伊曲康唑对胰腺癌细胞增殖和凋亡的影响

Effects of itraconazole on proliferation and apoptosis of pancreatic cancer cells

目的探讨B细胞淋巴瘤/白血病-2同源拮抗剂1(bak1)信号通路的活化在伊曲康唑抗胰腺癌中的作用及其机制。方法实验根据检测指标的不同分为对照和伊曲康唑组。用美国Merk公司提供的不同浓度伊曲康唑(0、10、30、50、70、90 mg/L)干预人胰腺癌MIA PaCa-2和CFPAC-1细胞,通过噻唑蓝(MTT)评价细胞增殖,流式细胞仪分析细胞周期,原位缺口末端标记法(TUNEL)免疫荧光检测细胞凋亡。同时,利用bak1小干扰RNA(siRNA)抑制bak1表达,评价伊曲康唑对胰腺癌细胞增殖和凋亡的作用。蛋白质印迹法(Western blot)分析bak1及其下游B细胞淋巴瘤/白血病-2相关X蛋白(bax)和半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3蛋白表达,应用SPSS 19.0统计软件分析,组间比较采用t检验分析,计量资料以均值±标准差(Mean±SD)表示,组间比较采用方差分析。结果10、30、50、70、90 mg/L伊曲康唑MIA PaCa-2的抑制率分别是6.211%、34.741%、63.226%、82.531%和89.112%,对CFPAC-1的抑制率分别是9.726%、47.322%、53.631%、72.629%和92.641%。50μmol/L伊曲康唑干预前后MIA PaCa-2 G0/G1期分别为43.142%和57.341%(t=21.762,P<0.05),差异有统计学意义,CFPAC-1 G0/G1期分别为40.107%和63.216%。50 mg/L伊曲康唑干预MIA PaCa-2细胞凋亡率分别3.406%、10.712%和22.626%(t=40.835,P<0.05),差异有统计学意义,CFPAC-1细胞凋亡率分别5.041%、16.135%和23.701%(t=36.761,P<0.05),差异有统计学意义。伊曲康唑可有效诱导bak1、bax及cleaved-Caspase-3高表达。bak1 siRNA抑制bak1表达后,伊曲康唑对胰腺癌的增殖抑制和诱导凋亡作用明显下降。结论伊曲康唑通过调节bak1信号通路的活化发挥抗胰腺癌作用。

Objective To investigate the role and mechanism of the activation of B cell lymphoma/leukemia-2-antagonist/killer 1(bak1)signaling pathway in anticancer effects of itraconazole on pancreatic cancers.Methods The experiment was divided into control group and itraconazole group according to the different detection indicators.Human pancreatic cancer MIA PaCa-2 and CFPAC-1 cells were treated with different concentrations of itraconazole(0,10,30,50,70,90 mg/L)bought from Merk’s company in USA.Cell proliferation was evaluated by 3-(4,5-dimethylhiazol-2-yl)-3,5-diphenyltetrazolium bromide(MTT)assay.Cell cycle was analyzed by flow cytometry,and cell apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL)immunofluorescence assay.Moreover,bak1 small interfering RNA(siRNA)was used to inhibit the expression of bak1,and then the proliferation and apoptosis of pancreatic cancer cells treated with itraconazole were evaluated.The expression of bak1 and its downstream bax and cysteinyl aspartate-specific protease(Caspase)-3 proteins was measured by Western blotting.SPSS 19.0 statistical software was used to analyze the measurement data.The mean soil standard deviation(Mean±SD)was used to express the measurement data.The analysis of variance was used for the comparison between groups.Results The inhibitory rates of 10,30,50,70 and 90 mg/L itraconazole Mia PaCa-2 were 6.211%,34.741%,63.226%,82.531%and 89.112%respectively,and the inhibitory rates of itraconazole on CFPAC-1 were 9.726%,47.322%,53.631%,72.629%and 92.641%respectively.Before and after 50μmol/L itraconazole intervention,Mia PaCa-2 G0/G1 phase was 43.142%and 57.341%(t=21.762,P<0.05),the difference was statistically significant;CFPAC-1 G0/G1 phase was 40.107%and 63.216%(t=17.186,P<0.05),the difference was statistically significant.50 mg/L itraconazole could effectively induce the expression of bak1,Bax and cleaved caspase-3 in Mia PaCa-2 cells,the difference was statistically significant(3.406%,10.712%and 22.626%,t=40.835,P<0.05),the difference was statistically significant(5.041%,16.135%and 23.701%,t=36.761,P<0.05).After the inhibition of bak1 expression by bak1 siRNA,the inhibition of proliferation and apoptosis induced by itraconazole decreased significantly.Conclusion Itraconazole exerts anti pancreatic cancer effect by regulating the activation of bak1 signaling pathway.