the PI3K inhibitor LY294002Flow cytometry assays indicated that the caspase-3 activityinduced by curcumin was repressed by addition of the PI3Kinhibitor LY294002 (Figure 5(d)) These results suggest thatPI3K activity might be associated with curcumin inducedupregulation of PTEN proteins and downregulation of nativeand phospho-AKT1 proteins in Hep3B cells

Evidence-Based Complementary and Alternative Medicine 3

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Figure 1 Curcumin treatments induces HepG2 and Hep3B cell death The concentration of curcumin used in the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin) 119909-axis indicatespropidium iodide (PI) intensities

35 FOXO4 Knockdown Reduces Curcumin Induced Apoptosisin Hep3B Cells To reveal the roles of FOXO family proteinsin curcumin induced Hep3B cell apoptosis specific siRNAswere used to knockdown FOXO1 FOXO3a and FOXO4As shown in Figures 6(a) and 6(b) knockdown of FOXO1and FOXO3a slightly enhances the curcumin induced Hep3Bcell apoptosis However knockdown of FOXO4 reducedsubG1 portion of Hep3B cell after curcumin treatments (Fig-ure 6(c)) Flow cytometry assays indicated that the caspase-8 caspase-9 and caspase-3 activities induced by curcumin

were reduced by knockdown of FOXO4 (Figure 6(d)) Theseresults suggest the role for FOXO4 protein in mediatingcurcumin induced Hep3B cell apoptosis

36 FOXO4 Overexpression Increases Curcumin InducedApoptosis in Hep3B Cells To confirm the role of theFOXO4 protein in curcumin-induce Hep3B cell apoptosisthe FOXO4 gene was cloned and overexpressed in Hep3Bcells (Figure 7(a)) As shown in Figures 7(b) and 7(c)

4 Evidence-Based Complementary and Alternative Medicine

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Figure 2 Flow cytometry analysis for caspase activity after curcumin and DMSO treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583M 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The data arerepresentative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

Evidence-Based Complementary and Alternative Medicine 5

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Figure 3 Western blot analysis for caspases and PARP-1 cleavages after curcumin treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583MThe data are representative of triplicate experiments

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Figure 4 Curcumin treatments induce AKT pathway in Hep3B cells (a)Western blot analysis for AKT activation after curcumin treatments(b) Western blot analysis for FOXO protein localization after curcumin treatments in Hep3B cells The concentration of curcumin used inthe experiments was 50120583MThe data are representative of triplicate experiments

6 Evidence-Based Complementary and Alternative Medicine

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Figure 5 Associations of PI3KAKT1PTEN and curcumin treatment in Hep3B cells (a) Western blot analysis for AKT1 activation afterPTEN knockdown (b) Hep3B cell viability after PTEN knockdown The ldquolowastlowastrdquo indicates Mann-Whitney 119880 test 119901 lt 001 (c) Western blotanalysis for AKT1PTENFOXOs protein expression in experiments with and without addition of PI3K inhibitor LY294002 before curcumintreatments (d) Flow cytometry analysis for caspase-3 activities in experiments with and without addition of PI3K inhibitor LY294002 beforecurcumin treatments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The concentration of curcuminused in the experiments was 50120583MThedata are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solventof curcumin) LY LY294002

Evidence-Based Complementary and Alternative Medicine 7

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8 Evidence-Based Complementary and Alternative Medicine

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Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

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S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

2 Evidence-Based Complementary and Alternative Medicine

23 Flow Cytometry For cell-cycle analysis cells were har-vested and fixed dropwise with 70 ethanol After incubationovernight at 4∘C ethanol was removed by centrifuge andRNase A was added followed by a propidium iodide (PI)solution The cell-cycle stages of stained cells were ana-lyzed by flow cytometry (Cytomics FC500 Beckman) Forcaspase analysis 25 120583L of FITC labeled caspase substrates(OncoImmuno Inc) was incubated with cells for 45minsPI solution was added and incubated for another 15minsCaspase substrate and propidium iodide were removed bycentrifuge and cells were washed by PBS Caspase intensitywas detected by flow cytometry

24 Immunoblotting Each well of polyacrylamide gel wasloaded with 55 120583g cell lysate After SDS-PAGE the stackinggel was discarded and separating gel was taken to thesemi-dryer blotting system Once blotting was finished thenitrocellulose (NC) membrane was washed with TTBS andwas blocked by 5 milk in TTBS solution Washed NCmembrane was incubated with specific primary antibodieswith shaking at 4∘C overnight Secondary antibody solutionwas added and incubated with shaking at room temperaturefor 1 hour NC membrane with secondary antibody waswashed with TTBS 3 times to remove nonspecific bindingEnhanced chemiluminescence (ECL) substrate solution wasadded to determine the results using luminescence readers

25 Cloning and Sequencing PCR primers of FOXO4 genewere designed by the Primer 3 software (httpbioinfouteeprimer3-040primer3) The forward primer sequence isFOXO4F 51015840-GAGCAGGAAGCTGAGTGAGAG-31015840 Thereverse primer sequence is FOXO4R 51015840-CCTAGTCCC-AGGACGCTAGTC-31015840 PCR products were subcloned intopcDNA 31 TOPO TA Expression Vector (Invitrogen) andvalidated by Sanger sequencing in the Genome Center ofNational Yang-Ming University

26 RNA Interference siRNAs of PTEN FOXO1 FOXO3aand FOXO4 genes were purchased from Invitrogen siR-NAs were transfected using Lipofectamine 2000 (Invitrogen)according to the instructions of the manufacturer The effec-tiveness of the siRNAs on protein expression was validated byWestern blotting

27 Statistics Nonparametric two sample tests were per-formed using the Mann-Whitney 119880 test by the wilcoxtestfunction of R 119901 values less than 005 were considered to bestatistically significant

3 Results

31 HepG2 and Hep3B Cells Exhibit Different Sensitivity inCurcumin Induced Cell Death The p53 protein expression inHepG2 and Hep3B cells was first checked by Western blotanalysis (Figure 1) As expected p53 protein is expressedin HepG2 cells but not expressed in Hep3B cells Flowcytometrywas used to analyze curcumin inducedHepG2 and

Hep3B cell death (Figure 1) The subG1 portions of HepG2cells were increased from 188 plusmn 15 245 plusmn 36 and339 plusmn 29 after 50 120583M curcumin treatment at 12 18 and24 hours respectively In contrast the subG1 portions ofHep3B cells were 173 plusmn 13 and 247 plusmn 31 after 50120583Mcurcumin treatment at 18 to 24 hours respectively Theseresults suggested that HepG2 cells (which express wild-typep53 protein) were more sensitive to curcumin treatment thanp53-null Hep3B cells

32 Curcumin Induces Apoptosis in Hep3B Cells To confirmthat the subG1 portion observed in Hep3B cells shown inFigure 1 was due to apoptosis caspase-3 caspase-8 andcaspase-9 activities were analyzed by flow cytometry Asshown in Figure 2 caspase-3 caspase-8 and caspase-9 wereactivated after 12 hours of curcumin treatments Westernblotting further confirmed the results of flow cytometryassays (Figure 3) The observations that both caspase-8 andcaspase-9 were activated indicate that curcumin inducedapoptosis may be mediated by both of the extrinsic andintrinsic pathways simultaneously

33 Treatments of Curcumin Activate AKTPTENFOXOPathway in Hep3B Cells Since the Hep3B cells are p53-nullthe p53p21 pathway for apoptosis is defective the responseof AKTPTEN pathway (for cell survival signal) to curcumintreatment was analyzed by Western blot analysis As shownin Figure 4(a) PTEN and phospho-PTEN proteins increasedaccompanied by the decreased native and phosphorylatedforms (p-AKT 308T and 473S) of the AKT1 protein aftertreatments of 50 120583M curcumin The FOXO1 and FOXO3aproteins were downregulated after 50120583M curcumin treat-ments (Figure 4(a)) In contrast the FOXO4 protein wasupregulated after 50 120583M curcumin treatments Subcellularfractionation revealed that the FOXO1 and FOXO3a proteinswere downregulated in both cytosol and the nucleus after50 120583M curcumin treatments (Figure 4(b)) On the otherhand the FOXO4 protein was translocated from cytosol tothe nucleus

34 PI3K Inhibitor Enhances Curcumin Induced Caspase 3Activation in Hep3B Cells Knockdown of PTEN by siRNAin Hep3B cells led to an increase in the native and phos-phorylation forms (p-AKT 308T and 473S) of AKT1 proteins(Figure 5(a)) and an increase in cell growth (Figure 5(b))To elucidate whether the downregulation of phospho-AKT1proteins and upregulation of PTEN proteins are associ-ated with PI3K activity the PI3K inhibitor LY294002 wasused to treat Hep3B cells with curcumin As shown inFigure 5(c) the effects of curcumin on AKT1 and PTENwere enhanced by addition of the PI3K inhibitor LY294002Flow cytometry assays indicated that the caspase-3 activityinduced by curcumin was repressed by addition of the PI3Kinhibitor LY294002 (Figure 5(d)) These results suggest thatPI3K activity might be associated with curcumin inducedupregulation of PTEN proteins and downregulation of nativeand phospho-AKT1 proteins in Hep3B cells

Evidence-Based Complementary and Alternative Medicine 3

G2M 210

S 152

G0G1 592

FL3 Lin0 1023

290

subG1 173

S 105

G2M 195

G0G1 500

FL3 Lin0 1023

169

subG1 247

S78

G2M 184

G0G1 410

FL3 Lin0 1023

151

S 103

G2M 286

G0G1 543

FL3 Lin0 1023

251

S 116

G2M 225

G0G1 578

FL3 Lin0 1023

345

S97

G2M 208

G0G1 612

FL3 Lin0 1023

363

S 118

G0G1 550

subG135

FL3 Lin0 1023

88

subG1 188

G2M 170

S91

G0G1 471

FL3 Lin0 1023

106

G2M 131

S 102

G0G1 488

subG1 245

FL3 Lin0 1023

91

subG1 339

G2M 130

S67

G0G1 430

FL3 Lin0 1023

78

G2M 185

S99

G0G1 666

FL3 Lin0 1023

331

G2M 160

S 119

G0G1 689

FL3 Lin0 1023

207

G2M 193

S 145

G0G1 631

FL3 Lin0 1023

190

G2M 185

S75

G0G1 698

FL3 Lin0 1023

180

S 114

G2M 212

G0G1 622

FL3 Lin0 1023

294

G2M

S 174

534

FL3 Lin0 1023

330

S 122

G2M267

G0G1 547

FL3 Lin0 1023

309

G2M181

S 127

FL3 Lin0 1023

214 G0G1

664

G0G1

250

G2M 203

Hep3B0 hr

Hep3BCUR

Hep3BCUR

Hep3BCUR

Hep3BCUR

3 hr 12 hr 18 hr 24 hr

12 hr 18 hr 24 hr

12 hr 18 hr 24 hr

Hep3BDMSO

Hep3BDMSO

Hep3BDMSO

Hep3BDMSO

3 hr

HepG2

H

P53

ep3B HepG2

0 hr

HepG2

CURHepG2

CURHepG2

CURHepG2

CUR

HepG2

DMSOHepG2

DMSOHepG2

DMSOHepG2

DMSO3 hr

12 hr 18 hr 24 hr3 hr

Figure 1 Curcumin treatments induces HepG2 and Hep3B cell death The concentration of curcumin used in the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin) 119909-axis indicatespropidium iodide (PI) intensities

35 FOXO4 Knockdown Reduces Curcumin Induced Apoptosisin Hep3B Cells To reveal the roles of FOXO family proteinsin curcumin induced Hep3B cell apoptosis specific siRNAswere used to knockdown FOXO1 FOXO3a and FOXO4As shown in Figures 6(a) and 6(b) knockdown of FOXO1and FOXO3a slightly enhances the curcumin induced Hep3Bcell apoptosis However knockdown of FOXO4 reducedsubG1 portion of Hep3B cell after curcumin treatments (Fig-ure 6(c)) Flow cytometry assays indicated that the caspase-8 caspase-9 and caspase-3 activities induced by curcumin

were reduced by knockdown of FOXO4 (Figure 6(d)) Theseresults suggest the role for FOXO4 protein in mediatingcurcumin induced Hep3B cell apoptosis

36 FOXO4 Overexpression Increases Curcumin InducedApoptosis in Hep3B Cells To confirm the role of theFOXO4 protein in curcumin-induce Hep3B cell apoptosisthe FOXO4 gene was cloned and overexpressed in Hep3Bcells (Figure 7(a)) As shown in Figures 7(b) and 7(c)

4 Evidence-Based Complementary and Alternative Medicine

A208

A116

FL1 log

FL3

log

103

102

101

100

103102101100

A2114

A116

A402

A300

A2 998

A100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

A202

A113

A207

A123

FL1 log

FL3

log

103

102

101

100

103102101100

A203

A120

FL1 log

FL3

log

103

102

101

100

103102101100

A204

A177

FL1 log

FL3

log

103

102

101

100

103102101100

A280

A1356

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

A2557

A1418

A203

A177

FL1 log

FL3

log

103

102

101

100

103102101100

A205

A165

A238

A1162

FL1 log

FL3

log

103

102

101

100

103102101100

A277

A1203

FL1 log

FL3

log

103

102

101

100

103102101100

A4512

A300

A2488

A100

A2913

A106

Caspase-8 Caspase-9 Caspase-30

hrD

MSO

12h

rD

MSO

24hr

CUR

12hr

CUR

24hr

2A20808

1A116

A418

A3959

2A2114

1A116

A4492

A3378

2A202

1A113

A406

A3978

2A207

1A123

A434

A3935

2A203

1A120

A4142

A3835

2A29133333

1A106

A481

A300

2A238

1A1162

A4112

A3688

2A277

1A1203

A4171

A3549

2A204

1A177

A400

A3918

2A280

1A1356

A413

A3551

2A2557

1A1418

A414

A311

2A203

1A177

A423

A3897

2A205

1A165

A402

A3928

Figure 2 Flow cytometry analysis for caspase activity after curcumin and DMSO treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583M 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The data arerepresentative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

Evidence-Based Complementary and Alternative Medicine 5

Pro-caspase-3

Caspase-8

Cleavage

Caspase-8

Pro-caspase-9

Cleavage

Caspase-9

Cleavage

Caspase-3

PARP-1

Full length

PARP-1

Cleavage

Curcumin

-Actin

0 hr 12 hr 18 hr 24 hr

Figure 3 Western blot analysis for caspases and PARP-1 cleavages after curcumin treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583MThe data are representative of triplicate experiments

AKT1

FOXO4

PTEN

p-PTEN

FOXO1

Curcumin

FOXO3a

p-AKT(473S)

p-AKT(308T)

0 hr 12 hr 18 hr

3 hr

-Actin

(a)

Cytosol NucleusCurcumin

FOXO1

FOXO3a

FOXO4

GAPDH

Histone H1

p-FOXO4(193S)

p-FOXO3a(ser318321)

p-FOXO1(ser256)

p-FOXO3a(253S)

0 hr 3 hr 8 hr 12 hr0 hr 3 hr 8 hr 12 hr

(b)

Figure 4 Curcumin treatments induce AKT pathway in Hep3B cells (a)Western blot analysis for AKT activation after curcumin treatments(b) Western blot analysis for FOXO protein localization after curcumin treatments in Hep3B cells The concentration of curcumin used inthe experiments was 50120583MThe data are representative of triplicate experiments

6 Evidence-Based Complementary and Alternative Medicine

PTEN siRNA

PTEN

p-PTEN

p-AKT (308)

p-AKT (473)

NS

AKT

-Actin

(a)C

ell n

umbe

r

PTENsiRNA

NS 0

50000

100000

150000

200000

250000lowastlowast

(b)

p-AKT (308)

p-AKT (473)

PTEN

p-PTEN

AKT

FOXO4

FOXO1

FOXO3a

DMSO CUR + LY CUR

-Actin

(c)

A4444

A359

A2493

A103

103

102

101

100

103102101100

103

102

101

100

103102101100

103

102

101

100

103102101100

A471

A300

A2929

A100

A201

A114

DMSO

CUR

CUR + Ly

A400

2A201

1A114

DMSOA400

A3985

FL1 log

FL1 log

FL1 log

FL3

log

FL3

log

FL3

log

(d)

Figure 5 Associations of PI3KAKT1PTEN and curcumin treatment in Hep3B cells (a) Western blot analysis for AKT1 activation afterPTEN knockdown (b) Hep3B cell viability after PTEN knockdown The ldquolowastlowastrdquo indicates Mann-Whitney 119880 test 119901 lt 001 (c) Western blotanalysis for AKT1PTENFOXOs protein expression in experiments with and without addition of PI3K inhibitor LY294002 before curcumintreatments (d) Flow cytometry analysis for caspase-3 activities in experiments with and without addition of PI3K inhibitor LY294002 beforecurcumin treatments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The concentration of curcuminused in the experiments was 50120583MThedata are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solventof curcumin) LY LY294002

Evidence-Based Complementary and Alternative Medicine 7

FOXO1

FOXO3a

FOXO4

NS FOXO1

siRNA

S + G2M 251

G0G1 516

subG1 208

FL3 Lin FL3 Lin

0 1023

78

G2M 134

S 118

G0G1 443

subG1 298

0 1023

66

FOXO1siRNA + CUR

NS + CUR

-Actin

(a)

NS FOXO3a

siRNA

G2M 177

S92

G0G1 490

F 219

75

G2M 162

S 100

G0G1 428

subG1 295

49

FOXO1

FOXO3a

FOXO4

FL3 Lin

0 1023

FL3 Lin

0 1023

FOXO3asiRNA + CUR

NS + CUR

-Actin

(b)

NS FOXO4siRNA

G2M 158

S 138

G0G1 480

subG1 215

85

subG1 123

G2M 200

S82

G0G1 583

85

FOXO1

FOXO3a

FOXO4

FL3 Lin0 1023

FL3 Lin0 1023

FOXO4siRNA + CUR

NS + CUR

-Actin

(c)

Figure 6 Continued

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Evidence-Based Complementary and Alternative Medicine 3

G2M 210

S 152

G0G1 592

FL3 Lin0 1023

290

subG1 173

S 105

G2M 195

G0G1 500

FL3 Lin0 1023

169

subG1 247

S78

G2M 184

G0G1 410

FL3 Lin0 1023

151

S 103

G2M 286

G0G1 543

FL3 Lin0 1023

251

S 116

G2M 225

G0G1 578

FL3 Lin0 1023

345

S97

G2M 208

G0G1 612

FL3 Lin0 1023

363

S 118

G0G1 550

subG135

FL3 Lin0 1023

88

subG1 188

G2M 170

S91

G0G1 471

FL3 Lin0 1023

106

G2M 131

S 102

G0G1 488

subG1 245

FL3 Lin0 1023

91

subG1 339

G2M 130

S67

G0G1 430

FL3 Lin0 1023

78

G2M 185

S99

G0G1 666

FL3 Lin0 1023

331

G2M 160

S 119

G0G1 689

FL3 Lin0 1023

207

G2M 193

S 145

G0G1 631

FL3 Lin0 1023

190

G2M 185

S75

G0G1 698

FL3 Lin0 1023

180

S 114

G2M 212

G0G1 622

FL3 Lin0 1023

294

G2M

S 174

534

FL3 Lin0 1023

330

S 122

G2M267

G0G1 547

FL3 Lin0 1023

309

G2M181

S 127

FL3 Lin0 1023

214 G0G1

664

G0G1

250

G2M 203

Hep3B0 hr

Hep3BCUR

Hep3BCUR

Hep3BCUR

Hep3BCUR

3 hr 12 hr 18 hr 24 hr

12 hr 18 hr 24 hr

12 hr 18 hr 24 hr

Hep3BDMSO

Hep3BDMSO

Hep3BDMSO

Hep3BDMSO

3 hr

HepG2

H

P53

ep3B HepG2

0 hr

HepG2

CURHepG2

CURHepG2

CURHepG2

CUR

HepG2

DMSOHepG2

DMSOHepG2

DMSOHepG2

DMSO3 hr

12 hr 18 hr 24 hr3 hr

Figure 1 Curcumin treatments induces HepG2 and Hep3B cell death The concentration of curcumin used in the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin) 119909-axis indicatespropidium iodide (PI) intensities

35 FOXO4 Knockdown Reduces Curcumin Induced Apoptosisin Hep3B Cells To reveal the roles of FOXO family proteinsin curcumin induced Hep3B cell apoptosis specific siRNAswere used to knockdown FOXO1 FOXO3a and FOXO4As shown in Figures 6(a) and 6(b) knockdown of FOXO1and FOXO3a slightly enhances the curcumin induced Hep3Bcell apoptosis However knockdown of FOXO4 reducedsubG1 portion of Hep3B cell after curcumin treatments (Fig-ure 6(c)) Flow cytometry assays indicated that the caspase-8 caspase-9 and caspase-3 activities induced by curcumin

were reduced by knockdown of FOXO4 (Figure 6(d)) Theseresults suggest the role for FOXO4 protein in mediatingcurcumin induced Hep3B cell apoptosis

36 FOXO4 Overexpression Increases Curcumin InducedApoptosis in Hep3B Cells To confirm the role of theFOXO4 protein in curcumin-induce Hep3B cell apoptosisthe FOXO4 gene was cloned and overexpressed in Hep3Bcells (Figure 7(a)) As shown in Figures 7(b) and 7(c)

4 Evidence-Based Complementary and Alternative Medicine

A208

A116

FL1 log

FL3

log

103

102

101

100

103102101100

A2114

A116

A402

A300

A2 998

A100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

A202

A113

A207

A123

FL1 log

FL3

log

103

102

101

100

103102101100

A203

A120

FL1 log

FL3

log

103

102

101

100

103102101100

A204

A177

FL1 log

FL3

log

103

102

101

100

103102101100

A280

A1356

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

A2557

A1418

A203

A177

FL1 log

FL3

log

103

102

101

100

103102101100

A205

A165

A238

A1162

FL1 log

FL3

log

103

102

101

100

103102101100

A277

A1203

FL1 log

FL3

log

103

102

101

100

103102101100

A4512

A300

A2488

A100

A2913

A106

Caspase-8 Caspase-9 Caspase-30

hrD

MSO

12h

rD

MSO

24hr

CUR

12hr

CUR

24hr

2A20808

1A116

A418

A3959

2A2114

1A116

A4492

A3378

2A202

1A113

A406

A3978

2A207

1A123

A434

A3935

2A203

1A120

A4142

A3835

2A29133333

1A106

A481

A300

2A238

1A1162

A4112

A3688

2A277

1A1203

A4171

A3549

2A204

1A177

A400

A3918

2A280

1A1356

A413

A3551

2A2557

1A1418

A414

A311

2A203

1A177

A423

A3897

2A205

1A165

A402

A3928

Figure 2 Flow cytometry analysis for caspase activity after curcumin and DMSO treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583M 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The data arerepresentative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

Evidence-Based Complementary and Alternative Medicine 5

Pro-caspase-3

Caspase-8

Cleavage

Caspase-8

Pro-caspase-9

Cleavage

Caspase-9

Cleavage

Caspase-3

PARP-1

Full length

PARP-1

Cleavage

Curcumin

-Actin

0 hr 12 hr 18 hr 24 hr

Figure 3 Western blot analysis for caspases and PARP-1 cleavages after curcumin treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583MThe data are representative of triplicate experiments

AKT1

FOXO4

PTEN

p-PTEN

FOXO1

Curcumin

FOXO3a

p-AKT(473S)

p-AKT(308T)

0 hr 12 hr 18 hr

3 hr

-Actin

(a)

Cytosol NucleusCurcumin

FOXO1

FOXO3a

FOXO4

GAPDH

Histone H1

p-FOXO4(193S)

p-FOXO3a(ser318321)

p-FOXO1(ser256)

p-FOXO3a(253S)

0 hr 3 hr 8 hr 12 hr0 hr 3 hr 8 hr 12 hr

(b)

Figure 4 Curcumin treatments induce AKT pathway in Hep3B cells (a)Western blot analysis for AKT activation after curcumin treatments(b) Western blot analysis for FOXO protein localization after curcumin treatments in Hep3B cells The concentration of curcumin used inthe experiments was 50120583MThe data are representative of triplicate experiments

6 Evidence-Based Complementary and Alternative Medicine

PTEN siRNA

PTEN

p-PTEN

p-AKT (308)

p-AKT (473)

NS

AKT

-Actin

(a)C

ell n

umbe

r

PTENsiRNA

NS 0

50000

100000

150000

200000

250000lowastlowast

(b)

p-AKT (308)

p-AKT (473)

PTEN

p-PTEN

AKT

FOXO4

FOXO1

FOXO3a

DMSO CUR + LY CUR

-Actin

(c)

A4444

A359

A2493

A103

103

102

101

100

103102101100

103

102

101

100

103102101100

103

102

101

100

103102101100

A471

A300

A2929

A100

A201

A114

DMSO

CUR

CUR + Ly

A400

2A201

1A114

DMSOA400

A3985

FL1 log

FL1 log

FL1 log

FL3

log

FL3

log

FL3

log

(d)

Figure 5 Associations of PI3KAKT1PTEN and curcumin treatment in Hep3B cells (a) Western blot analysis for AKT1 activation afterPTEN knockdown (b) Hep3B cell viability after PTEN knockdown The ldquolowastlowastrdquo indicates Mann-Whitney 119880 test 119901 lt 001 (c) Western blotanalysis for AKT1PTENFOXOs protein expression in experiments with and without addition of PI3K inhibitor LY294002 before curcumintreatments (d) Flow cytometry analysis for caspase-3 activities in experiments with and without addition of PI3K inhibitor LY294002 beforecurcumin treatments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The concentration of curcuminused in the experiments was 50120583MThedata are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solventof curcumin) LY LY294002

Evidence-Based Complementary and Alternative Medicine 7

FOXO1

FOXO3a

FOXO4

NS FOXO1

siRNA

S + G2M 251

G0G1 516

subG1 208

FL3 Lin FL3 Lin

0 1023

78

G2M 134

S 118

G0G1 443

subG1 298

0 1023

66

FOXO1siRNA + CUR

NS + CUR

-Actin

(a)

NS FOXO3a

siRNA

G2M 177

S92

G0G1 490

F 219

75

G2M 162

S 100

G0G1 428

subG1 295

49

FOXO1

FOXO3a

FOXO4

FL3 Lin

0 1023

FL3 Lin

0 1023

FOXO3asiRNA + CUR

NS + CUR

-Actin

(b)

NS FOXO4siRNA

G2M 158

S 138

G0G1 480

subG1 215

85

subG1 123

G2M 200

S82

G0G1 583

85

FOXO1

FOXO3a

FOXO4

FL3 Lin0 1023

FL3 Lin0 1023

FOXO4siRNA + CUR

NS + CUR

-Actin

(c)

Figure 6 Continued

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

4 Evidence-Based Complementary and Alternative Medicine

A208

A116

FL1 log

FL3

log

103

102

101

100

103102101100

A2114

A116

A402

A300

A2 998

A100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

A202

A113

A207

A123

FL1 log

FL3

log

103

102

101

100

103102101100

A203

A120

FL1 log

FL3

log

103

102

101

100

103102101100

A204

A177

FL1 log

FL3

log

103

102

101

100

103102101100

A280

A1356

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

FL1 log

FL3

log

103

102

101

100

103102101100

A2557

A1418

A203

A177

FL1 log

FL3

log

103

102

101

100

103102101100

A205

A165

A238

A1162

FL1 log

FL3

log

103

102

101

100

103102101100

A277

A1203

FL1 log

FL3

log

103

102

101

100

103102101100

A4512

A300

A2488

A100

A2913

A106

Caspase-8 Caspase-9 Caspase-30

hrD

MSO

12h

rD

MSO

24hr

CUR

12hr

CUR

24hr

2A20808

1A116

A418

A3959

2A2114

1A116

A4492

A3378

2A202

1A113

A406

A3978

2A207

1A123

A434

A3935

2A203

1A120

A4142

A3835

2A29133333

1A106

A481

A300

2A238

1A1162

A4112

A3688

2A277

1A1203

A4171

A3549

2A204

1A177

A400

A3918

2A280

1A1356

A413

A3551

2A2557

1A1418

A414

A311

2A203

1A177

A423

A3897

2A205

1A165

A402

A3928

Figure 2 Flow cytometry analysis for caspase activity after curcumin and DMSO treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583M 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The data arerepresentative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

Evidence-Based Complementary and Alternative Medicine 5

Pro-caspase-3

Caspase-8

Cleavage

Caspase-8

Pro-caspase-9

Cleavage

Caspase-9

Cleavage

Caspase-3

PARP-1

Full length

PARP-1

Cleavage

Curcumin

-Actin

0 hr 12 hr 18 hr 24 hr

Figure 3 Western blot analysis for caspases and PARP-1 cleavages after curcumin treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583MThe data are representative of triplicate experiments

AKT1

FOXO4

PTEN

p-PTEN

FOXO1

Curcumin

FOXO3a

p-AKT(473S)

p-AKT(308T)

0 hr 12 hr 18 hr

3 hr

-Actin

(a)

Cytosol NucleusCurcumin

FOXO1

FOXO3a

FOXO4

GAPDH

Histone H1

p-FOXO4(193S)

p-FOXO3a(ser318321)

p-FOXO1(ser256)

p-FOXO3a(253S)

0 hr 3 hr 8 hr 12 hr0 hr 3 hr 8 hr 12 hr

(b)

Figure 4 Curcumin treatments induce AKT pathway in Hep3B cells (a)Western blot analysis for AKT activation after curcumin treatments(b) Western blot analysis for FOXO protein localization after curcumin treatments in Hep3B cells The concentration of curcumin used inthe experiments was 50120583MThe data are representative of triplicate experiments

6 Evidence-Based Complementary and Alternative Medicine

PTEN siRNA

PTEN

p-PTEN

p-AKT (308)

p-AKT (473)

NS

AKT

-Actin

(a)C

ell n

umbe

r

PTENsiRNA

NS 0

50000

100000

150000

200000

250000lowastlowast

(b)

p-AKT (308)

p-AKT (473)

PTEN

p-PTEN

AKT

FOXO4

FOXO1

FOXO3a

DMSO CUR + LY CUR

-Actin

(c)

A4444

A359

A2493

A103

103

102

101

100

103102101100

103

102

101

100

103102101100

103

102

101

100

103102101100

A471

A300

A2929

A100

A201

A114

DMSO

CUR

CUR + Ly

A400

2A201

1A114

DMSOA400

A3985

FL1 log

FL1 log

FL1 log

FL3

log

FL3

log

FL3

log

(d)

Figure 5 Associations of PI3KAKT1PTEN and curcumin treatment in Hep3B cells (a) Western blot analysis for AKT1 activation afterPTEN knockdown (b) Hep3B cell viability after PTEN knockdown The ldquolowastlowastrdquo indicates Mann-Whitney 119880 test 119901 lt 001 (c) Western blotanalysis for AKT1PTENFOXOs protein expression in experiments with and without addition of PI3K inhibitor LY294002 before curcumintreatments (d) Flow cytometry analysis for caspase-3 activities in experiments with and without addition of PI3K inhibitor LY294002 beforecurcumin treatments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The concentration of curcuminused in the experiments was 50120583MThedata are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solventof curcumin) LY LY294002

Evidence-Based Complementary and Alternative Medicine 7

FOXO1

FOXO3a

FOXO4

NS FOXO1

siRNA

S + G2M 251

G0G1 516

subG1 208

FL3 Lin FL3 Lin

0 1023

78

G2M 134

S 118

G0G1 443

subG1 298

0 1023

66

FOXO1siRNA + CUR

NS + CUR

-Actin

(a)

NS FOXO3a

siRNA

G2M 177

S92

G0G1 490

F 219

75

G2M 162

S 100

G0G1 428

subG1 295

49

FOXO1

FOXO3a

FOXO4

FL3 Lin

0 1023

FL3 Lin

0 1023

FOXO3asiRNA + CUR

NS + CUR

-Actin

(b)

NS FOXO4siRNA

G2M 158

S 138

G0G1 480

subG1 215

85

subG1 123

G2M 200

S82

G0G1 583

85

FOXO1

FOXO3a

FOXO4

FL3 Lin0 1023

FL3 Lin0 1023

FOXO4siRNA + CUR

NS + CUR

-Actin

(c)

Figure 6 Continued

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Evidence-Based Complementary and Alternative Medicine 5

Pro-caspase-3

Caspase-8

Cleavage

Caspase-8

Pro-caspase-9

Cleavage

Caspase-9

Cleavage

Caspase-3

PARP-1

Full length

PARP-1

Cleavage

Curcumin

-Actin

0 hr 12 hr 18 hr 24 hr

Figure 3 Western blot analysis for caspases and PARP-1 cleavages after curcumin treatments in Hep3B cells The concentration of curcuminused in the experiments was 50120583MThe data are representative of triplicate experiments

AKT1

FOXO4

PTEN

p-PTEN

FOXO1

Curcumin

FOXO3a

p-AKT(473S)

p-AKT(308T)

0 hr 12 hr 18 hr

3 hr

-Actin

(a)

Cytosol NucleusCurcumin

FOXO1

FOXO3a

FOXO4

GAPDH

Histone H1

p-FOXO4(193S)

p-FOXO3a(ser318321)

p-FOXO1(ser256)

p-FOXO3a(253S)

0 hr 3 hr 8 hr 12 hr0 hr 3 hr 8 hr 12 hr

(b)

Figure 4 Curcumin treatments induce AKT pathway in Hep3B cells (a)Western blot analysis for AKT activation after curcumin treatments(b) Western blot analysis for FOXO protein localization after curcumin treatments in Hep3B cells The concentration of curcumin used inthe experiments was 50120583MThe data are representative of triplicate experiments

6 Evidence-Based Complementary and Alternative Medicine

PTEN siRNA

PTEN

p-PTEN

p-AKT (308)

p-AKT (473)

NS

AKT

-Actin

(a)C

ell n

umbe

r

PTENsiRNA

NS 0

50000

100000

150000

200000

250000lowastlowast

(b)

p-AKT (308)

p-AKT (473)

PTEN

p-PTEN

AKT

FOXO4

FOXO1

FOXO3a

DMSO CUR + LY CUR

-Actin

(c)

A4444

A359

A2493

A103

103

102

101

100

103102101100

103

102

101

100

103102101100

103

102

101

100

103102101100

A471

A300

A2929

A100

A201

A114

DMSO

CUR

CUR + Ly

A400

2A201

1A114

DMSOA400

A3985

FL1 log

FL1 log

FL1 log

FL3

log

FL3

log

FL3

log

(d)

Figure 5 Associations of PI3KAKT1PTEN and curcumin treatment in Hep3B cells (a) Western blot analysis for AKT1 activation afterPTEN knockdown (b) Hep3B cell viability after PTEN knockdown The ldquolowastlowastrdquo indicates Mann-Whitney 119880 test 119901 lt 001 (c) Western blotanalysis for AKT1PTENFOXOs protein expression in experiments with and without addition of PI3K inhibitor LY294002 before curcumintreatments (d) Flow cytometry analysis for caspase-3 activities in experiments with and without addition of PI3K inhibitor LY294002 beforecurcumin treatments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The concentration of curcuminused in the experiments was 50120583MThedata are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solventof curcumin) LY LY294002

Evidence-Based Complementary and Alternative Medicine 7

FOXO1

FOXO3a

FOXO4

NS FOXO1

siRNA

S + G2M 251

G0G1 516

subG1 208

FL3 Lin FL3 Lin

0 1023

78

G2M 134

S 118

G0G1 443

subG1 298

0 1023

66

FOXO1siRNA + CUR

NS + CUR

-Actin

(a)

NS FOXO3a

siRNA

G2M 177

S92

G0G1 490

F 219

75

G2M 162

S 100

G0G1 428

subG1 295

49

FOXO1

FOXO3a

FOXO4

FL3 Lin

0 1023

FL3 Lin

0 1023

FOXO3asiRNA + CUR

NS + CUR

-Actin

(b)

NS FOXO4siRNA

G2M 158

S 138

G0G1 480

subG1 215

85

subG1 123

G2M 200

S82

G0G1 583

85

FOXO1

FOXO3a

FOXO4

FL3 Lin0 1023

FL3 Lin0 1023

FOXO4siRNA + CUR

NS + CUR

-Actin

(c)

Figure 6 Continued

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

6 Evidence-Based Complementary and Alternative Medicine

PTEN siRNA

PTEN

p-PTEN

p-AKT (308)

p-AKT (473)

NS

AKT

-Actin

(a)C

ell n

umbe

r

PTENsiRNA

NS 0

50000

100000

150000

200000

250000lowastlowast

(b)

p-AKT (308)

p-AKT (473)

PTEN

p-PTEN

AKT

FOXO4

FOXO1

FOXO3a

DMSO CUR + LY CUR

-Actin

(c)

A4444

A359

A2493

A103

103

102

101

100

103102101100

103

102

101

100

103102101100

103

102

101

100

103102101100

A471

A300

A2929

A100

A201

A114

DMSO

CUR

CUR + Ly

A400

2A201

1A114

DMSOA400

A3985

FL1 log

FL1 log

FL1 log

FL3

log

FL3

log

FL3

log

(d)

Figure 5 Associations of PI3KAKT1PTEN and curcumin treatment in Hep3B cells (a) Western blot analysis for AKT1 activation afterPTEN knockdown (b) Hep3B cell viability after PTEN knockdown The ldquolowastlowastrdquo indicates Mann-Whitney 119880 test 119901 lt 001 (c) Western blotanalysis for AKT1PTENFOXOs protein expression in experiments with and without addition of PI3K inhibitor LY294002 before curcumintreatments (d) Flow cytometry analysis for caspase-3 activities in experiments with and without addition of PI3K inhibitor LY294002 beforecurcumin treatments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicates caspase activities The concentration of curcuminused in the experiments was 50120583MThedata are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solventof curcumin) LY LY294002

Evidence-Based Complementary and Alternative Medicine 7

FOXO1

FOXO3a

FOXO4

NS FOXO1

siRNA

S + G2M 251

G0G1 516

subG1 208

FL3 Lin FL3 Lin

0 1023

78

G2M 134

S 118

G0G1 443

subG1 298

0 1023

66

FOXO1siRNA + CUR

NS + CUR

-Actin

(a)

NS FOXO3a

siRNA

G2M 177

S92

G0G1 490

F 219

75

G2M 162

S 100

G0G1 428

subG1 295

49

FOXO1

FOXO3a

FOXO4

FL3 Lin

0 1023

FL3 Lin

0 1023

FOXO3asiRNA + CUR

NS + CUR

-Actin

(b)

NS FOXO4siRNA

G2M 158

S 138

G0G1 480

subG1 215

85

subG1 123

G2M 200

S82

G0G1 583

85

FOXO1

FOXO3a

FOXO4

FL3 Lin0 1023

FL3 Lin0 1023

FOXO4siRNA + CUR

NS + CUR

-Actin

(c)

Figure 6 Continued

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Evidence-Based Complementary and Alternative Medicine 7

FOXO1

FOXO3a

FOXO4

NS FOXO1

siRNA

S + G2M 251

G0G1 516

subG1 208

FL3 Lin FL3 Lin

0 1023

78

G2M 134

S 118

G0G1 443

subG1 298

0 1023

66

FOXO1siRNA + CUR

NS + CUR

-Actin

(a)

NS FOXO3a

siRNA

G2M 177

S92

G0G1 490

F 219

75

G2M 162

S 100

G0G1 428

subG1 295

49

FOXO1

FOXO3a

FOXO4

FL3 Lin

0 1023

FL3 Lin

0 1023

FOXO3asiRNA + CUR

NS + CUR

-Actin

(b)

NS FOXO4siRNA

G2M 158

S 138

G0G1 480

subG1 215

85

subG1 123

G2M 200

S82

G0G1 583

85

FOXO1

FOXO3a

FOXO4

FL3 Lin0 1023

FL3 Lin0 1023

FOXO4siRNA + CUR

NS + CUR

-Actin

(c)

Figure 6 Continued

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

8 Evidence-Based Complementary and Alternative Medicine

A400

A300

A2 721

A1 279

4A400

3A300

2A2721

1A1279

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

FL1 log

FL3

log

103

102

101

100

103

102

101

100

A432

A3905

A213

A151

A400

A300

A2403

A1596

4A400

3A300

2A24033

1A1596

A400

A300

A2689

A1311

4A400

3A300

2A2689

1A1311

A401

A209

A1192

4A4AAAA01

2A209

1A1192

A3798 A4

00A300

A2432

A1568

4A400

3A300

2A2432

1A1568

A400

A300

A2703

A1298

4A400

3A300

2A2703

1A1298

A203

A142

2A203

1A142

A431 A4

01A306

A2441

A1552

4A401

3A306666

2A2441

1A1552

NS + CUR FOXO4 siRNA + CURNS + DMSO

Casp

ase-

9Ca

spas

e-8

Casp

ase-

3

444AA4AAAA4AAA4A4AAAAA44AAAAAAAAAAAAAAA32333 2323223

3A3AAAAAAAA3333905

2A213

1A151

A3925

(d)

Figure 6 Effects of FOXO knockdown on curcumin induced Hep3B cell apoptosis (a) FOXO1 knockdown (b) FOXO3a knockdown (c)FOXO4 knockdown (d) caspase activities of FOXO4 knockdown experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axisindicates caspase activities The concentration of curcumin used in the experiments was 50120583M The data are representative of triplicateexperiments CUR curcumin DMSO dimethyl sulfoxide (solvent of curcumin)

overexpression of FOXO4 in Hep3B cells induced apoptosisMoreover Hep3B cells with overexpressed FOXO4 proteinsexhibited higher sensitivity to curcumin

4 Discussion

The expansion of tumor cell populations is dependent onboth the rates of cell proliferation and cell death Apoptosisis a major source of cell death Therefore agents that triggerapoptosiscell death could be the most promising candidatesof cancer therapies In the past years a number of studies havebeen published describing the anticancer effects of curcuminThese investigations could be divided into three categories (1)anti-inflammatory antioxidant and chemoprevention activ-ities [8 9] (2) antiproliferative and proapoptotic activities[11ndash13] (3) anti-invasive and antimetastasis activities [10]It has been shown that curcumin modulates various tar-gets including inflammatory pathway transcription factorsprotein kinases the cell survival pathway drug resistanceproteins adhesion molecules growth factor receptors and

the cell-cycle regulatory pathway [6] One of the major path-ways regarding antiproliferative and proapoptotic activities ofcurcumin is the p53-dependent (p53p21) pathway [13] Asshown in Figure 1 the HepG2 hepatoma cell that expresseswild-type p53 protein is more sensitive to curcumin inducedcell death than the p53-null Hep3B cell On the other handp53-independent pathways such as the PI3KPTENAKTpathway stress response pathways and the NF-kB pathwaywere also found to be activated by curcumin to induce cancercell death [11] In this study the PI3KPTENAKTFOXOpathway was shown to be activated to induce p53-null Hep3Bhepatoma cell apoptosis after curcumin treatment

Treatment with curcumin inhibits the PI3KAKT path-way in various types of cancer cells including breast cancer[17] lung cancer [18] colon cancer [19] renal cancer [20]ovarian carcinoma [21] pancreatic cancer [22] osteosarcoma[23] acute T cell leukemias [24] and liver cancer [25]The results of this study are consistent with these reportsHowever the consequence of PI3KAKT pathway inhibi-tion is different among different types of cancer cells For

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 9: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Evidence-Based Complementary and Alternative Medicine 9

pcDNA31FOXO4

FOXO4

pcDNA31FOXO4

-Actin

(a)

G2M 195

S 106

G1 394

subG1 292

FL3 Lin0 1023

41

S 110

G2M 136

G1 532

subG1 200

FL3 Lin0 1023

132

G2M 208

S 196

subG1 133

FL3 Lin0 1023

66

G2M 192

S77

G1 670

subG133

FL3 Lin0 1023

148

G1 452

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

(b)

A400

A300

A2803

A1198

A3395

A2129

A102

A4474

A400

A300

A1392

4A400

3A300

A1392

A2608

A235

A183

A235

A183

A486

A3796

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

103

102

101

100

pcDNA31

pcDNA31+ CUR

FOXO4

FOXO4

+ CUR

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

FL1 log

FL3

log

(c)

Figure 7 Effects of FOXO4 overexpression on curcumin induced Hep3B cell apoptosis (a) Western blot analysis of overexpressed andendogenous FOXO4 proteins in Hep3B cells (b) Effects of FOXO4 overexpression on Hep3B cell apoptosis with and without curcumintreatments (c) Caspase-3 activities in FOXO4 overexpression experiments 119910-axis indicates propidium iodide (PI) intensities 119909-axis indicatescaspase activities pcDNA 31 is a mammalian expression vector with the CMV promoter (Invitrogen) The concentration of curcumin usedin the experiments was 50120583MThe data are representative of triplicate experiments CUR curcumin DMSO dimethyl sulfoxide (solvent ofcurcumin)

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 10: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

10 Evidence-Based Complementary and Alternative Medicine

PTEN

PI3K

FOXO4

AKT

Apoptosis

TP53

Curcumin

FOXO1FOXO3a

Figure 8 A schematicmodel describing themechanism bywhich curcumin induces Hep3B cell apoptosis via the PI3KAKTFOXOpathway

example Zhao et al demonstrated that curcumin inhibitsthe PI3KAKT pathway and induces FOXO1 expression inpancreatic cancer cells [22] In contrast inhibition of thePI3KAKT pathway and induction of FOXO4 in curcumintreated p53-null hepatoma cell were observed in this studyThe diverse results among studies may be due to severalreasons The first possible reason is the tissue specificity ofgene (pathway) function Different cells (in different tissues)share common core signal transduction pathways with dif-ferent downstream effectors As a consequence activationof a common core signal transduction pathway leads todifferent responses in different type of cells Second differentgenetic backgrounds among cancer cells may cause differentresponses to curcumin As shown in Figure 1 the HepG2cell which expresses wild-type p53 protein exhibits differentsensitivity to curcumin compared with the p53-null Hep3Bcell Third the signal transduction pathways in cells arenetworks that perform crosstalk with and influence eachother However usually only one or two major pathways arefocused and observed in one study Therefore a very limitedscope is typically obtained in one study In this study theFOXO4 miRNA could not completely reverse the effects ofcurcumin treatment It has been shown that curcumin hasmultiple targets in the signal transduction pathway network[6ndash12] Therefore the results in this study could not rule outthat there are other pathwayswhich also respond to curcuminin Hep3B cells

Yang et al have shown that FOXO4 overexpressioninhibits AKT activity increases p27 Kip1 transcriptionand suppresses HER2-overexpressed tumors [26] Zhang etal showed that upregulation of cyclin-dependent kinaseinhibitors p21 (Cip1) and p27 (Kip1) enhanced transactiva-tion of FOXO4 factor and dysregulation of AKT signalingassociated with inhibition of cell proliferation and tumori-genicity of hepatoma cells [27] Xie et al have shown thatinhibiting the PI3KAKT signaling pathway enhanced the

transcriptional activity of FOXO4 associated with upregula-tion of the cyclin-dependent kinase (CDK) inhibitors p21Cip1and p27Kip1 and reduced proliferation of breast cancer cells[28] Roy et al showed that inhibition of the PI3KAKT path-way and activation of FOXO4 transcriptional activity led tocell-cycle arrest and apoptosis in pancreatic cancer cells [29]In contrast Chen et al have shown that downregulation ofFOXO4 by miR-421 induces cell proliferation and apoptosisresistance in human nasopharyngeal carcinoma [30] Li et alhave shown that targeting FOXO4 by miRNA-150 promotescervical cancer cell growth and survival [31] These studiesare consistent with results of this study namely FOXO4has antiproliferative and proapoptotic activities MoreoverYang et al showed that FOXO4 sensitized cells to apoptosisinduced by the chemotherapeutic agent 2-methoxyestradiol[26] A similar phenomenon was observed in this study asoverexpression of FOXO4 inHep3B cells increased sensitivityto curcumin

5 Conclusions

A model as shown in Figure 8 is proposed according toresults of this study In p53-null Hep3B cells treatmentwith curcumin upregulates PTEN proteins and downregu-lates AKT1 proteins As a consequence translocation of theFOXO4 protein into the nucleus leads to Hep3B cell apopto-sisThe results of this study revealed the AKTPTENFOXO4pathway as a potential candidate target for treatment of p53-null liver cancers

Conflicts of Interest

The authors declare that they have no conflicts of interestregarding the publication of this manuscript

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 11: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Evidence-Based Complementary and Alternative Medicine 11

Authorsrsquo Contributions

Chu-Wen Yang conceived and designed the experimentsanalyzed the data and wrote the paper An-Ting Liouand Mei-Fang Chen performed the experiments All of theauthors have read and approved the final version of the paper

References

[1] R X ZhuW-K Seto C-L Lai andM-F Yuen ldquoEpidemiologyof hepatocellular carcinoma in the Asia-Pacific regionrdquoGut andLiver vol 10 no 3 pp 332ndash339 2016

[2] K A McGlynn andW T London ldquoThe global epidemiology ofhepatocellular carcinoma present and futurerdquo Clinics in LiverDisease vol 15 no 2 pp 223ndash243 2011

[3] R L Siegel K D Miller and A Jemal ldquoCancer statistics 2016rdquoCA ACancer Journal for Clinicians vol 66 no 1 pp 7ndash30 2016

[4] B C Lewis D S Klimstra N D Socci S Xu J A Koutcherand H E Varmus ldquoThe absence of p53 promotes metastasisin a novel somatic mouse model for hepatocellular carcinomardquoMolecular and Cellular Biology vol 25 no 4 pp 1228ndash12372005

[5] M I F Shariff I J Cox A I Gomaa S A Khan W Gedroycand S D Taylor-Robinson ldquoHepatocellular carcinoma currenttrends in worldwide epidemiology risk factors diagnosis andtherapeuticsrdquo Expert Review of Gastroenterology and Hepatol-ogy vol 3 no 4 pp 353ndash367 2009

[6] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[7] M Pulido-Moran J Moreno-Fernandez C Ramirez-Tortosaand M Ramirez-Tortosa ldquoCurcumin and healthrdquo Moleculesvol 21 no 3 p E264 2016

[8] R Kotecha A Takami and J L Espinoza ldquoDietary phyto-chemicals and cancer chemoprevention a review of the clinicalevidencerdquo Oncotarget vol 7 no 32 pp 52517ndash52529 2016

[9] G B Maru R R Hudlikar G Kumar K Gandhi and M BMahimkar ldquoUnderstanding the molecular mechanisms of can-cer prevention by dietary phytochemicals From experimentalmodels to clinical trialsrdquoWorld Journal of Biological Chemistryvol 7 no 1 pp 88ndash99 2016

[10] D Bandyopadhyay ldquoFarmer to pharmacist Curcumin as ananti-invasive and antimetastatic agent for the treatment ofcancerrdquo Frontiers in Chemistry vol 2 article no 113 2014

[11] A R Pavan G D B da Silva D H Jornada et al ldquoUnravelingthe anticancer effect of curcumin and resveratrolrdquo Nutrientsvol 8 no 11 p E628 2016

[12] AVyas PDandawate S PadhyeAAhmad andF Sarkar ldquoPer-spectives on new synthetic curcumin analogs and their poten-tial anticancer propertiesrdquo Current Pharmaceutical Design vol19 no 11 pp 2047ndash2069 2013

[13] J Ravindran S Prasad and B B Aggarwal ldquoCurcumin andcancer cells how many ways can curry kill tumor cells selec-tivelyrdquo AAPS Journal vol 11 no 3 pp 495ndash510 2009

[14] M-C JiangH-F Yang-Yen J-K Lin and J J-Y Yen ldquoDifferen-tial regulation of p53 c-Myc Bcl-2 and Bax protein expressionduring apoptosis induced by widely divergent stimuli in humanhepatoblastoma cellsrdquo Oncogene vol 13 no 3 pp 609ndash6161996

[15] K S Bhullar A Jha andH PV Rupasinghe ldquoNovel carbocycliccurcumin analog CUR3d modulates genes involved in multipleapoptosis pathways in human hepatocellular carcinoma cellsrdquoChemico-Biological Interactions vol 242 pp 107ndash122 2015

[16] J Chen L Li J Su and T Chen ldquoNatural borneol enhancesbisdemethoxycurcumin-induced cell cycle arrest in the G2Mphase through up-regulation of intracellular ROS in HepG2cellsrdquo Food and Function vol 6 no 3 pp 740ndash748 2015

[17] F Guan Y Ding Y Zhang Y Zhou M Li and C WangldquoCurcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Aktdegradationrdquo PLoSONE vol 11 no 1 Article ID e0146553 2016

[18] H Jin F Qiao YWang Y Xu andY Shang ldquoCurcumin inhibitscell proliferation and induces apoptosis of human non-smallcell lung cancer cells through the upregulation of miR-192-5p and suppression of PI3KAkt signaling pathwayrdquo OncologyReports vol 34 no 5 pp 2782ndash2789 2015

[19] C Rana H Piplani V Vaish B Nehru and S N SanyalldquoDownregulation of PI3-KAktPTEN pathway and activationof mitochondrial intrinsic apoptosis by Diclofenac and Cur-cumin in colon cancerrdquo Molecular and Cellular Biochemistryvol 402 no 1-2 pp 225ndash241 2015

[20] H Zhang W Xu B Li et al ldquoCurcumin Promotes Cell CycleArrest and Inhibits Survival of Human Renal Cancer Cells byNegative Modulation of the PI3KAKT Signaling Pathwayrdquo CellBiochemistry and Biophysics vol 73 no 3 pp 681ndash686 2015

[21] Z Yu Y Wan Y Liu J Yang L Li and W Zhang ldquoCurcumininduced apoptosis via PI3KAkt-signalling pathways in SKOV3cellsrdquo Pharmaceutical Biology vol 54 no 10 pp 2026ndash20322016

[22] Z Zhao C Li H Xi Y Gao and D Xu ldquoCurcumin inducesapoptosis in pancreatic cancer cells through the induction offorkhead box O1 and inhibition of the PI3KAkt pathwayrdquoMolecular Medicine Reports vol 12 no 4 pp 5415ndash5422 2015

[23] P Angulo G Kaushik D Subramaniam et al ldquoNaturalcompounds targeting major cell signaling pathways a novelparadigm for osteosarcoma therapyrdquo Journal of HematologyOncology vol 10 no 1 p 10 2017

[24] A R Hussain M Al-Rasheed P S Manogaran et al ldquoCur-cumin induces apoptosis via inhibition of PI31015840-kinaseAKTpathway in acute T cell leukemiasrdquo Apoptosis vol 11 no 2 pp245ndash254 2006

[25] K Chiablaem K Lirdprapamongkol S KeeratichamroenR Surarit and J Svasti ldquoCurcumin suppresses vasculogenicmimicry capacity of hepatocellular carcinoma cells throughSTAT3 and PI3KAKT inhibitionrdquo Anticancer Research vol 34no 4 pp 1857ndash1864 2014

[26] H Yang R Zhao H-Y Yang and M-H Lee ldquoConstitutivelyactive FOXO4 inhibits Akt activity regulates p27 Kip1 stabilityand suppresses HER2-mediated tumorigenicityrdquoOncogene vol24 no 11 pp 1924ndash1935 2005

[27] T Zhang C Wan W Shi et al ldquoThe RNA-binding proteinSam68 regulates tumor cell viability and hepatic carcinogenesisby inhibiting the transcriptional activity of FOXOsrdquo Journal ofMolecular Histology vol 46 no 6 pp 485ndash497 2015

[28] G Xie J Li J Chen X Tang S Wu and C Liao ldquoKnock-down of flotillin-2 impairs the proliferation of breast cancercells through modulation of AktFOXO signalingrdquo OncologyReports vol 33 no 5 pp 2285ndash2290 2015

[29] S K Roy R K Srivastava and S Shankar ldquoInhibition ofPI3KAKT and MAPKERK pathways causes activation of

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 12: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

12 Evidence-Based Complementary and Alternative Medicine

FOXO transcription factor leading to cell cycle arrest andapoptosis in pancreatic cancerrdquo Journal of Molecular Signalingvol 5 p 10 2010

[30] L Chen Y Tang J Wang Z Yan and R Xu ldquoMiR-421induces cell proliferation and apoptosis resistance in humannasopharyngeal carcinoma via downregulation of FOXO4rdquoBiochemical and Biophysical Research Communications vol 435no 4 pp 745ndash750 2013

[31] J Li L Hu C Tian F Lu J Wu and L Liu ldquomicroRNA-150promotes cervical cancer cell growth and survival by targetingFOXO4rdquo BMC Molecular Biology vol 16 no 1 article no 242015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 13: Curcumin Induces p53-Null Hepatoma Cell Line Hep3B ...downloads.hindawi.com/journals/ecam/2017/4063865.pdf · Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


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