þ)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate ... Article (þ)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate Suppresses Solar UV-Induced Skin Carcinogenesis by Targeting PI3K,

Research Article

(þ)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl CaffeateSuppresses Solar UV-Induced Skin Carcinogenesis byTargeting PI3K, ERK1/2, and p38

Do Young Lim1, Mee-Hyun Lee1, Seung Ho Shin1,4, Hanyoung Chen1, Joohyun Ryu1, Lei Shan2,Honglin Li3, Ann M. Bode1, Wei-Dong Zhang2, and Zigang Dong1

AbstractFor decades, skin cancer incidence has increased, mainly because of oncogenic signaling pathways

activated by solar ultraviolet (UV) irradiation (i.e., sun exposure). Solar UV induces multiple signaling

pathways that are critical in the development of skin cancer, and therefore the development of compounds

capable of targeting multiple molecules for chemoprevention of skin carcinogenesis is urgently needed.

Herein, we examined the chemopreventive effects and the molecular mechanism of (þ)-2-(1-hydroxyl-4-

oxocyclohexyl) ethyl caffeate (HOEC), isolated from Incarvilleamairei var. grandiflora (Wehrhahn)Grierson.

HOEC strongly inhibited neoplastic transformation of JB6 Cl41 cells without toxicity. PI3K, ERK1/2, and

p38kinase activitieswere suppressed bydirect bindingwithHOEC in vitro.Our in silicodockingdata showed

that HOEC binds at the ATP-binding site of each kinase. The inhibition of solar UV-induced PI3K, ERK1/2,

and p38 kinase activities resulted in suppression of their downstream signaling pathways and AP1 andNF-

kB transactivation in JB6 cells. Furthermore, topical application ofHOEC reduced skin cancer incidence and

tumor volume in SKH-1 hairless mice chronically exposed to solar UV. In summary, our results show that

HOEC exerts inhibitory effects onmultiple kinase targets and their downstream pathways activated by solar

UV in vitro and in vivo. These findings suggest that HOEC is a potent chemopreventive compound against

skin carcinogenesis caused by solar UV exposure. Cancer Prev Res; 7(8); 856–65. �2014 AACR.

IntroductionSkin cancer is oneof themost common types of cancers in

the United States (1). Epidemiologic studies on cancerincidence show that in the last decade, skin cancer in menhas increased, which is in contrast to the decreases observedin 3 major cancers, prostate, lung, and colon (2, 3). In anytype of skin cancer, the major etiologic factor is solarultraviolet (solar UV, i.e., sunlight) irradiation (4, 5). SolarUV consists of UVA, comprising the largest portion (95%),

which has a relatively weaker effect on causing skin cancercompared with UVB. UVB comprises a smaller portion(5%) of solar UV, but is considered to be a completecarcinogen (6). UVA and UVB cause DNA damage (7) andactivate various signaling pathways for skin tumor promo-tion (8). Recent reports suggest that PI3K (9, 10) andmitogen-activated protein kinase (MAPK) family members,including ERKs (11–15) and p38 (14–18), aremajor factorsin skin carcinogenesis. This conclusion is based on researchfindings showing that these kinases activate oncogenictranscription factors, activator protein-1 (AP1) and nuclearfactor-kB (NF-kB; refs. 19 and 20). Thus, an effective strat-egy might be to directly suppress the activities of thesekinases or their common regulators (21–24) for chemo-prevention of skin cancer.

Elucidating the anticancer effects of plant-derived com-pounds might be a successful approach for chemopreven-tion (24). (þ)-2-(1-Hydroxyl-4-oxocyclohexyl) ethyl caffe-ate (HOEC; Fig. 1) is found in Incarvillea mairei var. gran-diflora (Wehrhahn) Grierson. Although the plants of theIncarvillea genus arewidely grown for ornamental purposes,especially in China, Incarvillea delavyi, one of the Incarvilleaspices, recently received attention for its anti-nociceptiveeffects (25). In particular, HOEC isolated from I. mairei isreported to exhibit anti-inflammatory effects on inflamma-tion-induced mice (26). However, the potential anticancereffects and molecular targets of the plant extract or its leadcompound have not yet been studied.

Authors' Affiliations: 1The Hormel Institute, University of Minnesota,Austin, Minnesota; 2Department of Natural Product Chemistry, SecondMilitary Medical University; 3School of Pharmacy, East China University ofScience & Technology, Shanghai, PR China; and 4Program in BiomedicalInformatics and Computational Biology, University of Minnesota, Minnea-polis, Minnesota.

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Current address forM-HLee: College of Pharmacy, TheCatholic Universityof Korea, Bucheon, Gyeonggi-do, South Korea.

Corresponding Authors: Zigang Dong, The Hormel Institute, University ofMinnesota, 801 16th Avenue NE, Austin, MN 55912. Phone: 1-507-437-9600; Fax: 1-507-437-9606; E-mail: [email protected]; and Wei-DongZhang, Department of Natural Product Chemistry, SecondMilitary MedicalUniversity, Shanghai, 325 Guohe Road, Shanghai 200433, PR China.Phone: 86-218-187-1244; Fax: 86-218-187-1245;E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-13-0286

�2014 American Association for Cancer Research.

CancerPreventionResearch

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Herein, we report that HOEC directly targets and inhibitsthe kinase activities of PI3K, ERK1/2, and p38 and down-regulates their downstream signaling pathways in solarUV–treated JB6 Cl41 mouse epidermal cells. The inhibition ofthese pathways leads to a reduction of AP1 and NF-kBtranscriptional activities. In an in vivo model of solar UV–induced skin carcinogenesis, HOEC significantly reducestumor volume and tumor number by inhibiting the PI3K,ERK1/2, and p38 signaling pathways.

Materials and MethodsReagentsHOEC is a natural compound isolated from the whole

plants of I. mairei and the compound used in this study wassynthesized according to previous reports (27). The purityof HOEC was assessed by high-performance liquid chro-matography (HPLC) and found to be greater than 97%.Active p110 (PI3K), active ERK1, active ERK2, active p38a,inactive RSK2, and inactive ATF2 recombinant proteins forkinase assays were purchased fromMillipore. Antibodies todetect phosphorylated tyrosines (p-Tyr, i.e., p-p110), phos-phorylated Akt (p-Akt S473), phosphorylated ERK1/2 (p-ERK1/2 Thr202/Tyr204), phosphorylated p38 (p-p38Thr180/Tyr182), phosphorylated RSK2 (p-RSK2 Ser380),phosphorylated MSK1 (p-MSK1 Ser376), phosphorylatedATF2 (p-ATF2 Thr69/71), phosphorylated S6 ribosomalprotein (p-S6 ribosomal protein Ser235/236), phosphory-lated c-Fos (p-c-Fos Ser32), phosphorylated c-Jun (p-c-JunSer63), total p110, total ERKs, total RSK, total Akt, totalATF2, totalMSK, total S6 ribosomal protein, total c-Fos, andtotal c-Jun were purchased fromCell Signaling Technology.The antibody against b-actin was from Santa Cruz Biotech-nology. CNBr-sepharose 4B beads were obtained fromAmersham Pharmacia Biotech. The luciferase assay sub-strate and the Cell Titer 96 Aqueous One Solution Reagent[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)]Kit for the cell proliferation assay were from Promega.

Cell cultureThe JB6 Cl41 murine epidermal cell line (a promotion-

sensitive cloneof the JB6Pþ cell line)was cultured inEagle’sminimum essential medium (MEM) and HaCaT humankeratinocytes were cultured in Dulbecco’s modified Eaglemedium (DMEM)/high glucose containing penicillin(100 units/mL), streptomycin (100 mg/mL), and 4% or10% fetal bovine serum (FBS; Gemini Bio-Products),

respectively. Cells were maintained in a 5% CO2, 37�C

humidified incubator. Cells were cytogenetically tested andauthenticated before being frozen. Each vial of frozen cellswas thawed and maintained in culture for a maximum of8 weeks.

In vitro kinase assayThe kinase assay was performed according to the instruc-

tions provided by Millipore. Briefly, for ERK1, ERK2, orp38a activity analysis, the relevant active protein (100 ng)was incubated with HOEC (0, 10, or 20 mmol/L) for 30minutes at 30�C. Then each reaction mixture was mixedwith isotope-unlabeled ATP and 10 mCi [g-32P] ATP witheach compound in 10 mL of reaction buffer containing 20mmol/L HEPES (pH 7.4), 10 mmol/L MgCl2, 10 mmol/LMnCl2, and 1 mmol/L dithiothreitol (DTT). After incuba-tion at 30�C for 30 minutes, the reaction was stopped byadding 5 mL protein loading buffer and the mixture wasseparated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). For measuring PI3K activity,an active p110aprotein (100ng)was incubatedwithHOEC(0, 10, or 20 mmol/L) for 30minutes at 30�C. Then, 20 mL of0.5mg/mL phosphatidylinositol (Avanti Polar Lipids) wereadded, and themixturewas incubated for 5minutes at roomtemperature. Reaction buffer (100 mmol/L HEPES, pH 7.6,50 mmol/L MgCl2, 250 mmol/L ATP) containing 10 mCi[g-32P] ATP was added, and the mixture was incubated foran additional 30minutes at 30�C. The reactionwas stoppedby the addition of 15 mL of 4 N HCl and 130 mL ofchloroform:methanol (1:1) and mixed by vortexing. Thelower chloroform phase (30 mL) was spotted onto a 1%potassium oxalate–coated silica-gel plate (previously acti-vated for 1 hour at 110�C) and subjected to thin-layerchromatography and autoradiography to visualize the32P-labeled phosphatidylinositol 3-phosphate (PIP) prod-uct. Each experiment was repeated twice and the relativeamounts of incorporated radioactivity were assessed byautoradiography.

In vitro and ex vivo pull-down assaysRecombinant p110a, ERK1, ERK2, or p38a (200 ng)

proteins or solar UV–treated JB6 Cl41 cell lysates (500 mg)were incubated with HOEC-sepharose 4B (or sepharose 4Bonly as a control) beads (50 mL 50% slurry) in reactionbuffer (50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA,150 mmol/L NaCl, 1 mmol/L DTT, 0.01% NP-40, and2 mg/mL bovine serum albumin). After incubation withgentle rocking overnight at 4�C, the beads were washed 3timeswith buffer (50mmol/L Tris, pH7.5, 5mmol/L EDTA,150 mmol/L NaCl, 1 mmol/L DTT, and 0.01%NP-40) andbinding was visualized by Western blotting.

ATP and HOEC competition assayTo determine whether HOEC inhibits a protein’s kinase

activity by interacting with the protein’s ATP-binding pock-et, we performed an ATP andHOEC competition assay. ATP(0, 1, 10, or 100 mmol/L) was mixed with active p110a,ERK1, ERK2, or p38a (200 ng) for 30 minutes. After

Figure 1. Chemical structure of HOEC.

Anti-skin Carcinogenic Mechanisms of HOEC

www.aacrjournals.org Cancer Prev Res; 7(8) August 2014 857




incubation, HOEC-sepharose 4B (or sepharose 4B only as acontrol) beads (50 mL 50% slurry) were added in reactionbuffer and kept at 4�C overnight. The beads were washedand proteins (p110, ERK1, ERK2, or p38) were detected byWestern blot analysis.

Solar UV irradiation systemThe solar UV source comprised UVA-340 lamps pur-

chased from Q-Lab Corporation. The UVA-340 lamps pro-vide the best possible simulation of sunlight in the criticalshort wavelength region from 365 nm down to the solarcutoff of 295 nmwith a peak emission of 340 nm (28). Thepercentage of UVA and UVB emitted from UVA-340 lampswas measured by a UV radiometer and was recorded as94.5% and 5.5%, respectively. Specifically, uncovereddishes of serum-starved cells were exposed toUV irradiationunder UVA-340 lamps. The dose of solar UV radiation was48 kJ/m2 UVA/2.9 kJ/m2 UVB, which was the same doseused in the animal study.

Western blot analysisCells were disrupted on ice for 40 minutes in cell lysis

buffer [20mmol/L Tris pH 7.5, 150mmol/LNaCl, 1mmol/LNa2EDTA, 1mmol/L EGTA, 1%TritonX-100, 2.5mmol/Lsodium pyrophosphate, 1 mmol/L b-glycerophosphate, 1mmol/L sodium vanadate, and 1 mmol/L phenylmethyl-sulfonyl fluoride (PMSF)]. After centrifugation at 12,000rpm for 10minutes, the supernatant fraction was harvestedas the total cellular protein extract. The protein concentra-tion was determined using the Bio-Rad protein assayreagent. Total cellular protein extracts were separated bySDS-PAGE and transferred to polyvinylidene fluoride(PVDF) membranes in 20 mmol/L Tris-HCl (pH 8.0),containing 150 mmol/L glycine and 20% (v/v) methanol.Membranes were blocked with 5% nonfat dry milk in 1 �TBS containing 0.05% Tween 20 (TBS-T) and incubatedwith antibodies against p-Tyr (p-p110), p110a, p-Akt,Akt, p-ERK1/2, ERK1/2, p-RSK, RSK, p-p38, p38, p-ATF2,ATF2, p-S6 ribosomal protein (p-S6), S6 ribosomal protein(S6), p-MSK1, MSK1, p-c-Fos, c-Fos, p-c-Jun, c-Jun, orb-actin. Blots were washed 3 times in 1� TBS-T buffer,followed by incubation with an appropriate horseradishperoxidase (HRP)–linked IgG. The specific proteins in theblots were visualized using the enhanced chemilumines-cence (ECL) detection reagent.

In silico target identification and molecular modelingTo identify the potential binding proteins of HOEC, a

shape similarity method (28) using the PHASE module ofSchr€odinger’s molecular modeling software package wasperformed to search forbiological targets ofHOECbasedonits structure. The atom-type parameter for volume was set atpharmacophore, which means the queries were used notonly to consider shape similarity but also to align potentialpharmacophore points between the queries and the targets.The target library was obtained from the Protein Data Bank(29) and our in-house library. To provide more structuralorientations for possible alignment, we set the maximum

number of conformers permolecule to be generated to 100,while retaining up to 10 conformers per rotatable bond.Wefiltered out conformers with similarity below 0.75. We thenobtained the PDB ID associated with each aligned targetmolecule and used these PDB IDs to search the onlineProtein Data Bank to identify the protein. Based on theshape similarity results, ERK1/2, PI3K and p38awere iden-tified as potential protein targets of HOEC. To study theHOEC interaction with PI2K, ERK1/2, and p38a, compu-tational docking and modeling studies were performed.First, an X-ray diffraction structure of p110with a resolution

of 2.8 A�

(PDB ID 3HHM, Chain A; ref. 30), an X-ray

diffraction structure of ERK1 with a resolution of 2.39 A�

(PDB ID 2ZOQ; ref. 31), an X-ray diffraction structure of

ERK2 with a resolution of 2.4 A�(PDB ID 2OJJ; ref. 32) and

an X-ray diffraction structure of p38with a resolution of 2.9

A�(PDB ID 3DS6; ref. 33) were downloaded. Hydrogen

atoms were added consistent with a pH of 7 and all watermolecules removed. Finally, an ATP-binding site–basedreceptor grid for docking with each respective kinase wasgenerated. HOEC was prepared for docking by defaultparameters using the LigPrep program in Schr€odinger. ThenHOEC-protein docking was accomplished with the pro-gram Glide using default parameters under the extra preci-sion (XP)mode allowing the acquisition of the best-dockedrepresentative structure.

Reporter gene activity of AP1 or NF-kB transactivationJB6 Cl41 cells stably transfected with an AP1 or NF-kB

luciferase reporter plasmid were seeded (1 � 104 viablecells/well) into a 24-well plate. Cells were incubatedovernight at 37�C in a humidified atmosphere of 5%CO2 and were starved in serum-free medium for another24 hours. The cells were exposed to solar UV (48 kJ/m2

UVA/2.9 kJ/m2) and then treated with HOEC (0, 10, or 20mmol/L). After 6 hours, cells were harvested and disruptedwith 100 mL of lysis buffer [0.1 mmol/L potassium phos-phate pH 7.8, 1% Triton X-100, 1 mmol/L dithiothreitol(DTT), and 2 mmol/L EDTA]. Then firefly luciferase activ-ities were measured by a luminometer (LuminoskanAscent, Thermo Fisher Scientific) using substrates provid-ed in the reporter assay system (Promega).

Solar UV–induced mouse skin carcinogenesis studyFemale SKH-1 hairlessmice, 6 weeks old, were purchased

fromCharles River andmaintained under conditions basedon the guidelines established by Research Animal Resourcesand the University of Minnesota Institutional Animal Careand Use Committee (IACUC). Skin carcinogenesis in micewas induced using UVA-340 lamps. Mice were divided into5 groups. In the control groups, the dorsal skinwas topicallytreated with acetone as vehicle (n ¼ 9) or HOEC (0.5 mg/mouse, n ¼ 12) in 200 mL acetone. In the solar UV–treatedgroup (n ¼ 21), the dorsal skin was topically treated with200 mL acetone after exposure to solar UV. The mice in 1group (0.5 mg/mouse, n¼ 20) received topical applicationof HOEC for a total of 30 weeks three times a week. The UV

Lim et al.

Cancer Prev Res; 7(8) August 2014 Cancer Prevention Research858




absorption spectra ofHOEC indicated thatHOECabsorbedUV in the solar UV range between 250 and 380 nm, whichmeans that HOEC could act as a sun-blocking agent. There-fore, to eliminate the UV blocking effect of HOEC, wetreated mice with HOEC for 12 weeks after solar UV expo-sure and for an additional 18 weeks without UV exposure.The HOEC (0.5 mg/mouse) was dissolved in 200 mL of

acetone, which is sufficient to cover all of the dorsal skinarea of eachmouse, whichmeasured approximately 2� 4.5cm2. The dose of solar UV was gradually increased by 10%each week for 6 weeks to acclimate the mice to the protocoland prevent sunburn and hyperplasia while maintainingthe tumor-promoting effect of solar UV. At week 1, the solarUV dose was 30 kJ/m2 UVA/1.8 kJ/m2 UVB given twice/week. At week 6, the dose was 48 kJ/m2UVA/2.9 kJ/m2UVBand this dosewasmaintained forweeks 6 to 12 and thenUVtreatment was discontinued and tumor growth was mon-itored for an additional 18weeks. A tumorwas defined as anoutgrowth of at least 1 mm in diameter that persisted for 2weeks or more. Mouse weight and tumor number andvolume were recorded every week until the end of theexperiment (30 weeks). Tumors and skin were frozen forWestern blot analysis. Tumor volume was calculatedaccording to the following formula: tumor volume (mm3)¼ length � width � height � 0.52.

Statistical analysisAll quantitative results are expressed asmeanvalues�SD.

Statistically significant differences were obtained using theStudent t test or by one-way ANOVA. A P-value of <0.05wasconsidered to be statistically significant.

ResultsHOEC binds with p110, ERK1, ERK2, or p38 at theirrespective ATP-binding siteTo better understand how HOEC interacts with p110a

(Fig. 2Aa-b), ERK1 (Fig. 2Ba-b), ERK2 (Fig. 2Ba, c), or p38a(Fig. 2Ca-b), we performed a computational docking studyusing the Glide docking program included in Schr€odingerSuite 2012. In the docked models, HOEC binds well atthe ATP-binding pockets of all 4 kinases (Fig. 2). Takentogether with the in vitro and ex vivo pull-down assayresults (Supplementary Fig. S1A–S1C), our data con-firmed that HOEC binds to the respective ATP-bindingsite and inhibits the kinase activity of p110a, ERK1,ERK2, or p38a. The images were generated using theUCSF Chimera program (34).

PI3K, ERK1, ERK2, andp38a aremajor targets ofHOECTo rule out the possibility that the inhibitory effect of

HOEC was because of toxicity, we conducted a cell toxicityassay and results indicated that HOEC had no cytotoxiceffect on normal JB6 Cl41murine epidermal cells or HaCaThuman keratinocytes (data not shown). To identify directtarget(s) of HOEC, we conducted several in vitro kinaseassays. HOEC selectively inhibited PI3K (10 mmol/L,48%; 20 mmol/L, 97%; Supplementary Fig. S1A-a), ERK1(10 mmol/L, 6%; 20 mmol/L, 16%; Supplementary Fig. S1A-

b), ERK2 (10 mmol/L, 8%; 20 mmol/L, 54%; SupplementaryFig. S1A-c), and p38a (10 mmol/L, 53%; 20 mmol/L, 68%;Supplementary Fig. S1A-d) kinase activities in a concentra-tion-dependent manner, but had no effect on EGFR or JNKskinase activity (data not shown). To confirm and identifyadditional kinase targets of HOEC,we used a commercial invitro kinase profiling system (Kinase Profiler, Millipore).The Kinase Profiler results showed that all other kinasestested were not affected by HOEC (Supplementary TableS1). To determine whether HOEC interacts directly withp110a, ERK1, ERK2, or p38a, we performed in vitro (Sup-plementary Fig. S1B-a) and ex vivo (Supplementary Fig. S1B-b) pull-down binding assays. Recombinant p110a, ERK1,ERK2, andp38a strongly interactedwithHOEC-conjugatedsepharose 4B beads (Supplementary Fig. S1B-a). In addi-tion, these proteins in solar UV–treated JB6 Cl41 cell lysateswere pulled down with HOEC-sepharose 4B beads (Sup-plementary Fig. S1B-b). To determine whether the bindingof HOEC to p110a, ERK, or p38a occurs in an ATP-com-petitive manner, we performed an ATP (0, 1, 10, or 100mmol/L) competitive kinase pull-down assay. Resultsshowed that the binding of HOEC to p110a, ERK1, ERK2,or p38a decreased in an ATP-competitive manner (Supple-mentary Fig. S1C). These results suggest that PI3K, ERK1,ERK2, and p38a are major targets of HOEC.

HOEC downregulates solar UV–inducedphosphorylation of PI3K, ERKs, and/or p38downstream signaling pathways

Our data showed that HOEC binds to the ATP-bindingsite of p110a, ERK1, ERK2, or p38a (Fig. 2) and suppressestheir respective activity (Supplementary Fig. S1A-a–S1A-d).Therefore, the inhibitory effects ofHOEC likely suppress therespective downstream signaling pathways. The PI3K/Aktand MAPKs pathways are highly activated in UV-inducedskin cancers (10, 11, 16). HOEC decreased solar UV–induced phosphorylation of PI3K downstream proteins,including Akt (10 mmol/L, 38%; 20 mmol/L, 62%) andS6 ribosomal protein (S6; 10 mmol/L, 5%; 20 mmol/L,62%) comparedwith the same kinases in solar UV–inducedHOEC, untreated JB6 Cl41 cells (Fig. 3A). ERK1/2 and p38are MAPK family members and HOEC affects these MAPKsignaling pathways. RSK2 and MSK1 are ERKs downstreamsignaling proteins and ATF2, c-Jun, and c-Fos act down-stream of p38. Results indicated that HOEC (20 mmol/L)suppressed solar UV–induced phosphorylation of RSK2 by85%, MSK1 by 64% (Fig. 3B), c-Fos by 29%, and c-Jun by64%(Fig. 3C). These findings suggest thatHOECsuppressessolar UV–induced activation of PI3K/Akt and MAPKs sig-naling pathways mediated by their well-known upstreamkinases, PI3K, ERK1, ERK2, or p38.

HOEC suppresses the transcriptional activity of AP1and NF-kB

After stimulationwithUV, the transcriptional activities ofAP1 andNF-kB are induced through the PI3K/Akt orMAPKssignaling pathways (8, 35). To examine the effect of HOECon the transcriptional activity ofAP1 andNF-kB, we exposed






JB6 Cl41 cells stably transfected with the AP1 or NF-kBluciferase reporter plasmid to solar UV (48 kJ/m2 UVA/2.9kJ/m2 UVB) and then treated cells with HOEC for another6 hours. HOEC (20 mmol/L) suppressed solar UV–inducedtransactivation of AP1 by 47% and NF-kB by 80% (Fig. 4).

HOEC attenuates solar UV–induced skincarcinogenesis in SKH-1 hairless mice

To determine the chemopreventive effects of HOEC invivo, we utilized a solar UV–induced skin carcinogenesismouse model. We treated mice with solar UV and then

Figure 2. Confirmation of the predicted interaction between HOEC and p110a, ERK1, ERK2, or p38a using a computer dockingmodel. A-a, HOEC can bind atthe ATP-binding pocket of p110a. A-b, enlarged view of HOEC binding with p110a at the ATP-binding pocket and formation of hydrogen bonds. B-a,HOEC can bind at the ATP-binding pocket of ERK1 or 2. Enlarged view of HOEC binding with ERK1 (B-b) or ERK2 (B-c) at the ATP-binding pocketand formation of hydrogen bonds. C-a, HOEC can bind at the ATP-binding pocket of p38a. C-b, Enlarged view of HOEC binding with p38a at theATP-binding pocket and formation of hydrogen bonds. For A to C, hydrogen bonding is shown as cyan-colored lines.

Lim et al.





topically applied vehicle or HOEC to the dorsal surface ofSKH-1 hairless mice. Volume (Fig. 5A) and number (Fig.5B) of tumors in mice treated with HOEC (0.5 mg/mouse)after solar UV exposure were significantly inhibited byHOEC (P < 0.05). At week 30, all mice were sacrificed andmouse skin and tumor tissues were collected for Westernblot analysis. Phosphorylation of p110, Akt, S6K, RSK,MSK, p38, ATF2, c-Jun, and c-Fos was induced by solar UVand HOEC strongly suppressed the phosphorylation (Fig.5C and Supplementary Fig. S2). The phosphorylation levelof HOEC-targeted kinases was quantified by densitometry(Fig. 5C). RSK2, MSK1, S6K40, and c-Jun are well-knownsubstrates of ERK1/2, p110, and p38, respectively. Thephosphorylation levels of RSK2, MSK1, S6K40, and c-Junwere significantly induced by solar UV treatment andstrongly inhibited by HOEC (40%, 50%, 77%, and 77%inhibition, respectively) in skin tissues (Fig. 5C). In addi-tion, phosphorylated Akt, ATF2, and c-Fos levels were alsodecreased by HOEC treatment (Supplementary Fig. S2).PCNA and the cleavage status of caspase-7 or PARP arerepresentative markers of cell growth or cell death, respec-tively. Moreover, HOEC substantially decreased PCNAlevels in skin tissues. However, HOEC did not affect cas-pase-7 or PARP cleavage. Note that HOEC was topicallyapplied after UV exposure to exclude the possibility of anysunblock effect of the HOEC compound (Supplementary

Fig. S3). To eliminate the possibility that residual HOECcould remain on the skin between UV exposures, we exam-ined the amount of HOEC remaining in the dorsal skintissues (500mg) frommice sacrificed at 0, 24, and 48 hoursafter HOEC treatment. Mass spectrometry (LC/MS) resultsindicated that skin tissues harvested at 24 or 48 hours afterHOEC treatment showed decreased HOEC levels of 50% or90%, respectively, compared with skin treated with HOECand harvested immediately (data not shown). This resultsupports the idea that residual HOEC does not blockpenetration of subsequent UV irradiation because most(90%) of the HOEC had disappeared within 48 hours.Overall, our data show that HOEC binds its target kinasesand inhibits their respective activities, blocking signal trans-duction to their downstream molecules resulting indecreased cell growth but not necessarily cell death. Theseresults suggest that HOEC is a robust multitargeted chemo-preventive agent against solar UV–induced skin carcino-genesis that acts through mechanisms other than, but notentirely excluding, a sunblock effect.

DiscussionCancer chemoprevention with natural compounds is a

promising strategy because of the ability of these com-pounds to inhibit multiple signaling pathways activatedby carcinogens (24) and tumor promotors. Solar UV,

Figure 3. HOEC downregulates solar UV–induced phosphorylation of PI3K, ERKs, and/or p38 downstream signaling pathways. A, HOEC inhibitssolar UV–induced phosphorylation of Akt and S6 ribosomal protein. B, HOEC inhibits solar UV–induced phosphorylation of RSK2 and MSK1. C, HOECsuppresses solar UV–induced phosphorylation of ATF2, c-Fos, and c-Jun. JB6 Cl41 cells were serum-deprived with MEM containing 0.01% FBS andtreated or not treated with solar UV (48 kJ/m2 UVA/2.9 kJ/m2 UVB) and HOEC (0, 10, or 20 mmol/L). Treated cells were disrupted 30 minutes after solarUV as described in Materials and Methods. The levels of phosphorylated and total proteins as indicated were determined by Western blot analysis.Representative blots from three independent experiments are shown. The relative abundanceof each band is normalized to its own total protein orb-actin andquantified. The value is shown above each respective lane in each blot. The untreated control levels were set at 1 and all lanes are compared with the control.






comprising both UVA and UVB, is a major factor in theincreased incidence of skin cancers, including nonmela-noma skin cancer and melanoma (36, 37). Moreover,multiple signaling pathways are activated by UV exposureand lead to the acquisition of gene alterations in skin cells(38, 39). Therefore, identifying chemopreventive com-pounds with multiple molecular targets against solar UV–induced skin carcinogenesis is crucial because of the con-tinued increasing incidence of this cancer. Several groupshave reported the skin photoprotective effects of variousphytochemicals (11, 22, 40). We used the natural com-pound, HOEC, which is a caffeic ester from I. mairei var.

grandiflora (Wehrhahn) Grierson (27, 41). HOEC wasrevealed to act as an anti-inflammatory agent targeting 5-LOX, which confirmed a key role of 5-LOX in the patho-genesis of inflammation, especially rheumatoid arthritis(26). HOEC may be a prodrug of caffeic acid because ofthe chemical relevance and biofunctional similarity. How-ever, the mechanism of the anticancer effects and directtargets of HOEC have not yet been elucidated.

In this study, we found that HOEC directly binds withPI3K, ERK1/2, and p38 and inhibits their kinases activities.PI3K, ERK1/2, and p38 are known to play critical roles inseveral cancers, including skin cancer (9, 12, 16, 39). ThePI3K/Akt signaling pathways are activated byUV irradiationand can induce apoptosis in human keratinocytes (42).ERK1/2 signaling pathways are also stimulated by UVirradiation and UVB irradiation of keratinocytes results inthe activation of ERK1/2 and p38 MAPK pathways(8, 14, 16, 39). Animal study results also revealed thattreatment of mouse skin with HOEC inhibits solar UV–induced skin carcinogenesis by attenuating PI3K, ERK1/2,and p38 activation leading to decreased downstream sig-nalings. The effects ofHOEConUV-inducedoxidative stressor UV-induced inflammation have not yet been elucidated.However, HOEC is a novel compound that suppresses solarUV–induced skin carcinogenesis by directly binding andinhibiting the activity of multiple kinases, resulting in theattenuation of UV-induced inflammation and skin carci-nogenesis (14). Thus, we suggest that HOEC is a novelcompound that functions as amultikinase inhibitor of solarUV–induced skin cancer.

The pursuit of molecular protein targets of naturalcompounds in skin cancer or other cancers is challenging.HOEC is a novel and unique compound because it targetsat least 3 important proteins that play significant rolesin solar UV–induced skin cancer, which makes it verypotent in suppressing solar UV–induced skin carcinogen-esis. Compared with taxifolin (43) or norathyriol (11),which each effectively suppressed solar UV–induced skincarcinogenesis in mice, HOEC has stronger effects on itstarget kinases’ activities and downstream transcriptionactivities. For example, the effect of HOEC against PI3K(IC50 ¼ 10.3 mmol/L), is 7.6-fold greater than that oftaxifolin (IC50 ¼ 78.4 mmol/L). The inhibition of ERK2and p38 by HOEC (IC50 ¼ 20.9 and 12.9 mmol/L, respec-tively) is 1.4- and 2.3-fold greater than the inhibition ofEGFR by taxifolin (IC50 ¼ 29.0 mmol/L). Moreover, theinhibition of AP1 and NF-kB transcription activity byHOEC was 1.4- and 2.8-fold greater than the effect ofnorathyriol (11).

UV-induced activation of these kinases leads to increasedAP1 and NF-kB transcription activities (43, 44). With UVirradiation, AP1 is stimulated in human keratinocytes andpromotes tumor development and UVB-induced photocar-cinogenesis is reduced by repression of AP1 and NF-kBactivation (45, 46). Because AP1 and NF-kB activate manyoncogenes in carcinogenesis, the inhibitory effect of HOEConAP1 andNF-kB transactivation activitymight account forits ability to suppress EGF-induced anchorage-independent

Figure 4. HOEC suppresses the transcriptional activity of AP1 or NF-kB. JB6 Cl41 cells stably transfected with an AP1 (A) or NF-kB (B)luciferase reporter plasmid were cultured and serum-deprived withMEM containing 0.01% FBS. Serum-deprived cells were treated withsolar UV (48 kJ/m2 UVA/2.9 kJ/m2 UVB) followed by treatment withHOEC (0, 10, or 20 mmol/L) for 6 hours. Luciferase activity wasmeasured as described in Materials and Methods. HOEC inhibits AP1(A) or NF-kB (B) transcriptional activity in JB6 Cl41 cells. Data,mean � SD of values from triplicate samples from three independentexperiments. �, indicates a significant difference between vehicle-treated and solar UV–induced transcriptional activity (P < 0.05), and ��,indicates a significant difference between untreated and HOEC-treated cells (P < 0.05).

Lim et al.





growth of JB6 Cl41 Pþ murine epidermal cells and HaCaThuman keratinocytes.This result is also supported by our in silico identifica-

tion and computational docking model. The theory ofshape-similarity screening is derived from the idea thatmolecules possessing similar shape and electrostatic capa-bilities might exhibit analogous biological activity.According to our in silico computer screening results,HOEC is similar in shape to well-known kinase inhibitorsof PI3K, ERK2, and p38. The specific similarity score ofHOEC to each kinase is 0.83 with (Z)-5-((5-(4-fluoro-2-hydroxyphenyl)furan-2-yl)methylene)thiazolidine-2,4-dione (47), which is a known ERK2 inhibitor; 0.83 with1-(4-bromophenyl)-2-(5-(furan-2-yl)-4H-1,2,4-triazol-3-ylthio)ethanone (48), which is a known PI3K a inhibitor;and 0.81 with 1-(5-tetra-butyl-2-methyl-2H-pyrazol-3-YL)-3-(4-chloro-phenyl)-urea (BMU; ref. 49), which isa known p38 inhibitor, respectively. Based on these

results, ERK1/2, PI3K, and p38 are potential proteintargets of HOEC.

Most studies focusing on UV-induced signaling haveinvolved mainly UVB irradiation. However, in this study,weused solarUV treatment,which comprises bothUVAandUVB, making it similar to our natural environment. Aneffective strategy to suppress solar UV–induced skin carci-nogenesismight include the use of amultitargeted inhibitorbecause solar UV activates multiple signaling pathways. Weandothers have found that determining themost importanttarget among many signals activated by UV is highly chal-lenging and to attempt to suppress only one signalingmolecule is probably not feasible (24). Notably, the che-mopreventive effects of HOEC are not because of its absorp-tion of UV (as described in theMaterials andMethods), butbecause of a direct inhibition of key signaling pathways.

In summary, HOEC attenuated solar UV–induced PI3K,ERK1/2, and p38 signaling pathways in JB6 Cl41 cells and

Figure 5. HOEC suppresses solarUV–induced skin carcinogenesis inSKH-1 hairless mice. Mice weredivided into 4 groups and treatedas described in the Materials andMethods. Groups are as follows: (i)vehicle and no solar UV; (ii) HOEC(0.5 mg/mouse) and no solar UV;(iii) solar UV þ vehicle; (iv) solar UVþ HOEC (0.5 mg/mouse). HOECwas applied topically in acetone onthe dorsal surface of each mouseafter exposure to solar UV(48 kJ/m2 UVA/2.9 kJ/m2 UVB).Tumor volume (A) and averagenumber (B) of tumors per mouseare shown. Data, mean � SD.�, indicates a significant differencebetween the vehicle-treated andHOEC-treated groups exposed tosolar UV (P < 0.05). C, the levels ofphosphorylated and total targetedproteins in mouse skin wereanalyzed by Western blot analysis.Quantitative analysis ofrepresentative results fromWestern blot assay was performedand is shown as means � SD(n¼ 3). The control (vehicle-treatedand no solar UV group) levels wereset at 100%. �, indicates asignificant difference between thevehicle-treated and HOEC-treatedgroups exposed to solar UV(P < 0.05) . ��, indicates a significantdifference between untreatedand HOEC-treated groups withinthe solar UV–exposed groups(P < 0.05).






decreased skin tumor formation in SKH-1 hairless miceexposed to solar UV. The chemopreventive effect of HOECon skin carcinogenesis in vitro and in vivo is because of directbinding and inhibition of PI3K, ERK1/2, and p38 activa-tion, resulting in suppression ofAP1 andNF-kB transactiva-tion. Treatment with HOEC resulted in decreased cellgrowthbut did not necessarily cause cell death inpreventingskin carcinogenesis. Based on our results, we conclude thatthe identification of HOEC as a chemopreventive agentagainst skin cancer might contribute to the further devel-opment of novel multitargeted agents to prevent solar UV–induced skin carcinogenesis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D.Y. Lim, H. Li, W.-D. Zhang, Z. DongDevelopment of methodology: M.-H. Lee, W.-D. Zhang, Z. DongAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): D.Y. Lim, S.H. Shin, L. Shan, W.-D. Zhang,Z. Dong

Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):D.Y. Lim, M.-H. Lee, S.H. Shin, H. Chen,J. Ryu, L. Shan, A.M. Bode, W.-D. Zhang, Z. DongWriting, review, and or revision of the manuscript:D.Y. Lim, M.-H. Lee,S.H. Shin, H. Chen, L. Shan, A.M. Bode, Z. DongAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.-H. Lee, W.-D. Zhang, Z. DongStudy supervision: A.M. Bode, W.-D. Zhang, Z. Dong

AcknowledgmentsThe authors thank T. Schuster and N. Oi for assistance with experiments

and N. Brickman for assisting with article submission.

Grant SupportThis work was supported by The Hormel Foundation and NIH

grants CA166011, CA172457, R37 CA081064, ES016548, and CA027502(to Z. Dong).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 1, 2013; revised May 14, 2014; accepted May 16, 2014;published OnlineFirst May 20, 2014.

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2014;7:856-865. Published OnlineFirst May 20, 2014.Cancer Prev Res Do Young Lim, Mee-Hyun Lee, Seung Ho Shin, et al. p38UV-Induced Skin Carcinogenesis by Targeting PI3K, ERK1/2, and (+)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate Suppresses Solar

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þ)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate ... Article (þ)-2-(1-Hydroxyl-4-Oxocyclohexyl) Ethyl Caffeate Suppresses Solar UV-Induced Skin Carcinogenesis by Targeting PI3K,