| Cancer is one of the major diseases to human health and the morbidity increased gradually. Chemotherapy is the effective means of cancer treatment. The occurance of multidrug resistance (MDR) impact the effect of chemotherapy. Therefore, the development of low toxicity, effective reversal agents has very vital significance. In this study, we selected chemical extracts of Herba Rabdosiae Rubescentis which has reversal effect, oridonin and rosmarinic acid as research materials to study reversal effect and reversal mechanism on SGC7901/ADR cells.The methods were as follows:(1) The cross-resistance assays of human gastric cancer SGC7901/ADR cells to adriamycin, cisplatin and vincristine were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. MTT assay was used to determine the cytotoxicity of drugs, in order to choose their non-cytotoxic doses. The reversal effect of drugs was also determined by MTT assay.(2) Flow cytometry was used to determine the effect of drugs on intracellular accumulation of adriamycin and Rhodamine123, the efflux of Rhodaminel23. The expression of P-glycoprotein was examined by immunofluorescence technique.(3) Semiquantitative RT-PCR analysis was used to determine the effect of drugs on the transcription of MDR1gene.(4) HPLC was used to determine the fingerprints of chloroform and ethyl acetate of Herba Rabdosiae Rubescentis.The results were as follows:(1) The resistance index (RI) to adriamycin, vincristine, cisplatin were11.68,2.89and2.97in SGC7901/ADR cells. Two non-toxic dose concentrations of extract of chloroform, extract of ethyl acetate, oridonin and rosmarinic acid were used in follow experiment, chloroform part:10μg/mL,50μg/mL; ethyl acetate part:12μg/mL,60μg/mL oridonin:2μM,10μM; rosmarinic acid:2.4Mm,12μM.10μM verapamil, extract of chloroform (10μg/mL,50μg/mL), extract of ethyl acetate (12μg/mL,60μg/mL), oridonin (2μM,10μM), and rosmarinic acid (2.4Mm,12μM) treated SGC7901/adr cells, we calculated the IC50were3.73,9.25,6.06,8.29,5.63,4.39,3.42,11.02,5.71, and the reversal fold were5.32,2.15,3.28,2.39,3.53,4.53,5.81,1.80,3.48.(2) The intracellular accumulation of adriamycin and Rhodamine123were increased remarkably and the efflux of Rhodamine123were decreased after SGC7901/ADR cells were incubated in the presense of extract of chloroform, extract of ethyl acetate, oridonin and rosmarinic acid. The MFI associated with adriamycin was2.58,2.52,4.25and3.75fold compared with SGC7901/Adr cells control group in the intracellular accumulation of adriamycin, the MFI associated with Rhodamine123was1.38,1.42,2.17and1.37fold compared with control group in the intracellular accumulation of Rhodamine123, and the MFI associated with Rhodamine123was1.16,2.15,3.01and2.30in the efflux of Rhodamine123.(3) The mRNA and protein expression (P-gp) of MDR1were inhibited by extract of chloroform, extract of ethyl acetate, oridonin and rosmarinic acid in SGC7901/ADR cells. P-gp decreased17.10%,14.17%,18.83%,18.06%and the mRNA of MDR1decreased59.37%,50.01%,59.60%,58.29%compared with control group.(4) The HPLC fingerprint chromatograms of Rabdosia rubescens’s ethyl acetate and chloroform sites are also very similar, and each site had eleven characteristic peaks. Oridonin is peak8in the fingerprint chromatograms, and the relative content were13.84%and15.46%respectively.Conclusion:Extract of chloroform, extract of ethyl acetate, oridonin and rosmarinic acid had the reversal effect significantly on SGC7901/ADR cells, and the mechanism of reversal effect might be associated with decreasing MDR1gene and P-glycoprotein. |
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