The Role Of Ezrin In The Invasiveness And Metastasis Of Pancreatic Adenocarcinoma And Effect Of Baicalein On Its Expression

Objective: To access the importance of membrane cytoskeletal crosslinker ezrin for the progression of pancreatic adenocarcinoma by detecting its expression in tumor tissues, and further elucidate the anti-tumor mechanism of baicalein by investigating the effect of baicalein on ezrin expression and its relation to PANC-1 cells proliferation, invasiveness and migration.Methods: Expression of ezrin in 46 pancreatic adenocarcinoma tissues and 13 normal tissues of pancreas from Huaihe Hospital, Henan University and Xiangya Hopital on the period from February 1997 to September 2005 was examined by immunohistochemical staining. The relationship of ezrin expression to clinicopathological characteristics and prognosis of patients after operation was analyzed. Four pairs of small interfering RNA ( siRNA) targeting Ezrin was transfected into the pancreatic adenocarcinoma cell line PANC-1. Western blotting was used to detect the transfection rates so as to screen the most effective siRNA which had been transfected into the PANC-1 cells. The biological behaviors of PANC-1 cells including cell proliferation, cell cycle and apoptosis , pseudopodia formation, cell invasion and migration ability were detected by MTT method , flow cytometry, scanning electron microscope, wound healing assay and transwell assay respectively when the expression level of ezrin was the lowest after successful transfection. Expressions of ezrin and phosphate-ezrin protein were detected by Western blotting and ezrin mRNA was detected by reverse transcriptase-polymerase chain reaction (RT- PCR) in PANC-1 cells treated by baicalein.Results: The positive rate of ezrin immunostaining in pancreatic adenocarcinoma tissues (89.1%) was significantly higher than in normal tissues of pancreas (53.8%) (P=0.009, Fisher's exact test). In pancreatic adenocarcinomas, the strong positive rate of ezrin immunostaining was 45.7%(21/46). The strong positive rate of ezrin immunostaining increased significantly with the worsening of histological grade (x~2 =7.725 , P=0.021).The strong positive rate of ezrin expression in lymph-node metastatic group (63.2%) was significantly higher than in without (33.3%) (x~2=3.989, P=0.046) . No significant correlation was found between the strong positive rate of ezrin expression and the age and gender of patients, the size and pathological staging of tumor. The median post-operative survival time was 16 months (range from 9 to 36 months) in the group with strong positive ezrin expression, and 24 months (range from 8 to 50 months) in the group with weak positive and negative ezrin expression(Log-rank test, x~2=4.860, P=0,0276).The second pair small interfering RNA (siRNA) targeting Ezrin was transfected successfully into the PANC-1 cells. Western blotting results revealed that Ezrin siRNA notably down-regulated the expression level of Ezrin protein. Down-regulation of Ezrin expression distinctly decreased the proliferation rates of the PANC-1 cells. After RNAi treatment, the cell proportion in S and G2/M phase decreased significanttly( P < 0. 05) and the cell proportion in G0/G1 phase increased slightly in PANC-1 cells. There was no significant difference in apoptosis rate between the two groups. The migration and invasion ability of PANC-1 cells significantly decreased after siRNA interference (P < 0. 01).The number of pseudopodia in PANC-1 cells significantly decreased after siRNA interference (P < 0. 01). Baicalein suppressed significantly pancreatic adenocarcinoma cell growth by inhibiting its proliferation in a time- and dose-dependent manner. Reverse transcriptase-polymerase chain reaction (RT- PCR) and Western blotting results revealed that baicalein notably down-regulated Ezrin expression at both mRNA and protein levels, and inhabited its phosphorylation as well as.Conclusion:1. The level of ezrin expression is closely correlated to invasion, metastasis and prognosis of pancreatic adenocarcinoma, which indicates that ezrin may be a valuable molecular marker of pancreatic adenocarcinoma.2. Pancreatic adenocarcinoma cells proliferation, migration, and invasiveness could be significantly inhibited with down-regulation of ezrin expression by siRNA, which indicates that ezrin may be a novel and effective therapeutic target for preventing pancreatic adenocarcinoma invasion and metastasis.3. Baicalein may suppress pancreatic adenocarcinoma cell growth by inhibiting its proliferation in a time- and dose-dependent manner, and down-regulate the expression level of ezrin mRNA and ezrin protein as well as its phosphorylation, which is related with the effects of baicalein on pancreatic adenocarcinoma cells. These results suggest that baicalein is likely to be valuable for the treatment of human pancreatic adenocarcinoma.