The Effect And Mechanism Of Anlotinib Against Pancreatic Adenocarcinoma

BackgroundPancreatic adenocarcinoma?PDAC?is the most common type of pancreatic cancer.The mutation KRAS proto-oncogene is the most common type of mutation in PDAC,and the most common type is KRAS G12D?44%?mutation.However,the treatment of targeting KRAS protein mostly ended in failure.The treatment of immune checkpoint inhibitors in PDAC did not show excellent efficacy.As another rookie in the field of tumor therapy,the antivascular targeting drugs bevacizumab,sorafenib,axitinib and sunitinib also did not show significant survival benefits.Anrotinib hydrochloride is a novel oral multi-target receptor tyrosine kinase inhibitor that can target both tumor blood vessels and inhibit tumor cells.Can anlotinib hydrochloride bring hope to the treatment of KRAS G12D mutant PDAC?ObjectiveTo identify on PDAC cell lines carrying KRAS G12D mutations to explore the role of anlotinib hydrochloride in anti-PDAC and its possible mechanisms,thus providing a new treatment for pancreatic cancer.Methods?1?Pancreatic cancer cells?PDAC?were treated with anlotinib at different concentrations,the PDAC cells viability was detected by CCK8 assay.?2?Pancreatic cancer cells?PDAC?were treated with anlotinib at different concentrations,the cell migration ability were measured by Transwell in vitro and migration assay were used to detect the pancreatic cancer cell?PDAC?movement ability.?3?Pancreatic cancer cells?PDAC?were treated with anlotinib at different concentrations,Flow cytometry was used to detect changes in cell cycle and apoptosis.?4?Pancreatic cancer cells?PDAC?were treated with anlotinib at different concentrations,Western blotting was used to detect the expression of epithelial-mesenchymal Transition?EMT?related molecules??-catenin,ZO-1,Slug,Vimentin,Snail,E-cadherin?in pancreatic cancer cells?PDAC?.?5?Using nude mice tumor formation experiments to observe the inhibitory effect of anlotinib on the growth of Transplanted tumors.?6?Statistical analysis was performed using SPSS 21.0.P?0.05 was considered statistically significant.Results1.Anlotinib inhibits the proliferation of PDAC cell in a dose-dependent manner:The CCK8 showed that anlotinib at different concentrations?0,1,2.5,5,10,20,40?M?has inhibitory effects of pancreatic cancer cells.This result indicates that anlotinib exerts inhibit the proliferation of PDAC in a certain concentration?P?0.01?.2.Anlotinib inhibis the in vitro motility of PDAC cell:the wound healing assay results indicated that anlotinib at different concentrations?0,2,4?M?had an inhibitory the in vitro movement of PDAC cell,and the inhibitory effect tends to increase as the concentration on the migration of pancreatic cancer cells?P?0.05?.The results of the Transwell migration experiment showed that anlotinib at different concentrations?0,2,4?M?had an inhibitory effect on the migration of PDACA cell,and the differences are statistically significant?P?0.05??P?0.001?.3.Anlotinib hydrochloride induces cell cycle arrest in phase G₂/M:The flow cytometry test cycle indicated that the effect of different concentrations of anlotinib?0,2,4?M?on the cell cycle,with the increase of drug concentration,the cell cycle was arrested in G₂/M phase in a dose-dependent manner?P?0.05?.4.Antinib hydrochloride induces apoptosis in PDAC cells:Apoptosis detected by flow cytometry suggested that after treatment of pancreatic cancer cells with different concentrations of anlotinib?0,2,4?M?,the apoptosis rate of the cells increased significantly with increasing drug concentration?P?0.001?.5.Western blotting results showed that treated with different concentrations of anlotinib for 48 h.the expression of the epithelial marker up-regulated the protein levels of ZO-1 and the expression of mesenchymal marker proteins were down-regulated the protein levels of Slug,?-catenin,Vimentin;There is no significant change in Snail and E-cadherin.6.Effect of anlotinib on xenograft tumor in nude mice:After AsPC-1 cells were inoculated into the axilla of nude mice,Observe the growth inhibitory effect of anlotinib at5mg/kg on the growth of transplanted tumors.The volume of nude mice was observed and recorded every 3 days,and they were sacrificed after 21 days.Results Compared with the control group,the volume of xenograft tumors in the anlotinib group was reduced?P?0.01?.Conclusion1.Anlotinib may inhibit the proliferation,inhibit migration,induce apoptosis,and arrest G₂/M cell cycle arrest by pancreatic cancer cells in vitro and in vivo.2.Anlotinib Hydrochloride Upregulates epithelial-like markers and down-regulated interstitial-like markers,suggesting that the anti-pancreatic effect of anlotinib may be inhibiting the transformation of epithelial-mesenchymal transition?EMT?.