.wnt / ¦¢-catenin Signaling Pathway And Pancreatic Cancer With Gemcitabine Resistance Preliminary Study Of The Relationship

BACKGROUNDPancreatic cancer is the fourth most common malignancy and the fourth leading cause of cancer-related death in the United States. Surgical resection offers the only chance of real cure; however, less than 20% of patients with pancreatic cancer are candidates for surgery because the disease is usually detected only in its later stages. Single-agent Gemcitabine is the standard therapy for advanced pancreatic cancer with low response rate of 17.3% and short progression-free survival of 3.7 month. Cellular resistance to gemcitabine can be intrinsic or acquired during gemcitabine treatment, which is the most possible reason for failure in chemotherapy. Gemcitabine has a complex pathway of metabolism, and there are many mechanisms that can contribute to gemcitabine cytotoxicity and/or chemoresistance. Wnt signaling pathway has been reported to be involved in cancer development and chemoresistance in leukemia and hepatocellular carcinoma. To date, there's few study on the relationship between Wnt signaling and gemcitabine resistance. In Partâ… andâ…¡of this preliminary study, the relevance between the canonical Wnt signaling pathway and gemcitabine resistance was studied. In partâ…¢, by FCM analysis we determined the percentage of CD133+and CD44+CD24+EpCAM+cells in pancreatic cell lines either with or without treatment.CHEMICALSGemcitabine was a commercial prodcut from Eli Lilly Pharmaceuticals. Recombinant Wnt3a and Dkk-1 were both from R & D.STATISTICAL ANALYSESData from reporter and MTT assays was analyzed using one-way ANOVA followed by Newman-Keul's multiple comparison test. P-values less than or equal to 0.05 were considered significant.METHODS AND RESULTSIn Partâ… of this study, four pancreatic cancer cell lines, SW1990, PANC-1, T3M4 and BxPC-3, were used for assessing the resistance against gemcitabine and the Wnt activity. The relative cytotoxicity of gemcitabine in each cell line was assessed with a MTT assay, in which cells were exposed to increasing concentrations of gemcitabine. Chemosensitivity was expressed as the drug concentration that inhibited cell proliferation by 50%(IC50 values). Reporter assays were used for Wnt activity, in which TOPflash or FOPflash, constructed in Dr. Moon R.T.'s Laboratory, was co-transfected into cells with renilla luciferase as internal control. Western blotting was performed for determining active P-catenin in cells. The results showed that SW1990 had the highest IC50 and Wnt activity, followed by PANC-1, T3M4 and BxPC-3, accordingly. Non-linear positive correlation was determined by Pearson's test between the canonical Wnt signaling pathway and gemcitabine resistance.Next in Part II, we chose SW1990 for alternating the Wnt signaling pathway activity. rWnt3a was used as agonist and rDkk-1 as antagonist. The results revealed that rWnt3a could enhance the Wnt activity, but rDkk-1 couldn't. The IC50 increased with significant difference after Wnt3a treatment, while IC50 slightly fell down without significant difference after rDkk-1 treatment, indicating that up-regulation of Wnt signaling could mediate gemcitabine resistance.In Part III, by FCM analysis we determined percentage of CD133+and CD44+CD24+EpCAM+cells in four pancreatic cancer cell lines (SW1990, PANC-1, T3M4 and SW1990/Gem). SW1990/Gem was a SW1990-derived cell line established after exposure to various concentrations of gemcitabine. The results showed that both SW1990 and SW1990/Gem had CD133 expression on cell surface, the proportion of which was 5.7% and 37.8%, respectively. PANC-1 and T3M4 had not any CD133 expression on cell surface. CD44+CD24+EpCAM+proportion was various in different cell lines, ranging from 10.9% to 72.1%. Here, we also detected Cd133 expression in SW1990 with additional temporary treatment by rWnt3a, rDkk-1 or gemcitabine, with CD133+fraction of 13.4%,3.0% and 5.6%, respectively.CONCLUSIONPancreatic cancer cell lines, SW1990, PANC-1, T3M4 and BxPC-3, have various IC50 value against gemcitabine, by which they can be simply divided into two groups: chemoresistant group (SW1990, PANC-1) and chemosensitive group (T3M4, BxPC-3). Activation of Wnt signaling pathway was not common in pancreatic cancer cell lines. Non-linear positive correlation was determined by Pearson's test between the canonical Wnt signaling pathway and gemcitabine resistance.rWnt3a and rDkk-1 are valid to regulate the Wnt activity. Up-regulation of Wnt signaling by rWnt3a can mediate gemcitabine resistance.There is a small fraction, or even no fraction, of CD133+cells in pancreatic cancer cell lines. CD 133 expression may be associated with gemcitabine resistance and Wnt signaling pathway activity.