Study On Anti-tumor Effects And Molecular Mechanism Of Ecliptasaponin A In Lung Cancer

Objective: Ecliptasaponin A(ES),which is a natural product,has been reported to have an anti-cancer effect in a variety of tumors,but its effect on lung cancer is still unknown.Therefore,this project systematically evaluated the anti-lung cancer effect of ES in vivo and in vitro and explored its anti-tumor molecular mechanism.Methods and Results:Section 1 The antitumor efficacy of ES in vitro and in vivo MTT colorimetry and colony cloning assay were used to determine the effect of ES on the proliferation ability of non-small cell lung cancer cells,and the cell cycle changes were detected by flow cytometry to confirm the reasons for the effect on cell proliferation ability.Hoechst 33342 staining was used to determine the apoptotic morphological changes of lung cancer cells by ES.Immunofluorescence assay was used to detect the effect of ES on autophagy in lung cancer cells.The effects of ES on migration and invasion of lung cancer cells were verified by the scratch test and the Transwell test.Then,Western Blot was used to determine the expression levels of the proteins related to the above cycle,migration,and invasion in lung cancer cells,to further verify the effect of ES on the anti-lung cancer effect in vivo.Finally,using the xenograft tumor model,the control group,the ES low-dose group,and the high-dose group were set up to verify the anti-tumor effect of ES A in vivo.The results showed that the survival rate of lung cancer cells decreased gradually with the increase of the concentration of ES and was time-dependent.G0/G1 phase arrest occurred in lung cancer cells at a certain concentration.ES can dose-dependently induce apoptosis and autophagy in lung cancer cells H460 and H1975.At the same time,it has also been proved that ES can inhibit cell migration and invasion.Cell cycle-related protein Cyclin D1,CDK6,P21,apoptosis-related proteins cleaved caspase 8,cleaved caspase 9,cleaved caspase 3,Bax,and Bcl-2,autophagy-related proteins LC3a/b,beclin-1,and p62,and migration and invasion related proteins E-cadherin,N-cadherin and Vimentin also altered.In the subcutaneous transplanted tumor model of human lung cancer H460 nude mice,it was found that the 50mg/Kg ES Group,given once drug every three days,could significantly inhibit the growth of the transplanted tumor,showing no significant weight loss in the mice.Section 2 The anti-tumor mechanisms of Ecliptasaponin A PART1 ES induced apoptosis and autophagy in lung cancer cells and their related mechanism Through flow cytometry combined with western blot assay,the tumor cells were induced to undergo apoptosis,which could be reversed by the Caspase inhibitor(Z-VAD-FMK).Flow cytometry combined with western blot assay showed that autophagy inhibitors 3-MA and CQ can block autophagy,thus reverse the apoptosis induced by ES.In order to further explore the correlation between apoptosis and autophagy induced by ES,it was found that ES promoted the apoptosis of lung cancer cells by activating ASK1/JNK pathway,ASK1 inhibitor GS-4997 and JNK inhibitor SP600125 could reverse the apoptosis induced by ES,and SP600125 could also inhibit the autophagy induced by ES.PART 2 Study on the molecular mechanism of endoplasmic reticulum stress in the antitumor effect of ES Western blotting experiments showed that the action of Ecliptasaponin A on H460 and H1975 cells induced ER stress,and the expression levels of Bip,IRE1α,XBP1 s,and c-Myc were increased.Flow cytometry showed that endoplasmic reticulum inhibitor 4-PBA attenuated the apoptosis of lung cancer cells induced by Ecliptasaponin A.Flow cytometry found IRE1α kinase inhibitors,kira6 can reverse the death caused by ES,the western blot found kira6 can reduce the phosphorylation expression level of IRE1α,ASK1 and JNK,illuminated the endoplasmic reticulum stress IRE1α/ASK1 / JNK pathway in ES promoting apoptosis in lung cancer cell.On the other hand,flow cytometry showed that the endonuclease of IRE1α inhibitor STF-083010 could promote the apoptosis induced by ES,and c-Myc inhibitor 10058-F4 could also promote the apoptosis induced by ES.Western blotting showed that STF-083010 could reduce the expression levels of XBP1 s and c-Myc,increase the expression level of cleaved caspase3,and elucidated the role of IRE1α /XBP1/ c-Myc pathway in promoting the survival of lung cancer cells.PART 3 ES promotes the apoptosis of lung cancer cells by inhibiting the expression of PKCα/STAT3 Western blot assay showed that ES could inhibit the expression of PKCα and STAT3 in lung cancer cells.By flow cytometry combined with western blot assay,plasmid transfection for continuous expressing PKCα can attenuate apoptosis in lung cancer cells induced by ES.Using an inhibitor of PKCα,Sotrastaurin can promote apoptosis induced by ES.And inhibition of PKCα can reduce the expression of STAT3.Further,IL-6 promoted the expression of STAT3,which could weaken apoptosis in lung cancer cells caused by ES.And S3I-201 inhibited STAT3,which could promote apoptosis caused by ES.Conclusion: This study confirmed that ES has a significant anti-lung cancer effect in vivo and in vitro,can inhibit the proliferation of lung cancer cells,make cell cycle arrest in G0/G1 phase,can induce tumor apoptosis and autophagy,while autophagy can promote cell apoptosis,JNK pathway can not only regulate cell apoptosis but also promote cell autophagy.Endoplasmic reticulum stress plays a "double-edged sword" role in the anti-tumor effect of ES.When IRE1α acts as a kinase,ES promotes cell apoptosis through the IRE1α /ASK1/JNK pathway.However,when IRE1α plays the role of endonuclease,ES promotes the survival of lung cancer cells through the IRE1α/XBP1/ c-Myc pathway.Further studies have also found that ES can inhibit the expression of STAT3 by inhibiting PKCα,thus promoting the apoptosis of lung cancer cells.