Demethylation Of Estrogen Receptor Gene And Its Re-expression In Estrogen Receptor-negative Human Breast Cancer Cells

Breast cancer is the most common cancer in women. Numerous experimental and clinical studies have established that estrogen plays a major role in the initiation and progression of breast cancer. Approximately two thirds of breast cancers express estrogen receptor(ER positive), and their growth is stimulated by estrogen. For these cancers, therapeutic strategies include estrogen ablation or anti-estrogen. However, the remaining one thirds of primary breast cancers lack the expression of ER protein(ER negative) and are rarely responsive to hormonal treatment.Apparently, these ER negative tumors lack ER gene expression, yet it is not due to mutations within the ER gene. Therefore, the loss of ER transcription is a potential mechanism for hormonal resistance. Studies have found that ER-negative breast cancer cells have a hypermethylated CpG islands in the ER gene promoter. Because methylation of CpGislands in the gene promoter is believed to directly inhibit gene expression. So extensive methylation of CpG islands of the ER gene may play a role in the absence of ER gene expression in ER negative human breast cancer cellsThe current study was designed to determine whether demethylation of the ER gene in the ER negative human breast cancer cell line MDA-MB-231 could reactivate estrogen receptor protein expression by 5-aza-2'-deoxycytidine and the functional status of re-expressed ER protein. We also explored the possibility and mechanism of re-expression of the ER protein in MDA-MB-231 cells by arsenic trioxide (As2O3). Materials and methods:(l)Human breast cancer cell lines (ER-positive MCF-7 and ER-negative MDA-MB-231) were acquired and routinely maintained. 5-aza-2'-deoxycytidine and arsenic trioxide were used to treat the ER-negative breast cancer cells. (2)Immunohistochemistry method was used to detect the expression of ER protein before and after 5-aza-2'-deoxycytidine treatment. (3)Western blot analysis was used to detect the expression of ER protein before and after 5-aza-2'-deoxycytidine or arsenic trioxide treatment respectively. (4)MTT assay was used to determine the effect of 5-aza-2'-deoxycytidine or arsenic trioxide treatment on the growth curve of MDA-MB-231 cells.(6) MTT assay was used to determine the effect of E2 and TAM onthe growth of MDA-MB-231 cells after 5-aza-2'-deoxycytidinetreatment.(7)Cell cycle and cell apoptosis were examined by PIfluorescence flow cytometry before and after 5-aza-2'-deoxycytidine orarsenic trioxide treatment.(8)All experimental data were processed bySPSS11.0. There was a statistically significant difference when P<0.05occurred.Results:(1) Immunohistochemistry method showed that the positive rate of ERprotein on MDA-MB-231 breast cancer cells treated by 5-AZA-dC wassignificantly higher than that of pretreatment (P < 0.05).(2)ER protein was detectable by Western blot analysis after5-aza-2'-deoxycytidine or arsenic trioxide treatment of MDA-MB-231cells, while ER protein was undetectable before treatment.(3)MTT showed that MDA-MB-231 cells, after 5-aza-2'-deoxycytidineor arsenic trioxide treatment, grew significantly more slowly thanpretreatment.(4) MTT showed that after 5-aza-2'-deoxycytidine treatment, the ODvalue of control group(5-aza-2'-deoxycytidine treated only) is 0. 1672±0. 0091; the OD value of TAM group (5-AZA-dC+ T A M) is 0. 1530± 0. 0168 (P < 0.05), the suppression rate is 8. 49%; the OD value of E2group (5-AZA-dC+E2) is 0.1677 + 0.0119 (P>0.05) .(5)Flow cytometry examination showed that after 5-aza-2'-deoxycytidineor arsenic tnoxide treatment of MDA-MB-231 cells, the percentage of Gl phase was remarkably reduced (P<0.05) and the percentage of S phase was remarkably increased (P<0.05), while the percentage of G2/M phase remained the similar level. And the examination did not show the typical subdiploid peak. Conclusion:(1) 5-aza-2'-deoxycytidine and arsenic trioxide both can reactivate the expression of the ER protein in ER-negative human breast cancer cell line MDA-MB-231.(2) 5-aza-2'-deoxycytidine and arsenic trioxide inhabit the growth of breast cancer cells, when they are inducing the expression of the ER protein of MDA-MB-231 cells.(3) After the expression of the ER protein of MDA-MB-231 cells, the percentage of S phase was remarkably increased with the influence of estradiol.(4) The re-expressed ER protein is functional, and TAM can suppress its estrogen-dependent stimulating effect on cell growth.