A Study On P-glycoprotein Inhibitory Activities Of Several Endophytic Fungi From Medicinal Plants

The overexpression of high level multidrug resistance（MDR）gene in tumor cells is the internal cause of tumor cell resistance and the main factor leading to the failure of tumor chemotherapy.Seeking an inhibitor that inhibits MDR activity is an very important way for the efficacy of anti-tumor drugs.The P-glycoprotein（P-gp）gene belongs to a family of MDR genes,and is a type of ATP-dependent drug molecular pump that pump intracellular drugs out of the cell via active transport.At the same time,P-gp is also an important medicinal target protein for anti-MDR.At present,although three generations of P-gp inhibitors have been developed,they are limited to clinical applications due to their cytotoxicity and other effects.To study the high-efficiency and low-toxicity P-gp inhibitors will help to overcome MDR in clinical application.In this study,internal salt method（MTS,modeling,cytotoxicity,in vitro reversal multiples）was used to evaluate the secondary metabolites of the strain and to obtain bioactive strain.Meanwhile real-time quantitative PCR（q PCR）and Western blot methods were employed to explore the molecular mechanism of the expression pattern of related genes reversing MDR in human breast cancer cells for the secondary metabolites from the bioactive strains.The specific results are as follows:1.Screening of the fungal metabolites from the bioactive strains that reverse tumor MDR.Constructing a human breast cancer（MCF-7）/adriamycin（ADM）drug-resistant cell line（MCF-7/ADM）model.A method of continuous induction at low-concentration was used to establish a human breast cancer drug-resistant cell line.The drug resistance measurement indicated that the IC50 values of human breast cancer cells and drug-resistant cells for doxorubicin were 19.453 and 99.39 respectively,and the drug resistance index RI was 5.11.The study also found that drug-resistant cell lines were also resistant to paclitaxel and cisplatin.Observing the morphology of the cells,it was found that the morphology of the cells induced by the drug changed significantly compared with the normal cells.A further complete evaluation of the established cells revealed that the expression of MDR genes ABCB1,ABCG2,ABCC1,ABCC2,ABCC3,ABCC4,ABCC5,ABCC6,ATM and CYP1A1 were significantly up-regulated after drug induction.In addition,it was found that the protein expression of P-gp in drug-resistant cells was significantly up-regulated via testing P-gp expression amount which indicated that this study has successfully constructed a human breast cancer resistant cell line with overexpression of P-gp.Testing the cytotoxicity of fungal metabolites.In order to exclude the secondary metabolites’P-gp direct inhibiting toxicity resulting the superposition of the cytotoxicity increasement of chemotherapeutic drugs,the cytotoxicity test of selected fungi was conducted;and the MTS method was used to detect the cytotoxicity of the secondary metabolites from 66 strains and found that all strains had low cytotoxicity at100μg·m L^-1,and their IC₅₀ values were higher than 100μg·m L^-1.This shows that the metabolites of the 66 strains all had low cytotoxicity and can be screened for the next step.Screening of fungal metabolites to reverse tumor MDR.The in vitro reversal multiple of the metabolites was tested by MTS method,and only 4 out of the 66 strains had good MDR reversal activity,Tubeufia rubra PF02-2、Cladosporium sp.02R2A、Nemania dendrobii S7S1A and Colletotrichum watphraense 03S2A,the reversal multiples are 3.79,3.04,3.04,3.29,respectively.2.Preliminary study on the mechanism of the bioactive strainsTo explore the possible mechanism of MDR reversal activity of the fungal secondary metabolites.The drug-resistant cell lines before/after treatment with secondary metabolites were tested at the transcription level for the expression patterns of related MDR genes（ABCB1,ABCC1,ABCC3,ABCC4,ABCG2,CYP1A1）.It was found that after treatment with PF02-2 secondary metabolites(90μg·m L^-1),the expressions of ABCB1,ABCG2,ABCC3,ABCC4 and CYP1A1 were down-regulated,and the expression of ABCC1 was up-regulated;At 20μg·m L^-1,the expressions of ABCB1,ABCG2,ABCC3 and ABCC4 were down-regulated,while that of ABCC1 and CYP1A1 were up-regulated.Treatment with 02R2A secondary metabolites showed that at 90μg·m L^-1,the expressions of ABCB1,ABCC1,ABCC3 and CYP1A1 were significantly down-regulated,and that of ABCG2 and ABCC4 were up-regulated,and at 20μg·m L^-1,the expressions of ABCB1、ABCG2、ABCC1、ABCC3、ABCC4CYP1A1 were down-regulated.Treatment with 03S2A secondary metabolites showed that at 90μg·m L^-1,the expressions of ABCB1,ABCG2,ABCC1,ABCC4 were significantly down-regulated,and ABCC3 expression was up-regulated;and at 20μg·m L^-1,the expressions of ABCB1 and ABCC3 was down-regulated,ABCG2 and ABCC4 had no significant difference;and the expressions of ABCC1 and CYP1A1 were up-regulated.Treatment with S7S1A secondary metabolites showed that at 90μg·m L^-1,the expressions of ABCB1,ABCC3 and CPY1A1 was down-regulated,the expressions of ABCC4 and ABCG2 was up-regulated,and ABCC4 had no significant difference;and at 20μg·m L^-1,the expressions of ABCB1,ABCC4 and ABCC3 was significant down-regulated,ABCG2 expression was up-regulated,and ABCC1 and CYP1A1 had no significant difference.At the protein level,the protein expression level of intracellular P-gp in drug-resistant cell lines before/after treatments with secondary metabolites was detected via Western blot.And it was found that at 20μg·m L^-1,the protein expression level of P-gp in the drug-resistant cell lines with the treatments of the secondary metabolites from all the bioactive strains was significantly reduced;while at 90μg·m L^-1,although the protein expression level of P-gp was reduced but less effective than a lower concentration.Based on the results above,it was found that the secondary metabolites of all the bioactive strains had different degrees of inhibitory expression effect on the ABCB1（P-gp）gene at different concentrations which indicated that these secondary metabolites had bioactive substance with inhibitory activity on P-gp.It is speculated that the secondary metabolites of the bioactive strains can reduce the efflux of drugs from the cell via inhibiting the expression patterns of multiple MDR related genes including P-gp gene and thus reverse MDR of tumor cells.