人工合成拟Smac多肽增强膀胱癌细胞化疗敏感性的实验研究

Experiment of promoting chemosensitivity of bladder cancer cell by synthetic Smac peptide

背景与目的:Smac是目前发现的惟一能直接同时抑制多个IAPs家族成员活性的凋亡相关蛋白,其N端的4个氨基酸残基AVPI(Ala-Val-Pro-Ile)是重要的促凋亡结构域,我们通过人工合成可穿透细胞的促凋亡融合多肽SmacN7,探讨其对膀胱癌化疗药物敏感性的促进作用。方法:噻唑蓝(MTT)检测SmacN7融合多肽对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞的相对存活率的影响:Annexin V/PI双标流式细胞术检测T24细胞的凋亡1;Western印迹检测SmacN7融合多肽与MMC联用后T24细胞内XIAP、Caspase-3的表达;同时检测Caspase-3活性及SmacN7融合多肽与MMC联用对T24细胞的杀伤作用。结果:SmacN7融合多肽能穿透细胞并与内源性XIAP结合,增加低剂量MMC诱导的T24细胞凋亡并呈时间和浓度依赖性:并能显著降低细胞内XIAP的表达水平,增强Caspase-3的表达及活性:在24 h和48 h,SmacN7+MMC组与单用MMCtg相比,T24细胞的存活率分别降低55%和72.7%。结论:人工合成可穿透细胞的促凋亡融合多肽SmacNT能促进化疗药物诱导的膀胱癌T24细胞凋亡,抑制细胞增殖,增强膀胱癌细胞对MMC的化疗药物敏感性。

Background and purpose： Smac/DIABLO was the only apoptosis-related protein that could inhibit IAPs directly and simultaneously. The four amino-residual AVPI（Ala-Val- Pro-Ile）in its N-terminal was the very important domain that could stimulate apoptosis. This study investigated the effect of synthetic Smac peptide （SmacN7） on chemotherapy sensitivity of bladder cancer cells. Methods： SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was adopted to evaluate the viability ofT24 cells induced by low-dosage of MMC. Flow cytometry was applied to analyze the proportion of apoptosis and Western blot was used to detect the expression of XIAP and caspase-3; The activity of caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. Results： SmacN7 penetratin peptide could successfully interact with endogenous XIAP and increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose- and timedependent manner. An obvious down-regulation of XIAP expression and up-regulation of caspase-3 was identified by Western blot. The activity of caspase-3 in experimental group was significantly increased as compared with that in the control group; Combining the treatment with SmaeN7 penetratin peptide,the viability ofT24 cells decreased to 55% and 72.7% in 24 hrs and 48 hrs respectively. Couclusion： SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. When combined with chemotherapy, it may be a very promising strategy for bladder cancer therapy.