The Molecular Pharmacological Mechanisms Of Dioscin To Reverse Multidrug Resistance Of Adriamycin And Enhance The Absorption Of Methotrexate

Drug resistance is a formidable obstacle in cancer chemotherapy.Tumor cells can develop resistance to structurally diverse and mechanistically unrelated anticancer drugs,a phenomenon termed MDR(multidrug resistance,MDR).MDR is associated with the overexpression of the MDR1(multidrug resistance 1,MDR1)protein.MDR1,a 170 KD plasma membrane glycoprotein encoded by the MDR1 gene,is a multidrug transporter that functions as an adenosine triphosphate-dependent drug effux pump.In tumor cells,MDR1 pumps out anticancer drugs,leading to drug resistance at the cellular level.The mechanism of MDR 1-mediated drug resistance is related to NF-κB(nuclear factor κ-B,NF-κB),a protein complex acting as a transcription factor.Phosphorylation of IκB-a(inhibitor κB-a,IκB-a)is required for NF-κB activation.A significant increase in p-IκB-a leads to the activation of NF-κB.MDR1-mediated drug resistance can be effectively overcome by either blocking MDR1 drug pump function or inhibiting its expression.Inhibition of MDR 1-mediated drug effux leads to the resensitization of MDR cancer cells to chemotherapeutic agents,allowing for successful chemotherapy in patients with multidrug-resistant tumors.Currently,many clinical anti-cancer drugs such as certain alkaloids,anthracycline antibiotics,and epipodophyllotoxin derivatives can induce MDR.The discovery and development of safe and effective MDR reversal agents is urgently required.Dioscin is a constituent of plants in the genus Discorea inclusive of medicinal plants such as 6ioscorea nipponica Makino and Dioscorea zingiberensis C.H.Wright(Dioscorea-ceae).Pharmacological research has demonstrated that dioscin has anti-inflammatory,lipid-lowering,anticancer,and hepatoprotective effects.Furthermore,it has been widely used as an important raw material for the synthesis of steroid hormone drugs.Whether dioscin can impact the activity of ADR in human leukemic cells and human breast cancer cells and methotrexate absorption is presently unclear.In the current study,we used K562 cells(a human leukemic cell line),K562/ADR((a ADR-resistant erythroleukemic cells derived from parental cells(K562)),Caco-2 cells(which express numerous characteristics of differentiated human small intestine cells,MCF-7 cells(a human breast cancer cell lines),MCF-7/ADR cells(a ADR-resistant erythroleukemic cells derived from parental cells(MCF-7))and intestine in rats.To understand the molecular mechanism involved,the effect of dioscin on MDR1 expression and the relationship between MDR1 expression and NF-κB activity were also investigated.Part I Dioscin restores the activity of the anticancer agent adriamycin in multidrug-resistant human leukemia K562/Adriamycin cells by down-regulating MDR1 via a mechanism involving NF-κB signaling inhibitionObjective:The purposes are to investigate the reversal effect of dioscin on multidrug resistance in K562/adriamycin cells and to clarify the molecular mechanisms involved.Methods:Human leukemic cell line K562 and its MDR counterpart K562/adr were used to examine the effect of dioscin on cytotoxicity of ADR by MTT assay.Effects of dioscin on MDR1 mRNA and protein expression and phospho-IκB-α expression in K562 and K562/adr cells were analyzed by Western blot or Q-RT-PCR method.Intracellular ADR retention was measured by flow cytometry.A dual-luciferase reporter assay system was used to determine the effect of dioscin on MDR1 or NF-κB promoter activity.Results:High level of MDR1 mRNA and protein and reduced adriamycin retention were investigated in K562/adr cells compared with their parental K562 cells.MDR1 mRNA,MDR1 protein expression,MDR1 promoter and NF-κB promoter activity in K562/adr cells were significantly inhibited by dioscin treatment.The suppression of MDR1 mRNA and MDR1 protein was accompanied by recovery of intracellular drug accumulation,suggesting that dioscin reversed the MDR phenotype by inhibiting the efflux function of MDR1 to ADR.Moreover,NF-κB activity and p-IκB-a expression were inhibited by dioscin,suggesting that dioscin inhibited NF-κB through down-regulating p-IκB-a expression.Conclusion:Dioscin could reverse the MDR induced by ADR in K562/adr cells by down-regulating MDR1 expression,at least in part,via inhibiting NF-κB signaling pathway activity.Part II Dioscin enhances methotrexate absorption by down-regulating MDR1 in vitro and in vivoObjective:To investigate the enhancing effect of dioscin on the absorption of methotrexate(MTX)and clarify the molecular mechanism involved in vivo and in vitro.Methods:In situ intestinal perfusions,in vitro everted intestinal sac preparation,histological examination of intestine slices in rats and bi-directional transport assay with Caco-2 cells,Western blotting,Quantitative RT-PCR,dual-luciferase reporter assay system were employed.Results:Dioscin increased MTX chemosensitivity and transepithelial flux in the absorptive direction,significantly inhibiting multidrug resistance 1 mRNA and protein expression and MDR1 promoter and nuclear factor κ-B activity in Caco-2 cells.Moreover,inhibitor κB-α degradation was inhibited by dioscin.Dioscin enhanced MTX transepithelial flux by down-regulating MDR 1 expression through a mechanism that involves NF-κB signaling pathway inhibition.Dioscin strengthened MTX absorption by inhibiting MDR1 expression in rat intestine.In addition,even though MTX is absorbed into the enterocytes,there was no increase in toxicity observed,and that,in fact,decreased toxicity was seen.Conclusions:The molecular mechanism for the enhancing effect of dioscin on the absorption of MTX is explained by its inhibitory effects on intestinal MDR1,at least in part,by down-regulating NF-κB activity.Part III Dioscin ameliorate the activity of the anticancer agent adriamycin in human breast cancer MCF-7 Cells and MCF-7/ADR Cells by Down-Regulating MDR1 via a Mechanism Involving NF-κB Signaling InhibitionObjective:To investigate the ameliorating effect of dioscin on activity of ADR in human breast cancer MCF-7 and MCF-7/ADR Cells and clarify the molecular mechanisms involved.Methods:Human breast cancer cell line MCF-7 and its MDR counterpart MCF-7/ADR were used to examine the effect of dioscin on cytotoxicity of adriamycin by MTT assay.Western blotting,Quantitative RT-PCR,dual-luciferase reporter assay system were employed to determine its molecular mechanisms.Results:Dioscin increased ADR chemosensitivity and transepithelial flux in the absorptive direction,significantly inhibiting multidrug resistance 1 mRNA and protein expression and MDR1 promoter and nuclear factor κ-B activity in MCF-7 and MCF-7/ADR Cells,respectively.Moreover,inhibitor κB-α degradation was inhibited by dioscin,suggesting that dioscin inhibited NF-κB through down-regulating p-IκB-αexpression.Conclusion:Dioscin increased ADR chemosensitivity in MCF-7 and MCF-7/ADR cells by down-regulating MDR1 expression by a mechanism that involves the inhibition of the NF-κB signaling pathway.These findings provide evidence to support the further investigation of the clinical application of dioscin as a chemotherapy adjuvant.