Aqueous Extract Of Taxus Chinensis Var.mairei Regulates The Hippo-YAP Pathway And Promotes Apoptosis Of Non-small Cell Lung Cancer Via ATF3 In Vivo And In Vitro

Objective To study the inhibitory effect of aqueous extract of Taxus chinensis var.mairei(AETC)in non-small cell lung cancer(NSCLC)cell lines,and its mechanism of promoting the apoptosis in NSCLC cell lines through the Hippo-YAP signaling pathway mediated by ATF3.What's more,this research provided basis for the clinical treatment of lung cancer with Taxus chinensis var.mairei and a new target for the treatment of NSCLC.Methods 1.Cell counting kit-8(CCK-8)method and flow cytometry were used to detect the proliferation and apoptosis after AETC treatment at different concentrations on NSCLC cell lines.Western blot was used to detect the expression of apoptosis proteins,such as cleaved caspase 3,cleaved caspase 8,cleaved caspase 9,and cleaved PARP.2.Transcriptome sequencing was used to analyze the target of AETC in promoting apoptosis of NSCLC lines,and ATF3 was identified as the up-regulated differentially expressed gene,which was verified by western blot,qRT-PCR,and immunofluorescence.3.Western blot,qRT-PCR,immunofluorescence,and TUNEL staining were used to detect the effect of ATF3 knockdown on caspase proteases expression and apoptosis induced by AETC.4.Western blot and TUNEL staining were used to detect the effects of AETC on the key proteins of Hippo-YAP signaling pathway(MOB1,p-MOB1,p-YAP(Ser397),p-YAP(Ser127)).YAP inhibitor verified that AETC promoted the apoptosis of NSCLC cells by activating Hippo signaling pathway.5.Western blot and TUNEL staining were used to detect the effects of AETC on the expression of MOB1,p-MOB1,p-YAP(Ser397),and p-YAP(Ser127)of Hippo-YAP signaling pathway in ATF3 knockout NSCLC cell lines.6.NSCLC tumor bearing nude mice were constructed and administrated continuous by gavage for 14 days.The general condition and tumor volume of tumor bearing nude mice was observed every two days and the tumor growth curve was drawn.After 14-day gavage,the tumor was dissected and weighed,and the inhibition rate was calculated.The expression of ATF3,MOB1,p-MOB1,p-YAP(Ser397),p-YAP(Ser127),and apoptotic protein in tumor tissue of nude mice were detected by western blot;The expression and localization of ATF3 were detected by immunohistochemistry;The apoptosis of tumor tissue was detected by TUNEL staining.Results 1.CCK-8 showed that AETC inhibited the proliferation of NSCLC cells in a time and dose-dependent manner.Flow cytometry showed that AETC promoted apoptosis.And western blot indicated that AETC increased the expression of cleaved caspase 3/8/9 and cleaved PARP in H1975 and A549 cells.2.The results of transcriptome sequencing showed that 119 genes were up-regulated and 20 genes were down regulated in H1975 treated with AETC,while 796 genes were up regulated and 666 genes were down regulated in A549 cells.ATF3 gene was up regulated by 1.61 times and 4.13 times in H1975 and A549.The results of western blot,qRT-PCR,and immunofluorescence were consistent with those of transcriptome sequencing.3.Western blot,qRT-PCR,and immunofluorescence were used to detect the expression of ATF3 in ATF3 knockdown cell lines.Compared with si-NC group,the expression of ATF3 protein and mRNA in si-ATF3 group was significantly decreased,and the expression of apoptotic proteins was decreased.Compared si-ATF3 group with si-ATF3+AETC group,the expression of ATF3 protein and mRNA had no change,and the expression of apoptotic proteins had no change.The results of TUNEL staining were consistent with western blot.4.Western blot analysis showed that p-MOB1,p-YAP(Ser397),and p-YAP(Ser127)were significantly up regulated in H1975 and A549 cells after AETC intervention.YAP inhibitor significantly increased the expression of apoptotic protein in NSCLC cells after AETC intervention.TUNEL staining also showed that YAP inhibitor could improve the ability of AETC to promote apoptosis.5.Western blot analysis showed that p-MOB1,p-YAP(Ser397),and p-YAP(Ser127)were significantly up regulated in H1975 and A549 cells after AETC intervention.YAP inhibitor significantly increased the expression of apoptotic protein in NSCLC cells after AETC intervention.TUNEL staining also showed that YAP inhibitor increased the apoptosis induced by AETC.6.Compared with the control group,the tumor growth and weight of low-dose and high-dose AETC groups were significantly decreased,and the inhibition rates of H1975 and A549 were 48.1%and 59.5%,respectively.Western blot analysis showed that ATF3 was significantly increased in AETC groups with both low-and high-dose manner,and the trend of immunohistochemistry was consistent with western blot;p-MOB1,p-YAP(Ser397),p-YAP(Ser127),and apoptotic protein expression were significantly up regulated in H1975 transplanted tumor,and p-YAP(Ser127)and apoptotic protein expression were significantly up-regulated in A549 transplanted tumor,and TUNEL staining results were consistent with western blot.Conclusion 1.AETC significantly inhibited the proliferation of NSCLC cell lines in a concentration-and dose-dependent manner,and promoted the apoptosis of A549 and H1975 cells.But the effect of AETC on apoptosis was not related to the dose.2.AETC significantly increased the expression of ATF3 protein and mRNA.ATF3 knockdown not only inhibited the apoptosis,but also weakened the apoptotic effect of AETC on NSCLC cells.3.AETC promoted the apoptosis of NSCLC cells by activating Hippo signaling pathway.ATF3 knockdown inhibited the activation of Hippo signaling pathway and the effect of AETC on the activation of Hippo signaling pathway.4.AETC promoted the apoptosis of NSCLC cells through the Hippo-YAP signaling pathway mediated by ATF3,which was one of the anti-tumor mechanisms of AETC.What's more,ATF3-Hippo-YAP signaling pathway can be a new target for the treatment of NSCLC.