| Background and objective:In recent years, the morbidity and mortality of breast cancer are growing rapidly. About 130 million women suffer from breast cancer and 50 million people died of breast cancer each year in the world. Although breast cancer has a low morbidity in our country, the incidence of breast cancer is rising obviously. We predict that it will be the highest incidence of malignant tumor in 20 years.Estrogen receptor (ER) is an important role in the development of breast cancer. In vivo, ER combine with estrogen and active the genes which contain estrogen response element and transcription factor response element, then lead to breast cancer. In general, we divide the breast cancer into ER negative breast cancer and ER positive breast cancer. Among about 20～35 percent of breast cancer patients, the estrogen receptor is negative. With the high malignant degree, rapid development, higher local recurrence, and metastases easily occurring in lung, liver, bone, brain and other important viscera, it has poor prognosis. Due to lack of corresponding receptor, the patients cannot apply endocrine and biological treatment. At present, the treatment of this kind of special type of breast cancer limits to chemotherapy, but it is far from perfect. Thus, as an important molecular marker, ER has vital significance to forecast the effect of endocrine therapy and evaluate the dependence of hormone in breast cancer.The main theories of ER gene expression silencing are DNA methylation and chromosome restructuring in ER negative breast cancer. Methylation occurs early in the process of cancer, it plays an important role in the occurrence and development of cancer. DNA methylation is a reversible process. By theoretically, if we reverse methylated oncogenes and tumor-suppressor genes with drugs, it will be made the gene re-expressed.Arsenic trioxide (AS2O3) is the main ingredient of arsenic's of Traditional Chinese medicine. In recent years, we find that it has better effects on many kinds of tumor cells, such as leukemia, liver cancer and so on. AS2O3 exert it's cytotoxicity by inducing cells apoptosis. Recent research suggests that it can demethylate.This study is designed that AS2O3 effect on ERa negative breast cancer cell MDA-MB-435. As2O3 can demethylate gene promoter CpG island partially, and made ERa re-expressed. Consequently, ERa negative breast cancer patients can restore the sensitivity of endocrine drugs.Materials and methods:(1) ERa negative human breast cancer cell line MDA-MB-435 planted at 37℃and 5% CO2. Adjust cell density of 5×105/ml and treat with 1640 containing AS2O3 0.5μmol/L, 1.0μmol/L,2.0μmol/L,4.0μmol/L respectively. Then plant for 72 hours. MDA-MB-435 without being treated with containing As2O3 was taken as negative control. Human breast cancer cell line MCF-7 (ER positive) was taken as positive control.(2) To detect the re-expression of the ERa mRNA by RT-PCR.(3) To detect the status of 5'CpG island methylation of ERa gene by Methylation Specific PCR(MSP).(4) To detect the re-expression of the ERa protein by Western blot.(5) To detect the re-expression of the ERa protein by immunohismchemistry.(6) MTT method tested whether Tamoxifen could induce the growth inhibition of MDA-MB-435 cell after treated with As2O3. (7) Using SPSS 16.0 statistics processing software andχ2-test for statistical analysis. Significant of difference standard is P<0.05.Result:(1) It is confirmed by the test of RT-PCR that expression of ERa mRNA were not found in group of negative control, neither in group of 0.5μmol/L. But ERa mRNA were detectable in groups of 1.0μmol/L,2.0μmol/L and 4.0μmol/L.(2) By MSP technique, methylation of ERa gene was found in group of negative control, methylation of ERa gene and demethylation of ERa gene were both found in groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L.(3) It is confirmed by the test of Western blot that expression of ERa protein were not found in group of negative control and in group of 0.5μmol/L. But ERa protein were detectable in groups of 1.0μmol/L,2.0μmol/L and 4.0μmol/L.(4) By immunohistochemistry technique, we found that, in group of negative control, the positive rate was 7.4%. In groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L, the positive rate is respectively 7.8%,43.8%,78.2% and 41.5%.(5) By the test of MTT, we found that tamoxifen can effect on the growth of MDA-MB-435 cells treated with AS2O3. We found that, in group of negative control, the inhibition rate was 10.7%. Inhibition rate was respectively 14.9%,30.7%,43.1% and 56.6% in groups of 0.5μmol/L, 1.0μmol/L,2.0μmol/L and 4.0μmol/L.Conclusion:A proper concentration of AS2O3 could effect on a part cells through demethylation and induce ERa gene re-expression in human breast cancer cell line MDA-MB-435. And it can restore to sensitivity of endocrine therapy drug(tamoxifen). Consequently, this study provides the reliable theory base for treating ERa negative breast cancer patients with endocrine therapy. |
PDF Full Text Request