The Study Of Pim-3Regulating Growth And Gemcitabine Chemoresistance In Human Pancreatic Cancer Cells

[Background] As one of the most lethal cancers in gastrointestinal tract, pancreatic cancer owns poor prognosis with an overall5-year survival less than5%. Gemcitabine remains the current first-line chemotherapeutic agent available for treatment of advanced pancreatic cancer. However, single gemcitabine treatment has a response rate of less than20%. Chemoresistance becomes a major reason for failure in treatment. Chemoresistance of tumour cells can be provoked by limited drug delivery and uptake with augmenting expression P-gp and dysregulationof apoptosis due to increase of tumour suppressor gene Bcl-XL. Pim-3is a member of proto-oncogene expressing serine/threonine kinase activity, and Pim-3can inactivate Bad by phosphorylating Bad at Ser112and maintain the expression of Bcl-XL and thus prevent apoptosis of human pancreatic cancer cells. To date, the effects and mechanisms of Pim-3in pancreatic gemcitabine chemoresistance are not clear.[Purpose] In this research we aimed to identify the role of Pim-3on tumor growth and angiogenesis in orthotopic nude mouse models of human pancreatic cancer, and study the relationship between Pim-3and pancreatic cancer gemcitabine chemoresistance as well as molecular mechanisms of chemoresistance induced by Pim-3.[Methods]1. We established orthotopic model of pancreatic cancer to estimate Pim-3roles in tumor growth and angiogenesis. MRI was applied to measure the tumor growth, PCNA, CD31and VEGF were examined by immunohistochemistry; Pim-3and VEGF protein expression were detected by Western immunoblot.2. The cells stable overexpression or downregulation of Pim-3were treated by gemcitabine and evaluated cytotoxicity, cell cycle distribution and apoptosis by CCK8kit and flow cytometry. Miapaca-2cells that overexpressed Pim-3or Pim-3kinase dead (K69M-Pim-3) were transplanted into the mice, then the mice were adminiserted with gemcitabine. To assess the influences of Pim-3on gemcitabine resistantance, the tumor volume was measured, PCNA and CD31were detected by immunohistochemistry and cell apoptosis were determined with Tunnel staining kit. Western immunoblot was performed to investigate the expression of some indicators involved in chemoresistance of human pancreatic cancer.3.A gemcitabine-resistant pancreatic cancer cell line (Miapaca-2-res) was obtained by gradient increase of concentration.It’s biological properties and activity were detected by CCK8kit> flow cytometry and orthotopic nude mouse models. To observe the interaction between Pim-3and gemcitabine resistantance, the mRNA and protein expression of Pim-3were detected respectively by RT-PCR and Western blot. For the further aim to study the molecular mechanisms underlying in Pim-3roles in chemoresistant, Pim-3was knockdowed by RNAi interference, then Westernblot was used to investigate the expression of some indicators involved in chemoresistance of pancreatic cancer.[Results]1. Pim-3promoted the human pancreatic cancer cell proliferation in vitro and in vivo. Moreover, compared to the parental cells, the overexpression of Pim-3increased tumor vessels formation with increasing expression of VEGF.2. RT-PCR and Western blot analysis showed that gemcitabine significantly increased expression of Pim-3mRNA and protein in Miapaca-2and PCI55cells, suggesting the activation of Pim-3pathway. In comparision to parental cells, Miapaca-2-Pim-3cells that forced expression Pim-3, treated with gemcitabine displayed less influences on growth inhibition, cell cycle arrest and apoptosis.In orthotopic model, overexpression of Pim-3protected pancreatic cancer cells from gemcitabine-induced growth inhibition and apoptosis. In contrst, PCI55-Pim-3shRNA cells that knockouted expression Pim-3, treated with gemcitabine demostrated synergistic effects on growth inhibition, cell cycle arrest and apoptosis. Western blot analysis showed that cells treatment with gemcitabine augments anti-apoptotic molecule Bcl-XL and cell cycle regulators Cdc25A and Cdc25C protein expression level. These results suggested that Pim-3, which may regulate cell proliferation, cell cycle and apoptosis, involved in gemcitabine resistance in human pancreatic cancer.3. Stable gemcitabine-resistant Miapaca-2-res cells were established and owned resistant property that protected cells from gemcitabine-induced growth inhibition, cell cycle arrest and apoptosis in vitro and in vivo.RT-PCR and Western blot analysis found Pim-3expression was higher in Miapaca-2-res cells than in Miapaca-2cells with a dose-dependent manner. Knockouted expression Pim-3of Miapaca-2-res cell enhanced the chemosesitivty of gemcitabine. Western blot analysis shown P-gp、Bcl-XL、Cdc25A、Cdc25C were upregulated in Miapaca-2-res cells along with Pim-3, and these makers were downregulated after the Pim-3expression of Miapaca-2-res cells was knockouted.[Conclusion]1. Pim-3mediated cell proliferation and angiogenesis in human pancreatic cancer with reference to the expression of VEGF.2. Pim-3expression in pancreatic cancer cells was induced by gemcitabine, and Pim-3protected cells from gemcitabine-induced growth inhibition, cell cycle arrest and apoptosis by upregulating Bcl-XL. Cdc25A and Cdc25C eapression.3. Stable gemcitabine resistant Miapaca-2-res cell strain could be obtained by intermittent increasing concentration of gemcitabine. Pim-3may play a role in the process of gemcitabine resistance through boosting levels of Bcl-XL and P-gp.