Effects Of Paris Polyphylla Ⅱ On Angiogenesis Of Tumour Induced Viaautophagy By PI3K/Akt Signaling Pathway

In recent years,the innovation of traditional Chinese medicine has widened the range and effect of tumor therapy.It is a hot spot and breakthrough to search new anti-tumor angiogenesis drugs from natural plants or traditional Chinese medicine.Tumor vessels are complex pathophysiological processes regulated by many growth factors that form new vessels from the original vascular bed.Tumor angiogenesis plays an important role in the occurrence,development and progression of tumor.The research group found that Paridonin II,an effective component of Rhizoma Paridis,can inhibit the angiogenesis of melanoma cells and induce autophagy,the purpose of this study was to investigate the role of PI3K/Akt signaling pathway in the induction of autophagy and anti-melanoma angiogenesis by Paris Saponin II.In this study,we designed a model to simulate the angiogenesis of human umbilical vein endothelial cells(HUVECs)and melanoma cells(B16F10)in vitro,to investigate whether Parisaponin II can block the formation and migration of HUVECs in cultured human umbilical vein endothelial cells induced by supernatant of B16F10 cells,and whether the molecular mechanism of Parisaponin II inhibiting tumor angiogenesis is related to autophagy,it provides a theoretical and experimental basis for the further study of the anti-tumor mechanism of Paris Polyphylla Saponin II and provides a good start for the development of new anti-tumor angiogenesis drugs from traditional Chinese medicine.Objective: To study the effect of Paris Polyphylla Saponins II on the induction of autophagy and anti-angiogenesis of melanoma B16F10 cells by PI3K/Akt signaling pathway and related molecular mechanism.Methods: Human Umbilical Vein endothelial cells(HUVECs)and B16F10 cells(B16F10 cells)were co-cultured in a non-direct-contact culture system,and conditioned medium was prepared to simulate the angiogenesis of melanoma in vitro;MTT assay to detect the effects of Paris Polyphylla Saponin II on HUVECs and B16F10 cells in different time and concentration,and the effects of Paris Polyphylla Saponin II on HUVECs and B16F10 cells with different inhibitors;To observe the changes of HUVECs and B16F10 cells under an inverted microscope;To observe the effect of different concentrations of Paris Polyphylla Saponins II on the lumen formation and scratch rate of HUVECs;Positive staining of the perikaryon region was observed by MDC staining;Acid region was observed by AO staining;Electron microscopic observation of cell ultrastructureand autophagy;Western blot to detect expression of LC3(LC3-Ⅰ、LC3-Ⅱ)、P62 and beclin 1 protein;RT-q PCR analyse of beclin 1 mRNA 、Atg5 mRNA、HIF-1α mRNA、and VEGF mRNA gene expression levels.Autophagy inhibitor 3-MA and CQ treated cells,lumen formation experiment was conducted to investigate the effect of Rhizoma Paridis on angiogenesis of HUVECs after autophagy,The lumen formation experiment was conducted to investigate the effect of inhibiting autophagy on Angiogenesis of HUVECs;The effects of different inhibitors on the migration of B16F10 cells were examined by scratch and migration assay;The combination of PP II and LY294002,western blot detect the change of expression of VEGF,HIF-1,AKT,p-AKT,m TOR,p-m TOR,PI3 K and p-PI3 K proteins.Results:1.The effect of Paris Saponin II on angiogenesis of melanoma cells(1)MTT results showed that PP II significantly inhibited the proliferation of HUVECs and B16F10 cell,and the cell survival rate decreased with the increase of concentration and time of Paris Saponin II;(2)Morphological observation showed that with the increase of the concentration of Paris Polyphylla Saponin II,the refractive index of HUVECs decreased,the cell adhered to the wall badly and the morphology began to shrink compared with the control group;(3)PP II significantly inhibited the lumen formation of HUVECs,and the inhibitory effect was dose-dependent;(4)Migration test showed that PP II significantly inhibited the cell migration of HUVECs,and the inhibitory effect was dose-dependent;(5)Cell scratch test showed that PP II could significantly inhibit the migration of B16F10 cells.The results were consistent with the migration test.2.Studies on autophagy induced by Paris Polyphylla Saponin II in B16F10 cells;(1)MDC staining results showed that PP II increased the accumulation of autophagic vacuoles in B16F10 cells;(2)AO staining results were observed,PP II probably induced autophagy in B16F10cells;(3)The results of transmission electron microscope assay showed that PP II changed the mitochondrial morphology of B16F10 cells,but when used in combination with3-MA,the mitochondrial morphology of B16F10 cells was improved;(4)Western blot results showed that,PP II could down-regulate the expression levels of LC3,Beclin 1 and P62,while the combined autophagy inhibitor 3-MA and CQ were increased.3.The inhibitory effect of Paris Saponins II on tumor angiogenesis by regulating autophagy(1)Compared with PP II 4m group,the inhibitory effect of PP II combined with CQ(10 m)and 3-MA(5 mm)on the proliferation of B16F10 cells was decreased.(2)Combined with autophagy inhibitor CQ and 3-MA,the structural integrity of HUVECs was restored to some extent.(3)The results of scratch test showed that the scratch healing area of PP II 4μM group was slower than that of PP II 4μM,compared with that of PPII 4μM,PP II combined with autophagy inhibitors CQ and 3-MA improved the healing rate of the scratch area.(4)Transwell test was consistent with the results of cell scratch test.(5)Western Blot Assay showed that the expression of VEGF protein in B16F10 cells decreased with the increase of PP II,but the expression of VEGF protein in B16F10 cells increased after PP II combined with autophagy inhibitor CQ and 3-MA.(6)The results of fluorescence quantitative q RT-PCR were consistent with those of Western blot.4.The role of PI3K/Akt signaling pathway in the inhibition of autophagy induced by Paris Saponins II.In order to explore the pathway of inhibition of PP II,the cells were pretreated with LY294002,a PI3 K inhibitor,the expression of p-PI3 K,p-Akt and p-m TOR protein was significantly inhibited after the combination of PP II and LY294002,but the expression of Akt,PI3 Kand m TOR protein had no obvious changes.Conclusion:Paris Saponins II inhibited the proliferation and migration of tumor cells B16F10 and HUVECs,and induced autophagy of melanoma B16F10 cells by regulating the PI3K/AKT signal pathway.