PKCι对CIK杀伤胰腺癌细胞的影响及作用机制探讨

The Effects of PKCι on Anti-tumor Activity of Cytokine-induced Killer Cells Against Pancreatic Cancer Cells and the Possible Underlying Mechanisms

目的研究非典型蛋白激酶C异构体ι(protein kinase C,PKCι)对细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)作用于胰腺癌细胞的杀伤作用的影响,并探讨其作用机制。方法用白介素-2(IL-2)、干扰素(IFN)、分化抗原簇三分子单克隆抗体(CD3 mAb)等细胞因子诱导健康人外周血中单个核细胞,制备成CIK细胞。将胰腺癌细胞MiaPaCa、PANC-1和胰腺上皮细胞HPDE6-C7分为对照组、化学抑制剂硫代苹果酸钠(ATM组)、与CIK共培养组、ATM+CIK共培养组。采用细胞计数检测各组细胞1~8 d的生长情况;各组细胞培养48 h后采用流式仪检测细胞死亡率;使用针对人PKCι的小发夹RNA(small hairpin RNA,shRNA)敲低胰腺癌细胞中PKCι的表达,胰腺细胞转染过表达PKCι的重组质粒,分别采用免疫印迹检测PKCι蛋白表达和实时荧光定量PCR(qRT-PCR)检测PKCι对下游信号分子中转化生长因子-β(transforming growth factor-β,TGF-β)基因表达的影响;并在MiaPaCa和PANC-1与CIK共培养中加入不同质量浓度TGF-β(1、10、20 ng/mL),48 h后流式仪检测细胞死亡情况,以探讨PKCι影响CIK细胞杀伤活性的相关机制。结果 ATM和CIK可抑制胰腺癌细胞MiaPaCa、PANC-1的生长,诱导癌细胞凋亡和死亡,ATM可增强CIK对癌细胞的杀伤作用。敲低胰腺癌细胞中PKCι的表达,可下调其下游信号分子TGF-β基因的表达,而胰腺细胞过表达PKCι,可上调TGF-β mRNA表达,并且在胰腺癌细胞中加入TGF-β10、20 ng/mL,癌细胞的死亡率较对照组下降(P<0.05)。结论降低胰腺癌细胞中PKCι的表达可以抑制胰腺癌细胞的生长,增强CIK对癌细胞的杀伤作用。PKCι的作用机制可能是通过调控TGF-β水平影响肿瘤细胞的免疫逃逸。

Objective To study the impact of atypical protein kinase Cι (PKCι)isoform PKC on the pancreatic cancer cells towards the tumoricidal effect of cytokine-induced killer(CIK)cells and explore its mechanisms.Methods CIK cells were prepared by inducing mononuclear cells isolated from the peripheral blood of healthy people with interleukin-2(IL-2),interferon(IFN)and CD3 mAb and subsequently co-cultured with pancreatic epithelial cell HPDE6-C7,pancreatic cancer cells MiaPaCa and PANC-1 with or without PKC inhibitor named sodium thiomalate(ATM).All cells were divided into control group,ATM group,co-culture group with CIK and co-culture group with CIK+ATM.Cell count was used to detect the growth of each group from 1 to 8 d.Flow cytometry was used to detect the death rate of the cell lines after 48 hcell culture in each group.The small hairpin RNA(shRNA)was used for PKCιknockdown and the recombinant plasmid transfection was for PKCιoverexpression in pancreatic cancer cells.Western blot and real-time fluorescent quantitative PCR(qRT-PCR)were utilized to determine the expression of PKCιprotein and the impact on gene expression of transforming growth factor-β(TGF-β),a downstream effector modulated by PKC.Different mass concentrations of TGF-β(1,10,20 ng/mL)were added into the co-culture of MiaPaCa and PANC-1 with CIK.The cell death rate was detected by flow cytometry 48 hlater,so as to explore the possible mechanisms of the impact of PKCιon the tumoricidal effects of CIK cells.Results ATM and CIK were shown to suppress the growth and induce apoptosis or death of pancreatic cancer cells,meanwhile,ATM can enhance the tumoricidal effect of CIK on pancreatic cancer cells.Moreover,we found that PKCιknockdown in pancreatic cancer cells can down-regulate the gene expression of TGF-β.In return,PKCιoverexpression in pancreatic cancer cells can increase the gene expression of TGF-β.The death rate of cancer cells with 10,20 ng/mL TGF-βwas lower compared with the control group(P<0.05).Conclusions PKCιknockdown in pancreatic cancer cells can not only inhibit the growth of pancreatic cancer cells,but also enhance the tumoricidal effects of CIK on cancer cells.The possible mechanism of PKCιis to affect the immune escape of tumor cells by regulating the expression of TGF-β.