自噬在吉西他滨诱导胰腺癌细胞凋亡中的作用

目的:研究自噬在吉西他滨(gemcitabine,Gem)诱导胰腺癌细胞SW1990凋亡中的作用,并以氯喹特异性抑制自噬,以探讨其可能机制。方法:采用CCK8法检测Gem对胰腺癌细胞增殖的影响,并用实时定量PCR检测自噬相关基因LC3的表达,以p62免疫荧光染色检测细胞内自噬泡,通过Western印迹法检测自噬相关蛋白LC3、Beclin 1的表达,并用AnnexinⅤ/PI流式细胞方法检测Gem诱导后细胞凋亡的变化。进一步通过氯喹抑制自噬,检测自噬抑制前后细胞增殖、自噬及凋亡的变化。结果:Gem对胰腺癌细胞SW1990增殖具有部分抑制作用,Gem作用能迅速激活细胞自噬从而产生耐药。LC3的mRNA表达升高1.4~2.2倍(P0.05),LC3-Ⅱ蛋白水平升高1.5~2.7倍(P0.05),Beclin 1蛋白含量升高1.2~1.3倍(P0.05)。Gem和氯喹联合用药组较Gem单药组细胞凋亡蛋白Caspase-3表达升高,细胞存活率从64.3%±3.1%降低为35.2%±3.4%(P0.05)。结论:自噬在Gem诱导胰腺癌细胞凋亡的过程中可能起到保护作用,氯喹抑制自噬后可增强Gem的促凋亡作用。

Role of autophagy in gemcltabine-induced apoptosis of pancreatic cancer cell

Objective To study the role of autophagy in gemcitabine-induced apoptosis of pancreatic cancer cell SW1990 and the mechanism. Methods The cell proliferation of pancreatic cancer cell treated by gemcitabine was measured with CCK-8 assay at the setting time points. The mRNA expression of autophagy associated gene LC3 was analyzed by real-time PCR. The present of autophagie vacuoles was evaluated by immunofluorescenee using protein p62. Western blot was used to examine the expression of autophagy related proteins Beclin 1 and LC3. The gemcitabine- induced apoptosis of cell was detected by Annexin V/PI flow cytometry. The gemcitabine induced autophagy and apoptosis and the proliferation of cell were also analyzed with the inhibition of autophagy by chloroquine. Results Gemcitabine had partial inhibitory effect on the proliferation of pancreatic cancer cell SW1990 and induced autophagy of the cells which generated resistance of drugs. There was the elevated expression of LC3 mRNA （1.4-2.2, P〈0.05）, LC3- Ⅱ protein （1.5-2.7, P〈0.05） and Beclin 1 protein （1.2-1.3, P〈0.05）. The expression of apoptosis associated protein Caspase-3 elevated in combined group （gemcitabine and chloroquine） compared with that in gemcitabine group, with the inhibited cell proliferation from 64.3%±3.1% to 35.2%±3.4% （P〈0.05）. Conclusions Autophagy may play a protective role in gemcitabine-induced apoptosis in pancreatic cancer cells and the inhibition of autophagy by chloroquinecould enhance gemcitabine-induced cell apoptosis.