Comparison Of Expression Effect Of ER-a Induced By Arsenic Trioxide And Hydrazine In Vitro

Background and ObiectiveIncidence rate of breast cancer is gradually advancing year by year in Asia where used to be lower,especially in bei jing and shang hai since 1970. Breast cancer is the most common cancer in women. Estrogen receptor is usually an important molecule marker to evaluate hormonal dependence and predict the effect of endocrine therapy. According to the difference of ER phaenotype,breast cancer is divided into two types:ER negative and ER positive. Two thirds of primary breast cancers have the expression of ER and the growth of tumor cell is hormonal dependent. The clinical thrapy of breast cancer patients(ER positive) is endocrine therapy which is equal to chemotherapy.The mechanism of estrogen receptor(ER) alpha gene expression silencing is not clear in ER alpha negative breast cancer. Recently study discovers that estrogen receptor(ER) alpha gene expression silencing is not due to generearrangement,mutation,absence and so on and it's probably associated with the CpG island methylation in the core promotor of ER alpha gene. Medcine being proved with demethylation is mostly arsenic trioxide,5-AZA-CdR and hydrazine. Arsenic trioxide is a kind of traditional Chinese medicine with anti-tumor. At present it has been used to cure neoplastic hematologic disorder. Meanwhile it could demethylate the CpG island in estrogen receptor(ER) alpha gene. Hydrazine is a conventional dyazide. Because of its side effect,it is used to treat hypertensive disease of pregnancy and rarely other diseases.This present study is designed to compare the effect of re-expression of the silenced estrogen receptor (ER-a) gene in human breast cell line MDA-MB-231 and MDA-MB-435 by arsenic trioxide (As₂O₃) and hydrazine.Methods and materials(1) Human breast cell line MDA-MB-231 and MDA-MB-435 were growing at 37℃and 5% CO₂. MDA-231 and MDA-MB-435 were plated at density of 5×10⁵ cells/100-mm dish and were treated with L-15 containing As₂O₃ 2.0μmol/L and hydrazine 10μmol/L. The media containing the drug was maintained during the 3 days of treatment. MDA-MB-231 and MDA-MB-435 without being treated with containing As₂O₃ were taken as negative control.(2) The MDA-MB-231 and MDA-MB-435 cell line in which the estrogen receptor gene is silenced due to hypermethylation was treated with As₂O₃ and hydrazine, expression levels of ER-a genes were determined with RT-PCR technique and analyzed result with imagej software.(3) Immunohistochemistry was performed for ERa protein after the MDA-MB-231 and MDA-MB-435 cell line in which the estrogen receptor gene is silenced due to hypermethylation was treated with As₂O₃ and hydrazine.(4) experimental data are disposed by SPSS13.0 statistics soft ware,deploying analysis of variance ,significance of difference standard (a=0.05).Results(1) Detectable expression of ERa protein was found in the MDA-MB-231cells treated with As₂O₃ and hydrazine determined with immunohistochemistry technique positive rate(%) of As₂O₃ is 52.04±2.38,positive rate(%) of hydrazine is 51.07±1.43 (P>0.05). Detectable expression of ERa protein was found in the MDA-MB-435 cells treated with As₂O₃ and hydrazine determined with immunohistochemistry technique positive rate(%) of As₂O₃ is 55.15±6.56,positive rate (%)of hydrazine is 54.11±5.36 (P>0.05).(2) There is no conspicuous difference between MDA-MB-231and MDA-MB-435 (P >0.05).(3) Detectable expression of ERa mRNA was found in the MDA-MB-231 and MDA-MB-435 cells treated with As₂O₃ and hydrazine determined with RT-PCR technique. Result analyzed by imagej software was not conspicuous difference(P > 0.05).Conclusion(1) At their optimal concentration,the effect of re-expression of the silenced estrogen receptor (ER-a) gene was not diffent in the MDA-MB-231.(2) At their optimal concentration,the effect of re-expression of the silenced estrogen receptor (ER-a) gene was not diffent in the MDA-MB-435.(3) The result provide theory evidence for the selection of demethylation drugs,richen the basic research and provide a new method for endocrine therapy.