Ganoderma Extract Reverse Multidrug Resistance And Its Mechanism Of Sgc7901 / Adr Cells Research

Purpose:The tumor is one of the major diseases that endanger human health. Chemotherapy play an important role in therapy of gastric cancer. However, multidrug resistance(MDR) of tumors resulted in that process have become the main obstacle in chemotherapeutic treatment of tumor cells. P-glycoprotein is the most well known of the transmembrane efflux transporters. As the development of modern pharmaceutical technology, the P-gp inhibitors have caused the attention of people. G. lucidum often used as an adjuvant drugs of chemotherapy in cancer therapy and have good efficacy in clinical treatment of tumor. Erg is an important plant sterols and rich in G. lucidum. Erg itself has other physiological activity, such as anti-inflammatory, anti-tumor, anti-aging etc. In our research, we focused on the MDR reversal effect of Ganoderma Lucidum extracts on human gastric cancer SGC7901/ADR cells, the mechanism of its MDR reversing effect is also explored. It can provide the theoretical basis and experimental evidence for discovering and researching MDR reversal agent from Ganoderma Lucidum.Methods:(1) The determination of cell toxicity and reversal effectApply MTT method to measure direct cell toxicity on SGC7901/ADR cells by each drug treatment group. ethyl acetate part(GE), butanol part(GB), ergosterol(Erg);60%Ethyl acetate part(GER). To observe the reversal effect of each drug treatment group which has sreened for nontoxicity concentration to SGC7901/ADR cells by MTT. Using MTT method measured the reversal effect on SGC7901and SGC7901/ADR cells by ADR, VCR and DDP.(2) The determination of Rh123/ADRimycin accumulation and the P-gp expression detectionApply Flow cytometry to detect accumulatiom of intracellular adriamycin and Rh123. Apply Flow cytometry to survey expression of P-gp in SGC7901/ADR cells which were treated with each drug treatment group through indirect fluorescent labeling method.(3) The transcription of MDR1in cells by RT-PCR The specific gene transcription level was determined semiquantitatively by calculating the ratio of density metric value from specific genes expressed in retation to the internal standard (MDR1gene expression/β-actin gene expression).(4)Macroporous resin methodExtract from Ganoderma lucidum’s ethyl acetate sites were isolated using macroporous resin method.(5) The detection of active sites by Hing performance liquid chromatography(HPLC)Apply Hing performance liquid chromatography to detect the fingerprints of Ganoderma lucidum’s active chemical extracts.Results:(1) MTT assay was used to detec the cytotoxicity of three sites extracts on SGC7901/ADR cells, choose two concentrations for each as a non-toxic doses for the experiment:GE:20μg/mL,100μg/mL; GB:4μg/mL,20μg/mL; Erg:1μM,5μM; GER:4μg/mL,20μg/mL.Being treated by verapamil, GE(20μg/ml,100μg/ml), GB(4μg/ml,20μg/ml), Erg(1μM,5μM); GER(4μg/mL,20μg/mL), we can calculate the reversing multiple were4.23,3.71,4.53,2.64,2.12,3.92,4.84,3.33,3.64;(2)Intracellular Rh123and ADRiamycin were increased by GE(20μg/ml,100μg/ml), GB(4μg/ml,20μg/ml), Erg(1μM,5μM); GER(4μg/mL,20μg/mL);The expression levels of P-gp in SGC7901cells and SGC7901/ADR cells treated with GE(20μg/ml,100μg/ml), GB(4μg/ml,20μg/ml), Erg(1μM,5μM); GER(4μg/mL,20μg/mL) were analyzed by flow cytometry. The expression level of P-gp was decreased by21.5%,32.7%,20.5%,27.8%,20.4%,32.7%,29.4%,37.0%.(3) The MDR1gene expression level in SGC7901/ADR cells treated with GE(20μg/ml,100μg/ml), GB(4μg/ml,20μg/ml), Erg(1μM,5μM); GER(4μg/mL,20μg/mL) were decreased by32.16%,50.27%,25.34%,41.53%,36.56%,57.63%,24.69%,50.18%.(4) The HPLC fingerprint chromatograms of Ganoderma Lucidum’s ethyl acetate and butanol sites are very similar, and it is twelve characteristic peaks. On the basis of ergosterol standards, which is peak12in the fingerprint chromatograms. Quantitative assay results showed that the ergosterol in Ganoderma Lucidum’s ethyl acetate sites1.70%and in butanol sites1.91%.Conclusion:GE, GB, Erg, GER could significantly inhibit the role of P-glycoprotein and increase the sensitivity of SGC-7901/ADR cells for anticancer drugs to reverse MDR. It can supply a new effective drug for MDR targeted to P-gp.