肼苯哒嗪对人骨肉瘤细胞的生长抑制及对WWOX基因的调控作用研究

Effects of Hydralazine and 5-Aza-CdR on cell proliferation of human osteosarcoma cell line MG-63 and expression of gene WW domain-containing oxidoreductase

目的通过观察肼苯哒嗪、5’-氮杂-2’-脱氧胞嘧啶核苷酸（5’-aza-2’deoxycytidine，5-Aza-CdR）及其联合使用对人成骨肉瘤细胞MG-63的生长抑制作用，并检测对骨肉瘤细胞中WWOX基因表达的调控作用，初步明确肼苯哒嗪对骨肉瘤细胞的作用机制。方法取对数生长期的人骨肉瘤细胞株MG-63制成细胞悬液，计数板计数5×10^4个，ml，加入96孔板。分为肼苯哒嗪组（药物浓度为0．1、1．0、10μmol／L）、5-Aza-CdR组（药物剂量为5、10、20μmol／L）、肼苯哒嗪联合5-Aza-CdR组f药物剂量为0．1μmol／L＋5μmo／L、1．0μmol／L＋10／μmol／L、10μmol／L＋20μmol／L）及对照组（培养基）。噻唑蓝（M1Tr）比色法检测药物对MG．63细胞生长抑制作用；流式细胞仪检测药物对细胞周期分布作用及凋亡影响；Real-TimePCR技术检测WWOXmRNA表达改变情况；Western-blot检测药物处理后各组细胞中WWOX蛋白表达水平。结果肼苯哒嗪、5-Aza-CdR及其联合应用对MG-63细胞的增殖均有明显抑制作用，且呈浓度依赖性并与作用时间相关；联合应用较单用抑制作用明显增强，且各组差异有统计学意义。肼苯哒嗪、5-Aza-CdR可诱导骨肉瘤MG-63细胞凋亡，联合应用时诱导凋亡作用加强。肼苯哒嗪、5-Aza-CdR对骨肉瘤MG-63细胞中WWOX基因表达有促进作用，联合作用使WWOX表达明显增强。结论肼苯哒嗪可明显抑制骨肉瘤细胞的增殖并诱导其凋亡，并促进WWOX基因的表达，其作用机制可能为肼苯哒嗪能够使抑癌基因WWOX基因甲基化状态逆转，表达增加，从而抑制MG-63细胞的增殖。

Objective To investigate the growth inhibition of human osteosareoma cell line（MG-63） intervened by Hy- dralazine and 5＇ -aza- 2＇ -deoxycytidine （5-Aza-CdR）, and the effect on the mRNA expression of gene WW domain-containing oxi- doreductase （WWOX）. Methods Certain volume of 5 × 10^4/ml of human osteosarcoma cell line MG- 63 in logarithmic growth phase were added into 96-well plate. There were Hydralazine group （drug concentration, 0.1, 1.0, 10 μLmol/L）, 5-Aza-CdR group （drug concentration, 5, 10, 20 Ixmol/L）, Hydralazine combined with 5-Aza-CdR group （drug concentration, 0.1 μmo/L ＋ 5 Ixmol/L, 1.0μmol/L ＋ 10 μmol/L, 10 μmol/L ＋ 20 μmol/L） and control group （culture medium）. Methyl thiazol tetrazolium（MTr） colorimet- ric methods were used to test the growth inhibition of MG-63 cells intervened by different concentrations of Hydralazine and 5＇-aza -2＇ -deoxycytidine （5-Aza-CdR）. Flow cytometry AnnexinV-FITC/PI methods were used to assay the effects of Hydralazine and 5- Aza-CdR inducing apoptosis in osteosarcoma cells in vitro. Real-time polymerase chain reaction （Real-Time PCR）methods were used to detect amplification of WWOX mRNA induced by Hydralazine combined with 5-Aza-CdR or alone. Western-blotting meth- ods were used to examine the expression of WWOX in MG-63 cells. Results Hydralazine and 5-Aza-CdR effectively inhibited the growth of MG-63 cells in a concentration and time-dependent manner. Combined effect was more obvious. Further more the ex- pression levels of WWOX mRNA and protein were increased significantly in combined groups as compared with other groups. Conclusion Hydralazine and 5-Aza-CdR could effectively inhibit the proliferation of MG-63 cells and induce apoptosis which is concurrent with the promotion of the expression of WWOX. The mechanism may be that Hydralazine/5-Aza-CdR effectively cause the demethylated of WWOX gene CpG-rich promoter regions, leading to the high expression of WWOX and inhibit the growth of MG-63 cells. The use of hvdralazine in the treatment of osteosarcoma is worthy of further investigation.