Study Of The Relationship And Mechanism Of P-gp Expression On The Sensitivity Of Gemcitabine In Patients With Pancreatic Adenocarcinoma

Objective:(1) To investigate whether P-gp levels can predict response to gemcitabine(GEM) in patients with resectable Pancreatic carcinoma.(2) To observe whether TNF-related apoptosis inducing ligand (TRAIL) can modulate the sensitivity of SW1990/GEM to gemcitabine, and explore the related mechanisms.Methods:(1) SP(streptavidin-peroxidase) immunohistochemical method was used to detect the expressions of P-gp in carcinoma specimens from 79 patients with resectable pancreatic ductal adenocarcinoma (PDAC) separately. The levels of P-gp expression were examined for correlations with GEM sensitivity by Kaplan-Meier survival analysis and multivariate Cox model analysis.(2) To identify human pancreatic adenocarcinoma gemcitabine-resistant sub-strains, CCK8 assay is used to examine the growth inhibition rate of cells.(3) Examine how TRAIL and gemcitabine intervention affects the proliferation and apoptosis rate of SW1990/GEM in vitro:SW1990/GEM was treated with combined TRAIL and gemcitabine or TRAIL, then CCK8 assay and flow cytometry were used to examine the growth inhibition rate and apoptosis rate of cells.(4) Examine how TRAIL and gemcitabine intervention affects the apoptosis and chemoresistance related protein expression of SW1990/GEM in vitro:SW1990/GEM was treated with combined or single TRAIL and gemcitabine, then the expression of the active cleaved Caspase-3、P-gp、Bax and Bcl-2, all of which are apoptosis and chemoresistance related proteins, was examined by using Western Blot.Results:(1) The P-gp was mainly in cytoplasm and with some staining on cell membranes. The positive expression rates of P-gp were 69.62%(55/79) in pancreatic carcinoma tissues. The log-rank test detected no significant differences in both overall survival(OS) and disease-free survival (DFS) between GEM-treated patients with positive and negative P-gp levels.(2) Multivariate Cox model analysis showed that P-gp expression were signifiant independent prognostic factors for DFS(P=0.050). On the contrary, P-gp expression did not independently inflence patient’s OS(P=0.142).(3) The IC50 of SW1990/GEM is 178.70±20.32 μmol/L.(4) CCK8 results showed:comparing with separate use, combined use of TRAIL and gemcitabine significantly inhibit the growth rate of SW1990/GEM cell (P<0.05), the two drugs have a synergistic effect.(5) Western Blot showed:TRAIL can inhance the sensitivity of SW1990/GEM to gecitabine via upregulating Cleaved-caspase-3、Bax and downregulating P-gp、Bcl-2.Conclusion:(1) P-gp may be as a key predictive biomarkers of GEM sensitivity in patients with resectable PDAC. These findings begin to identify P-gp as a marker to guide the estimation of the prognosis and chemotherapy choice in PDAC patients.(2) TRAIL can sensitize SW1990/GEM to gemcitabine in vitro;(3) TRAIL may sensitize the SW1990/GEM to gemcitabine by promoting apoptosis. The mechanism might involved in upregulating of Cleaved-caspase-3、Bax and downregulating of P-gp、Bcl-2.