5-氮杂胞嘧啶核苷联合hepaCAM腺病毒通过抑制p-AKT诱导膀胱癌细胞T24凋亡

Inducing apoptosis of bladder cancer cell T24 by 5-azacytidine combined with adenovirus-hepaCAM via inhibiting p-AKT

目的:研究5-氮杂胞嘧啶核苷(5-azacytidine,azac)联合肝细胞黏附分子(hepatocyte cell adhesion molecule,hepaCAM)腺病毒对膀胱癌细胞T24凋亡的影响及可能机制的探讨。方法:7种不同因素分别处理培养的膀胱癌细胞T24,hoechst 33258染色检测各处理组细胞凋亡率,流式细胞仪(flow cytometry,FCM)检测细胞早期凋亡率,RT-PCR检测hepaCAM及B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,bcl-2)mRNA的表达,Western blot检测hepaCAM、p-AKT、total-AKT、bcl-2蛋白的表达。结果:hoechst 33258显示,azac联合hepaCAM腺病毒组细胞凋亡率为(45.21±0.06)%,明显高于hepaCAM腺病毒组(15.60±0.02)%和azac组(26.30±0.02)%,差异有统计学意义(P=0.003,P=0.008);FCM显示,azac联合hepaCAM腺病毒组细胞早期凋亡率为(16.05±0.06)%,明显高于hepaCAM腺病毒组(3.90±0.02)%和azac组(9.67±0.02)%,差异有统计学意义(P=0.002,P=0.043);RT-PCR显示,与单独hepaCAM腺病毒组和azac组相比,azac与hepaCAM腺病毒联用可以上调hepaCAM mRNA的表达(P=0.004,P=0.000),下调bcl-2 mRNA的表达,差异有统计学意义(P=0.026,P=0.030);Western blot显示,与单独hepaCAM腺病毒组和azac组相比,azac联合hepaCAM腺病毒组可上调hepaCAM蛋白的表达(P=0.003,P=0.003),同时下调p-AKT(P=0.001,P=0.005)和bcl-2(P=0.000,P=0.002)蛋白的表达,差异有统计学意义。结论:azac联合hepaCAM腺病毒可能通过抑制p-AKT的磷酸化水平加速诱导T24细胞凋亡,可为膀胱癌的治疗提供新的思路。

Objective：To explore the effect of 5-azacytidine（azac）combined with adenovirus-hepatocyte cell adhesion molecule（adhepaCAM）on the apoptosis of bladder cancer cells T24 and the possible mechanisms. Methods：Bladder cancer cells T24 were treated with seven different factors. Apoptosis rate of each treatment group was detected by the kits of hoechst 33258. Cell apoptotic ratio at early stage was detected by flow cytometry. The mRNA expression of hepaCAM and bcl-2 was measured by RT-PCR. The protein expression of hepaCAM,p-AKT,total-AKT and bcl-2 was measured by Western blot. Results：Higher apoptosis rate in azac combined with ad-hepaCAM group（45.21±0.06）% than in adenovirus-hepaCAM group（15.60±0.02）% and azac group（26.30±0.02）% was observed based on hoechst 33258（P=0.003,P=0.008）. Higher apoptosis rate at early stage in azac combined with ad-hepaCAM group group（16.05±0.06）% than in adenovirus-hepaCAM group（3.90±0.02）% and azac group（9.67±0.02）% was observed based on FCM（P=0.002,P=0.043）. Higher expression of hepaCAM mRNA（P=0.004,P=0.000）and lower expression of bcl-2 mRNA（P=0.026,P=0.030）in azac combined with ad-hepaCAM group than in adenovirus-hepaCAM group and azac group was observed based on RT-PCR. Higher expression of hepaCAM（P=0.003,P=0.003）and lower expression of p-AKT（P=0.001,P=0.005）and bcl-2（P=0.000,P=0.002）in azac combined with ad-hepaCAM group than in adenovirus-hepaCAM group and azac group was observed based on Western blot. Conclusion：Azac combined with ad-hepaCAM can induce the apoptosis of T24 by inhibiting p-AKT,which may provide a new therapy for bladder cancer.