Study On Regulation Of Human Sex Hormone Receptors Gene Expression By AZA And TSA In Breast Cancer Cells

BackgroudBreast cancer was known as a systemic disease because it often caused earlier hematogeneous metastasis,which heavily threatened women’s health. Accordingly,great change happened in the therapy strategy of breast cancer ,from the past local treatments alone to local and systemic treatment in combination. Endocrine therapy as one of systemic treatments was well welcomed because of its factual effects ,less adverse-effects and convenience. There were about 45% breast cancer patients would have lost the chance of endocrine therapy because estrogen receptor alpha ( ERa ) was negative.So we could drow that human sex hormone reccpters (SHR) were the main prognostic indicators of endocrine therapy. DNA methylation and histone acetylation are important covalent chromatin modification in epigenetic modification, which might regulate the expression of SHR gene. However,very little is known about the molecular mechanisms of the regulation of SHR gene expression itself.To better understand transcriptional and translational regulation of SHR gene expression,we carded out this study and the major results are summarized below.ObjectiveTo investigate whether DNMT inhibitor 5-AZA-CdR (AZA) and HDAC inhibitor tri- chostatin A(TSA) in combination can synergistically induce ERa and AR reexpression in initially Era- and AR- negative breast cancer cells SKBR-3 and restore its response to estrogen and endocrine therapy.Moreover,to investigate whether TSA can induce the chan- ge of ERa and AR expression level in SKBR-3 or MCF-7 cells and change their prolife- ration after chemical treatment.Methods1. The mRNA expression of ERa、PR、AR、pS2 and PSA in SKBR-3 cells treated with TSA and AZA in combination or TSA alone was detected by RT-PCR.2. Human breast cancer cells SKBR-3 were treated with TSA and AZA in combina- tion,breast cancer cells SKBR-3 or MCF-7 were treated with TSA alone,and then there are six treatment groups established:①Group (Vehicle) SKBR-3,②Group (AZA+TSA) SKBR-3,③Group (Vehicle) MCF-7,④Group (TSA) MCF-7,⑤Group (Vehicle) SKBR-3,⑥Group (TSA) SKBR-3. The protein expression of ERa、PR、AR、pS2 and PSA in the treated SKBR-3 and MCF-7 cells was detected by immunocytochemistry(ICC) method.3. The water-soluble tetrazolium salt (WST) method was used to analyze the cells proliferation.(1) SKBR-3 cells proliferation treated with TSA and AZA in combination was ana- lyzed in the different concentration of megestrol acetate (MA):①vehicle,②lnmol/L,③10nmo1/L④l00nmol/L⑤1μmo1/L⑥l0μmo1/L.(2) In the study of the combination treatment by AZA and TSA, there were six treat- ment groups in breast cancer cells SKBR-3 after the optimal concentration of megestrol acetate (MA) was established:①vehicle,②AZA+TSA,③AZA+TSA+E2,④AZA+TSA +E2+TAM,⑤AZA+TSA+E2+MA,⑥AZA+TSA+E2+TAM+MA.(3)After the treatment by TSA alone ,there were four treatment groups established in breast cancer cells SKBR-3 and MCF-7:①(Vehicle)SKBR-3,②(TSA)SKBR-3,③(Vehicle)MCF-7,④(TSA)MCF-7.The proliferation rate of every group was analyzed at different time :12h, 24h, 48h , 72h, 96h.Results1. After treatment by AZA and TSA in combination or TSA alone, ERa、AR、PR、pS2 and PSA mRNA reexpressed in the initially ERa- and AR-negative breast cancer cells SKBR-3, whereas the mRNA expression level in the one chemical treatment was less than that in the combination treatment(P<0.05),and both of them was much less than that in MCF-7 cells(P<0.01).2. In the protein expression analysis:(1) After the combination treatment, ERa、PR、AR、pS2 and PSA protein re-expre- ssed in the initially ER- and AR-negotive SKBR-3 cells .The protein expression level indu- ced by AZA and TSA was much less than that in MCF-7 cells(P<0.01). In per field of high multiple vision(×400)in the glass plate where human breast cancer line SKBR-3 was se- eded,the number of cells present of AZA and TSA was less than that absent of the two che- micals.No cell death was found.(2) After treatment by the one chemical, ERa、PR、AR、pS2 and PSA protein re-ex- pressed in the initially ERa- and AR- negative breast cancer cells SKBR-3 whereas they were suppressed in the initially ERa-and AR- positive breast cancer cells MCF-7.However, the protein expression level in SKBR-3 cells treated with TSA was less than the remainder in MCF-7 cells treated with TSA(P<0.05).More cells death was found in the glass plate where human breast cancer line SKBR-3 was seeded than that of breast cancer cells MCF -7.3. In the proliferation rate analysis:(1) Breast cancer cells SKBR-3 induced by AZA and TSA was treated for 96 hours by Megestrol acetate (MA) at different concentrations (1nmol/L–10μmol/L).The dose-resp- onse study demonstrated lower proliferation rate at the higher concentration of megestrol acetate (MA),the optimal concentration of MA was 10μmol/L.(2) The growth of SKBR-3 cells treated with AZA and TSA was suppressed signifi- cantly compared with the vehicle control(P<0.05);and then 1nmol/L 17β-estrodial added, the proliferation rate of the induced cells was upregulated again but not significantly(p> 0.05); TAM or MA added respectively again ,the growth of cells treated with AZA +TSA +E2+ MA as well as AZA +TSA +E2+TAM was suppressed compared with AZA +TSA +E2 group (P < 0.05); At last, the growth of the induced cells treated with TAM and MA in combination was further suppressed than that of cells treated with TAM or MA alone (P<0.05).(3)The growth of both SKBR-3 and MCF-7 cells was significantly suppressed by TSA alone compared with the vehicle group (P<0.05). In addition, the suppression of SKBR-3 cells proliferation rate by TSA happened much earlier and was much more than that of MCF-7 cells (24h vs 48h, OD 1.1437 vs 0.6387).Conclusion1.After treatment with AZA and TSA,ERa- and AR-negative SKBR-3 cells can ex- press functional ERa and AR and become responsive to estrogen and endocrine therapy , suggesting that the two chemicals may play important roles in restoring sensitivity of the SHR-negotive breast cancer to endocrinetherapy.Moreover,AZA and TSA could suppress SKBR-3 cells proliferation , suggesting that they could inhibits the growth of breast can- cer by changing its biology.2.Breast cancer cells SKBR-3 chemically induced ERa and AR re-expression res- tored dose-response to MA, which demonstrated lower proliferation rate in higher concen- tration of megestrol acetate (MA). In addition, the combination treatment with TAM and MA could synergistically inhibit the growth of induced breast cancer cells via functional ER and AR ,respectively, suggesting that small dose of MA(8mg/d)often used in hormone replacement therapy (HRT) maybe a potent plus for postmenopausal patients treated with long-term TAM to prevent the most unpleasant adverse effect of TAM,raised risk of endo- metrial cancer. However, this hypothesis is just from an experiment in vitro and must be further certified by clinical trials.3.TSA can not only improve sensitivity of the initially ERa and AR negative breast cancer cells to endocrine therapy by chemical induction of ERa and AR re-expression but also inhibit ERa and AR positive breast cancer cells growth by down-regulation of ERa and AR expression. It was more important to infer that TSA could not only induce the ex- pression of tumor suppressor genes but also down-regulate the expression of oncogenic protein to inhibit breast cancer cells proliferation by induction of their diffrention and apo- ptosis.