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Arsenic Induce Functional Re-expression of Estrogen Receptor α by Demethylation in Estrogen Receptor-negative Human Breast Cancer Cells

Author: DuJuan
Tutor: LiZhong；WangShouLin；LiuQiZhan
School: Nanjing Medical University
Course: Nutrition and Food Hygiene
Keywords: Arsenic trioxide Estrogen receptor α Demethylation CpG island methylation Estrogen receptor-negative breast cancer cells
CLC: R737.9
Type: Master's thesis
Year: 2010
Downloads: 31
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Abstract


Breast cancer is the highest incidence of malignancy in women worldwide. Estrogen receptor α (ERα) markers of breast cancer response to endocrine therapy. About 2/3 of patients with breast cancer, breast cancer tissue ERα, these tumor cells generally differentiated growth and metastasis slow against endocrine therapy sensitive, estrogen-dependent. But ERα expression in the breast tissue of about 1/3 of patients, these tumors are poorly differentiated, high growth fraction and poor clinical outcome, confrontation not sensitive to endocrine therapy. With the progress of the tumor, the original ERα-positive breast cancer patients will eventually lose ERα expression. Therefore, ERα re-expression and restoration of the sensitivity of the new strategy for endocrine therapy is particularly important. ERα loss of expression of the mechanism is unclear, may be related to the promoter region CpG island methylation. Recently, there are reported arsenic trioxide demethylation. This with arsenic trioxide metabolic process in consumption caused by methyl donor methyl donor in the process of DNA methylation relative or absolute lack methyltransferase enzymes (DNMTs) related to its inhibition of DNA methylation required. Therefore, trivalent arsenic may make certain gene through the channels demethylation and re-expression, but arsenic whether the lack of ERα in breast cancer cells can be re-expression, as well as the exact mechanism has not been reported in the literature. Objective: In this study, the use of estrogen-receptor negative breast cancer cells (MDA-MB-231 cells) as an in vitro model, trivalent arsenic (arsenic trioxide) induced ERα re-expression explore different concentrations of arsenic trioxide-induced MDA-MB-231 cell line ERα the possibility of re-expression and its possible mechanism. Is the arsenic used clinically to improve the lack of ERα provide a theoretical basis for sensitivity to endocrine therapy in breast cancer. Methods: RT-PCR and Western blotting, arsenic trioxide the ERαmRNA and protein expression in MDA-MB-231 cells; using MTS assay (the detection cell proliferation), RT-PCR (detection ERα target gene expression) and luciferase reporter genetic experiments (detection ERα transcriptional activity) to detect whether the re-expression of ERα function; 5mC-immunofluorescence experiments, methylation-specific PCR (MSP) and bisulfite modification - sequencing PCR (BSP) assay arsenic trioxide MDA Start the methylation status of the promoter region;-MB-231 cell genome and ERα by Western blotting detection of arsenic trioxide methyltransferase (DNMTs) MBD2 expression; final ERα repressor total promoter ChIP experiments detected arsenic trioxide member of the complexes of DNMT1. The result: a certain concentration of arsenic trioxide can induce ER-negative breast cancer cells ERαmRNA and re-expression of the protein, RT-PCR experiments showed that arsenic trioxide in the presence of estrogen can induce ERα target gene pS2, GREB1 expression; MTS and dual luciferase report gene experiments showed that arsenic trioxide can make the original of MDA-MB-231 cells do not respond to endocrine therapy of estrogen and ER antagonist hydroxy tamoxifen and ICI182, 780 become sensitive. Suggesting that arsenic trioxide induced re-expression of ERα function. The 5mC-immune assay, MSP and BSP experiments show that portion of arsenic trioxide can cause the genome of MDA-MB-231 breast cancer cells and ERα promoter region demethylation. Methyl donor SAM can reduce arsenic caused ERα re-expression and ERα promoter region, the degree of de-methylation. Western blotting experiments showed that arsenic trioxide inhibition of MDA-MB-231 cells DNMT1, DNMT3A protein expression induced MBD2 protein enhanced. ChIP experiments showed that DNMT1 protein in MDA-MB-231 cells with the ERα promoter combination of arsenic trioxide inhibition. Prompted arsenic trioxide ERα promoter demethylation of leaving the silent ERα re-expression, and restore the ERα receptor function. Conclusion: compete with DNA methylation, a certain concentration of arsenic trioxide donor SAM, inhibition DNMT1, DNMT3A protein expression, inducing increased expression of demethylation protein MBD2, while suppressing the total binding repressor complexes DNMT1 promoter and ERα common cause demethylation ERα start the decrease in the degree of methylation of the promoter region, so high methylation silencing ERα re-expression, and to restore the sensitivity of unopposed estrogen therapy.

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