丹参酮ⅡA对人膀胱癌J82细胞增殖与凋亡效应及作用机制

The effect and mechanism of tanshinone ⅡA on proliferation and apoptosis of human bladder cancer J82 cells

目的探讨丹参酮ⅡA对人膀胱癌J82细胞增殖与凋亡效应及作用机制。方法以不同浓度(1、2、3、5、8μmol/L)的丹参酮ⅡA处理J82细胞48 h,先后采用细胞计数试剂盒(CCK-8)检测法检测梯度剂量的丹参酮ⅡA对J82细胞半数抑制率(IC50)的测定及细胞增殖的影响,流式细胞术及Annex V/PI染色检测法用于检测丹参酮ⅡA对J82细胞凋亡及细胞周期的影响,免疫印迹法(Western-blotting)检测与细胞;周亡相关的蛋白cyclin Dl、Bel-xL、caspase3、caspase8、caspase9、P65表达的差异。结果丹参酮ⅡA能够抑制人膀耽癌J82细胞的活性,且具有浓度依赖性(P<0.05),半数抑制浓度(IC50)为4.16μmol/L。1、3μmol/L丹参酮ⅡA处理组的G1期细胞百分比分别为(47.98%±1.49%)、(54.94%±3.39%),均高于对照组(42.22%±0.48%)(均P<0.05),且3μmol/L组高于1 pmol/L组(P<0.05);3μmol/L组的S期细胞百分比(33.19%±4.17%)低于对照组(46.32%±2.14%)(P<0.05)。1、3、5μmol/L组的总凋亡率分别为11.23%、14.84%、18.42%,均高于对照组的3.26%(P<0.05),且呈剂量依赖性(P<0.05)。1、3 pmol/L组的caspase3蛋白相对表达量分别为(0.39±0.02)、(0.52±0.03),均高于对照组的0.26±0.02(P<0.05);caspase9蛋白相对表达量分别为(0.18±0.03)、(0.33±0.12),均高于对照组的0.02±0.02(P<0.05);3μmol/L组两种蛋白的表达量均高于1μmol/L组(P<0.05)。结论丹参酮ⅡA在体外可抑制J82细胞增殖,G1/S期阻滞可能是其中的机制之一,同时可能通过线粒体途径诱导J82细胞凋亡。

Objective To investigate the effect and mechanism of tanshinone ⅡA on proliferation and apoptosis of human bladder carcinoma J82 cells.Methods The J82 cells was treated with different concentrations of tanshinone ⅡA(1、2、3、5、8μmol/L)for 48 hours.Cell counting kit(CCK-8)was used to detect the effect of gradient dose of tanshinone on IC50 of J82 cells and cell proliferation;Flow cytometry and Annex V/PI staining were used to detect the effect of tanshinone ⅡA on apoptosis and cell cycle of J82 cells.Western blotting was adopted to observe the protein expression differences of cyclin Dl Bcl-xL、caspase3、caspase9、P65 related to cell apoptosis.Results The proliferation of J82 cells was inhibited by tanshinone ⅡA in a dose-dependent manner(P<0.05),and the half maximal inhibitory concentmtion(IC50)was 4.16μmol/L.The percentage of G1 phase cells in 3μmol/L group and in 1μmol/L group were(54.94%±3.39%)and(47.98%±1.49%)respectively,both of them were higher than that in control group(42.22%±0.48%)(P<0.05)and the percentage of G1 phase cells in 3μmol/L group was higher than that in 1μmol/L group(P<0.05);the percentage of S phase cells was lower in 3μmol/L group(33.19%±4.17%)than in control group(46.32%±2.14%)(P<0.05).The total apoptosis rates of J82 cells in 1μmol/L group(l 1.23%),3 mol/L group(14.84%)and 5μmol/L group(18.42%)were all higher than that in control group(3.26%)(P<0.05),and the apoptosis rate was dose-dependent(P<0.05).The relative expression levels of caspase 3 protein in 1 and 3μmol/L groups were(0.39+0.02)and(0.52+0.03),respectively,and both of them were higher than the control group 0.26±0.02(P<0.05).The relative expression levels of caspase 9 protein were 0.18±0.03 and(0.33±0.12),respectively,(0.18±0.03),(0.33±0.12),and both of them were higher than the control group 0.02±0.02(P<0.05).The expression levels of both proteins in 3μmol/L group were higher than those in 1μmol/L group(P<0.05).Conclusion Tanshinone ⅡA can significandy inhibit proliferation and induce apoptosis of human bbdder carcinoma J82 cells in vitro,probably via arresting cell cycle in GI/S phase and activating mitochondrial pathway,respectively.