| Background:Pancreatic cancer carries a uniformly poor prognosis with low surgical resection rate and short survival time. For decades, there was no significant therapeutic improvement, even for resectable cases. Recently, adjuvant therapy for pancreatic cancer develops. Gemcitabine is used as the first-line chemotherapy drug in adjuvant treatment of pancreatic cancer, but with limited ability to improve the prognosis of patients with pancreatic cancer. Poor efficacy of gemcitabine is due to the chemoresistance characteristics of pancreatic cancer cells. The chemoresistance mechanisms are complicated; several drug resistance associated genes and signaling pathways have been identified. Nowadays, a large number of drugs have been designed to target these drug resistance associated genes and signaling pathways. However, the vast majority of these new drugs exhibited poor efficacy. Therefore, it is necessary to explore new gemcitabine-resistance mechanisms in pancreatic cancer for new targets. Vasohibin-2 (Vash2) is a newly identified gene. During the past two years, Vash2 was found to be highly expressed and associated with malignant behavior in a number of tumors, but the mechanism needs further study.Objectives:1 To prepare the rabbit polyclonal antibody against human Vash2.2 To identify the intracellular localization of Vash2.3 To investigate the relationship between the Vash2 expression and prognosis in pancreatic cancer tissues.4 To study the gemcitabine-sensitivity in pancreatic cancer cells regulated by Vash2.5 To investigate the mechanism of inhibited gemcitabine-sensitivity in pancreatic cancer cells induced by Vash2.Methods:1 The recombinant protein and synthetic polypeptides were used as immunogens, the animals were immunized, and the anti-serums were purified to harvest the antibodies. Western blotting, immunofluorescence and immunohistochemistry were used to test the antibodies.2 The cytoplasmic and nuclear cellular extracts were isolated and used to determine intracellular distribution of Vash2 by Western blotting. The tagged Vash2 proteins were detected by the tag immunofluorescence to identify the localization of Vash2 in the cell.3 105 pairs of pancreatic cancer and their adjacent tissues microarray were established with relevant clinical and follow-up data. Immunohistochemical detection of Vash2 was performed. The correlation of Vash2 expression and survival time was analyzed.4 Vash2 overexpression/interference stably transfected pancreatic cancer cell models were established. CCK8 method was used to detect the IC50 values. Apoptotic cells were detected by flow cytometry. Vash2 overexpressing/interference stably transfected pancreatic cancer cells were inoculated into nude mice, and the tumor suppressor factors (Vash2 was considerd as intervention factor) were calculated. TUNEL assay was used to detect the apoptosis in xenograft tumor from the nude mice.5 Quantitative PCR was used to detect gene expression changes related to the gemcitabine metabolism in Vash2 overexpressing/interference stably transfected pancreatic cancer cells. Western blot was used for further verification. And the pancreatic cancer tissue microarray was also used for further verification.Results:1 The rabbit polyclonal antibodies against human Vash2 were made and available for western blotting, immunofluorescence and immunohistochemistry.2 Different protein isoforms of Vash2 were within different intracellular localization, and Vash2 could be divided into karyo and cytoplasmic type.3 We successfully completed the pancreatic cancer tissue microarray. Immunohistochemical results showed that the cytoplasmic Vash2 was associated with pancreatic cancer tissue differentiation and tumor vascular involvement; cytoplasmic Vash2 down-regulated the gemcitabine-sensitivity in pancreatic cancer cells.4 Vash2 overexpressing decreased the gemcitabine-sensitivity while Vash2 interference increased the gemcitabine-sensitivity in pancreatic cancer cells.5 Overexpression of Vash2 increased the mRNA and protein levels of ribonucleotide reductase M2 (RRM2); interference of Vash2 decreased the mRNA and protein levels RRM2. Clinical pancreatic cancer tissue microarray of Vash2 and RRM2 showed the positive correlation between them.Conclusion:1 Vash2 proteins can be divided into karyo type and cytoplasmic type.2 Cytoplasmic Vash2 was associated with differentiation of pancreatic cancer.3 Cytoplasmic Vash2 reduces the sensitivity of pancreatic cancer cells to gemcitabine.4 The expression of cytoplasmic Vash2 raises gemcitabine-sensitivity via up-regulating RRM2 in pancreatic cancer cells. |
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