Research On The Growth Inhibition Of Breast Cancer And Reversion Of ERα Hympermethylation By Trichosanthin

Breast cancer is the most common cancer in women,of which the morbidity increases year by year.Many researches presented estrogen is important in breast cancer.Two thirds of primary breast cancer have the expression of estrogen receptor(ER) and the growth of tumor cell is hormonal dependent.The function of estrogen depends on binding with its receptor and promoting the progression of breast tumor.The clinical therapy of breast cancer patients(ER positive) usually includes endocrinotherapy,which is equal to chemotherapy.However,one third breast cancer patients(ER negative) are not sensitive to endocrine therapy.Estrogen receptor alpha(ERα) gene is located on the chromosome 6q24-27.The proteinum expressed by ERαgene is usually an important molecule marker to evaluate hormonal dependence and predicts the effect of endocrine therapy on breast cancer.Current researches report the mechanism of ERαgene expression silencing is not due to gene mutation, but probably associate with the genetic transcription.Because of the 5'CpG island methylation in the core promotor of ERαgene,the genetic transcription is suppressed which induce to the silence of ERαgene.Trichosanthin(TCS) is a kind of traditional Chinese medicine,which we choose as experimental drug of demethylation.The purpose is to investigate the the effect of TCS on proliferation of MDA-MB-231 and MCF-7 breast cancer cell,the demethylating effect on ERαgene in MDA-MB-231 breast cancer cell,the relationship between demethylation and gene expression;meanwhile,to explore a new therapeutic strategy for breast cancer by demethylating treatment.The design of the study include:(1) Investigate the effect of TCS acting on breast cancer cell MDA-MB-231 and MCF-7;(2) Measure the demethylation of ERαmRNA and protein.Analyze the relationship between ERαmRNA expression and methylation;(3) Explore the demethylating effect of TCS on MDA-MB-231 breast cancer cell and the sensitivity to endocrine therapy;(4) Study the suppressive effect of TCS on transplanted human breast cancer in nude mice and the security in vivo.The Partâ… Objective:Investigate the effects of TCS on proliferation,apoptosis of MDA-MB-231 and MCF-7 breast cancer cell.Methods:(1) Human breast cancer cell lines MDA-MB-231 and MCF-7 were growing at 37℃and 5%CO₂.The morphological change of cells were observed by optical microscope after treated with TCS(90μg/ml) for 72 hours;(2) MTT was performed for the proliferation of MDA-MB-231 and MCF-7 cell after treated with TCS;(3) The period and apoptosis rate of MDA-MB-231 and MCF-7 cell were tested by Flow Cytometry method(FCM) after treated with TCS(90μg/ml) for 48 hours.Results:(1) The cellular morphology changed after treated with TCS.Denaturation and necrosis occurred in some cells;(2) TCS could inhibit the growth of MDA-MB-231 and MCF-7 cell by MTT test;(3) TCS could suspend MDA-MB-231 and MCF-7 cell generation at G0/G1 period.It also induced MDA-MB-231 and MCF-7 cell apoptosis by FCM test.Conclusion:TCS could inhibit MDA-MB-231 and MCF-7 cell proliferation in a time- and concentration-dependent manner.TCS could induce MDA-MB -231 and MCF-7 cell apoptosis and suspend the cell generation at G0/G1 period.The Partâ…¡Objective:Explore the relationship between ERαgene expression silencing and hypermethylation.Study the effect of TCS on ERαgene expression and 5'CpG island methylation in MDA-MB-231 breast cancer cell.Test the possibility that TCS may induce MDA-MB-231 breast cancer cell sensitive to endocrine therapy. Methods:We chose MDA-MB-231 breast cancer cell(ERαnegative) and MCF-7 breast cancer cell(ERαpositive).MDA-MB-231 cell was treated with TCS(90μg/ml) or 5-Aza-C(5μM).(1) The ERαmRNA level of the cells was measured by RT-PCR;(2) The 5'CpG island methylation of ERαgene was tested by Methylation Specific PCR(MSP) test;(3) Western blot method was performed for ERαprotein in the cells;(4) MTT method tested whether Tamoxifen could induce the growth inhibition of MDA-MB-231 cell after treated with TCS or 5-Aza-C.Results:(1) The expression of ERαmRNA in MDA-MB-231 cell was silencing by RT-PCR test,while the expression of ERαmRNA in MCF-7 cell was positive.The re-expression of ERαmRNA in MDA-MB-231 cell treated with TCS or 5-Aza-C was shown by RT-PCR; (2) Methylation of ERαgene was found in MDA-MB-231 cell with MSP technique.Demethylation of ERαgene was found in MCF-7 cell and MDA-MB-231 cell treated with TCS or 5-Aza-C;(3) The expression of ERαprotein was negative in MDA-MB-231 cell,but positive in MCF-7 cell determined by western blot method,and re-expressed when treated with TCS or 5-Aza-C;(4) Tamoxifen could inhibit the proliferation of MDA-MB-231 cell treated with TCS efficiently through MTT method and the suppressive effect of TCS was stronger than 5-Aza-C(p＜0.05).Conclusion:ERαgene expression silencing was related to the hypermethylation of ERαgene.TCS could induce the re-expression of ERαmRNA and protein in MDA-MB-231 cell by demethylating ERαgene.TCS could induce MDA-MB-231 breast cancer cell sensitive to Tamoxifen.The Partâ…¢Objective:Study the suppressive effect of TCS on transplanted human breast cancer in nude mice and effect on the expression of ERα.Methods:(1) 20 nude mice were transplanted with human breast cancer MDA-MB-231 cell to construct tumor models.Once the tumor model in nude mice was established,18 nude mice were devided into 3 groups:control group(without treatment),TCS group and saline group. TCS(0.4mg/kg) and sodium chloride were injected around the tumor.A total of 7 injections were given,once every 2 days;(2) Observations were made on tumor suppression rate,tumor volume and weight,liver and renal function,blood routine test;(3) The ERαmRNA level in transplanted neuplasma was measured by RT-PCR assays;(4) The methylation of ERαgene in transplanted neuplasma was performed by MSP test;(5) Immunohistochemistry and western blot were used to detemine the expression of ERαprotein in the 3 groups.Results:(1) The subcutaneous tumor model in nude mice was successfully established(18/20);(2) The tumor volume and weight in TCS group decreased(p＜0.05);(3) Renal function and blood system were normal(P＞0.05) in TCS group.SGPT in TCS group was a little higher than control group(P＜0.05);(4) The re-expression of ERαmRNA was shown by RT-PCR in TCS group;(5) Demethylation of ERαgene was found in transplanted neuplasma treated with TCS by MSP test;(6) The expression,of ERαprotein was found in transplanted neuplasma treated with TCS by immunohistochemistry and western blot test.Conclusions:TCS could effectively suppress human breast cancer cells growth rate in subcutaneous tumor of nude mice and it was relatively safe to use it.TCS could induce the re-expression of ERαmRNA and protein in vivo by demethylating ERαgene.