Roles Of MICA In Anti-pancreatic Cancer Mediated By NK Cells And Effects Of Gemcitabine On MICA Protein Ectodomain Shedding In Pancreatic Cancer Cells

Objective:To investigate the clinical significance of MICA and NKG2D receptor on NK cells in pancreatic cancer.Methods:MICA mRNA in 10 pancreatic cancer tissues,6 chronic pancreatitis tissues and 6 normal tissues of pancreas was determined by RT-PCR analysis.Expression of MICA in 64 pancreatic cancer tissues, 13 chronic pancreatitis samples and 11 normal tissues of pancreas was examined by immunohistochemistry. The serum levels of sMICA and NKG2D expression on NK cells of 53 pancreatic cancer patients,7 patients with chronic pancreatitis and 20 healthy volunteers was assessed by ELISA and Flow cytometry analysis.Results:There was a significant increase in MICA mRNA levels in the pancreatic cancer samples compared with the chronic pancreatitis and normal tissues of pancreas(P<0.01).The positive rate of MICA immunostaining in pancreatic cancer tissues was 89.1%,whereas fewer was expressed in inflammatory and normal pancreatic tissues(P<0.01). Significant difference was noted between the MICA expression with respect to the histological grade(P=0.042),distant metastasis(P=0.028) and TNM stages (P=0.003).MICA expression was found to be a prognostic factor in resected pancreatic cancer (P=0.019).The serum levels of sMICA were frequently elevated in patients with pancreatic cancer (P<0.01).The level of sMICA was significantly higher inⅢ/Ⅳstage pancreatic cancer patients than that in I/II stage patients (P<0.01).Notably, our data also showed that sMICA levels correlated significantly with the presence of metastasis(P<0.01).NKG2D expression on NK cells from sMICA-positive pancreatic cancer patients was markedly reduced compared to that from sMICA-negative pancreatic cancer patients, chronic pancreatitis patients or healthy volunteers (P<0.01).Significant inverse correlation between NKG2D expression and levels of sMICA was observed in pancreatic cancer patients (r=-0.499,P<0.01).The successful radical resection of tumor significantly decreased the serum levels of sMICA and increased the NKG2D expression on NK cells.Conclusions:MICA is induced and expressed widely in the early stage of pancreatic cancer.With the progression of pancreatic cancer, MICA can be released into the bloodstream or tissue culture medium as sMICA.The elevation of sMICA may be associated with down-regulated NKG2D expression on NK cells.The successful radical resection of tumor significantly decreased the serum levels of sMICA and increased the NKG2D expression on NK cells. Objective:To investigate the role of MICA on anti-human pancreatic cancer cell line PANC-1 mediated by NK cells.Methods:NK cells were purified by negative depletion using NK cell isolation kit by Vario MACS system.Studyed the reaction between MICA and NKG2D by purified-antibody blockade.Cultured NK cells from healthy donors with sMICA-positive serum for 24 h and assessed the NKG2D expression and Cytotoxicity of NK cells to PANC-1 cells.Results:1.Cytotoxicity of NK cells to PANC-1 cells was decreased because of NKG2D or MICA antibody blockade(P<0.01)2.The expression of NKG2D was decreased after NK cells cultured with sMICA-positive serum.NK cells pre-treated with sMICA-positive serum were not capable of efficiently killing PANC-1 cells.Conclusions:MICA-NKG2D play a critical role in cytotoxicity of NK cells to PANC-1 cells.sMICA reduce the expression of NKG2D,and impaire NK cell-mediated imnune surveillance in pancreatic cancer.Part 3 ADAM10 is involved in MICA shedding and effects of gemcitabine on MICA protein ectodomain shedding in PANC-1 cellsObjective:To investigate the role of ADAM 10 on MICA protein ectodomain shedding and observe whether gemcitabine affect MICA protein shedding from PANC-1 cells.Methods:1.Expression of ADAM 10 in 64 pancreatic cancer tissues and 11 normal pancreas tissues was examined by immunohistochemistry.2. PANC-1 cells were transfected with ADAM10 siRNA or control siRNA for 48 h. The expression of membrane-bound MICA was evaluated by flow cytometry,and sMICA production in the culture supernatant was evaluated by ELISA.3.The cytotoxicity of gemcitabine to PANC-1 cells was evaluated by MTT.4.PANC-1 cells were treated with a nontoxic dose of 5ng/ml gemcitabine for 24 h,and their expression of ADAM 10 were evaluated by RT-PCR and Western blot.The expression of membrane-bound MICA and MICA mRNA were evaluated by flow cytometry and RT-PCR, respectively. At the same time,24h culture supernatants were subjected to the analysis of sMICA levels by ELISA.5.PANC-1 cells were transfected with ADAM10 siRNA or control siRNA for 24 h and further cultured with 5ng/ml gemcitabine (equal culture medium for control)for 24 h.The expression of membrane-bound MICA was evaluated by flow cytometry,and sMICA production in the culture supernatants was evaluated by ELISA。 Results:1.The positive rate of ADAM 10 immunostaining in pancreatic cancer tissues(87.5%)was significant higher than in normal tissues of pancreas(P<0.01)2.Knockdown of ADAM10 for PANC-1 cells clearly upregulated MICA expression on their cellular surface and downregulated sMICA levels in their culture supernatants(P<0.01)3.When the concentration of gemcitabine was lower than 7ng/ml, gemcitabine maked little effect on the growth of PANC-1 cells in 24h.4.Gemcitabine suppresses ADAM10 expression in PANC-1 cells (P<0.01)5.Gemcitabine(5ng/ml)treatment led to an increase in membrane-bound MICA expression and a decrease in sMICA production in PANC-1 cells(P<0.01).The mRNA levels of MICA did not change after PANC-1 cells exposure to gemcitabine(P>0.05)6.PANC-1 cells were transfected with ADAM10 siRNA and then treated with gemcitabine.Neither upregulation of surface MICA nor downregulation of soluble MICA levels was observed.Conclusions:ADAM 10 is involved in MICA shedding of PANC-1 cells.Gemcitabine inhibits MICA ectodomain shedding through suppression of ADAM10.