Roles And Possible Mechanisms Of Heme Oxygenase-1 Influencing The Multidrug Resistance In Drug-resistant Hepatic Carcinoma Bel/FU Cell Line

Multidrug resistance (MDR) is a significant obstacle to providing effective chemotherapy to many Hepatic Carcinoma patients.Once the cells gain MDR,they will describe the phenomenon of simultaneous resistance to a broad of unrelated drugs,which affect the chemotherapeutic effectiveness and prognosis of the patients.Some molecular targets and proteins are gradually found,but the mechanisms of mutidrug resistance are not yet fully understood.Much effort needed has been extended to advance the mechanism in cancer cells,especially in the upstream signal pathways.In recent years,there reported some products in the degradation of heme catalyzed by heme oxygenase-1(HO-1),which can protect the cells from apoptosis. Elevated HO-1 expression and activity was found in various tumor tissues. The studies suggest that HO-1 exerts a role in controlling cell growth in a cell-specific manner which could be a new molecular target and upstream mechanism of MDR.In our studies,some methods are adopted to affect the HO-1 expression in Drug-resistant Hepatic Carcinoma Bel/FU Cells,then explores the effect and relevant mechanism of Heme Oxygenase-1 on chemosensitivity of the cells.Part one Effect of Heme Oxygenase on Multidrug Resistance of Drug-resistant Hepatic Carcinoma Bel/FU Cell LineObjective:To study the effect and relevant mechanism of Heme Oxygenase-1 on chemosensitivity of drug-resistant hepatic carcinoma Bel/FU cell line.Methods:①:Bel/FU cells were collected and planted in 96 well plates,cells were divided into three groups,two induced separately with 60uM Hemin and 10uM ZnPPⅨ,the other planted in normal condition.After several gradient concentrations of drugs(ADM,MMC,5-FU) are added into the wells,the drug sensitivity test of Bel/FU cells was carried out with MTT assay.②:After induced with different gradient concentrations of Hemin(15umol/L,30umol/L,60umol/L,120umol/L,240umol/L)and ZnPPⅨ(2umol/L, 6umol/L,10umol/L,14umol/L,18umol/L)for 24h,heme oxygenase-1 (HO-1),multidrug resistance-1(MDR-1) and Glutathione S-transferase(GST-π)mRNA level were determined by RT-PCR technique;P-glycoprotein(P-GP),GST-πprotein level were detected by Western blot analysis;Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of P-GP.Results:After Bel/FU cells being dealt with Hemin for 24h,there were significant differences in 50% inhibiting concentration (IC50) of chemical drugs between control groups and contrast group.IC50 of control groups increased obviously. Meanwhile,the mRNA expression of HO-1 and MDR-1 or GST-πin the cells of control group were observably higher than those in contrast group,of which were both consistent with the dose-effect curve. P-GP and GST-πprotein level expression appeared the analogous results. The curve of Rhodamine123 retention fluorescent intensity in the control cells removed left award from the place of contrast group,which indicated that the efflux pump function of P-GP was strengthened.The control groups by ZnPPⅨ,however,showed a contrary result. Lineal correlation analysis showed that the HO-1mRNA levels were significantly correlated with mRNA and protein expression of MDR-1 and GST-π.Conclusions:Inpacting heme oxygenas-1 can influence the chemosensitivity of Bel/FU cell line,which relates with its action in up(or down)-regulation of MDR-1 and GST-πexpression and efflux pump function of P-GP.The enzyme of HO-1 may be a new target of reversing multidrug resistance in Hepatic Carcinoma cells. Part two Effect of HO-1 Small Interfering RNA on Chemosensitivity of Adriamycin in Drug-resistant Hepatic Carcinoma Bel/FU Cell LineObjective:To investigate the chemosensitivity of Adriamycin and reverse mechanism of drug resistance in hepatic carcinoma Bel/FU cell line after heme oxygenase-1 being blocked with siRNAs.Methods:Design and prepare the small interfering RNAs aiming at HO-1 andβ-actin gene.The cells were divided into 4 groups as normal group(only in nutritive medium),blank contrast group(medium contain blank liposome),RNA intefering group(medium contain HO-1 siRNAs liposome)and negative group(medium containβ-actin siRNAs liposome).After intubating for 24 hours, HO-1 andβ-actin mRNA level were determined by RT-PCR technique;P-GP,GST-πprotein level were detected by Western blot analysis;Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of P-GP.In additively, different gradient concentrations of Adriamycin were added into plant wells,then MTT assay was carried out to detect the chemosensitivity of Bel/FU cells.Results:After Bel/FU cells being dealt with different methods for 24h, HO-1 mRNA level almost disppeared in RNA intefering group while other groups had no change; P-GP protein level by Western blot analysis deceased in RNA intefering group contrasted with those of another three groups; The curve of Rhodamine123 retention fluorescent intensity in the control cells removed right award from the place of contrast group,which indicated that the efflux pump function of P-GP was weakened; MTT assay indicated that the cell activity decreased significantly and 50% inhibiting concentration (IC50) of Adriamycin decreased obviously in the control group. Conclusions:After Bel/FU cells being dealt with HO-1 siRNAs liposome, the Adriamycin chemosensitivity of Drug-resistant Bel/FU cells increased,which achieved by down-regulation of protein level expression and efflux pump function of P-GP. Part three Effect of Astragulus Injection on Chemosensitivity of Drug-resistant Hepatic Carcinoma Bel/FU Cell LineObjective:To study the effect and relevant mechanism of Astragulus injection on chemosensitivity of drug-resistant hepatic carcinoma Bel/FU cell line.Methods:The drug sensitivity test of Bel/FU was carried out with MTT assay.The distribution of P-glucoprotein(P-GP) in the cells were determined by immunohistochemical stainning.Rhodamine123 retention fluorescent intensity measured by flow cytometry was used to detect the function of P-GP. HO-1mRNA level was determined by RT-PCR technique.Results:After Bel/FU cells being dealing with Astragulus injection for 24h,there were significant differences in 50% inhibiting concentration (IC50) of chemical drugs between control groups and contrast group(P<0.01).IC50 of control groups decreased obviously.Immunohistochemistry showed that P-GP expression were significantly lower in the cells treated with 8ul/ml Astragulus injection than those of contrast group(Z=-2.363, P=0.018).The curve of Rhodamine123 retention fluorescent intensity in the control cells removed right award from the place of contrast group,which indicated that the efflux pump function of P-GP was weakened.Meanwhile,the mRNA expression of HO-1 in the cells of control group were observably lower than those of in contrast group(0.316±0.08vs0.624±0.173, P<0.01).Conclusions:Astragulus injection can enhance the chemosensitivity of Bel/Fu cell line,which may relate with its action in decreasing protein expression and efflux pump function of P-GP concomitant with the decrease of HO-1mRNA level.