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

Abstract

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 h cell culture in each group. The small hairpin RNA (shRNA) was used for PKCι knockdown and the recombinant plasmid transfection was for PKCι overexpression in pancreatic cancer cells. Western blot and real-time fluorescent quantitative PCR (qRT-PCR) were utilized to determine the expression of PKCι protein 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 h later, so as to explore the possible mechanisms of the impact of PKCι on 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 PKCι knockdown in pancreatic cancer cells can down-regulate the gene expression of TGF-β. In return, PKCι overexpression in pancreatic cancer cells can increase the gene expression of TGF-β. The death rate of cancer cells with 10, 20 ng/mL TGF-β was lower compared with the control group (P < 0.05).  Conclusions  PKCι knockdown 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 PKCι is to affect the immune escape of tumor cells by regulating the expression of TGF-β.

Keywords: CIK cells, Pancreatic cancer, PKCι, TGF-β