The Molecular Mechanism And Active Ingredients Of Marsdenia Tenacissima Injection Induces Apoptosis Of Prostate Cancer Via GSK3β/STAT3 Signaling Axis

Objective:To explore the molecular mechanism and active ingredients of Marsdenia tenacissima injection,which main active components is Marsdenia tenacissima extract（MTE）anti-prostate cancer（PCa）,so as to provide a preliminary research basis for clinical application of MTE in the treatment of PCa.Methods:1.The effects of MTE on PC3,DU145 and RM-1 cells proliferation were detected by CCK8 assay.2.Colony formation experiments were conducted to observe the effect of MTE on clone formation of PC3 and DU145 cells.3.Flow cytometry was used to detect the effect of MTE on cell cycle,apoptosis and the mitochondrial membrane potential of DU145 cells.4.The endogenous mitochondrial pathway apoptosis-related proteins were detected by Western-blot（WB）.5.Experiment of NOD-SCID mouse model of DU145 cells xenotransplantation was conducted to observe the effect of MTE on the growth of DU145 cells in vivo;Apoptosis-related proteins in tumor tissues were detect-ed by WB.6.Network pharmacological methods were used to predict the ingredients,targets and related pathways of MTE.7.The key proteins such as PI3K,AKT and GSK3βin the PI3K-AKT signaling pathway and the downstream proteins STAT3 were verified by WB.8.Molecular docking technology was used to predict the potential active components of MTE inducing apoptosis of DU145 cells.9.The chemical components in MTE were analyzed and identified by HPLC-CAD-QTOP/MS and UPLC-QTOP/MS technologies.Results:1.CCK8 assay showed that MTE could inhibit the proliferation of PC3,DU145 and RM-1 cells.After treated with MTE for 72 h,the half maximal inhibitory concentration values of PC3,DU145,RM-1 are 84.59,77.93,44.92mg/m L,respectively.2.Colony formation experiment verified that MTE inhibit the colony formation of PC3 and DU145 cells in a dose-dependent manner.3.Flow cytometry assy showed that MTE had no significant regulation effect on cells cycle,but could induce apoptosis of DU145 cells.4.The results of mitochondrial membrane potential experiment showed that MTE could significantly reduce the mitochondrial membrane potential of DU145 cells.5.The expression of endogenous mitochondrial pathways proapoptosis-related proteins,such as Bax,Cytochrome C,Cleaved Caspase 3and Cleaved Caspase 7 were up-regulated by MTE.6.The results of subcutaneous tumor transplantation in mice showed that MTE inhibited the growth of DU145 cells in vivo and have no significant effect on body weight;WB assay showed that MTE up-regulated Cleaved Caspase 3expression in tumor tissues.7.Based on literature reviewing and public database related to network pharmacology searching,196 active ingredients of MTE were collected,which corresponded to 655 targets,and 709 targets of prostate cancer were retrieved.The potential targets of MTE anti-PCa were discovered by comparing MTE genes with prostate cancer genes,and 10 candidate targets VEGFA,EGFR,AKT1,CASP3,SRC,GSK3B,IL6,STAT3,HSP90AA1and MTOR were obtained after screening.Pathway enrichment of 10 core targets revealed that HIF-1,PI3K-AKT and Erb B were closely related to tumor signaling pathways.8.WB assay was used to verify the key proteins in the PI3K-AKT signaling pathway,and the results showed that MTE could significantly up-regulate the expression of PI3K,p-AKT and p-GSK3β^Ser9in DU145 cells.Furthermore,the expression of STAT3,the downstream core target of GSK3β,was detected,and the results showed that MTE significantly down-regulated the expression of p-STAT3^Tyr705in DU145 cells.WB assay also showed that MTE could significantly incease the expression of p-GSK3β^Ser9and down-regulate the expression of p-STAT3^Tyr705in tumor tissues.These results suggest that MTE induces apoptosis of DU145 cells through endogenous mitochondrial pathway via GSK3β/STAT3 signaling axis.9.Molecular docking results showed that a total of 13 compounds could well bind with GSK3βand STAT3.Dresgenin,Marstenacigenin A and Marstenacigenin B can be well binding with GSK3β.Besides,Marstenacisside C3,TenacissimosideH,Daucosterol,MarsdenosideD,12β-O-Acetyl-3-O-（6-deoxy-3-O-methyl-β-D-allopyranosyl-（1→4）-D-olean-dr-onyl）-11α-O-isobutyryltenacigenin B,Marsdenoside H,Marstenacisside A4,Tenacigenoside I,Tenacigenoside D and Tenacissoside B can be well bind with STAT3.10.HPLC-CAD-QTOP/MS and UPLC-QTOF/MS results confirmed that Marstenacisside C3,12β-O-Acetyl-3-O-（6-deoxy-3-O-methyl-β-D-allopyrano-syl-（1→4）-D-oleandronyl）-11α-O-isobutyryltenacigenin B,Tenacissimoside H,Marstenacisside A4,Tenacissoside D,Tenacissoside B,Marsdenoside D and Marsdenoside H exist in MTE.These 8 components might be the potential ingredients of MTE anti-PCa.Conclusion:MTE induces apoptosis of DU145 cells through endogenous mitochondrial pathway via GSK3β/STAT3 signaling axis.Marstenacisside C3,12β-O-Acetyl-3-O-（6-deoxy-3-O-methyl-beta-D-allopy-ranosyl-（1→4）-D-olean-dronyl）-11-α-O-isobutyryltenacigenin B,Tenacissimoside H,Marstenacisside A4,Tenacissoside D,Tenacissoside B,Marsdenoside D and Marsdenoside H might be the active ingredients of MTE induces apoptosis of DU145 cells.