Study On Multidrug Resistance Reversal And Molecular Mechanism Of Uncaria Alkaloid

Multidrug resistance(MDR)has become the important cause of the failure of chemotherapy,and the increase of drug effluxcaused by over-expression of ATP binding cassette(ABC)transfer family proteins is one of thecauses of MDR,thus,developing ABC-mediated reversal agents becomesan effective mean to overcome MDR.However,at present,all of three generations of ABC-mediated reversal agents failed to come to the market because of their low specificity,big toxicity and weak body affinity.In recent years,screening high effective and low toxic MDR reversal agents from the traditional Chinese medicineshas become the hot topic study.Uncaria alkaloids are isolated from traditional Chinese medicine uncaria which has been used in clinic for a long time.Rhynchophylline(Rhy),Isorhynchophylline(Irhy),Corynoxeine(Cory)and Isocorynoxeine(Icory)are the major constituents in uncaria oxindolealkaloids,and Hirsutine(HT)and Hirsuteine(HST)are the major constituents in uncaria non-oxindolealkaloids.Some studies have reported that uncaria alkaloid extracts and the monomer compositionscould inhibit proliferation of variouscancer cells and promote apoptosis of variouscancer cells,but there has been little reported research in uncaria alkaloids reversal activity.This study demonstrate that uncaria oxindole alkaloid extracts(U1),uncaria non-oxindole alkaloidextracts(U2)and their six monomer compositionscan reverse MDR,and Icory with lower toxicity and most effective reversal MDR activity is chosen as representative to study the the molecular mechanism of uncaria alkaloid.Objective:To evaluate the reversal MDR activity of uncaria alkaloids,and screening the most active monomer composition in order to further evaluateits reversal MDR activity and study its molecular mechanism.Thus,to provide scientific evidence for the developinguncaria alkaloids intopotential reversal agents.Method:1.The evaluation of the MDR reversal activity of uncaria alkaloids:(1)Using MTT method to determine the cytotoxicity of U1,U2,Rhy,Irhy,Cory,Icory,HT and HST in HepG2 cells and HepG2 / ADM cells,respectively;(2)Using MTT method to evaluate the reversal MDR activity of U1,U2 and six monomer compounds,and to screenthe most effective compound;(3)Using MTT method to further evaluate the reversal MDR activity of Icory in ABCB1-overexpressing cell lines and its sensitive cell lines;(4)Using MTT method to further evaluate the reversal MDR activity of Icory in ABCC1-and ABCG2-overexpressing cell lines and their sensitive cell lines;2.The molecular mechanisms study of Icory:(1)Using microplate reader and fluorescence image to detect intracellular accumulation of DOX affected by Icory;(2)Using microplate reader to detect intracellular efflux of DOX affected by Icory;(3)Using qPCR and Western Blot methods to detect the expressions of ABCB1 mRNA and protein affected by Icory;(4)Using Pgp-GloTM ATPase kits to defect ABCB1 ATPase activity affected by Icory;(5)Using MTT method to detect the sustainability of MDR activity by Icory;(6)Using Docking method to analyze the bindingmode and binding sites between Icory and ABCB1.Result:1.U1,U2,Rhy,Irhy,Cory,Icory,HT and HST have reversalMDR activity,and Icory exhibits the strongest activity;2.Icory can increase the sensitivity of DOX,Vincristine(VCR)and Paclitaxel in ABCB1-overexpressing cell lines(HepG2/ADM and MCF-7/ADR cells),but can not increase the sensitivity of Cisplatin in HepG2/ADM and MCF-7/ADR cells;3.Icory can not increase the sensitivity of antineoplastic drugs in ABCC1-overexpressing HEK293/MRP1 cells and ABCG1-overexpressing S1-M1-80 cells;4.Icory can increase the accumulation ofDOX in HepG2/ADM and MCF-7/ADR cells;5.Icory can inhibit the efflux of DOX in HepG2/ADM cells;6.Icory has no obvious influence on the expression of ABCB1;7.Icory can stimulate the ABCB1 ATPase activity;8.The reversal effect of Icory depends on its concentration;9.Icory occupiesthe important binding sites of ABCB1 substrates,and Icory binds toABCB1 at the same site as VRP but that the binding site does not completely overlap with that ofVRP.Conclusion:Uncaria alkaloids have reversal MDR activity,and Icory reverse specifically ABCB1-mediated MDR,but dose not reverse ABCC1-and ABCG2-mediated MDR;Icory does not affect the expression of ABCB1,but stimulate ABCB1 ATPase to competitively inhibitABCB1 pumping function,and further to reduce the efflux of antineoplastic drugs and increase the intracellular drug accumulation whichresults in MDR.