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Demethylation of ER-α Gene in MDA-MB-231 Cell Line by Arsenic Trioxide

Author: LiuJian
Tutor: WangLiuXing；LuShiXin
School: Zhengzhou University
Course: Oncology
Keywords: Breast Cancer Estrogen Receptor Arsenic trioxide DNA methylation
CLC: R737.9
Type: Master's thesis
Year: 2007
Downloads: 150
Quote: 0
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Abstract


ER-α gene is located on chromosome 6q24-27, ER-α protein expression often used as an important molecular markers used to assess the treatment of breast cancer, hormone-dependent and prediction of endocrine treatment effect; ER-α gene loss of expression (ER -α-negative) breast cancer patients are not sensitive to endocrine therapy, the prognosis is poor. According to the literature, the ER-α gene expression silencing and gene rearrangements, mutations, deletions, and other independent and may be related to the promoter region CpG island methylation. Therefore, for the loss of expression of ER-α gene demethylation treatment to become a new way of estrogen receptor-negative breast cancer treatment. Subject to this end in the direction of experimental research. In this experiment, the earliest reports of the original chemicals for induction therapy (As 2 O 3 ) as the demethylation of experimental drugs, to carry on the ER-α gene methylation of breast cancer cell line MDA-MB-231 cells to the experimental study of the methylation mechanism to explore of As 2 O 3 MDA-MB-231 breast cancer cells norepinephrine the effect of the base of its relationship with gene expression to study As to 2 O 3 relationship with the breast cancer cell DNA methylation and demethylation therapy Possible mechanisms of As 2 O 3 as demethylation agent to provide experimental basis. Materials and methods: (1) selection of the estrogen receptor (ER) conventional culture-negative human breast cancer cell line MDA-MB-231, MDA-MB-231 cells in log phase will adjust the cell concentration of 5 × 10 5 / mL, change containing of As 2 O 3 L-15 culture medium to As 2 O 3 final concentration to 0.5μmol / L, 2.0μmol / L, 4.0μmol / L, continuous culture for 72 hours. MDA-MB-231 cells cultured in the same period without drug as a negative control. Cultured in RPMI-1640 environment to the same period of the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 as a positive control. (2) the use of the ER-α gene REP-PCR was used to detect the expression of exon I (containing hypermethylation of CpG Island). (3) using the Western Blot detection by different concentration of As 2 O 3 treatment of MDA-MB-231 cells ER-α protein expression. (4) MTT to detect different concentrations of As 2 O 3 treatment of MDA-MB-231 cells, and then after tamoxifen treatment, the inhibition rate of cell proliferation differences. (5) the experimental data by the SPSS11.0 statistical software processing, using one-way ANOVA to, α = 0.05 significantly different standards. The results: (1) REP-PCR assay the without of As 2 O 3 treatment of MDA-MB-231 cells before and after by NotI enzyme to digest, can expand the The Clear exon Ⅰ fragment, suggesting that ER-α gene methylation positive; certain concentration (2.0μmol / L 4.0μmol / L) As 2 O 3 After 72 hours, again digested amplified no purpose strip Step ER-α gene methylation negative; to MCF-7 cells as a positive control, with NotI enzyme excess after digestion also not amplified ER-α gene exon Ⅰ fragment. (2) Western Blot analysis without the of As 2 O 3 treatment of MDA-MB-231 cells did not show ER-α protein with 0.5μmol / L As 2 O 3 treatment of MDA-MB-231 cells did not show ER-α protein band, but 2.0μmol / L 4.0μmol / L of As O 3 treatment of MDA-MB-231 cells visible ER-α protein band. (3) MTT assay tamoxifen role in arsenic trioxide treated MDA-MB-231 cells was significantly inhibited cell proliferation. The concentration 2.0μmol / L around As 2 O 3 should be more suited to the dose of the MDA-MB-231 cell line demethylation in vitro experiments, although As 2 O 3 MA-MB-231 cells after treatment of TAM sensitivity less than MCF-7 cells, but significantly better than the untreated MA-MB-231 cells, prompted TAM As 2 O 3 MDA-MB-231 cells treated with significant cytotoxicity. Conclusion: 1. A certain concentration of As 2 O 3 enables the ER-α gene in the cell line MDA-MB-231 CpG island go methylation. 2. A certain concentration of As 2 O 3 MDA-MB-231 cell lines can be induced ER-α protein expression. 3. The As 2 O 3 induced ER-α expression in MDA-MB-231 cells are functional. 4. As 2 O 3 by demethylation of the estrogen receptor-negative breast cancer cells express functional estrogen receptor, which is estrogen receptor-negative breast cancer patients receiving endocrine therapy provide a theoretical basis.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Breast tumor
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