RG108 induces the apoptosis of endometrial cancer Ishikawa cell lines by inhibiting the expression of DNMT3B and demethylation of HMLH1

L. Yang, J. Hou, X.-H. Cui, L.-N. Suo, Y.-W. Lv

Department of Obstetrics and Gynecology, PLA 463 hospital, Shenyang, China. yanglee2000@126.com

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• Oncology

OBJECTIVE: The effects of DNA methyltransferase (DNMT) inhibitor RG108 on the proliferation and apoptosis of endometrial cancer was investigated, and whether its mechanism was related to the inhibition of DNMT3B, thereby affecting the human mutL homolog 1 (hMLH1) methylation status and its expression, was further studied.

MATERIALS AND METHODS: Culture of human endometrial cancer Ishikawa cell lines: cells grew adhering to the wall in Roswell Park Memorial Institute-1640 (RPMI-1640) medium (supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamic acid). After the cells were treated with RG108, the changes in cell viability were detected via methyl thiazolyl tetrazolium (MTT) assay. The effect of RG108 on cell cycle was detected via flow cytometry, and its effect on cell apoptosis was detected via flow cytometry and TUNEL. Moreover, the methylation status of hMLH1gene in endometrial cancer cells was detected via methylation specific-PCR (MSP), and the changes in DNMT3Band hMLH1 expressions were detected via RT-PCR and Western blotting, respectively.

RESULTS: MTT results showed that RG108 inhibited the cell viability in a dose-dependent and time-dependent manner. Flow cytometry revealed that RG108 blocked the cell cycle in G2/M phase and promoted the apoptosis, and TUNEL assay further proved that RG108 promoted the apoptosis. It was found in the detection via MSP that the methylated hMLH1 gene was significantly reduced after 72 h of treatment with RG108. Besides, RT-PCR and Western blotting showed that RG108 inhibited the DNMT3B expression and activated the hMLH1 expression.

CONCLUSIONS: The demethylation drug RG108 can significantly inhibit the proliferation of endometrial cancer cells, block the cell cycle in the G2/M phase and induce the cell apoptosis, which is a new candidate drug in the treatment of endometrial cancer. RG108 realizes the hMLH1 demethylation and increases the hMLH1 expression through inhibiting the expression of DNMT3B.

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