The Experimental Study On The Biological Efficacy Of Apatinib And Its Related Mechanism In Pancreatic Cancer

Pancreatic ductal adenocarcinoma is a highly lethal tumor,with 5-year survival rate of less than 5% and poor prognosis,mainly because the late diagnosis ?early and progressive invasion and metastasis and the lack of effective treatment.It is reported that about 50% patients occur metastasis when diagnosed with pancreatic cancer.In recent years,although the treatment of pancreatic cancer has developed rapidly,but the mortality rate is still high.Although gemcitabine is the standard treatment for pancreatic cancer,it does not significantly prolong the survival of patients,so we need to explore new treatments and molecular targeted drugs to improve the prognosis of patients with pancreatic cancer.Angiogenesis has been demonstrated to be a promising therapeutic target through many VEGF inhibitors in many tumors.As we all know,pancreatic cancer is a tumor of deficient blood supply.Previous studies of VEGF signaling inhibitors such as bevacizumab,Axitinib,and Sorafenib have a significant effect in II / III clinical trials of pancreatic cancer,but the results are not satisfactory.At present,a number of clinical trials have confirmed that apatinib has a good effect on patients with advanced pancreatic cancer.We know that apatinib is a kind of TKI inhibitor of small molecule that targets angiogenesis and selectivly inhibit VEGFR2.Compare to Sorafenib and other TKI inhibitors,apatinib predominantly compete intracellular ATP binding sites of VEGFR2.Apatinib have good anti-tumor activity and good inhibitory effect on tumor angiogenesis.Studies have shown that apatinib has good antitumor activity,with no toxic effects and is well tolerated in many human tumor transplant models when apatinib was administered alone or in combination with chemotherapeutic agents.In addition,apatinib can significantly inhibit tumor cell proliferation,migration and can promote apoptosis in a variety of tumor cells such as gastric cancer cells?cholangiocarcinoma cells.However,the study of apatinib in pancreatic cancer has not been seen.Based on the above,the experimental study on the biological efficacy of apatinib and its related mechanism in pancreatic cancer was studied by in-vitro and in-vivo study of BXPC-3,ASPC-1 and PANC-1 pancreatic cancer cells to lay the foundation for the study of the clinical application of apotinib.ObjectivesTo investigate the effect of apatinib through in-vitro and in-vivo study on the biological efficacy of apatinib and its related mechanism in pancreatic cancer.Methods1?We inhibit the expression of VEGF-VEGFR2 pathway in human BXPC-3,ASPC-1 and PANC-1 pancreatic cancer cells by using of apatinib in different concentrations(20,40?mol / L).The proliferation was measured by CCK8 method.The effects of apatinib on the growth cycle of pancreatic cancer cells were detected by flow cytometry.Apoptosis was detected by flow cytometry.Transwell tumor invasion assay was used to observe the effect of apatinib on the migration and invasion of pancreatic cancer cells.The expression of autocrine VEGF was detected by ELISA.2?When human BXPC-3,ASPC-1 and PANC-1 pancreatic cancer cells were treated with apatinib(20,40?mol / L)for 24 hours,western blot was used to detect the phosphorylation of VEGFR2 / PI3 K / AKT pathway and the expression of anti-apoptotic protein Bcl-2.3?Human BXPC-3 pancreatic cancer cells were cultured in vitro and 100 ?l mixed solution of cells and PBS(containing 1 × 107 cells)were inoculated into the right armpit of male and 6-week-old BALB / C nude mice.When the lumps(about 0.5cm or so),mice were randomly divided into control group and apatinib group and each group contains eight mice.Apatinib(200 mg / kg / d)and placebo were administered orally.And then we measure the length and width of a tumor and the weight of nude mice every three days.After 3 weeks of administration,the nude mice were sacrificed and the tumor tissue was removed and weighed.The expression of CD31 and Ki67 in the two groups was detected by immunohistochemistry.The apoptosis of the two groups was detected by Tunel method.Results1?Compared with the control group,the growth of BXPC-3,ASPC-1,PANC-1 pancreatic cancercells were significantly inhibited when treated with different concentrations of apatinib(20,40?mol / L).The results of cell cycle showed that S phase and G2 / M phase in ASPC-1 and PANC-1 cells were reduced by low concentration of apatinib and apatinib slow down cell proliferation.High concentrations of apatinib mainly caused G2 / M arrest to inhibit proliferation.But in BXPC-3 cells,low concentrations of apatinib mainly cause G2 / M arrest to inhibit proliferation.The level of autocrine VEGF is also significantly reduced when apatinib blocks VEGF / VEGFR2.The higher the concentration of apatinib,the less the secretion of VEGF by pancreatic cancer cells.Apatinib had a pro-apoptotic effect on BXPC-3 ?ASPC-1 pancreatic cancer cells,but had no significant effect on PANC-1 cells.In addition,apatinib could also inhibit the invasion and migration of BXPC-3 ?ASPC-1 cells,whereas in PANC-1,there was no significant change in the experimental group compared with the control group.2?Western-blot showed that the phosphorylation of VEGFR2 was reduced in each group and the phosphorylation of PI3 K / AKT was also decreased.VEGF / VEGFR2 / PI3 K / AKT signal transduction pathway may be involved in the effect of apatinib on the biological behavior of pancreatic cancer.The expression of anti-apoptotic protein BCL-2 was down-regulated in BXPC-3 cells in a dose-dependent manner,but no significant changes were observed in ASPC-1 and PANC-1 cells.3?In-vivo experiments show that apatinib can significantly delay the growth of transplanted tumors.Compared with the control group,the expression of CD31 and the proportion of Ki67 positive cells in the apatinib group were significantly lower,indicating that apatinib could decrease the vascular density and the proliferation of cancer cells.ConclusionsIn-vitro and in-vivo studies have shown that apatinib has an inhibitory effect on pancreatic cancer.Apatinib can significantly inhibit the proliferation,migration and invasion of pancreatic cancer cells and induce apoptosis in a concentration-dependent manner.It is possible that the role of apatinib is related to reducing the phosphorylation of VEGF / VEGFR2 / PI3 K / AKT pathway and the VEGF secretion.Apatinib may have a certain treatment prospects,the specific clinical benefits worth further exploration.