Study On The Reversal Of P-glycoprotein-mediated Tumor Multidrug Resistance By Ginsengenins Derivatives

Malignant tumors pose a serious threat to human health.Multidrug resistance is one of the major reasons of cancer treatment failure.The typical multidrug resistance transporter protein,P-glycoprotein(P-gp),has been proven to be associated with multidrug resistance.Paclitaxel,doxorubicin,vincristine,and tyrosine kinase inhibitors are all P-gp substrates.That is,the drug will be actively transported outside the of cell through P-gp-mediated transport,which could lead to the reduction of intracellular accumulation,and the reduction of drug efficacy,and ultimately lead to drug resistance.In recent years,great progress has been made in the research of P-gp inhibitors,but there are still some problems such as poor efficacy,high toxicity and undesirable pharmacokinetics.Therefore,it is necessary to develop novel drugs with high efficiency and low toxicity.Natural products and their derivatives have the characteristics of safety,effectiveness,stability and controllable quality.In recent years,more and more scholars have devoted themselves to screening active substances from natural products and their derivatives.It has been reported in the literatures that ginsengenins have good antimultidrug resistance activity.For example,protopanaxadiol(PPD),(20R)-panaxadiol(PD),protopanaxatriol(PPT),(20R)-panaxatriol(PT),Pyxinol,(24S)-Pyxinol all have the effect of reversing P-gp-mediated multidrug resistance in tumors.In order to further enhance the activity of above ginsengenins in reversing multidrug resistance of tumors,a series of amino acid ester derivatives of ginsengenins were prepared using structural modification techniques;derivatives with potential antimultidrug resistance activity were virtually screened using molecular docking technology;A549 cells and MCF-7 cells were used to determine the reversal effect of potential active derivatives on multidrug resistance of tumor,and the active derivative was selected;the mechanism of the active derivatives was explored at the protein and metabolite levels by using western blot,intracellular Rh123 accumulation assay,P-gp ATPase activity assay and differential endogenous metabolite identification.The main findings in this dissertation are as follows:1.Synthesis of amino acid ester derivatives of ginsengeninsIn order to improve the water solubility of ginsengenins and to enhance the activity to reverse tumor drug resistance,11 amino acids(glycine,valine,methionine,lysine,glutamic acid,aspartic acid,phenylalanine,tyrosine,tryptophan,threonine,proline)were selected to be esterified with 6 ginsengenins(PPD,PD,PPT,PT,Pyxniol,(24S)-Pyxinol)at the C-3 position,and a total of 66 amino acid ester derivatives were prepared.All the derivatives were separated and purified by silica gel column chromatography and recrystallization,and their structures were identified by physicochemical property analysis,Ultra Performance Liquid Chromatography Quadrupole-Time of Flight Mass Spectrome(UPLC-Q/TOF-MS)and Nuclear Magnetic Resonance(NMR)techniques.Among them,19 derivatives were new compounds synthesized for the first time.2.Virtual screening of P-gp inhibition by amino acid ester derivatives of ginsengeninsThe applicability of docking software such as Auto Dock Vina,Auto Dock,and Discovery Studio was investigated by using P-gp as a receptor protein,P-gp protoligand as a ligand molecule,and scoring functions,Root Mean Square Deviation(RMSD),visual analysis,and Key Interaction Score System(KISS)as standards.The results showed that Auto Dock Vina was more suitable for docking P-gp receptor protein with ligand molecules.Using Auto Dock Vina docking software,6 ginsengenins,66 amino acid ester derivatives and 36 urea derivatives previously reported by the research group as ligands were used for molecular docking with P-gp receptor.Using docking binding energy,binding sites and key residues numbers as screening criteria,the antitumor multidrug resistance activities of each ligand were virtually screened.The results showed that 5amino acid ester derivatives(1g,3f,5j,6g,6j)and 5 urea derivatives(7m,8d,8l,8m,8q)were potential antitumor multidrug resistance active substances.3.Study on the reversal effect of ginsengenins derivatives on multidrug resistance of tumor(1)Evalution of the activity of reversing multidrug resistance in tumorsCCK-8 method was used to evaluate the toxicity of the above 10 potential active compounds against human lung cancer sensitive cells(A549)and their drug-resistant cells(A549/Tax),human breast cancer sensitive cells(MCF-7)and their drug-resistant cells(MCF-7/DOX).and the non-toxic dosage of each compound was all 10 μM.The potential active compounds were co-administered with antitumor drugs(paclitaxel or doxorubicin),and the half inhibition concentration(IC50)of the antitumor drugs in drug-resistant cells before and after the intervention of the potential active compounds were determined,and the Resistance Index(RI)and Reversal Fold(RF)were calculated.The results showed that compound 6g((24S)-3-L-threonyl-Pyxinol)had the strongest activity in reversing multidrug resistance in A549/Tax cells,with a RF of 15.31-fold,followed by compound 8m((24S)-3β-[3,5-bis(trifluoromethyl)phenylureido]-Pyxinol)with RF of 9.90-fold.(2)Effect of compound 6g on P-gpA549 and A549/Tax cells were used to investigate the effect of compound 6g on P-gp expression by western blot assay;intracellular rhodamine 123 accumulation assay was performed to detect the effect of compound 6g on P-gp efflux function by using live cell staining;the effect of compound 6g on P-gp ATPase activity was detected using Pgp-GloTM kit.The results showed that compound 6g had no significant effect on P-gp protein expression level,suggesting that its reversal effect was not achieved by inhibiting P-gp expression;compound 6g could dose-dependently enhance the accumulation of P-gp substrate(rhodamine 123)in drug-resistant cells and effectively inhibit the efflux function of P-gp,and it could stimulate the consumption of P-gp ATPase.It is suggested that compound 6g could act as a substrate of P-gp and inhibit the efflux function of Pgp by competitively binding to the binding sites of antitumor drugs,thereby exerting the effect of reversing multidrug resistance of tumor.(3)Metabolomic study of compound 6g in drug-resistant lung cancer cellsUsing metabolomics based on UPLC-Q/TOF-MS technology,14 endogenous metabolites(Chenodeoxycholic acid glycine conjugate,Phytosphingosine,Sphinganine,5,6-Epoxy-8,11,14-eicosatrienoic acid,Lyso PC(18:1(9Z)/0:0),9,10-Epoxyoctadecenoic acid,PC(18:1(9Z)e/2:0),Lyso PC(O-18:0/0:0),Arachidonic acid,3a,7a,12a-Trihydroxy-5β-cholestan-26-al,Linoleic acid,PC(16:0/16:0),3β,7aDihydroxy-5-cholestenoate,PE(P-18:0/20:4(5Z,8Z,11 Z,14Z))and 6 metabolic pathways(Linoleic acid metabolism,Glycerophospholipid metabolism,Arachidonic acid metabolism,Primary bile acid biosynthesis,Ether lipid metabolism,Sphingolipid metabolism)involved closely associated with the reversal of tumor multidrug resistance effects of compound 6g were identified from A549/Tax cells.Among these,7metabolites,including Phytosphingosine,Sphinganine,Lyso PC(18:1(9Z)/0:0),PC(18:1(9Z)e/2:0),Lyso PC(O-18:0/0:0),PC(16:0/16:0),PE(P-18:0/20:4(5Z,8Z,11 Z,14Z))and their involved Sphingolipid metabolism pathway and Glycero-phospholipid metabolism pathway are closely related to P-gp ATPase activity and could alter P-gp lipid environment,thereby inhibiting the transport function of P-gp and acting to reverse the multidrug resistance in tumor.