紫草素促进caspase-9活化诱导大肠癌细胞凋亡的分子机制研究

目的探讨紫草素对大肠癌(CRC)细胞LoVo的抗肿瘤作用及其机制。方法以不同紫草素浓度梯度(0、2、4、6μmol/L)处理CRC细胞LoVo 24 h,以4μmol/L紫草素处理不同时间梯度(0、12、24、48 h)的CRC细胞LoVo。流式细胞技术结合Annexin V-FITC/PI双染色测定细胞凋亡率,Western blot检测细胞中caspase-9蛋白的表达及切割情况。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果与0μmol/L处理CRC细胞LoVo 24 h比较,2、4、6μmol/L的细胞凋亡率(6.94±1.02)﹪比(10.61±1.12)﹪、(15.55±1.35)﹪、(36.51±1.46)﹪]均升高;与4μmol/L紫草素处理0 h的CRC细胞LoVo比较,12、24、48 h的细胞凋亡率(1.33±0.59)﹪比(19.23±1.24)﹪、(22.24±1.41)﹪、(28.41±1.52)﹪]均升高,差异具有统计学意义(P均<0.001)。当紫草素剂量≥2μmol/L,处理时间≥12 h时,caspase-9蛋白表达上调并被诱导活化,而caspase-9抑制剂(Z-LEHD-FMK)预处理后,LoVo细胞凋亡率下降38.7﹪,差异有统计学意义(P<0.05)。结论紫草素可以通过caspase-9蛋白的表达及其切割活性诱导CRC细胞凋亡。

The molecular mechanism of shikonin in promoting caspase-9 activation and inducing apoptosis in colorectal cancer cells

Objective To investigate the anti-tumor effect and mechanism of shikonin on colorectal cancer cell LoVo.Methods The colorectal cancer cell LoVo was treated with different concentration gradients(0,2,4,6μmol/L)for 24 hours.Besides,the other colorectal cancer cell LoVo was treated with 4μmol/L of shikonin according to different time gradients(0,12,24,48 h).The apoptosis rate was determined by flow cytometry combined with Annexin V-FITC/PI double staining.The expression and cleavage of caspase-9 protein were detected by Western blot.ANOVA and LSD-t tests were conducted to compare the average values between the control and experimental groups.Results Compared with the colorectal cancer cell LoVo treated with 0μmol/L for 24 hours,the apoptosis rate of colorectal cancer cell LoVo treated with 2,4,6μmol/L for 24 hours were increased(6.94±1.02)﹪vs(10.61±1.12)﹪,(15.55±1.35)﹪and(36.51±1.46)﹪].Compared with the colorectal cancer cell LoVo treated with 4μmol/L for 0 hours,the apoptosis rate of colorectal cancer cell LoVo treated with 4μmol/L for 12 hours,24 hours and 48 hours were increased(1.33±0.59)﹪vs(19.23±1.24)﹪,(22.24±1.41)﹪and(28.41±1.52)﹪],differences were statistically significant(P<0.001).In the mean time,caspase-9 protein was up-regulated and activated when shikonin dose was≥2μmol/L and treatment time was≥12 h,while caspase inhibitor(Z-LEHD-FMK)pretreatment could reduced the apoptosis rate significantly(P<0.001),by approximately 38.7﹪(P<0.05).Conclusion Shikonin could induce apoptosis of colorectal cancer cells by regulating the expression of caspase-9 protein and its cleavage activity.