Hydralazine inhibits human cervical cancer cell growth in vitro in association with APC demethylation and re-expression

Abstract

Purpose: The tumor suppressor adenomatous polyposis coli (APC) is frequently silenced by promoter hypermethylation in human cervical cancer. Clinically, it has been approved that DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (5-Aza-dC), can reverse APC promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. Hydralazine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. The purpose of this study was to evaluate the effects of hydralazine on APC reactivation and the inhibition of human cervical cancer cells in vitro.

Methods: Expression of APC gene, and methylation status were analyzed by RT-PCR, quantitative real time RT-PCR, and methylation-specific PCR methods. beta-Catenin protein that correlates closely with APC was detected by immunohistochemistry method after treatment with hydralazine. MTT and FCM assays were used to observe the changes of proliferation activity, cell cycle, and apoptosis of the cells.

Results: Methylated APC was not expressed in HeLa cell, hemimethylated APC was expressed in CaSki cells, and unmethylated APC was expressed normally in SiHa cells. Hydralazine induces APC expression and promotes demethylation in HeLa and CaSki cells. After treatment with 40 mumol/L hydralazine for 72 h, growth inhibitive rates (%) of HeLa, CaSki, and SiHa cell lines were 52.12 +/- 3.78, 44.31 +/- 2.59, and 47.73 +/- 4.73, respectively. On the contrary, the normal cell ECV304 growth inhibitory rate was only 27.18 +/- 0.79. The expression of APC mRNA in HeLa, CaSki, and SiHa cell lines increased 10.35-, 11.40-, and 0.73-fold, respectively. HeLa and CaSki cells were arrested in S phase of the cell cycle by hydralazine, and the percentage of apoptotic cells in the two cell lines treated with hydralazine was increased significantly compared to the untreated cells (P < 0.01). The expression of beta-catenin protein in the cell membrane was observed after the treatment with hydralazine.

Conclusions: Hydralazine, an effective inhibitor of APC methylation and promoter of APC re-expression, can inhibit cell growth in human cervical cancer in vitro and be potentially used for the clinical treatment of human cervical cancer.