Effects Of Ganoderma Lucidum Polysaccharides Peptide On P-glycoprotein

OBJECTIVETo study the mechanism of interaction between Ganoderma lucidum polysaccharides peptide and P-glycoprotein by assessing the effects of Ganoderma lucidum polysaccharides peptide on the function and expression of P-glycoprotein, and investigating the effect of these componets on the Rhodamine-123bidirectional transport though Caco-2cells monolayer.METHODS1Cell culture and morphological validated of cellsCaco-2cell and ECV304cell were cultured with DMEM (Dulbecco’s Modified Eagle’s Medium) and RPMI-1640(Roswell Park Memorial Institute) respectively which contains10%FBS(Fetal Bovine Serum) in a humidified atmosphere of95%air and5%CO2at37℃. The expression of P-glycoprotein in the cells was identified by immunofluorescence assy.2Cytotoxicity studies in vitroMTT (diphenyltetrazolium bromide) assay was used to find the non-cytotoxicity dosage of Ganoderma lucidum polysaccharides peptide、 cyclosporine and verapamile in Caco-2and ECV304cells. The dosage at which the survival rate of cells is above90%was considered as the highest non-cytotoxic dosage.3Effect of Ganoderma lucidum polysaccharides peptide on P-glycoprotein functionCompared with the negative group, The effect of Ganoderma lucidum polysaccharides peptide on P-glycoprotein function was analyzed using Rh-123assay. Verapamile was used as positive control of down-regulation effect on the function of P-glycoprotein, flowcytometry was used to determine the intracellular Rh-123concentration.4Effect of Ganoderma lucidum polysaccharides peptide on P-glycoprotein expressionCyclosporine was used as positive control of up regulation effect on the expression of P-glycoprotein, and Caco-2cells without drug dealing were used as negative control. Flowcytometry was used to measure the expression of P-glycoprotein in Caco-2cells.5Effect of Ganoderma lucidum polysaccharides peptide on Rhodamine-123bidirectional transport through Caco-2cells monolayerThe Caco-2cells monolayer model was builded、identified, and verapamile was used as positive control drug to investigate the inhibitory effect on the function of P-glycoprotein. Then we investigated the effects of Ganoderma lucidum polysaccharides peptide on Rhodamine-123bidirectional transport though Caco-2cells monolayer.RESULTS1Cell culture and morphological validated of cellsBoth Caco-2cells and ECV304cells grew well in the experimental conditions.The expression of P-glycoprotein is plentiful in Caco-2cell but sparse in ECV304cell. Caco-2cells is suitable for studying Ganoderma lucidum polysaccharides peptide on the function and expression of P-glycoprotein, and ECV304cells is fit to be the negative control.2Cytotoxicity assay studies in vitroGanoderma lucidum polysaccharides peptide at concentrations of10-100μg/mL and verapamile at concentration of0.01-100μmol/mL and Cyclosporine at concentration of0.01-10μmol/L were found to be non-cytotoxic towards the Caco-2cells, these compounds were non-toxic to ECV304at the experimental concentrations.3Effect of Ganoderma lucidum polysaccharides peptide on P-glycoprotein functionCompared with the negative group, after incubated with cells for1h, The efflux quantity of Rhodanmine-123was reduced by8%,16%,19%and33%(p<0.05), compared with that of controls when being treated with Ganoderma lucidum polysaccharides peptide at10,20,50,100μg/mL. GLPP showed the concentration-dependent inhibitory effect on P-glycoprotein.4Effect of Ganoderma lucidum polysaccharides peptide on P-glycoprotein expressionAt the protein level, long term (72h) co-incubation of the Caco-2cells with different drugs, the fluorescent intensity of Ganoderma lucidum polysaccharides peptide group was reduced by10.6%、7.6%(p<0.05), compared with that of controls when being treated with Ganoderma lucidum polysaccharides peptide at50、100μg/mL. The lower group showed slight effect on the expression of P-glycoprotein. These results indicated that there were some synergistic reactions between the effects of the expression and function of P-glycoprotein. 5Effect of Lycium barbarum polysaccharide on Rhodamine-123bidirectional transport through Caco-2cells monolayerThe result of Rhodamine-123bidirectional transport was that the Papp of rhodamine-123from BL to AP was decreased by Ganoderma lucidum polysaccharides peptide, compared with the negative group. The ER of Rhodanmine-123was reduced by15%,18%when treated with GLPP at50,100μg/mL. The result of Rhodamine-123bidirectional transport accorded with the result of P-glycoprotein function.CONCLUSION.1Ganoderma lucidum polysaccharides peptide can inhibit the function of P-glycoprotein in1hour, and decrease the expression of P-gp after being incubated with Caco-2cells for72h.Therefore when used together with other drugs that are P-glycoprotein substrate, it may cause drug interactions. Since Ganoderma lucidum polysaccharides peptide can increace the concentration of P-glycoprotein substrate in tumor cells, we can suggest that Ganoderma lucidum polysaccharides peptide can reverse multidrug resistance (MDR) by downregulating the expressions of P-gp for the treatment of cancer.2The higher concentration of Ganoderma lucidum polysaccharides peptide can decreace the value of ER of Rhodamine-123, and the lower group showed slight effect on Rhodamine-123bidirectional transport. It suggest that Ganoderma lucidum polysaccharides peptide has a relative low affinity to P-gp, further research should be done for the identification of Ganoderma lucidum polysaccharides peptide as P-gp substrate.