桦木醇对人结肠癌SW480细胞增殖和凋亡的影响

目的: 研究桦木醇对人结肠癌SW480细胞增殖及凋亡的影响,并探讨其相关作用机制。方法: 培养结肠癌SW480细胞,传代后用于实验。SW480细胞分为对照组和不同剂量桦木醇(1、5、10、20、50和100 mg·L^-1)组,MTT法测定各组细胞的增殖抑制率;以不加桦木醇的SW480细胞为对照组,DAPI染色观察15 mg·L^-1桦木醇作用SW480细胞6、12和24h后的形态学变化;流式细胞术分析Sub-G₁细胞百分比即细胞凋亡率,体外caspase活力测定法检测凋亡相关蛋白caspase-3、caspase-8和caspase-9的激活情况,Western blotting法检测各组细胞caspase-3底物多聚(ADP-核糖)聚合酶(PARP)断裂和凋亡蛋Bcl-2、Bcl-xL和cytochrome C的表达情况。结果: MTT法检测,与对照组比较,随着桦木醇剂量(1、5、10、20、50和100 mg·L^-1)增加SW480细胞的增殖抑制率逐渐增加(P<0.05或P<0.01),其增殖抑制率呈剂量效应关系,半数抑制浓度(IC₅₀)为12.875 mg·L^-1。DAPI染色,与对照组比较,15 mg·L^-1桦木醇组正常细胞数量减少,在12和24 h时呈现出典型的细胞凋亡特征(膜气泡,核固缩、变形及染色质浓缩等)。流式细胞术检测,与对照组比较,随着作用时间的延长,15 mg·L^-1桦木醇组SW480细胞中处于Sub-G₁期的细胞逐渐增多,即细胞凋亡率逐渐增加。caspase活力测定,与对照组比较,caspase-3和 caspase-9活性明显增加(P<0.05或P<0.01)。免疫印迹检测,与对照组比较,15 mg·L^-1桦木醇组SW480细胞中PARP出现断裂,凋亡蛋白Bcl-xL表达下调,cytochrome C表达上调,而Bcl-2蛋白表达无明显变化。结论: 桦木醇可抑制结肠癌SW480细胞增殖并诱导其凋亡,其作用机制可能是通过下调Bcl-xL表达启动线粒体介导的cytochrome C释放进而激活caspase-9凋亡途径实现。

Influence of betulin in proliferation and apoptosis of human colon cancer SW480 cells

Objective To study the influence of betulin in the proliferation and apoptosis of human colon cancer SW480 cells, and to explore its related mechanism. Methods The SW480 cells were cultured in Dulbecco's modified Eagle's medium and used in this experiment after passages.The SW480 cells were divided into control group and different doses (1, 5, 10, 20, 50, 100 mg·L^-1) of betulin groups.MTT colorimetric assay was used to determine the inhibitory rates of proliferation of SW480 cells in various groups.The morphological changes of SW480 cells were detected by DAPI staining after treated with 15 mg·L^-1 betulin for different time(6, 12, 24 h).The apoptotic cells were quantified by flow cytometry (FCM);the activities of caspase-3, caspase-8 and caspase-9 were determined through Caspase Assay in vitro;Western blotting method was used to detect the breakage of poly (ADP-ribose) polymerase (PARP) and the protein expression of Bcl-2, Bcl-xL and cytochrome C. Results The MTT results showed that the inhibitory rates of proliferation of SW480 cells were gradually increased with the increasing of betulin dose (1, 5, 10, 20, 50, 100 mg·L^-1) in a dose-dependent manner compared with control group(P<0.05 or P<0.01).The 50% inhibitory concentration (IC₅₀) of SW480 cells were 12.875 mg·L^-1.The typical morphological changes of apoptosis were observed in SW480 cells with DAPI staining after induction in 15 mg·L^-1 betulin group such as membrane bubbles, chromatin condensation, nuclear condensation and deformation, especially at 12 or 24 h;compared with control group, the number of normal cells in the experimental group (12, 24 h)was reduced.The FCM results showed the percentage of cells at sub-G₁ arrest in 15 mg·L^-1 betulin group was increased compared with control group.Compared with control group, the activities of caspase-3 and caspase-9 were significantly increased (P<0.05 or P<0.01).The Western blotting method results showed that poly- (ADP-ribose) polymerase (PARP) was cleaved in 15 mg·L^-1 betulin group, the expression of apoptotic protein Bcl-xL was down-regulated, and the expression of cytochrome C was up-regulated, but the expression of Bcl-2 had no changes. Conclusion Betulin can inhibit the proliferation and induce the apoptosis of SW480 cells.This effect may be related to Bcl-xL degradation through caspase-9 related mitochondrial-dependent signal pathway.