LupeolSuppressesCisplatin-InducedNuclearFactor ... · 30-gauge hypodermicneedle. Themice were imagedon days 0, 7,and37 aftertumorinoculation.Micewereanesthetizedwithaketamine/xylazine

Lupeol Suppresses Cisplatin-Induced Nuclear Factor-KB Activation

in Head and Neck Squamous Cell Carcinoma and Inhibits Local

Invasion and Nodal Metastasis in an Orthotopic Nude Mouse Model

Terence K. Lee,1Ronnie T.P. Poon,

1Jana Y. Wo,

1Stephanie Ma,

2Xin-Yuan Guan,

2

Jeffrey N. Myers,3Peter Altevogt,

4and Anthony P.W. Yuen

1

Departments of 1Surgery and 2Clinical Oncology, The University of Hong Kong, Pokfulam, Hong Kong, China; 3Department of Head andNeck Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; and 4Tumor Immunology Programme,D010 German Cancer Research Center, Heidelberg, Germany

Abstract

A poor prognosis in head and neck squamous cell carcinoma(HNSCC) patients is commonly associated with the presence ofregional metastasis. Cisplatin-based chemotherapy concur-rent with radiation therapy is commonly used in the treatmentof locally advanced HNSCC. However, the result is dismal dueto common acquisition of chemoresistance and radioresist-ance. Epidemiologic studies have shown the importance ofdietary substances in the prevention of HNSCC. Here, we foundthat lupeol, a triterpene found in fruits and vegetables,selectively induced substantial HNSCC cell death but exhibitedonly a minimal effect on a normal tongue fibroblast cell linein vitro . Down-regulation of NF-KB was identified as the majormechanism of the anticancer properties of lupeol againstHNSCC. Lupeol alone was not only found to suppress tumorgrowth but also to impair HNSCC cell invasion by reversal ofthe NF-KB–dependent epithelial-to-mesenchymal transition.Lupeol exerted a synergistic effect with cisplatin, resulting inchemosensitization of HNSCC cell lines with high NF-KBactivity in vitro . In in vivo studies, using an orthotopicmetastatic nude mouse model of oral tongue squamous cellcarcinoma, lupeol at a dose of 2 mg/animal dramaticallydecreased tumor volume and suppressed local metastasis,which was more effective than cisplatin alone. Lupeol exerteda significant synergistic cytotoxic effect when combined withlow-dose cisplatin without side effects. Our results suggestthat lupeol may be an effective agent either alone or incombination for treatment of advanced tumors. [Cancer Res2007;67(18):8800–9]

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixthmost common human neoplasm, with an estimated annualworldwide incidence of 500,000 new cases (1, 2). Despite advance-ments in surgery, chemotherapy, radiotherapy, and combinations oftreatment modalities, the long-term survival of patients withHNSCC has remained 50% for the past 30 years (3). Therefore,investigations of potential alternative treatments for HNSCC withgreater efficacy and fewer associated toxicities are urgently needed.

Human papilloma virus infection and alcohol and tobacco useare each known to play a role in the pathogenesis of HNSCC (4).Further, the combined effects of alcohol and tobacco use have amultiplicative risk in the development of HNSCC (5, 6). Cigarettesmoke and alcohol consumption can cause significant oxidativestress (7, 8), which increases DNA damage (9) and, consequently,leads to the malignant transformation of normal cells (10).Epidemiologic studies have attributed the lower incidence ofHNSCC in the United States to the high intake of fruits andvegetables, suggesting that some dietary substances have aprotective effect against HNSCC (11, 12). In recent years,substances obtained from fruits and vegetables have gainedconsiderable attention for the prevention and/or treatment ofcertain cancers (13–15). Lup-20(29)-en-3h-ol (lupeol), a triterpenefound in fruits (e.g., olive, mango, strawberry, grapes, and figs),vegetables, and medicinal plants, recently showed antitumoractivity in a two-stage model of mouse skin carcinogenesis andapoptotic effects in androgen-dependent prostate carcinomas(16, 17). Lupeol possesses strong antioxidant, anti-inflammatory,antiarthritic, antimutagenic, and antimalarial activities in in vitroand in vivo systems by inhibiting Ras and Fas signaling pathways(18–21). It has also been shown that lupeol induces differentiationand inhibits growth of mouse melanoma and human leukemia cells(22, 23). In view of the wide range of pharmacologic activities oflupeol, we exploited the therapeutic efficacy of lupeol in thetreatment of HNSCC.

Cisplatin-based chemotherapy concurrent with radiation thera-py was commonly used in the treatment of locally advancedHNSCC (24). However, the result is unsatisfactory due to theacquisition of chemoresistance by tumor cells. Multiple mecha-nisms for chemoresistance have been described in various cancercells (25–27). Chemotherapeutic compounds that induce apoptosisare also known to activate nuclear factor-nB (NF-nB) in theprotection of genotoxic stress. However, the role of NF-nB–relatedchemoresistance remains open and controversial (28). Therefore,we set out first to understand the molecular mechanism governingthe chemoresistance of HNSCC and then to examine the potentialchemosensitization effect of lupeol in combination with cisplatintreatment with the aim of developing an effective treatment for thisdeadly disease.

In the present study, lupeol selectively induced substantialHNSCC cell death and exhibited minimal effects on a normaltongue epithelial cell line in vitro. In vitro , down-regulation ofNF-nB is the major mechanism of the anticancer properties oflupeol against HNSCC, as indicated by chemosensitization of thetongue carcinoma cell line CAL27. We observed that lupeol drama-tically decreased tumor volume and suppressed local metastasis in

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Anthony P.W. Yuen, Department of Surgery, The Universityof Hong Kong Queen Mary Hospital, 102 Pokfulam Road, Hong Kong. Phone: 852-2855-3641; Fax: 852-2819-3464; E-mail: [email protected].

I2007 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-0801

Cancer Res 2007; 67: (18). September 15, 2007 8800 www.aacrjournals.org

Research Article

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our animal model. Cisplatin alone did not satisfactorily shrink thetumor and it resulted in toxicity by causing a decrease in bodyweight. Combination therapy consisting of a low dose of cisplatinand lupeol showed a synergistic effect on the suppression of tumorgrowth and metastasis, accompanied by inhibition of NF-nBwithout toxicity. Our results provide solid evidence that lupeolmay be a novel therapeutic agent that is clinically applicable for thetreatment of cancers in which NF-nB plays a significant role.

Materials and Methods

Cell lines and cell culture. The human oral squamous cell carcinoma

cell lines TU159 and MDA1986 were obtained from the laboratory of Dr.

Gary L. Clayman (The University of Texas M. D. Anderson Cancer Center,Houston, TX; ref. 29). The tongue fibroblast cell line Hs 677.Tg (CRL-7408)

and the human tongue carcinoma cell line CAL27 were obtained from

American Type Culture Collection (Manassas, VA). They were maintained inDMEM with high glucose (Life Technologies, Inc.) supplemented with 10%

heat-inactivated fetal bovine serum (Life Technologies), 100 mg/mL

penicillin G, and 50 Ag/mL streptomycin (Life Technologies) at 37jC in a

humidified atmosphere containing 5% CO2.Plasmids. IKKhWT was provided by Dr. D.Y. Jin (Department of

Biochemistry, University of Hong Kong, Hong Kong, China; ref. 30).

Antibodies and reagents. Antibodies against h-actin, NF-nB/p50, NF-nB/p65, histone H, Sm-actin, and vimentin were purchased from Santa CruzBiotechnology. Antibodies to E-cadherin and a-cadherin were purchased

from Zymed Laboratories. Antibodies against caspase-3 and cleaved

caspase-3 were purchased from Cell Signaling Technologies. Lupeol was

purchased from Sigma. Cisplatin was purchased from David BullLaboratories.

Treatment of cells. A stock solution of lupeol (30 mmol/L; MW, 426.72)

was prepared by resuspension in warm alcohol and dilution in DMSO at a1:1 ratio. For dose-dependent studies, the cells (50% confluent) were treated

with lupeol (1–30 Amol/L) for 48 h in complete cell medium. The final

concentrations of DMSO and alcohol were 0.25% and 0.075%, respectively, in

all treatment protocols. After 48 h of treatment with lupeol, the cells wereharvested and cell lysates were prepared and stored at �80jC for later use.

Cell transfection. For transient transfection, CAL27 cells were trans-

fected with 2 Ag of plasmid IKKhWT or empty vector as a control using

FuGENE 6 according to the manufacturer’s protocol (Roche). For generationof CAL27 cells harboring luciferase, a lentiviral vector harboring the

luciferase gene was constructed and transfected using the Lentiviral RNAi

Expression System (Invitrogen). Stable transfectants were generated from apool of >20 positive clones, which were selected with blasticidin at a

concentration of 2 Ag/mL.

Immunofluorescence. Cells cultured on chamber slides were permea-

bilized with 0.1% Triton X-100 and fixed with 4% paraformaldehyde in PBS.The cells were incubated with monoclonal antibody against E-cadherin.

The secondary antibody was TRITC-conjugated goat anti-mouse immuno-

globulin G (Molecular Probes), and the cells were counterstained with 4¶,6-diamidino-2-phenylindole. For phalloidin staining, the same fixationprocedure was used as described above except that cells were incubated

with fluorescein phalloidin (Molecular Probes) in 1% bovine serum albumin

(dilution factor of 1:50) at 37jC for 1 h. All images were visualized byconfocal microscopy and photographs were taken at �600 magnification.

Cell cycle analysis. After lupeol treatment, the DNA content and cell

cycle distribution of CAL27 cells grown in six-well plates were determined

by flow cytometry. The cells were plated at a low density (5 � 104 per well)and were harvested at 0, 6, 12, 24, and 48 h. Another set of controls that

lacked lupeol treatment was used. Cells were trypsinized and washed once

in PBS. They were then fixed in cold 70% ethanol and stored at 4jC. Beforetesting, ethanol was removed and the cells were resuspended in PBS. Thefixed cells were then washed with PBS, treated with RNase (1 Ag/mL), and

stained with propidium iodide (50 Ag/mL) for 30 min at 37jC. Cell cycleanalysis was done in an EPICS profile analyzer using ModFit LT2.0 software

(Coulter Electronics).

Immunostaining. Immunohistochemistry was done as previouslydescribed (31). To detect NF-nB/p65 and E-cadherin, the antibodiesdescribed above were used.

Luciferase promoter assay. Cells were plated onto 24-well culture

plates and allowed to grow for 24 h; the NF-nB-luciferase–based constructand pRL-CMV-Luc were cotransfected into CAL27 cells using FuGENE 6

reagent (Roche Diagnostics). Cells were lysed 48 h after transfection andwere assayed for luciferase activity using the Dual-Luciferase Reporter AssaySystem (Promega). Firefly luciferase activity was measured at 48 h after

transfection and the reading was normalized to Renilla luciferase activity,which served as an internal control for transfection efficiency. Eachexperiment was done at least thrice in duplicate wells and each data point

represented the mean and SD. The mean percentage increase (or decrease)in luciferase activity was presented as the final result and the SD of the

means was used as the error bar.Western blot. The Western blots were developed using the enhanced

chemiluminescence kit (Amersham Biosciences) and was done with the

antibodies described previously (32).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.CAL27, TU159, MDA1986, and CRL-7408 were seeded onto 96-well plates

and appropriate concentrations of lupeol, ranging from 5 to 125 Amol/L, or

cisplatin, ranging from 0.2 to 5 Ag/mL, were then added. After 4 to 24 h,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye, at

a concentration of 5 mg/mL (Sigma-Aldrich), was added and the plates were

incubated for 12 h in a moist chamber at 37jC. Absorbance was determined

by eluting the dye with DMSO (Sigma-Aldrich) and the absorbance wasmeasured at 570 nm. At least three independent experiments were done.

Quantification of apoptosis. The terminal deoxyribonucleotidyl

transferase–mediated dUTP nick end labeling (TUNEL) technique was

done to detect apoptotic cells using the in situ cell death detection kit

(Roche Diagnostics). Briefly, paraffin-embedded tissues were fixed with

15 Ag/mL proteinase K in 10 mmol/L Tris-HCl (pH 7.4). The slides were then

incubated with the TUNEL reaction mixture for 1 h at 37jC. After washing,the slides were incubated with horseradish peroxidase–conjugated anti-

fluorescein antibody for 30 min at 37jC. For cytofluorometric apoptosis

analysis, CAL27 cells (5 � 105) were inoculated into each well of a six-well

plate and treated with 1% DMSO and different doses (1, 5, and 10 Amol/L)

of lupeol in 10% fetal bovine serum-DMEM for 48 h. The cells were

then labeled with Annexin V-FITC (BD Biosciences Pharmingen) and

analyzed with FACSCalibur (Becton Dickinson Immunocytometry Systems).

Unstained cells were used as a negative control.

Wound healing assay and invasion assay. Cell migration was assessed

by measuring the movement of cells into a scraped acellular area created by

a 200-AL pipette tip (time 0) and the speed of wound closure was monitoredafter 24 h. Invasion assays were done with 24-well BioCoat Matrigel

Invasion Chambers (Becton Dickinson) using 5 � 104 cells in serum-free

DMEM that were plated onto either control or Matrigel-coated filters.Conditioned medium from CAL27 cells was placed in the lower chambers

as a chemoattractant. After 22 h in culture, cells were removed from the

upper surface of the filter by scraping with a cotton swab. Cells that had

invaded through the Matrigel and were adherent to the bottom of themembrane were stained with crystal violet solution. The cell-associated dye

was eluted with 10% acetic acid and the resultant absorbance at 595 nm

was determined. Each experiment was done in triplicate and the mean

values (F SE) are presented.Animal studies. A total of 5 � 105 CAL27 cells were harvested from

subconfluent cultures and injected directly into anterior tongue using a1-mL tuberculin syringe (Hamilton Co.). A week later, the nude mice wererandomized into four groups and each group consisted of 10 nude mice.They were treated with i.p. injection of lupeol (2 mg/animal) in 0.2 mL ofcorn oil, 5 mg/kg cisplatin, lupeol (2 mg/animal) plus 1 mg/kg cisplatin,or corn oil alone as the control group, twice per week for 30 days. Theadministration protocol of lupeol was followed according to Saleem et al.(16, 17).

Animal charge-coupled device experiments. A total of 5 � 105

CAL27-luciferase–expressing cells in 30-AL HBSS were injected directly intothe anterior tongue using a 1-mL tuberculin syringe (Hamilton Co.) with a

Lupeol Suppressed Tumor Growth and Metastasis in HNSCC

www.aacrjournals.org 8801 Cancer Res 2007; 67: (18). September 15, 2007



30-gauge hypodermic needle. The mice were imaged on days 0, 7, and 37

after tumor inoculation. Mice were anesthetized with a ketamine/xylazine

mix (4:1). Imaging was done using a Xenogen IVIS 100 cooled charge-coupled device (CCD) camera (Xenogen). The mice were injected i.p. with

200 AL of 15 mg/mL D-luciferin for 15 min before imaging, after which they

were placed in a light-tight chamber. The acquisition time ranged from 3 s

to 1 min. The images shown are pseudoimages of the emitted light inphotons/s/cm2/Sr, superimposed over the gray-scale photographs of the

animal.

Histologic analysis. Sections of different tissues (4 Am) were cut and

stained with H&E as previously described (32).Statistical analysis. Continuous data were expressed as median and

range and compared between groups using the Mann-Whitney U test.

Categorical variables were compared using the m2 test (or Fisher’s exact test

where appropriate). A Pearson test was used for bivariate correlationcomparison. All statistical analyses were done using statistical software

(SPSS 9.0 for Windows, SPSS, Inc.). P < 0.05 was considered statistically

significant.

Results

Growth inhibition of HNSCC cell lines with lupeol. CAL27 is atongue squamous cell carcinoma cell line, whereas TU159 and

MDA1986 are primary andmetastatic oral squamous cell carcinomacell lines, respectively. A human tongue fibroblast cell line, CRL-7408, served as the control. The MTT assay was done to determineits IC10 and IC50 doses. The MTT assay showed that lupeoltreatment induced dramatic cell death in HNSCC cell lines in adose-dependent manner (Fig. 1A). There was no significantdifference in drug sensitivity (IC10 range, 13.7–22.2 Amol/L) amongthe three HNSCC cell lines (CAL27, MDA1986, and TU159). Toexamine its toxicity, the effect of lupeol on CRL-7408 cells was alsoinvestigated. The results showed that CRL-7408 is less sensitive tolupeol, with an IC10 up to 41.2 Amol/L. The effect of lupeol on cellcycle distribution was evaluated by flow cytometry. When lupeolwas administered at the IC10 dose, CAL27 cells exhibited G1 arrest( from 53.2% to 69.9%) in a time-dependent manner (Fig. 1B). Theeffect of lupeol on cell apoptosis was evaluated by cytofluorometricapoptosis analysis. Lupeol induced CAL27 cell apoptosis at dosesthat ranged from 15 to 30 Amol/L in CAL27 cells (Fig. 1C). The effectof lupeol on apoptosis and cell cycle distribution in another HNSCCcell line, MDA1986, was found to be similar (data not shown).

ReducedNF-kB expression inCAL27 cells on lupeol treatment.To determine whether the growth-suppressive effect of lupeol is

Figure 1. Lupeol selectively induced cell death of HNSCC cell lines. Cytotoxicity of CRL-7408, TU159, CAL27, and MDA1986 cells following lupeol treatment.To evaluate drug sensitivity, the MTT assay was done in cells at 48 h after lupeol treatment. A, lupeol treatment induced dramatic cell death in HNSCC cell lines in adose-dependent manner. B, flow cytometry analysis of CAL27 cells following lupeol treatment. The cells were harvested at 0, 6, 12, 24, and 48 h after treatment with anIC10 dose of lupeol. CAL27 cells exhibited G1 arrest in a dose-dependent manner. C, the effect of lupeol on apoptosis was analyzed by Annexin V staining aftertreatment of CAL27 cells at doses of 15, 20, 25, and 30 Amol/L. The percentage of cell apoptosis increased in a dose-dependent manner at 8.59%, 11.44%, 24.91%,and 27.62%, respectively, as detected by Annexin staining and fluorescence-activated cell sorting analysis.

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mediated through inhibition of NF-nB, we first examined theNF-nB promoter activity in CRL-7408 and three HNSCC cell lines(TU159, MDA1986, and CAL27). By promoter assay, CAL27 showedthe highest NF-nB activity whereas CRL-7408 showed the lowest(Fig. 2A). NF-nB promoter activity was evaluated in CAL27 andMDA1986 cells with the highest NF-nB activity treated with lupeolat the dose of IC10 for different time periods ( from 6 to 48 h;Fig. 2B). There was a dramatic decrease in NF-nB after 6 h, whichwas correlated with increased growth inhibition shown in Fig. 1B .Consistently, decreased nuclear p65 and p50 protein levelsaccompanied by inhibition of InBa phosphorylation were foundby Western blot (Fig. 2C). To further confirm the central role of

NF-nB in lupeol-induced growth suppression, we transientlytransfected CAL27 and MDA1986 cells with either empty vectoror IKKhWT and examined the difference in the effects of lupeol ongrowth suppression. As expected, NF-nB promoter activity wasincreased f3.5- and 3.1-fold, respectively (Fig. 2D). By MTT assay,IC10 and IC50 were increased in IKKhWT when compared with theempty vector control, suggesting that InBa phosphorylation plays acentral role in growth suppression of CAL27 and MDA1986 cells(Fig. 2D).

Lupeol and cisplatin synergistically inhibited cell growththrough inhibition of NF-KB activity. Given the potential role ofNF-nB in chemoresistance of some tumor types (28), we examined

Figure 2. Lupeol induced cell death by down-regulation of NF-nB activity. The NF-nB activity in various HNSCC cell lines was evaluated by a promoter assay. A, of thethree HNSCC cell lines, NF-nB activity was found to be highest in CAL27. All three HNSCC cell lines exhibited elevated NF-nB expression when compared with thenormal tongue fibroblast cell line. There is an f13-fold increase in NF-nB activity in CAL27 cells when compared with CRL-7408 cells. B, by a promoter assay,lupeol was found to inhibit NF-nB promoter activity in CAL27 and MDA1986 cells in a time-dependent manner at the IC10 dose. C, consistently, nuclear p65 and p50protein levels were decreased in a time-dependent manner with inhibition of InB phosphorylation. Either empty vector or IKKhWT was transfected into CAL27 andMDA1986 cells. D, IKKhWT transfection into CAL27 and MDA1986 cells conferred resistance to lupeol treatment when compared with the empty vector control.





whether lupeol sensitized the effect of cisplatin. First, wedetermined the IC10 and IC30 of CAL27 and MDA1986 cells inresponse to cisplatin by the MTT assay. Cisplatin was found toinhibit CAL27 and MDA1986 cell growth in a dose-dependentmanner (Fig. 3A). The respective treatments of CAL27 andMDA1986 cells that have high levels of NF-nB activity were eithercisplatin (0.15 and 0.57 Ag/AL; 0.21 and 0.68 Ag/AL) or lupeol (13.7and 32.3 Amol/L; 15.1 and 36.4 Amol/L). These treatments resultedin 10% to 30% inhibition of cell growth. When lupeol and cisplatinwere combined at their IC30 doses, growth was inhibited by 88%and 80% in CAL27 and MDA1986 cells, respectively. This effect wassimilar to that exerted by an 8.4-fold and 6.2-fold higherconcentration of cisplatin (4.8 and 4.2 Ag/mL, respectively;Fig. 3B). A weaker effect was seen in TU159 cells that showedlow NF-nB activity (data not shown). The CAL27 cell line waschosen to study whether lupeol and cisplatin synergisticallyinhibited cell growth through inhibition of NF-nB activity. InFig. 3C , it is shown that cisplatin induced NF-nB activity of CAL27

cells in a time-dependent manner. Cisplatin increased NF-nBpromoter activity from 6 to 48 h at the IC10 dose, accompanied byan increase in nuclear p65 and p50 protein levels. At the IC10 dose,cleaved caspase-3 was barely detectable. When lupeol and cisplatinwere combined, NF-nB promoter activity decreased from 6 to 48 h.Consistently, nuclear p65 and p50 protein levels decreased in atime-dependent manner. Apoptosis was evidenced by an increasedcleaved caspase-3 protein level at 24 h after combined treatmentwith lupeol and cisplatin.

Lupeol suppressed cell motility and invasiveness by reversalof epithelial-to-mesenchymal transition. Increased invasion andcell motility is a major factor contributing to a poor prognosis ofHNSCC (2). To examine the antimetastatic effect of lupeol onCAL27 and MDA1986 cells, we carried out assays for wound healingand invasion. As shown in Fig. 4A , similarly sized wounds wereintroduced into monolayers of CAL27 and MDA1986 cells at 0 h. Inthe control cells, the gap of the wound was filled gradually bymigrating cells, and at 24 h after wound induction, the gap was

Figure 3. Lupeol exerted a synergistic effect with cisplatin in the treatment of HNSCC cells with high NF-nB activity. A, IC10, IC30, and IC50 of CAL27 and MDA1986cells in response to cisplatin treatment were determined by MTT assay. B, effects of lupeol, cisplatin, and their combination on the growth of CAL27 and MDA1986cells expressing high levels of NF-nB. The cells were exposed for 48 h to different concentrations of lupeol, cisplatin, and their combination as indicated. Columns,mean from three individual experiments done in duplicate; bars, SD. Differences in cell growth after exposure to lupeol and cisplatin, alone and in combination,were determined with the one-way ANOVA test. *, P < 0.05. C, cisplatin increased NF-nB promoter activity together with an increase in nuclear p65 and p50 proteinwhen CAL27 cells were treated with an IC10 dose of lupeol at different time points, but cleaved caspase-3 was not detected. When exposed to both lupeol andcisplatin, NF-nB activity was found to decrease from 12 h after treatment, with an increase in cleaved caspase-3 protein.

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almost closed (solid arrow). In contrast, after exposure to lupeol atdoses ranging from 5 to 15 Amol/L, the speed of wound closure wasmuch slower and the wound was still widely open at 24 h afterexposure. As the speed of wound closure reflects the migrationability of cancer cells, these results indicate that lupeol treatmentinhibits cell migration. Lupeol also exhibited a dramatic effect onthe suppression of cell invasion. As indicated by an invasion assay,lupeol suppressed cell invasion by 1.7- to 6.6-fold and by 1.69- to5.05-fold at doses ranging 5 to 15 Amol/L in CAL27 and MDA1986cells, respectively (Fig. 4B). Cell motility is dependent onrearrangement of the actin cytoskeleton in invasive cells (33). Inthe untreated CAL27 cells, bundles of actin filaments were wellorganized as evidenced by the strong staining of stress fiberformation, intact network, and protruding morphology (Fig. 4C).However, after exposure of CAL27 cells to lupeol, a reduction anddisruption of the actin stress fiber network and a lack of filopodiawere observed (Fig. 4C). EMT, transdifferentiation from epithelialtype to mesenchymal phenotype, is one of the major events duringacquisition of the invasive phenotype in tumors of epithelial origin(34, 35). This process is often accompanied by expression ofmesenchymal markers and loss of epithelial markers, especiallyE-cadherin (36–38). We therefore studied whether the inhibitoryeffect of lupeol on cell invasion of CAL27 and MDA1986 cells was

mediated through reversal of EMT. We found that the morphologyof lupeol-treated cells changed from a more elongated fibroblast-like morphology to a round and packed appearance of epithelialcells (Fig. 5A). In addition, expression of epithelial markers suchas E-cadherin and g-cadherin was increased by lupeol at IC10

concentrations in a time-dependent manner (Fig. 5B). In contrast,expression of mesenchymal markers such as a-smooth muscleactin and vimentin was reduced (Fig. 5B). Most importantly, wefound intense membrane staining of E-cadherin in lupeol-treatedHNSCC cells compared with the controls, indicating activation ofthis protein, whereas weakly positive E-cadherin staining wasmainly observed in the cytoplasm of untreated controls (Fig. 5C).

IKKBWT abolishes lupeol-induced chemosensitization andEMT reversal. To examine whether down-regulation of NF-nB isthe major mechanism for lupeol-induced chemosensitization andreversal of EMT, we also transfected CAL27 cells with an NF-nBactivator, IKKhWT, to examine whether the effect of lupeol can bereversed. Introduction of IKKhWT stimulated NF-nB activity by3.5-fold as described above. The expression of IKKhWT abolishedthe chemosensitization effect of lupeol. Cleaved caspase-3 wasobserved at 48 h after the addition of both cisplatin and lupeol atIC10 doses to CAL27 control cells, but it was absent in IKKhWTtransfectants (Supplementary Fig. S1). To examine whether

Figure 4. Lupeol suppressed cell motility and invasion of HNSCC cells in vitro. Similarly sized wounds were introduced into confluent monolayers of cells andvarious concentrations of lupeol were added. The speed of wound closure was monitored at 24 h. Note that the speed of wound closure was suppressed by lupeol inCAL27 and MDA1986 cells in a dose-dependent manner (A ). In addition, the effect of lupeol on cell invasiveness was evaluated by a Matrigel invasion assay.Suppression of cell invasion was also observed in a dose-dependent manner following lupeol treatment (B ). The effect of lupeol on stress fiber formation was evaluatedby phalloidin staining. Lupeol treatment destroyed the actin network and reduced the protrusion ability of CAL27 cells.





reversal of EMT by lupeol is NF-nB dependent, we examinedalternations of both epithelial and mesenchymal markers in eitherpcDNA-CAL27 or IKKhWT-CAL27 transfectants after 48 h oflupeol administration at the IC10 dose. By Western blot, there wereno apparent changes in epithelial or mesenchymal markers inIKKhWT-CAL27 transfectants, suggesting that lupeol-inducedreversal of EMT is NF-nB dependent (Supplementary Fig. S1).

Combined lupeol and cisplatin treatment significantlysuppressed tumor growth and metastasis in an orthotopicmetastatic nude mouse model of HNSCC. The in vivotherapeutic effect of lupeol was examined using the metastaticorthotopic tongue carcinoma nude mouse model. Based on thesignificant correlation between CCD camera signal and tumor sizein vivo (39), the CCD camera provided a novel and noninvasive toolfor evaluation of tumor size in vivo . CAL27 cells were first stablytransduced with the luciferase gene by lentiviral infection and5 � 105 cells were injected directly into the anterior tongue of nudemice, as shown in Fig. 6A . On day 37 after tumor inoculation, localinvasion of the lower jaw and regional lymph node metastasisdeveloped and were detected by CCD camera (Fig. 6A). To confirmlocal invasion and regional metastasis, the animals were sacrificedand dissected (Fig. 6B). The tissues of the lower jaw and lymphnode were further examined by H&E staining, which showedmassive tumor cell infiltration into these tissues (Fig. 6B). Usingthis animal model, we examined the effect of lupeol alone and in

combination with cisplatin using continuous lupeol administrationat a dose of 2 mg/animal (group B), cisplatin alone (5 mg/kg; groupC), lupeol (2 mg/animal) + cisplatin (1 mg/kg; group D), or cornoil (group A) as a control. The treatment began 7 days after theorthotopic implantation of CAL27 cells (Fig. 6C). During the expe-riment, there was a significant decrease in body weight in thecontrol group (15 F 2.4 g) and cisplatin alone group (15.2 F 3.2 g)when compared with the lupeol (23.9 F 2.5 g) and lupeol +cisplatin (22.9 F 3.2 g) groups. This result indicated that bothlupeol-treated and lupeol combined with low-dose cisplatin groupsshowed no signs of toxicity (infection, diarrhea, or loss of bodyweight). Histologic sections of normal organs like tongue, heart,liver, spleen, lung, and kidney showed no substantial cell deathafter H&E staining (data not shown). The tumor volumes in thesefour groups of animals were documented with a CCD camera.Figure 6C shows the optical CCD signals from representativegroups on day 30 after treatment and Fig. 6D shows graphs of theresults from cohorts using the same photonic scale. Lupeol wasfound to decrease the tumor volume significantly in a manner thatis much more effective than cisplatin treatment (P < 0.05). Lupeolexerted a synergistic effect with low-dose cisplatin resulting indramatic shrinkage of the tumor. By TUNEL assay, there was aremarkable difference in the number of apoptotic nuclei andpatchy necrosis in tumor tissues treated with lupeol (Supple-mentary Fig. S2B) when compared with the untreated samples

Figure 5. Lupeol induced reversal of NF-nB–dependent EMT. CAL27 and MDA1986 cells were treated with an IC10 dose of lupeol for 48 h and photos were takenthrough a phase-contrast microscope at �400 magnification. A, lupeol treatment results in morphologic changes. B, expression of epithelial markers E-cadherin andg-cadherin and mesenchymal markers a-smooth muscle actin (a-SMA ) and vimentin were evaluated in CAL27 cells at various time points C, representativeimmunofluorescent images of CAL27 and MDA1986 cells stained for E-cadherin in the control and lupeol-treated cells under a confocal microscope at �400magnification.

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(Supplementary Fig. S2A). Combined lupeol and cisplatin treat-ment significantly induced tumor cell apoptosis and necrosis whencompared with cisplatin alone (Supplementary Fig. S2C and D).Lupeol not only shrunk the tumor volume but also suppressed localinvasion of jaw and nodal metastasis. Five of five nude miceexhibited local invasion of the jaw and/or nodal metastasis in thecontrol group. However, an absence of jaw invasion and nodalmetastasis was found in both lupeol and lupeol + cisplatin groups.Although cisplatin can exert a limited apoptotic effect on tumorcells, two of five nude mice exhibited local metastasis. From ourin vitro study, we found that lupeol suppressed NF-nB–dependentEMT. By immunostaining, NF-nB expression was found to beabundant in both the nucleus (active form) and cytoplasm in thecontrol group (Supplementary Fig. S3). After lupeol treatment, lessnuclear and cytoplasmic staining of NF-nB protein was observed inthe CAL27 treatment group (Supplementary Fig. S3). Accompaniedwith decreased NF-nB protein in the treatment group, increasedmembranous staining of E-cadherin was also observed, whichindicates a functionally active protein (Supplementary Fig. S3).

Discussion

Despite multiple modalities of treatment, such as surgery,radiation, and chemotherapy, head and neck cancers continue to

have one of the lowest 5-year survival rates (3). The recentpreclinical success of green tea in growth suppression of HNSCCcell lines suggested the potential use of dietary substances in thetreatment of HNSCC (40). The major advantage of dietarysubstances over the conventional chemotherapy is its minimaltoxicity to the body. In addition, natural agents that induceapoptosis may provide an opportunity for minimal acquired drugresistance and decreased mutagenesis (41). In the present study, wefound that lupeol effectively and selectively inhibited cellularproliferation of head and neck cancer in vitro and in vivo . Usedalone or in combination with cisplatin, lupeol induced tumor cellapoptosis and suppressed metastasis through modulation of NF-nBactivity and was well tolerated in mice.

A striking observation from Fig. 1A suggests a selective responseof HNSCC cell lines to lupeol compared with CRL-7408 cells. Ourdata are significant because, in recent years, emphasis has beenplaced on natural diet-based agents that selectively or preferen-tially eliminate cancer cells by inhibiting cell cycle progressionand/or by causing apoptosis. Studies have shown that inhibition ofNF-nB could result in suppression of tumor growth (28, 42). Onemajor mechanism of NF-nB activation is through inhibition of InBphosphorylation (43). Lupeol prevented this phosphorylation andthus resulted in reduced NF-nB activation. Our data support the

Figure 6. The effect of cisplatin and lupeol in an orthotopic metastatic nude mouse model of oral tongue squamous cell carcinoma. CAL27 cells were transducedwith a luciferase gene and 5 � 105 cells were injected into the anterior tongues of nude mice. A, tumor formation and metastases were monitored from day 0 today 37 by a CCD camera. B, to confirm metastases, paraffin-embedded tissue sections of the lower jaw and lymph node were analyzed by H&E staining.C, tumor formation and metastases before and after treatment in the four groups of animals. D, histogram of the average basal signals of tumors from the fourgroups of animal in photons/s/cm2/Sr. Columns, average basal level signals on day 30 following different treatments; bars, SD. *, P < 0.05.





idea that lupeol exerts its effects in HNSCC through inhibition ofthe NF-nB pathway. This finding explains the selective eliminationof HNSCC cells, which showed elevated NF-nB activity whencompared with normal tongue fibroblast cells.

Recent data suggested that chemotherapy including cisplatinactivates the NF-nB activity, which results in chemoresistance (28).To test this hypothesis experimentally in HNSCC, we examinedNF-nB activity in CAL27 cells on cisplatin treatment. Interestingly,cisplatin was able to increase NF-nB promoter activity in a time-dependent manner, accompanied with an increase in nuclear p65and p50 protein levels. Activation of NF-nB by cisplatin couldcontribute to the acquired chemoresistance of HNSCC to cisplatinin a clinical situation (44). The role of NF-nB in chemoresistance ofHNSCC to cisplatin was further confirmed by transfection ofIKKhDN into CAL27 cells, resulting in chemosensitization (datanot shown). In Fig. 3B , lupeol synergistically augmented the growthinhibitory effect of cisplatin. The underlying mechanism mostprobably involves down-regulation of NF-nB activity. The role ofNF-nB in lupeol-induced chemosensitization was further con-firmed by transfection of IKKhWT into CAL27 cells (Supplemen-tary Fig. S1). This finding provided solid confirmation that lupeolcan potentially be used for combination therapy with chemother-apeutic agents and radiation that activate the NF-nB activity.

Recently, NF-nB was shown to be involved in tumor progressionvia EMT, a central process governing both morphogenesis (45) andcarcinoma progression in multicellular organisms (31). Due to theinhibitory effect of lupeol on NF-nB activation, we examinedwhether lupeol inhibited CAL27 cell motility and invasion bysuppression of NF-nB–dependent EMT. In Fig. 4A and B , lupeolinhibited cell motility and invasion in a dose-dependent manner,which is supported by disruption of F-actin structure. Accompaniedwith decreased invasiveness, lupeol induced morphologic changesfrom fibroblastic to epithelial appearance, which was accompaniedwith a gain of the epithelial marker vimentin and loss ofmesenchymal markers. In addition, these changes were accompa-nied with increased translocation of E-cadherin from the membraneto the cytoplasm, indicating functional activation. To furtherexamine whether reversal of EMT is NF-nB dependent, wetransfected IKKhWT into CAL27 cells. Activation of NF-nB byIKKhWT offset the effect of lupeol on reversal of EMT. Our work isthe first demonstration that lupeol suppresses tumor cell invasive-ness by reversal of EMT via the down-regulation of NF-nB activity.

To gain further support for our hypothesis, the in vivo effects oflupeol were examined in an orthotopic metastatic nude mousemodel of oral tongue squamous cell carcinoma. In this animalmodel, tongue carcinoma cells invade the lower jaw andmetastasize to the neck lymph node after 37 days of tumorinoculation. On day 37, the body weight of nude mice is decreaseddue to a feeding problem. Lupeol was i.p. administered on day 7after tumor inoculation rather on the day 0 time point. Lupeolshrunk the tumor volume by induction of apoptosis and necrosis asshown in Supplementary Fig. S2. Lupeol showed no sign of toxicity.Interestingly, no local metastasis was detected in lupeol-treatednude mice, likely due to reversal of NF-nB–dependent EMT.Cisplatin alone (5 mg/kg) was able to partly suppress tumorgrowth; however, only three of five nude mice showed no jawinvasion or nodal metastasis. In addition, cisplatin has severe sideeffects in terms of decreasing body weight. Our data showed thatlupeol is surprisingly more potent than cisplatin by f3-fold interms of decrease in tumor volume. Lupeol combined with low-dosecisplatin (5-fold less than cisplatin alone) can effectively suppresstumor growth and metastasis. Most importantly, combinationtherapy with a low dose of cisplatin is f40-fold more potent thancisplatin alone and has no side effects in this animal model.

Taken together, our present study showed, both in vitro andin vivo , the anticancer and antimetastatic efficacy of lupeol, whichacts by down-regulating NF-nB activity, against HNSCC with highNF-nB expression without any toxicity in nonneoplastic tonguefibroblast cells. Our experiments using our animal model showedthat lupeol is much more potent than cisplatin in the treatment ofHNSCC in terms of efficacy, specificity, and toxicity. Lupeol exerteda significant synergistic cytotoxic effect when combined with low-dose cisplatin without side effects. Our tongue model systemprovides a strong basis for the development of lupeol as a singleagent or for use in combination for the treatment of different typesof cancers in which NF-nB plays a significant role.

Acknowledgments

Received 3/2/2007; revised 6/16/2007; accepted 7/17/2007.Grant support: Betty and Kadoorie Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. Hasan Mukhtar for critical reading of the manuscript.

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Correction

Correction: Lupeol Suppresses Cisplatin-InducedNuclear Factor-kBActivation inHeadand Neck Squamous Cell Carcinoma andInhibits Local Invasion and Nodal Metastasisin an Orthotopic Nude Mouse Model

In this article (Cancer Res 2007;67:8800–9), which appeared in the September 15,2007 issue of Cancer Research (1), there are errors in Figs. 2C and 5B; incorrect gelimages belonging to a different experiment were inadvertently used. The affecteddata are total p65 and total p50 in Fig. 2C and a-SMA in Fig. 5B for CAL27 cells.The data shown for MDA1986 cells are accurate. The authors were unable toretrieve the original data, so they repeated results as shown in these three gelphotos as well as other unaffected gel photos within the panels of Figs. 2C and 5Bin CAL27cells but not MDA1986 cells. From the new data, the authors observedsimilar findings as described in the article that lupeol has no effect on total p65and p50 and exerted significant suppressive effect on a-SMA in a time-dependentmanner. For the result in nuclear p65, a discrepancy was found in the time pointfor the decrease in nuclear NF-kB expression upon lupeol administration for thenew data; the authors believe that this discrepancy may be due to differentpassages of CAL27 and a different batch of lupeol used in the new experiment.Overall, the authors found the suppressive effect of lupeol on NF-kB activation.The corrected figures appear below.

The authors believe that the effect of lupeol on NF-kB and EMT represents the truephenomenon and are confident that these inadvertentmistakes infigure assembly donot affect the conclusions drawn or the validity of the underlying research andapologize to the community for any confusion they have caused.

Figure 2.

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Cancer Res; 76(7) April 1, 20162052

Reference1. Lee TK, Poon RTP, Wo JY, Ma S, Guan XY, Myers JN, et al. Lupeol suppresses cisplatin-induced

nuclear factor-kB activation in head and neck squamous cell carcinoma and inhibits local invasionand nodal metastasis in an orthotopic nude mouse model. Cancer Res 2007;67:8800–9.

Published online April 1, 2016.doi: 10.1158/0008-5472.CAN-16-0381�2016 American Association for Cancer Research.

Figure 5.

Correction

www.aacrjournals.org Cancer Res; 76(7) April 1, 2016 2053

2007;67:8800-8809. Cancer Res Terence K. Lee, Ronnie T.P. Poon, Jana Y. Wo, et al. Orthotopic Nude Mouse ModelInhibits Local Invasion and Nodal Metastasis in anActivation in Head and Neck Squamous Cell Carcinoma and

BκLupeol Suppresses Cisplatin-Induced Nuclear Factor-

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LupeolSuppressesCisplatin-InducedNuclearFactor ... · 30-gauge hypodermicneedle. Themice were imagedon days 0, 7,and37 aftertumorinoculation.Micewereanesthetizedwithaketamine/xylazine