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Research Article 3,6-Dihydroxyavone Suppresses Breast Carcinogenesis by Epigenetically Regulating miR-34a and miR-21 Xiaoli Peng 1,2 , Hui Chang 1 , Yeyun Gu 1 , Junli Chen 1 , Long Yi 1 , Qi Xie 1 , Jundong Zhu 1 , Qianyong Zhang 1 , and Mantian Mi 1 Abstract Our previous study selected a promising chemopreventive agent 3,6-dihydroxyavone (3,6-DHF) and found that 3,6-DHF signicantly upregulates miR-34a and downregulates miR-21 in breast carcinogenesis, yet the upstream and downstream events of the anticancer mechanism remain unclear. The present study showed that 3,6-DHF cotreatment effectively inhibits carcino- gens-induced breast carcinogenic transformation in human breast epithelial MCF10A cells. The data revealed the signicant down- regulation of miR-34a and upregulation of miR-21 in breast carcinogenesis, which could be mitigated by 3,6-DHF treatment. Methylation-specic PCR detections showed that 3,6-DHF inhi- bits the hypermethylation of the miR-34a promoter. Further studies indicated that 3,6-DHF is an effective methyltransferase (DNMT)1 inhibitor, docking to the putative cytosine pocket of the protein, and thus decreases the DNMT activity in a dose-depen- dent manner. Moreover, the ChIP-qPCR analysis for histone modications showed that 3,6-DHF treatment signicantly low- ers the H3K9-14ac on the miR-21 promoter. In addition, our study revealed that 3,6-DHF represses the PI3K/Akt/mTOR sig- naling pathway in breast carcinogenesis in vitro and in vivo. Inhibition of miR-34a or overexpression of miR-21 signicantly reduced the effects of 3,6-DHF on Notch-1 and PTEN, and consequently weakened the suppression of 3,6-DHF on PI3K/ Akt/mTOR. We concluded that 3,6-DHF upregulates miR-34a via inhibiting DNMT1 and hypermethylation, whereas downregu- lates miR-21 by modulating histone modication, and conse- quently suppresses the PI3K/Akt/mTOR signaling pathway in breast carcinogenesis. Cancer Prev Res; 8(6); 19. Ó2015 AACR. Introduction Breast cancer is one of the most serious health threats to women worldwide. In the United States, 230,480 new cases of invasive breast cancer were diagnosed in 2011. The lifetime risk of being diagnosed with breast cancer is 12.5% for women in the United States, and approximately 39,520 women are expected to die from this disease annually (1). Despite advances in screening, diagno- sis, and therapy, breast cancer continues to pose an enormous global healthcare problem. miRNAs are a class of 20- to 25-nucleotide-long noncoding RNAs that negatively regulate gene expression by binding to the 3 0 untranslated region (3 0 UTR) of target messenger RNAs, causing translational repression or degradation. miRNAs have been well established to be aberrantly expressed in carcinogenesis and progression, where they function as tumor suppressors or act as oncogenes. Our study has revealed the global upregulation of miR-21 and downregulation of miR-34a in breast carcinogenesis (2). As a proapoptotic transcriptional target of p53, miR-34a has been shown to behave as a tumor suppressor by repressing genes involved in various oncogenic signaling pathways. On the con- trary, miR-21 is a notable oncogene, which is consistently over- expressed in a wide range of cancers and plays a key role in resisting programmed cell death in cancer cells (3). We have previously selected a promising anticancer agent, the avonol 3,6-dihydroxyavone (3,6-DHF), in pharmacodynamic experi- ments, and demonstrated that it is a potent natural chemopreven- tive agent against breast carcinogenesis. IN addition, the previous study also revealed that 3,6-DHF treatment signicantly upregu- lates miR-34a and downregulates miR-21 in breast cancer cells, yet the upstream and downstream events of the anticancer mecha- nism remain unclear. Increasing evidence supports that the epigenetic variability at specic transcription regulation sites appears to be susceptible to modulation by diet and nutrition; some dietary components may suppress tumorigenesis and development by affecting the process of DNA methylation and histone modications, which modulates the expression of certain key genes and the activation of crucial signaling pathways (4). In the last two decades, the study of epigenetic modication emerged as one of the major areas of cancer treatment. The key processes responsible for epigenetic regulation are DNA methylation and modications in chromatin and posttranscriptional gene regulation by noncoding RNA. Thus, we hypothesized that 3,6-DHF may regulate the expressions of miR-34a and miR-21 by modulating DNA methylation or histone modication. The PI3K/Akt/mTOR signaling pathway is critical to normal cellular physical progress and also commonly overactivated in cancer progression, especially in breast carcinogenesis and 1 Research Center for Nutrition and Food Safety,Third Military Medical University; Chongqing Key Laboratory of Nutrition and Food Safety, Chongqing, China. 2 Department of Public Health, School of Preclinical Medicine, Chengdu Medical College, Chengdu, China. Corresponding Authors: Hui Chang, Research Center for Nutrition and Food Safety, 30 Gaotanyan Street, Chongqing 400038, China; Phone: 8602368752291; Fax: 8602368752291; E-mail: [email protected]; or Mantian Mi, [email protected] doi: 10.1158/1940-6207.CAPR-14-0357 Ó2015 American Association for Cancer Research. Cancer Prevention Research www.aacrjournals.org OF1 for Cancer Research. on March 1, 2019. © 2015 American Association cancerpreventionresearch.aacrjournals.org Downloaded from Published OnlineFirst March 17, 2015; DOI: 10.1158/1940-6207.CAPR-14-0357
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Research Article

3,6-Dihydroxyflavone Suppresses BreastCarcinogenesis by Epigenetically RegulatingmiR-34a and miR-21Xiaoli Peng1,2, Hui Chang1, Yeyun Gu1, Junli Chen1, Long Yi1, Qi Xie1, Jundong Zhu1,Qianyong Zhang1, and Mantian Mi1

Abstract

Our previous study selected a promising chemopreventiveagent 3,6-dihydroxyflavone (3,6-DHF) and found that 3,6-DHFsignificantly upregulates miR-34a and downregulates miR-21 inbreast carcinogenesis, yet the upstream and downstream events ofthe anticancer mechanism remain unclear. The present studyshowed that 3,6-DHF cotreatment effectively inhibits carcino-gens-induced breast carcinogenic transformation in humanbreastepithelial MCF10A cells. The data revealed the significant down-regulation of miR-34a and upregulation of miR-21 in breastcarcinogenesis, which could be mitigated by 3,6-DHF treatment.Methylation-specific PCR detections showed that 3,6-DHF inhi-bits the hypermethylation of the miR-34a promoter. Furtherstudies indicated that 3,6-DHF is an effective methyltransferase(DNMT)1 inhibitor, docking to theputative cytosine pocket of the

protein, and thus decreases the DNMT activity in a dose-depen-dent manner. Moreover, the ChIP-qPCR analysis for histonemodifications showed that 3,6-DHF treatment significantly low-ers the H3K9-14ac on the miR-21 promoter. In addition, ourstudy revealed that 3,6-DHF represses the PI3K/Akt/mTOR sig-naling pathway in breast carcinogenesis in vitro and in vivo.Inhibition of miR-34a or overexpression of miR-21 significantlyreduced the effects of 3,6-DHF on Notch-1 and PTEN, andconsequently weakened the suppression of 3,6-DHF on PI3K/Akt/mTOR. We concluded that 3,6-DHF upregulates miR-34a viainhibiting DNMT1 and hypermethylation, whereas downregu-lates miR-21 by modulating histone modification, and conse-quently suppresses the PI3K/Akt/mTOR signaling pathway inbreast carcinogenesis. Cancer Prev Res; 8(6); 1–9. �2015 AACR.

IntroductionBreast cancer is one of themost serious health threats towomen

worldwide. In the United States, 230,480 new cases of invasivebreast cancer were diagnosed in 2011. The lifetime risk of beingdiagnosed with breast cancer is 12.5% for women in the UnitedStates, and approximately 39,520women are expected to die fromthis disease annually (1). Despite advances in screening, diagno-sis, and therapy, breast cancer continues to pose an enormousglobal healthcare problem.

miRNAs are a class of 20- to 25-nucleotide-long noncodingRNAs that negatively regulate gene expression by binding to the 30

untranslated region (30UTR) of target messenger RNAs, causingtranslational repression or degradation. miRNAs have been wellestablished to be aberrantly expressed in carcinogenesis andprogression, where they function as tumor suppressors or act asoncogenes. Our study has revealed the global upregulation ofmiR-21 and downregulation of miR-34a in breast carcinogenesis(2). As a proapoptotic transcriptional target of p53, miR-34a has

been shown to behave as a tumor suppressor by repressing genesinvolved in various oncogenic signaling pathways. On the con-trary, miR-21 is a notable oncogene, which is consistently over-expressed in a wide range of cancers and plays a key role inresisting programmed cell death in cancer cells (3). We havepreviously selected a promising anticancer agent, the flavonol3,6-dihydroxyflavone (3,6-DHF), in pharmacodynamic experi-ments, and demonstrated that it is a potent natural chemopreven-tive agent against breast carcinogenesis. IN addition, the previousstudy also revealed that 3,6-DHF treatment significantly upregu-latesmiR-34a anddownregulatesmiR-21 inbreast cancer cells, yetthe upstream and downstream events of the anticancer mecha-nism remain unclear.

Increasing evidence supports that the epigenetic variability atspecific transcription regulation sites appears to be susceptible tomodulation by diet and nutrition; some dietary components maysuppress tumorigenesis and development by affecting the processof DNAmethylation and histonemodifications, whichmodulatesthe expression of certain key genes and the activation of crucialsignaling pathways (4). In the last two decades, the study ofepigenetic modification emerged as one of the major areas ofcancer treatment. The key processes responsible for epigeneticregulation are DNA methylation and modifications in chromatinand posttranscriptional gene regulation by noncoding RNA. Thus,we hypothesized that 3,6-DHF may regulate the expressions ofmiR-34a andmiR-21 by modulating DNAmethylation or histonemodification.

The PI3K/Akt/mTOR signaling pathway is critical to normalcellular physical progress and also commonly overactivated incancer progression, especially in breast carcinogenesis and

1Research Center for Nutrition and Food Safety,Third Military MedicalUniversity; Chongqing Key Laboratory of Nutrition and Food Safety,Chongqing,China. 2Department of Public Health, School of PreclinicalMedicine, Chengdu Medical College, Chengdu, China.

Corresponding Authors: Hui Chang, Research Center for Nutrition andFood Safety, 30 Gaotanyan Street, Chongqing 400038, China; Phone:8602368752291; Fax: 8602368752291; E-mail: [email protected]; or MantianMi, [email protected]

doi: 10.1158/1940-6207.CAPR-14-0357

�2015 American Association for Cancer Research.

CancerPreventionResearch

www.aacrjournals.org OF1

for Cancer Research. on March 1, 2019. © 2015 American Associationcancerpreventionresearch.aacrjournals.org Downloaded from

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metastasis (5, 6). In fact, this signaling pathway acts as a keyintegration point between the extrinsic and intrinsic cellularenvironments (7, 8); it regulates a broad spectrum of cellularprocesses and consequently plays an important role in breasttumorigenesis and development (9, 10). Currently, targeting thePI3K/AKT/mTOR signaling pathway has become an effectivestrategy for chemoprevention and cancer treatment (11, 12).Studies indicated that miR-34a and miR-21 play an importantrole in regulating the PI3K/Akt/mTOR signaling pathway. Thetumor suppressor gene PTEN acts as the principal negative reg-ulator of the PI3K pathway. Notably, PTEN is a direct target ofmiR-21, which negatively regulates its expression (13). Converse-ly, Notch-1, a direct target of miR-34a, is also known to interactwith the PI3K/Akt signaling pathway (14). The aberrant activationof Notch signaling is an early event in breast cancer (15, 16).

In the present study, we investigated the underlying epigeneticmechanisms by which 3,6-DHF regulates the expression of miR-34a andmiR-21 in breast carcinogenesis and in breast cancer cells.Furthermore, we determined the effects of 3,6-DHF on the PI3K/Akt/mTOR signaling pathway in vivo and in vitro, and explored theroles of miR-34a and miR-21.

Materials and MethodsChemicals and reagents

3,6-DHF was purchased from Alfa Aesar; DMEM/F12 medi-um and FBS were purchased from HyClone; Trizol reagent,horse serum, gentamicin, insulin, Lipofectamine 2000, andOpti-Mem were purchased from Invitrogen; epidermal growthfactor (EGF) was purchased from PeproTech Inc.; all antibodieswere purchased from Cell Signaling Technology. 1-methyl-1-nitrosourea (MNU), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[a]pyrene (B[a]P), cholera enterotoxin,hydrocortisol, protein A/G-agarose, dimethylsulfoxide(DMSO), PBS, and other chemicals were purchased from Sig-ma-Aldrich. The miRNA first-strand cDNA synthesis Kit andmiRNA Real-Time PCR Assay Kit were purchased from aidlab.Anti–miR-34a oligonucleotides were purchased from Exiqon.pcDNA3-miR21 was purchased from Addgene.

Animals and treatmentFemale Sprague-Dawley (SD) rats (aged 42–48 days, 145–165

g) and BALB/c nude mice (aged 42–48 days, 15–20 g) wereobtained from the Medical Experimental Animal Center of theThird Military Medical University [SCXK-(army)-2007-015].These animals were bred and maintained in accordance with ourinstitutional guidelines for the use of laboratory animals. Theanimal roomsweremaintained at 25�Cwith 50% relative humid-ity and a 12-hour light/12-hour dark cycle. All of the animalprocedures were approved by the Animal Ethics Committee of theThird Military Medical University. Experimental models of carci-nogenesis in rats and xenografted MDA-MB-231 cells in athymicmice were processed as reported previously (2)

Cells and cultureHuman breast epithelial MCF-10A cells were maintained in

complete medium (DMEM/F12 medium supplemented withmitogenic additives, including 100 ng/mL cholera enterotoxin,10 mg/mL insulin, 0.5 mg/mL hydrocortisol, 20 ng/mL EGF, and5% horse serum). Breast cancer cell MDA-MB-231 and MCF-7were grown inDMEM/F12medium,MDA-MB-453were grown in

RPMI 1640 medium, supplemented with 10% FBS. All the cul-tures were maintained in a humidified atmosphere of 5%CO2/95% air at 37�C. All cell lines were purchased from theInstitute of Biochemistry and Cell Biology, Chinese Academy ofSciences (Shanghai, China) in October, 2013. All cell lines havebeen tested and authenticated by DNA (short tandem repeatgenotyping) profiling in May, 2014.

Chronic cellular breast carcinogenesisThe cellular breast carcinogenesis model was processed as

reported previously (17). Briefly, MCF10A cells were treated withNNK and B[a]P (each at 100 pmol/L) and with different con-centrations of 3,6-DHF (0, 5, 10, and 20 mmol/L); the cells weresubcultured every 3 days. The cancer-associated properties of thetreated cells were evaluated using the reduced dependence ongrowth factors (RDGF) assay, anchorage-independent cell growth(AIG) assay, and scratch/wound-healing assay.

mTOR kinase assay and p-Akt ELISAThe activities of mTOR and p-Akt (S473) weremeasured with a

colorimetric K-LISA mTOR activity assay kit (Calbiochem) and aPathScan Phospho-Akt (S473) ELISA kit (Cell Signaling Technol-ogy), respectively. Briefly, 107 treated cells were prepared andmeasured. The assays were performed as per the manufacturer'smanual.

Western blot analysisThe cell lysateswere preparedusingRIPAbuffer; equal amounts

of cellular proteins were resolved by electrophoresis in 10% or12% SDS-polyacrylamide gels for Western immunoblotting withspecific antibodies. The antigen–antibody complexes on thefilterswere detected by chemiluminescence.

ImmunofluorescenceBriefly, slides were rinsedwith PBS and cells were fixedwith 2%

paraformaldehyde andpermeabilized inmethanol. Afterwashingwith PBS, slides were blocked with 2% donkey serum. Primaryand secondary antibodies were incubated in 5% donkey serum.Then the slides were immediate analyzed by a laser confocalscanning microscopy.

ImmunohistochemistryXenografted breast tumors of MDA-MB-231 cells in athymic

mice, breast tissues, and the tumors of MNU-treated rats were allobtained from the previous study (2). The tissue sections (4-mm-thick) were placed onto treated slides, heat-fixed, deparaffinized,rehydrated, boiled in citrate buffer for antigen retrieval, andtreated with 3% hydrogen peroxide to block the endogenousperoxidase activities. After washing with PBS, the slides wereblocked with 2% donkey serum for 0.5 hours and then incubatedwith an antibody against p-Akt(T308) (dilution 1:200) at 4�Covernight. The primary antibody was omitted for the negativecontrols. The secondary biotinylated antibody was then applied,and the signal was developed using a modified avidin–biotincomplex immunoperoxidase staining procedure. Counterstain-ing was performed with Trypan blue or Harris hematoxylin.Immunostaining density was quantified using Image J analysis.

Plasmids, oligonucleotides, and transfectionsAnti–miR-34a oligonucleotides (50-ACAACCAGCTAAGA-

CACTGCC-30) were obtained from Exiqon. The pcDNA6.2-

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GW/miR-21 (pcDNA3-miR21) (Plasmid 21114) plasmid wasprovided by Addgene. The cells were transfected using Lipofecta-mine 2000 inOpti-Memaccording to themanufacturer's protocol.Themediumwas replaced8hours later, and the cellswere collectedfor the subsequent experiments 48 hours after transfection. Thefinal concentrations of oligonucleotides were 100 nmol/L.

qRT-PCR analysisThe total RNA was extracted using Biozol reagent. The miRNA

first-strand cDNA synthesis Kit and miRNA Real-Time PCR AssayKit (aidlab) were used to quantify the miRNA transcripts in ourstudy following the manufacturer's instructions. Each reactionsample was run in triplicate. The expression of U6 small nucleolarRNA was used as an internal control. The relative expression levelfor each miRNA was calculated with the comparative CT method(2�DDCt).

Bisulfite modification and methylation-specific PCRThe genomic DNA was subjected to sodium bisulfite modifi-

cation using the EZDNAMethylation-Gold Kit (ZYMOResearch)according to the manufacturer's instructions. The modified DNAwas eluted at a final volume of 10 mL, and 1.5 mL were used formethylation-specific PCR (MSP). The PCR primers used to detectthe CpG-methylation of the miR-34a promoter were previouslyestablished. MSP primer sequences for CarT and MDA-MB-231cells: M: Forward 5'-GGTTTTGGGTAGGCGCGTTTC-3', Reverse5'-TCCTCATCCCCTTCACCGCCG-3'; U: Forward 5'-IIGGTTTT-GGGTAGGTGTGTTTT-3', Reverse 5'-AATCCTCATCCCCTTCAC-CACCA-3'. MSP primer sequences for MCF-7 and MDA-MB-453cells: M: Forward 5'-ATGAGGATTAGGATTTCGGAG, Reverse 5'-AACGCATAAAAACGACGACAA; U: Forward 5'-GGGGATGAG-GATTAGGATTTT, Reverse 5'-CAAACAAAACACATAAAAACAACA.The reactionswere carriedout using the iQ5Multicolor Real-TimePCRDetection System (BioRad) at 95�C for 10minutes; followedby 35 cycles of 30 seconds at 95�C, 30 seconds at 60�C, 30 secondsat 72�C (30 cycles); and a final 7-minute extension at 72�C. Eachamplificationproduct (5mL)was resolved in a 2.5%agarose gel byelectrophoresis (Invitrogen). The electrophoresis conditions usedwere 80V for 60minutes. The gel was directly visualized underUVillumination after electrophoresis.

DNMT activity assay and DNMT1 inhibitor testingFor the DNAmethyltransferase (DNMT) activity assay, nuclear

extracts of 106 treated cells were prepared using a nuclear extrac-tion reagent (Pierce) following the manufacturer's instructions.The DNMT activity was determined in the nuclear extracts usingthe EpiQuik DNAMethyltransferase Activity Assay Kit (EpigentekInc.) following the manufacturer's protocol. For DNMT1 inhib-itor testing, 1 mL of DNMT1 enzyme (Epigentek Inc.) was incu-bated with different concentrations of 3,6-DHF at 37�C for 60minutes, and the DNMT activity was determined using the Epi-Quik DNAMethyltransferase 1 Activity/Inhibitor Screening AssayCore Kit (Epigentek Inc.).

Docking studyThe blind docking was performed with Autodock4 by setting

grid sizes. The receptor sitewas preparedwith Sybyl (Tripos) usingthe NMR structure 2NPU model 1 from the Protein Data Bank(www.pdb.org). The grid size for the docking sitewas expanded toinclude the entire DNMT1 molecule, and 3,6-DHF was docked.

ChIP-qPCR analysisChromatin immunoprecipitation (ChIP)was carriedout accord-

ing to the instructions of the EZ-ChIP Chromatin immunoprecip-itation Kit (Millipore). After ChIP, the DNA precipitated by thetarget antibodywas detected with qRT-PCR. PCRwas conducted ina final volume of 25 mL containing 12.5 mL of 2� SYBR Mix, TaqDNA Polymerase (BioEasy), 1 mL each of forward primer andreverse primers (10 mmol/L), and 6 mL of DNA template underthe following conditions: the template was first denatured at 94�Cfor 10 minutes, then subjected to 50 cycles of amplification (94�Cfor 20 seconds, 60�C for 1minute), 95�C for 2minutes, 72�C for 1minute, 95�C for 30 seconds, and 55�C for 10 seconds (repeat 80times), 30�C for 1 minute. After real-time PCR, relative dataquantification was performed using the 2�DDCt method, and theresultwas calculated in the formof% Input,whichwas givenby thefollowing formula: %Input ¼ 2(Ctinput�CtChIP) � input dilutionfactor � 100. Real-time PCR primers were synthesized by SBSGenethech Co., Ltd. The purified DNAs were amplified with thefollowing primer pairs: 50-TGC TGTTTGGTCTCAGTA-30and 50-GGCAAGTTAACGAAAAGAA-30 for miR-21.

Statistical analysisThe results are presented as the mean � SD from at least three

independent experiments. The tumor incidences were comparedusing the c2 test. The other data were analyzed by one-wayANOVA followed by the Tukey test for multiple comparisons.Differences were considered significant at P < 0.05.

Results3,6-DHF suppresses carcinogens-inducedbreast carcinogenesis

Using cancer-associated properties as the target endpoints, weassessed the effects of 3,6-DHF on the chronic breast carcinogen-esis induced in vitro by the carcinogens NNK and B[a]P. As shownin Fig. 1A and B, human breast epithelial MCF10A cells treatedwith carcinogens (CarT) for 30 days showed aberrantly increasedcell survival adapted to RDGF and AIG, indicating cellular carci-nogenic transformation. Compared with CarT, cells cotreatedwith carcinogens and 3,6-DHF (C-DHF) for 30 days exhibiteda significantly lower acquisition of RDGF and AIG. Similarly, thewound-healing assay showed that CarT cells required apparentlyincreased mobility and proliferation to heal the wound, whichcan be suppressed by 3,6-DHF cotreatment (Fig. 1C). These datasuggest the chemopreventive effects of 3,6-DHF on carcinogen-induced breast carcinogenic transformation.

3,6-DHF epigenetically regulates the expressions of miR-34aand miR-21

The qRT-PCR data revealed the significant downregulation ofmiR-34a and upregulation ofmiR-21 in cellular breast carcinogen-esis,which couldbemitigatedby3,6-DHFcotreatment (Fig. 2AandB). The data also showed that 3,6-DHF treatment effectively mod-ulates the expressions ofmiR-34a andmiR-21 in breast cancer cellsMDA-MB-231, MCF-7, and MDA-MB-453 (Fig. 2C and D).

The inactivation of miR-34a in multiple types of malignancies,including breast cancer, has been attributed to the hypermethyla-tion of the promoter. We detected the effect of 3,6-DHF on themethylation status of the miR-34a promoter in breast cancer cellsand in carcinogenesis. TheMSPdata showed that the hypermethy-lation of miR-34a promoter was significantly inhibited in 30-dayC-DHF and 3,6-DHF–treated breast cancer cells compared with

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CarT and the control, respectively (Fig. 3A). Because DNMTscatalyze the process of DNA methylation, we assessed whether3,6-DHF changed the DNMT activity in breast cancer cells. Ourstudy found that 3,6-DHF acted in a dose-dependent manner todecrease the DNMT activity in breast cancer cells (Fig. 3B). Wethen performed the DNMT1 inhibitor testing, and the data (Fig.3C) showed that 3,6-DHF is an effective DNMT1 inhibitor thatcan significantly inhibit the DNMT1 activity in a dose-dependentmanner. Furthermore, we blind-docked 3,6-DHF to the DNMT1target using Autodock4 by setting grid sizes that included theentire DNMT1molecule. As shown in Fig. 3D, 3,6-DHF docked tothe putative cytosine pocket with the lowest binding energies of�7.1 kcal/mol. This pocket is the active region that facilitatesbinding with hemimethylated DNA, a process that is required forsubsequent methylation.

In addition, we performed ChIP-qPCR analysis for histonemodifications associated with the active transcription state

(H3K9-14ac and H3K27ac) and the inactive transcription state(H3K27me3) on the miR-21 promoter. The results showed that3,6-DHF treatment significantly lowers the H3K9-14ac on themiR-21 promoter, while it does not apparently affect otherhistone modifications (Fig. 3E). These findings indicated that3,6-DHF upregulates miR-34a expression via demethylation,whereas downregulates miR-21 expression by modulating his-tone modification.

3,6-DHF represses the PI3K/Akt/mTOR signaling pathwayGiven the important role of the PI3K/Akt/mTOR signaling path-

way in tumorigenesis, we evaluated the effect of 3,6-DHF on thesignaling in breast carcinogenesis. Western blot detections (Fig. 4A)showed that the levels of PI3K, p-Akt, and p-mTOR in CarT cellssignificantly increased in a time-dependent manner, indicatingthe dramatic promotion of the PI3K/Akt/mTOR signaling pathwayin carcinogenesis. Cotreatment with 3,6-DHF could effectively

Figure 2.3,6-DHFmodulates the expressions ofmiR-34a and miR-21 in breastcarcinogenesis. The levels of miR-34a(A) and miR-21 (B) in MCF10A, CarT,and C-DHF (20 mmol/L 3,6-DHF) cellsin the cellular breast carcinogenesismodel. Effects of 3,6-DHF (20mmol/L)treatment for 24 hours on the levels ofmiR-34a (C) and miR-21 (D) in breastcancer cell MDA-MB-231 (M231), MCF-7 cells (M7), MDA-MB-453 cells(M453), MDA-MB-231 cells transfectedwith blank plasmids (mock control,MC), MDA-MB-231 cells transfectedwith anti–miR-34a oligonucleotides(TCanti-34a), and MDA-MB-231 cellstransfected with pcDNA6.2-GW/miR-21 (TCmiR-21). The data are presentedas the mean � SD (n ¼ 3). �, P < 0.05and �� , P < 0.01 compared withMCF10A cells; #, P < 0.05 and##, P < 0.01 compared with CarT cellsor the control; &, P < 0.05 and&&, P < 0.01 compared with M231.

Figure 1.3,6-DHF suppresses carcinogens-induced chronic cellular breast carcinogenesis. 3,6-DHF effectively suppresses carcinogens NNK and B[a]P-induced cellularbreast carcinogenic transformation. Cancer-associated properties were evaluated by RDGF assay (A), AIG assay (B), and scratch/wound-healing assay (C) inMCF10A cells, cells treatedwith carcinogens (CarT) for 30 days, and cells cotreatedwith carcinogens and 3,6-DHF (C-DHF) for 30 days. The data are presented as themean � SD (n ¼ 3). � , P < 0.05 and

��, P < 0.01 compared with MCF10A cells; #, P < 0.05 and ##, P < 0.01 compared with CarT cells.

Peng et al.

Cancer Prev Res; 8(6) June 2015 Cancer Prevention ResearchOF4

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suppress the cancer-promoting signaling pathway. We then mea-sured the activity of Akt and mTOR kinase with an ELISA-basedkinaseactivity assay. In accordancewith theWesternblot detections,thedata (Fig. 4BandC) confirmedthat the activityofAkt andmTORin CarT cells significantly increased in a time-dependent manner,which could be counteracted by 3,6-DHF cotreatment. Further-more, we detected the p-Akt level of breast tissues and tumors inMNU-treated rats via immunohistochemistry. The results (Fig. 5A)showed that the p-Akt level significantly increased in breast carci-nogenesis in vivo, which could be effectively inhibited by 3,6-DHFadministration [20 mg/kg, intragastric (i.g.) administration].

We also evaluated the effect of 3,6-DHF on the PI3K/Akt/mTOR signaling pathway in breast cancer cells in vitro and invivo. As shown in Fig. 6A, 3,6-DHF treatment could significantlydownregulate the levels of PI3K (p85, p110), p-Akt (Thr308,Ser473), and p-mTOR (S2448, S2481) and consequentlyreduce the activity of Akt (Fig. 6B) and mTOR (Fig. 6C) in adose-dependent manner. In accordance with these findings, theimmunohistochemistry for p-Akt(T308) in xenografted breasttumors of MDA-MB-231 cells showed that 3,6-DHF adminis-tration (20 mg/kg, i.g.) could significantly suppress the activa-tion of Akt in vivo (Fig. 5B). Furthermore, to support our

Figure 4.3,6-DHF suppresses the PI3K/Akt/mTOR signaling pathway in breast carcinogenesis. A,Western blot detections for the levels of PI3K(p85, p110), p-Akt(T308, S473),p-mTOR(S2448, S2481) in CarT and C-DHF (20 mmol/L 3,6-DHF) cells in the cellular breast carcinogenesis model. The changes in the Akt activity (B) and inthe mTOR kinase activity (C) in MCF10A, CarT, and C-DHF (20 mmol/L 3,6-DHF) cells in the cellular breast carcinogenesis model. The data are presentedas the mean � SD (n ¼ 3). � , P < 0.05 and

��, P < 0.01 compared with MCF10A cells; #, P < 0.05 and ##, P < 0.01 compared with CarT cells.

Figure 3.3,6-DHF regulates miR-34a and miR-21 via epigenetic mechanisms. A, the methylation status of miR-34a promoter detected by MSP in 30-day CarT and30-day C-DHF (20 mmol/L) cells; effect of 3,6-DHF (20 mmol/L) treatment for 24 hours on the methylation status of miR-34a promoter in MDA-MB-231 cells (M231),MCF-7 cells (M7), and MDA-MB-453 cells (M453). m, methylation; u, unmethylation. B, effect of 3,6-DHF treatment for 24 hours on the DNMT activity inM231, M7, and M453 cells. C, DNMT1 inhibitor testing of 3,6-DHF. D, blind docking of 3,6-DHF to the DNMT1 target performed with Autodock 4. E, effects of 3,6-DHFtreatment (20 mmol/L) for 24 hours on the histone modifications of H3K9-14ac, H3K27me3, and H3K27ac on miR-21 promoter in MDA-MB-231 cells. Thedata are presented as the mean � SD (n ¼ 3). �, P < 0.05 and

��, P < 0.01 compared with the control.

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findings, we further investigated the effects of 3,6-DHF on thedownstream targets of the PI3K/Akt/mTOR signaling pathway.As expected, the detections showed that 3,6-DHF treatmentcould significantly suppress the activation of the downstreamtargets, including ribosomal p70-S6 kinase (S6K70), ribosomalprotein S6 (rpS6), eukaryotic translation initiation factor (eIF)4B, and eukaryotic translation initiation factor 4E-bindingprotein (4EBP) 1 (Figs. 5C and 6D).

MiR-34a andmiR-21 play crucial roles in the 3,6-DHF–inducedrepression of PI3K/Akt/mTOR

We detected the effects of 3,6-DHF on the levels of PTEN,Notch-1, and Hes-1 because they are targets of miR-21 and miR-34a. Notably, PTEN is the key inhibitor of the PI3K/Akt signalingpathway; on the contrary, Notch-1 is an activator of this signaling.The results indicated that 3,6-DHF treatment significantly upre-gulates PTEN and downregulates Notch-1 and Hes-1 in carcino-genesis (Fig. 7A) and in breast cancer cells (Fig. 7B).

To explore the roles of miR-34a and miR-21, we blocked theeffects of 3,6-DHF on the two miRNAs by transfecting the cellswith a specific inhibitor or plasmid. As shown in Fig. 2C and D, alocked nucleic acid oligonucleotide complementary to the miR-34a sequence (TCanti-34a) blocked miR-34a function, while thepcDNA6.2-GW/miR-21 plasmids led to the substantial produc-tion of miR-21 in MDA-MB-231 cells. The results indicated thatthe inhibition of miR-34a or overexpression of miR-21 signifi-cantly reduced the effects of 3,6-DHF on Notch-1 and PTEN, andconsequently weakened the suppression of 3,6-DHF on the

activation of Akt and mTOR (Fig. 7C and D). Taken together,these findings indicated that 3,6-DHF suppresses the PI3K/Akt/mTOR signaling pathway partially by modulating miR-34a andmiR-21.

DiscussionEpidemiologic studies and systematic analyses indicated that

diets rich in fruits and vegetables are associatedwith a reduced riskof breast cancer (18, 19). Dietary flavonoids, a group of poly-phenolic compounds, have been identified as potential cancer-preventive components of fruits and vegetables (20, 21). Flavo-noids can be categorized into six major subclasses based on theirrange and structural complexity: flavonols, flavones, flavan-3-ols,flavanones, anthocyanins, and isoflavones. Flavonolsmainly existin onions, broccoli, tea, and various common fruits; flavones arecommon in aromatic herbs, celery, and chamomile tea; flavan-3-ols are present in cocoa, red wine, grapes, apples, green tea, andother fruits; flavanones can be found in oranges and other citrusfruits; anthocyanidines are abundant in colored berries, blackcurrants; and isoflavones are found in soy food (22, 23). In fact,our previous study revealed that flavonols and flavones, but notthe total flavonoids, flavan-3-ols, flavanones, or anthocyanins,reduce breast cancer risk (24). Further studies should be devel-oped to select effective anticancer compounds from flavonoidsand uncover the mechanisms of their relevance to cancer preven-tion. The findings will provide useful insight and evidence fordietitians and other healthcare professionals when discussing dietand cancer prevention.

Figure 5.3,6-DHF decreases the level of p-Akt and p-4EBP1. 3,6-DHF administration (20 mg/kg, i.g.) significantly decreases the level of p-Akt(T308) in breast tissues [0, 2weeks (w)] and tumors (18 w) in MNU-treated rats (A), as well as in xenografted breast tumors of MDA-MB-231 cells in athymic mice (B), detected byimmunohistochemistry. Immunostaining of p-Akt(T308) protein was indicated by arrows (dark brown color). C, immunofluorescence analysis for the levelof p-4EBP1(S65) inMDA-MB-231 cells treatedwith different doses of 3,6-DHF (0, 5, 10, and 20 mmol/L) for 24 hours. Immunostaining density or immunofluorescencewas quantified using Image J analysis. The data are presented as the mean � SD (n ¼ 3). � , P < 0.05 and �� , P < 0.01 compared with the control.

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Our preliminary screening of anticancer compounds fromflavonoids identified 3,6-DHF as a promising anticancer agentand an effective natural chemopreventive agent against breastcarcinogenesis. Based on these findings, the present study furtherrevealed that 3,6-DHF epigenetically regulates miR-34a andmiR-21, and consequently represses the PI3K/Akt/mTOR signalingpathway. Our study suggested a new strategy for suppressing thecritical cancer-promoting signaling pathway in the treatment ofcancer viamodulatingmiRNAs.Moreover, our findings will likelyprovide useful insight and evidence that can be used by registereddietitians and other healthcare professionals when discussing dietand cancer prevention with patients. However, the anticancereffects of 3,6-DHF were only demonstrated in animal and cell

culture studies. Because human clinical trials examining thechemopreventive potential of 3,6-DHF have not been conducted,more studies are warranted to confirm the results.

DNAmethylation is responsible for regulating gene expressionand interacting with the nucleosomes that control DNA packag-ing. This process can affect entire domains of DNA. The genesilencing of tumor suppressors is frequently associated with thehypermethylation of theCpG islands in promoter regions, but theinitial trigger of hypermethylation is unknown. CpG islandstypically span the promoter region and first exon of approximate-ly 60% of all genes. The majority of these CpG islands areunmethylated in the normal cell, which correlates with activegene transcription, in comparison with the bulk of the genome,

Figure 6.3,6-DHF represses the PI3K/Akt/mTOR signaling pathway in breast cancer cells. A, Western blot detections for the level of PI3K(p85, p110), p-Akt(T308, S473),p-mTOR(S2448, S2481) in MDA-MB-231 cells (M231), MCF-7 cells (M7), and MDA-MB-453 cells (M453) treated with different doses of 3,6-DHF (0, 5, 10, and20 mmol/L) for 24 hours. B, effect of 3,6-DHF treatment (0, 5, 10, and 20 mmol/L) for 24 hours on Akt activity in M231, M7, and M453 cells. C, effectof 3,6-DHF treatment (0, 5, 10, and 20 mmol/L) for 24 hours on mTOR kinase activity in M231, M7, and M453 cells. D, effects of 3,6-DHF treatment (0, 5, 10,and 20 mmol/L) for 24 hours on the phosphorylation of S6K70, rpS6, eIF4B, and 4EBP1 in M231, M7, andM453 cells. The data are presented as themean� SD (n¼ 3).� , P < 0.05 and

��, P < 0.01 compared with the control.

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which is methylated. However, these methylation states are oftenreversed in cancer cells: the global levels of DNA methylationbecome hypomethylated in conjunction with the DNA hyper-methylation of the CpG islands associated with gene promoters,especially tumor suppressors. MiR-34a was demonstrated to be atumor suppressor, a direct transcriptional target of p53, and acomponent of the p53 transcriptional network (25, 26). Notably,the epigenetic silencing of miR-34 by aberrant CpG methylationfrequently occurs in various types of human cancer, includingbreast cancer (27, 28). Our data indicated that 3,6-DHF demethy-lates miR-34 and increases its expression in breast cancer cells, amechanism that may be associated with the direct inhibition ofDNMT1 activity.

Next, the histone modifications in the chromatin structurealso play an important role in gene regulation and carcino-genesis. Histone modifications are specifically characterizedby the genomic regulatory regions, such as inactive promotersthat are enriched in trimethylated H3 at lysine 27(H3K27me3) or trimethylated H3 at lysine 9 (H3K9me3)and active promoter regions that are enriched in acetylatedH3 at lysine 27 (H3K27ac; ref. 29). Our study showed that3,6-DHF significantly affects the histone modification inbreast cancer cells, lowering the H3K9-14ac on the miR-21promoter, which may contribute to the downregulation ofmiR-21. A deep investigation of the underlying mechanism isinteresting.

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

Authors' ContributionsConception and design: H. Chang, M. MiDevelopment of methodology: X. Peng, Y. GuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Peng, H. Chang, Y. Gu, J. Chen, Q. XieAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. ChangWriting, review, and/or revision of the manuscript: X. Peng, H. Chang, M. MiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Yi, J. Zhu, Q. ZhangStudy supervision: M. Mi

AcknowledgmentsThe authors thank Elsevier WebShop for the English language editing of the

article.

Grant SupportThis work was supported by the grant from the National Natural Science

Foundation of China (81372974) and Chongqing Fundamental and AdvancedResearch Project (cstc2013jcyjA10083; to H. Chang).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 14, 2014; revised February 11, 2015; accepted March 9,2015; published OnlineFirst March 17, 2015.

Figure 7.miR-34a and miR-21 play crucial roles in the 3,6-DHF–induced repression of PI3K/Akt/mTOR. A, Western blot detections for the levels of PTEN, Notch-1,and Hes-1 in CarT and C-DHF (20 mmol/L 3,6-DHF) cells in the cellular breast carcinogenesis model. B, effects of 3,6-DHF treatment for 24 hours on thelevels of PTEN, Notch-1, andHes-1 inM231, M7, andM453 cells, on the levels of Notch-1, p-Akt(T308), and p-mTOR(S2448) in TCanti-34a cells, and on the levels of PTEN,p-Akt(T308), and p-mTOR(S2448) in TCmiR-21 cells. Effects of 3,6-DHF treatment (20 mmol/L) for 24 hours on Akt activity (C) and mTOR kinase activity (D)in MDA-MB-231, TCanti-34a, and TCmiR-21 cells. The data are presented as the mean � SD (n ¼ 3). #, P < 0.05 and ##, P < 0.01 compared with the control; &, P < 0.05and &&, P < 0.01 compared with M231.

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