Study On The Reversal Of Multidrug Resistance Of Human Hepatocellular Carcinoma And Related Mechanisms

Multidrug resistance (MDR) is the protection of tumor cell population against numerous drugs differing in structure and mechanisms of influence on the cells. MDR is also one of the major causes of failure of chemotherapy of human maligancies. It is related to many active mechanisms. Often several different mechanisms are switched on in the cells, but usually one major mechanism is operating. The active mechanisms read as follows: a) activation of transmembrane proteins effluxing different chemical substances from the cells (P-glycoprotein is the most known efflux pump); b) activation of the enzymes of glutathione detoxification system; alteration of the genes and the proteins involved into the control of apoptosis (especially P53 and Bcl-2); changes of activity and quantity of topoisomerases. The tumor cell MDR and looking for little toxic and more effective chemotherapy drug has long been an interesting subject. The first part of this paper deals with study of Bupleurun Chinese DC (BCDC) on the MDR reversal of human hepatocellular carcinoma Bel-7402 cell line and related mechanisms. The reversal action of BCDC, IC50 value and synergic action of vincristine (VCR) and BCDC were determined by MTT assay. The reversal action of BCDC was also observed by clone forming text. The intracellular VCR concentration was examined by high performance liquid chromatography (HPLC) and the change of [Ca2+] I in Bel-7402 was measured by double wavelength spectrofluorometer. Induction of cell cycle arrest and apoptosis were determined by flow cytometry (FCM) and light microscopy. MDR1 expression of Pgp was examined by immunochemical assay and determination of gene expression of MDR1 ,MRP, glutathione S-transferase(GSTpi) and topoisomerase Ⅱαof Bel-7402 cell line were investigated using reverse transcription polymerase chain reaction (RT-PCR).The second part of this paper consists of study of P53 gene on the MDR reversal of human hepatocellular carcinoma Bel-7402 cell line and related mechanisms. First the cloning plamid pMD-18-T-P53 was constructed and then plasmids pCR3.1 and pMD-18-T-P53 were digested by restriction endonucleases BamH I and EcoR I simultaneously. The eukaryotic expression plasmid pCR 3.1-P53 was constructed by inserted the coding sequence of P53 from pMD-18-T-P53 into the multi cloning sites of eukaryotic expression vector pCR 3.1.The recombinant plasmid pCR 3.1-P53 was confirmed by restriction endonuclease digestion, PCR and seqencing. The pCR 3.1-P53 was introduced into the Bel-7402 cell line by lipofectamine transfection reagent. The cell proliferation and chemosensitivity after the treatment of VCR were measured by MTT assay. The apoptosis of transfection Bel-7402 cell line was observed by morphologic method and FCM. MDR1 expression of Pgp was examined by immunochemical assay and determination of gene expression of MDR1 ,MRP, glutathione S-transferase(GSTpi) and topoisomerase Ⅱαof transfection cell line were investigated using reverse transcription polymerase chain reaction(RT-PCR).The results are as follows:1. BCDC on the MDR reversal of human hepatocellular carcinoma Bel-7402 cell lineThe reversal effect of BCDC was evaluated using Bel-7402 cell line with innate resistance in this study. The IC50 value of BCDC crude extract was 2000.68μg/ml. BCDC at 390μg/ml concentration was no toxic but had a toxic effect on Bel-7402 cell line with VCR. The cytotoxicity of 390μg/ml BCDC and VCR to Bel-7402 cell line was more remarkable than only with VCR(P<0.01). The fold reversal of BCDC combining with VCR was 7.8 but it was lower than with verapamil of 15.6 fold. While BCDC concentrations lower than 390μg/ml had less toxic, it had no synergetic effect with VCR. BCDC concentrations higher than 400μg/ml were not suitable due to cytotoxicitiy to tumor cells. The reversal effect of BCDC was confirmed by clone forming text. The inhibited effect of BCDC on Bel-7402 cell line correlated well with BCDC concentration. However, Bel-7402 cell line treated with VCR and BCDC showed significantly...