百里醌联合吉西他滨对胰腺癌BxPC-3细胞体外生长的影响

Thymoquinone Potentiates Antitumor Activity of Gemcitabine in Pancreatic Cancer BxPC-3 Cells in vitro

目的探讨百里醌联合吉西他滨对胰腺癌BxPC-3细胞增殖和凋亡的影响,并探讨其作用机制。方法采用CCK-8法检测细胞相对活性,Hoechst染色法及流式细胞术检测细胞凋亡情况,Western blot法检测凋亡相关蛋白(Bcl-2、Bax、XIAP)、半胱天冬酶(cleaved-caspase-3、cleaved-caspase-9)、PTEN、Akt及phospho-Akt蛋白的表达。结果百里醌呈浓度依赖性地抑制胰腺癌BxPC-3细胞增殖,诱导细胞凋亡。吉西他滨组(GEM)、百里醌组(TQ)、百里醌联合吉西他滨组(TQ+GEM)、百里醌与吉西他滨序贯给药组(TQ-GEM)相对细胞活性分别为83.7%±4.1%、51.8%±6.0%、48.25%±6.50%、33.3%±3.9%;早期凋亡率分别为12.2%±3.8%、30.4%±4.3%、43.5%±5.7%、58.3%±6.1%;各组相对细胞活性均明显低于对照组(P<0.05),而细胞凋亡率显著增高(P<0.05);与GEM组比较,TQ-GEM组与TQ+GEM组相对细胞活性明显下调(P<0.001),凋亡率明显上调(P<0.001);TQ-GEM组较TQ+GEM组出现更明显的增殖受抑(P<0.001),更高的细胞凋亡率(P<0.001)。百里醌-吉西他滨序贯给药作用于胰腺癌BxPC-3细胞后,BxPC-3细胞中Bax蛋白表达明显下调,而Bcl-2、XIAP、cleaved caspase-3、cleaved caspase-9蛋白表达明显上调。百里醌可显著上调BxPC-3细胞PTEN的表达,明显抑制Akt磷酸化。结论百里醌可明显增强吉西他滨对体外胰腺癌细胞生长抑制作用,可能是通过上调PTEN,Akt去磷酸化,促使Bcl-2、XIAP表达上调及Bax表达下调,活化caspase-3、caspase-9诱导细胞凋亡而实现。

Objective To investigate the effect of thymoquinone on the sensitization of pancreatic cancer cells to gemcitabine and explore its mechanism.Methods Human pancreatic cancer PANC-1 cells were cultured in vitro and treated with thymoquinone,gemcitabine,thymoquinone combined with gemcitabine,and sequential administration of thymoquinone and gemcitabine.The relative cell viability,cell apoptosis,expression of Bcl-2,Bax,XIAP,cleaved caspase-3,-9,PTEN and the phosphorylated Akt of BxPC-3 cells were determined respectively by Cell Counting Kit 8 （CCK-8） assay,flow cytometry,and western blotting assay.Results Thymoquinone dose-dependently inhibited the proliferation and increased the apoptosis of pancreatic cancer BxPC-3 cells.The relative cell viabilities of gemcitabine treated group （GEM）,thymoquinone treated group （TQ）,the group with combination treatment of the both （TQ ＋ GEM）,and group of sequential administration of the two drugs （TQ-GEM） were 83.7％ ± 4.1％,51.8％ ± 6.0％,48.25％ ± 6.50％,33.3％ ± 3.9％ respectively and the early apoptosis of which were 12.2％ ± 3.8％,30.4％ ± 4.3％,43.5％ ± 5.7％,58.3％ ± 6.1％ respectively.The relative cell viabilities in every treated groups were significantly lower than the control untreated group （P ＜ 0.05）,while the early apoptosis increase noticeably （P ＜ 0.05）.Compared with GEM group,the proliferation was suppressed and the apoptosis was aggrandized significantly in TQ ＋ GEM and TQ-GEM groups （P ＜ 0.001）.The cells of TQ-GEM group havd higher apoptosis rate and lower proliferation index than TQ ＋ GEM group （P ＜ 0.001）.Thymoquine up-regulated expressions of Bcl-2,XIAP,cleaved caspase-3,cleaved caspase-9 and suppressed Bax.Thymoquinone noticeably up-regulated PTEN expression and inactivated the phosphorylation of Akt in BxPC-3 cells.Conclusion Thymoquinone enhances the chemosensitivity of BxPC-3 cell to gemcitabine with more apoptosis through up-regulation of PTEN expression and inactivation of Akt.