Study On The Anti-leukemia Drug Resistance And Molecular Mechanism Of The Total Saponins From Solanum Nigrum L.

Aim:Solanum nigrum L.is a traditional Chinese medicine in China,and steroidal saponins are the main active ingredients.In our former study,the steroidal saponins of S.nigrum exhibited significant anti-proliferative activity in multidrug resistant leukemia cells,but the molecular mechanism is still unclear.Therefore,this study clarified the potential anti-proliferative activity and anti-drug resistance mechanism of the total steroidal saponins（SN）and the steroidal saponins monomer（S-20）of S.nigrum in vitro and in vivo.Methods:In vitro,cytotoxicity of SN and S-20 against different types of leukemia cells（K562,K562/ADR,HL-60,U937,Jurkat）were determined by CCK-8 assay.Finally,K562 and K562/ADR cells were selected as the target cell lines in follow-up experiments.Apoptosis induced by SN or S-20 was measured by Annexin V-FITC/PI staining.Cell cycle was performed by flow cytometric analysis.The effect of SN or S-20 on drug resistance protein function in K562/ADR cells was detected by Rhodamine 123 fluorescence assay.Western blot was used to detect the expression of drug resistance,apoptosis,autophagy and other related proteins.In addition,to confirm the biological role of autophagy and apoptosis in overcoming the drug resistance,the effect of cell transfection and the related inhibitors was measured.In vivo,to evaluate the effect of SN on the drug resistance and growth of tumors,a xenograft model was established by injecting K562/ADR cells into nude mice.Result:In vitro,SN and S-20 had broad spectrum cytotoxic activity against various human leukemia cancer cell lines,especially in K562 and K562/ADR cells.Western blot results showed that SN and S-20 inhibited the expression of drug resistance-related proteins in K562/ADR cells.Further research found that SN inhibited the proliferation of K562/ADR and K562 cells by inducing cell cycle arrest in G₀/G₁ phase.In addition,SN and S-20 induced caspase-dependent apoptosis in K562/ADR and K562 cells.More importantly,SN induced autophagy in K562/ADR cells through PI3K/Akt/m TOR signaling pathway to overcome the drug resistance,and ultimately induced autophagic cell death.In vivo,the inhibition rates of tumor weight were 12.92%and 27.63%at the dose of 125 mg/kg and 250 mg/kg in SN-treated groups,and these doses had no effect on the bodyweight of the nude mice.The down-regulation of immunohistochemical positivity of BCRP and Ki67 of tumor tissues showed that SN overcame drug resistance and inhibited the proliferation of tumors.In addition,western blot analysis showed SN significantly decreased the expression of drug resistance-related proteins,p-m TOR and increased the conversion of LC3-I to LC3-II in K562/ADR xenografts.The above results speculated that SN may overcome drug resistance and inhibited proliferation of tumor by regulating autophagy in vivo.Conclusion:These results indicated that the steroidal saponins of S.nigrum exhibited significant anti-leukemia activity in vivo and in vitro.In addition,SN induced autophagy in K562/ADR cells through PI3K/Akt/m TOR signaling pathway to overcome drug resistance,and ultimately induced autophagic cell death in K562/ADR cells.Therefore,our findings emphasize that the steroidal saponins of S.nigrum are the potential candidates for overcoming tumor resistance.