肼苯哒嗪与APC去甲基化和重新表达相关，在体外抑制人宫颈癌细胞生长。

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

作者信息

Song Yinhong, Zhang Changju

机构信息

Department of Immunology, Medical College of Three Gorges University, Yichang, Hubei, People's Republic of China.

出版信息

Cancer Chemother Pharmacol. 2009 Mar;63(4):605-13. doi: 10.1007/s00280-008-0773-z. Epub 2008 Jun 3.

DOI:10.1007/s00280-008-0773-z

PMID:18521605

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.

摘要

目的

肿瘤抑制基因腺瘤性息肉病 coli（APC）在人类宫颈癌中常因启动子高甲基化而沉默。临床上，已证实 DNA 甲基化抑制剂，如 5-氮杂-2'-脱氧胞苷（5-aza-dC），可逆转 APC 启动子甲基化，但这些抑制剂的广泛临床应用受到其毒性和在水溶液中不稳定性的限制。肼屈嗪是一种稳定的 DNA 甲基化抑制剂，在体外和体内具有最小的毒性。本研究的目的是评估肼屈嗪对 APC 重新激活和体外抑制人宫颈癌细胞的作用。

方法

采用 RT-PCR、实时定量 RT-PCR 和甲基化特异性 PCR 方法分析 APC 基因的表达及甲基化状态。用肼屈嗪处理后，通过免疫组织化学方法检测与 APC 密切相关的β-连环蛋白。采用 MTT 和 FCM 检测观察细胞增殖活性、细胞周期和凋亡的变化。

结果

甲基化的 APC 在 HeLa 细胞中不表达，半甲基化的 APC 在 CaSki 细胞中表达，未甲基化的 APC 在 SiHa 细胞中正常表达。肼屈嗪可诱导 HeLa 和 CaSki 细胞中 APC 的表达并促进去甲基化。用 40μmol/L 肼屈嗪处理 72 小时后，HeLa、CaSki 和 SiHa 细胞系的生长抑制率（%）分别为 52.12±3.78、44.31±2.59 和 47.73±4.73。相反，正常细胞 ECV304 的生长抑制率仅为 27.18±0.79。HeLa、CaSki 和 SiHa 细胞系中 APC mRNA 的表达分别增加了 10.35 倍、11.40 倍和 0.73 倍。肼屈嗪使 HeLa 和 CaSki 细胞停滞在细胞周期的 S 期，与未处理细胞相比，用肼屈嗪处理的这两种细胞系中的凋亡细胞百分比显著增加（P<0.01）。用肼屈嗪处理后观察到细胞膜中β-连环蛋白的表达。