隐丹参酮对人膀胱癌J82细胞增殖、凋亡的影响及其机制

Effects of cryptotanshinone on proliferation and apoptosis of human bladder carcinoma J82 cell line and its mechanisms

目的探讨隐丹参酮在体外对人膀胱癌J82细胞增殖、凋亡的影响及其机制。方法以浓度为0、0.5、1.0、2.0、3.0、4.0、5.0、8.0μmol/L的隐丹参酮处理J82细胞48 h后,采用CCK-8法检测其对J82细胞增殖的影响。Annexin V-FITC/PI双标记流式细胞术检测J82细胞凋亡情况。免疫印迹法(Western blotting)检测细胞增殖及凋亡相关蛋白p65、caspase 3、caspase 8、caspase 9表达的差异。结果CCK-8法结果显示各组的450 nm吸光度(A)值:对照组(1.77±0.06),0.5μmol/L组(1.78±0.08),1.0μmol/L组(1.64±0.05),2.0μmol/L组(1.48±0.12),3.0μmol/L组(1.20±0.07),4.0μmol/L组(0.93±0.10),5.0μmol/L组(0.76±0.02),8.0μmol/L组(0.05±0.01),各组的450 nm A值差异有统计学意义(F=329.83,P=0.00),除0.5μmol/L组外其余各两组间的差异均有统计学意义(均P<0.05)。流式细胞术检测结果显示三组的早期凋亡率和总凋亡率在总体上差异均有统计学意义(F=32.49,P=0.00;F=6.39,P=0.03);两个实验组的早期凋亡率均高于对照组(均P<0.05),且3.0μmol/L组比1.0μmol/L组更高[(11.83±1.12)%比(7.01±1.84)%,t=3.73,P<0.05]。Western blotting结果显示隐丹参酮处理后p65蛋白表达减少,caspase 3、caspase 9蛋白表达增加。结论隐丹参酮在体外可能通过抑制NF-κB信号转导通路来抑制人膀胱癌J82细胞增殖,并通过线粒体途径诱导其凋亡。

Objective To study the effects of cryptotanshinone on proliferation and apoptosis of human bladder carcinoma J82 cells in vitro and its mechanisms.Methods The J82 cells were treated with cryptotanshinone of different concentrations including 0,0.5,1.0,2.0,3.0,4.0,5.0 and 8.0μmol/L.After 48-hour treatment,the proliferation of J82 cells was determined by CCK-8 assay method.Flow cytometric analysis with Annexin V-FITC/PI staining was used to evaluate the apoptosis of J82 cells,and Western blotting was adopted to observe the protein expressions of p65,caspase-3,caspase-8 and caspase-9 related to proliferation and apoptosis.Results CCK-8 results showed that the A values of 450nm-wavelength were as following:the control group(1.77±0.06),0.5μmol/L group(1.78±0.08),1.0μmol/L group(1.64±0.05),2.0μmol/L group(1.48±0.12),3.0μmol/L group(1.20±0.07),4.0μmol/L group(0.93±0.10),5.0μmol/L group(0.76±0.02),8.0μmol/L group(0.05±0.01),and the A values were significantly different among the three groups(F=329.83,P=0.00),there were statistically significant differences between any two groups except the 0.5μmol/L group(all P<0.05).The early and total apoptosis rates were both significantly different among the three groups(F=32.49,P=0.00;F=6.39;P=0.03),the early apoptosis rates of 3.0,1.0μmol/L group were higher than that of the control group(all P<0.05),and the early apoptosis rate in the 3.0μmol/L group was higher than that in the 1.0μmol/L group[(11.83±1.12)%vs.(7.01±1.84)%,t=3.73,P<0.05].The expression of p65 protein decreased,while both the expressions of caspase-3 and caspase-9 proteins increased after treatment with cryptotanshinone.Conclusion Cryptotanshinone can significantly inhibit proliferation and induce apoptosis of human bladder carcinoma J82 cells in vitro,probably via suppressing NF-κB signal pathway and activating mitochondrial pathway,respectively.