The In-vitro Studies Of TSA Enhanced Killing Effects Of CIK To Pancreatic Cancer Cell Lines

Background：In China, the incidence of pancreatic cancer increased year by year. According tostatistics, the median survival time of the whole R0resection of pancreatic cancer patientswas1.7years, and the5-year survival rate was5%。The median survival of unresectablepancreatic cancer was6months, even Metastatic carcinoma was less than3months. In orderto improve the clinical efficacy and long-term results, academia currently believes the realway is the comprehensive treatment. Biological treatment of immunotherapy is an importantcomponent, it represents a comprehensive treatment of pancreatic cancer. cytokine inducedkiller cell(CIK) effect on tumor cells with high efficiency and non-MHC restricted features, ithas also strong tumoricidal activity and broad spectrum anti-tumor. On the other hand, CIKcell has well auxiliary treatment with surgery, chemotherapy, molecular targeted therapysynergistic. But it is not optimistic that some papers reported to the killing rate is only51%with CIK cells to pancreatic cancer cell line ASPC-1in vitro experiments on100︰1targetefficiency ratio (CIK cell︰tumor cells). Research shows that in the progression ofpancreatic cancer, MICA/B-NKG2D immune surveillance mediated disorders has played animportant role in promoting. Specifically, the reducing of MICA/B expression for pancreaticcancer cells is one of the main mechanisms of immune escape. However, there were reportedthat trichostatin A (TSA) can effectively increase the MICA/B expression of tumor cell. Sincepancreatic cells showed low expression of MICA/B molecules, we try to use TSA to increaseMICA/B expression. So as to enhance the MICA/B-NKG2D mediated immune response, andthe cells killing viability of the CIK cells to pancreatic cancer cell. That will become anothertreatment for pancreatic cancer.Objectives：To study on pancreatic cancer cell lines CIK killing effect; explore TSA mechanisms toenhance its lethal effect. Methods：1．PANC-1, BXPC-3, CFPAC-1pancreatic cancer cell lines were respectively used.2．The pancreatic cancer cell lines were set a without drug group and the trichostatin A(TSA) groups.3．The groups of TSA was effected for24hours, but with different concentrations(0,50nmol/L,100nmol/L,200nmol/L,400nmol/L); and was effected for the sameconcentration of200nmol/L, but with different time (12hours,24hours,36hours,48hours).Using PE-labeled anti-MICA/B labeled pancreatic cancer cell lines.4．Flow cytometry were used to calculate the percentage of positive expression ofMICA/B expression of the cell lines, in order to find the optimal concentration of TSA toincrease pancreatic cancer cell lines MICA/B expression was200nmol/L, the optimal reactiontime was24hours. Follow-up experiments were used the best reaction time, optimalconcentration.5． The expression of membrane type MICA/B was measured in two groups ofpancreatic cancer cell lines by immunofluorescence staining and Western bolt technicalmethods.6．With the10：1proportion of CIK cells and pancreatic cancer cell，the killing rateof CIK cell for two groups was measured by LDH release method.7．Bivariate correlation analysis found the relationship of killing rates of CIK cell andMICA/B expression for two groups.Results：1．The rates of MICA/B expression in PANC-1， BXPC-3， CFPAC-1cell lines weremeasured2.2%、61.2%、31.2%by flow cytometry. However，the MICA/B expressionrates of the other group who was acted by200nmol/L TSA for24hours were measured13.8%，86.6%，76.1%（P <0.01）.2．On the other hand，after TSA treatment，immunofluorescence staining showed thatthe cells were stained significantly increased and western bolt detected the membrane proteinMICA/B expression of pancreatic cancer cell lines was enhanced.3．With the10：1proportion of CIK cells and pancreatic cancer cells，the killing ratesof CIK cell for PANC-1、BXPC-3、CFPAC-1were measured7.66%、12.03%，12.15%， and the rates were24.32%，58.63%，49.25%of the other group who was acted by200nmol/L TSA for24hours（P <0.01）.4．The correlation coefficient were0.959,0.964,0.972with the killing rates of CIK cellsto PANC-1, BXPC-3, CFPAC-1cell lines and surface membrane type MICA/B expression ofthe cell lines. P﹤0.01. Correlation analysis showed it positively correlated with theincreasing of the MICA/B expression of pancreatic cancer cell lines and CIK cell killing rates.Conclusions：TSA can enhance CIK killing function through increased the MICA/B expression ofpancreatic cancer cell lines，so as to improve the effect the cellular immunotherapy ofpancreatic cancer.