Study On The Mechanism Of GSTM2 In Chemoresistance Of Pancreatic Cancer

Glutathione-S-transferases(GSTs)show not only cytoprotective role but also involvement in the development of anticancer drug resistance.However,the role of GST isoforms in chemotherapy resistance remains unclear in pancreatic cancer.In current study,expression of GSTM2 in pancreatic cell lines treated with gemcitabine and pancreatic cancer patients with neoadjuvant therapy was assessed by western blot,quantitative RT-PCR and IHC,respectively.SiRNA and shRNA were used to silence GSTM2 gene in pancreatic cell lines and orthotopic pancreatic tumor mice models,respectively.Correlation of clinical outcome with GSTM2 expression was analyzed by Kaplan-Meier overall survival curve.Here,we demonstrated that gemcitabine treatment increased the GSTM2 expression in pancreatic cancer cell lines.Silence of GSTM2 by siRNA elevated apoptosis and decreased viability of pancreatic cancer cells treated with gemcitabine.Moreover,In vivo experiments further showed that shRNA induced GSTM2 silence enhanced drug sensitivity of gemcitabine in orthotopic pancreatic tumor mice.We also found that in tumor tissues GSTM2 expression was lower than that in normal tissues and higher GSTM2 expression was significantly associated with better prognosis.In conclusion,our findings indicate that GSTM2 expression is essential for the survival of pancreatic cancer cells undergoing gemcitabine treatment and leads to chemo resistance,so gemcitabine treatment can increase the GSTM2 expression in pancreatic cancer cell lines as well.Silence of GSTM2 in pancreatic cancer may benefit gemcitabine treatment.GSTM2 expression in patients also shows significant correlation with overall survival.Thus,our study suggests that GSTM2 is a potential target for chemotherapy optimization and prognostic biomarker of pancreatic cancer.1.GSTM2 expression in pancreatic cancer cell lines with gemcitabine treatment and pancreatic cancer patients receiving neoadjuvant therapyObjective: To analysis of the effect of gemcitabine on the expression of GSTM2 in pancreatic cancer cells and tissues.Materials and methods: The expression level of GSTM2 in pancreatic cancer cell lines before and after treating by gemcitabine was tested by qRT-PCR and western blot.The tissue specimens of pancreatic cancer patients undergoing surgery after neoadjuvant chemotherapy with gemcitabine and only surgical treatment were collected.The different expression level of GSTM2 in each tissue specimens was determined by Immunohistochemical staining.Results: The expression level of GSTM2 in the pancreatic cancer cell line treated by gemcitabine was significantly higher than that of the control group without gemcitabine;the expression of GSTM2 in the tissues of pancreatic cancer patients receiving gemcitabine chemotherapy was significantly higher than that of only surgical patients.Conclusions: Gemcitabine could elevate the expression level of GSTM2 in pancreatic cancer cell line and tissues.It is preliminarily confirmed that GSTM2 is involved in the chemotherapeutic drug resistance of gemcitabine in pancreatic cancer.2.The effects of GSTM2 silence on the chemosensitivity of pancreatic cancer in vitroObjective: To determine effect of siGSTM2 on the chemosensitivity of pancreatic cancer in vitro.Materials and methods: The siRNA was used to silence the expression of GSTM2 in four pancreatic cancer cell lines BXPC-3,Panc-1,L3.6pl and MPanc96.The qRT-PCR and western blot were used to test the expression level of GSTM2.After treating with Gemcitabine on above cell lines,the Flow cytometry assay was used to determine the cell apoptosis and the MTT analysis was used to determine the cell viability.Results: The results of qRT-PCR and western blot indicated that siRNA could interfere the expression of GSTM2 successful in above four pancreatic cancer cell lines.The silent efficiency of GSTM2 was relatively high,and more than 60%.After treating with Gemcitabine on above cell lines,the percentage of apoptosis of pancreatic cancer cells transfected to siGSTM2 was significantly higher than it in siControl group.And,the cell activity of pancreatic cancer cells transfected to siGSTM2 was significantly decreased,compared to it in siControl group.Conclusions: The siGSTM2 could effectively silence the expression of GSTM2 in the pancreatic cancer cell lines,which could enhance the sensitivity of pancreatic cancer cells to gemcitabine.3.The effects of GSTM2 silence on the chemosensitivity of pancreatic cancer in vivoObjective: To determine effect of shGSTM2 on the chemosensitivity of Orthotopic pancreas xenografts in nude mice.Materials and methods: The shGSTM2 was transfected to pancreatic cancer cell line Mpanc96.The effect of shGSTM2 was tested by qRT-PCR and western blot.The Mpanc96 cell of successful transfection of shRNA was injected into the pancreas of nude mice.The effect of tumorigenicity on nude mice was evaluated by bioluminescence of IVIS imaging system.After treating with gemcitabine,the tumor volume was determined by Living Image tool.Results: The results revealed that shRNA could interfere the expression of GSTM2 successful in pancreatic cancer cell line Mpanc96.The silent efficiency of GSTM2 was relatively high.Biofluorescence imaging showed that there were fluorescent signals in the position of the pancreas in nude mice,and the transplanted tumor was tumorigenic and the modeling was successful.The volumes of tumor in nude mice were the smallest in shGSTM2 combined with gemcitabine treatment group.Conclusions: To silence the expression of GSTM2 could enhance the sensitivity of gemcitabine chemotherapy in pancreatic cancer in vivo.4.The study of the correlation between the expression of GSTM2 in the patients with pancreatic cancer and the clinical prognosisObjective: The study of the correlation between the expression of GSTM2 in the patients with pancreatic cancer and the clinical prognosis.Materials and methods: Specimens of 50 patients with pancreatic cancer in our hospital were collected.The paracancerous tissue and health human pancreatic tissue samples were as control.The difference in expression level of GSTM2 mRNA was detected.According to the average protein expression of GSTM2,pancreatic cancer samples were divided into GSTM2 low expression group and GSTM2 high expression group,and the difference between the two groups of survival periods was analyzed.Results: The expression of GSTM2 in pancreatic tissue samples of pancreatic patients was significantly lower than that of the paracancerous tissue and normal human pancreatic tissue samples.The overall survival rate of the patients in the low expression group of GSTM2 was low and the prognosis was poor.Conclusions: The expression of GSTM2 in normal people was higher than that in pancreatic cancer patients,and the high expression of GSTM2 was significantly related to the high survival rate and good prognosis of the patients with pancreatic cancer.Summing up the above research,we can come to the conclusions as follows:1.Gemcitabine could elevate the expression of GSTM2 in pancreatic cancer.2.Silence of GSTM2 can promote the apoptosis and decrease the viability of the pancreatic cancer cells treated by gemcitabine.3.Silence of GSTM2 can reduce the volume of the tumor in situ of the pancreas in nude mice receiving gemcitabine chemotherapy and enhance the sensitivity of Gemcitabine chemotherapy in vivo.4.The expression of GSTM2 in normal people was higher than that of pancreatic cancer,and the high expression of GSTM2 was significantly related to the high survival rate and good prognosis of the pancreatic cancer patients.5.GSTM2 was a potential biomarker to screen for accurate treatment of highly sensitive pancreatic cancer patients in gemcitabine and to predict the prognosis of pancreatic cancer.