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Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR)in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma (PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Westem blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR)alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate)was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ,in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ,activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.
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Aim To observe whether the inhibition of PI3K/PKB signal pathway by LY294002[PI3K pathway inhibitor] could improve the sensitivities of human gastric carcinoma cell line SGC7901 and SGC7901/VCR to anti-cancer drugs.Methods The sensitivities of SGC7901 and SGC7901/VCR to chemotherapeutic drug VCR were detected by MTT.The MDR1 and XIAP mRNA expression levels were evaluated by semi-quantitative RT-PCR,and their protein levels were detected by immunocytochemistry.The PKB and phospho-PKB protein levels were detected by Western blot and the apoptosis ratio was detected by flow cytometry.Results 2?10-5mol?L-1LY294002 enhanced the sensitivities of SGC7901 and SGC7901/VCR cells to VCR(P
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AIM: To investigate the effect of neferine (Nef) on human gastric carcinoma cell line with multidrug resistance (MDR). METHODS: The cytotoxic effect of vincristine (VCR) was evaluated by MTT assay. The cell apoptosis induced by VCR was determined by flow cytometry, and the expression of P-glycoprotein (P-gp) and multidrug-resistance-associated protein (MRP) in cells was examined by immunofluorescence flow cytometry. RESULTS: MTT assay showed that Nef at the concentration of 5 ?mol?L~(-1) to 10 ?mol?L~(-1) have no cytotoxicity to parent human gastric carcinoma cell line (SGC7901) and its VCR-resistant variant cell line (SGC7901/VCR). The IC_(50) value of VCR to SGC7901 cell line was 0.06 mg?L~(-1)and that of to SGC7901/VCR cell line was 2.32 mg?L~(-1), which indicated SGC7901/VCR cell line were 39 times more resistant to VCR in comparison with the parent SGC7901 cell line. After treatment with Nef at the concentrations of 2.5, 5 and 10 ?mol?L~(-1), the IC_(50) value of VCR to SGC7901/VCR cell line decreased to 0.34, 0.12 and 0.05 mg?L~(-1), respectively and those increased by 6.8-, 18.1- and 43.8- fold in the chemosensitivity, respectively. Flow cytometry showed that SGC7901/VCR cells were resistant to apoptosis induced by VCR. After 24 h treatment with Nef (2.5, 5 and 10 ?mol?L~(-1)) and VCR, the apoptosis of SGC7901/VCR cells increased, which indicated Nef could abolish resistance of SGC7901/VCR cells to VCR-induced apoptosis. Furthermore, the action of Nef was more potent than verapamil. The expression of P-glycoprotein and multidrug resistance associated protein was strongly positive in SGC7901/VCR cells, and the expression level of P-gp and MRP in SGC701/VCR cells was significantly down-regulated at 24 h after treatment with Nef (10 ?mol?L~(-1)). CONCLUSIONS: Nef can reverse MDR in multidrug-resistant human gastric carcinoma SGC7901/VCR cell line. Its mechanism might be associated with down-regulation of expression of P-gp and MRP in SGC7901/VCR cells.
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Objective To investigate the reversing effects on drug resistance of gastric carcinoma by suppressing PI3K/PKB signal pathway,and explore its implicated mechanism.Methods MTT assay was used to test the inhibitory effects of VCR alone or VCR in combination with PI3K/PKB inhibitor,LY294002 on SGC7901/VCR cells.The SGC7901 treated with or without LY294002 served as control.The protein levels of PKB and phospho-PKB in the above VCR cells were determined by Western blot analysis.The mRNA expressions of MDR1,Bax and Bcl-2 were evaluated by semi-quantitative PCR with ?-actin as the inner control.The apoptosis was detected by flow cytometry.Tumor volume on the tumor-bearing mice transplanted by SGC7901/VCR cells or cells treated by VCR,LY294002 and their combination respectively was measure for the in vivo effect of LY294002.Results LY294002 enhanced the sensitivities of SGC7901/VCR cells to VCR significantly,and promoted the apoptosis rate induced by VCR prominently,with their corresponding drug resistant index decreasing from 40.45 to 8.50.The protein level of phospho-PKB was reduced,and the mRNA expression levels of MDR1 and Bcl-2 were inhibited(P
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Objective To investigate the reversal effect on mdr1 gene mediated multidrug resistance in gastric cancer SGC7901/VCR cells by small interfering RNA(siRNA).Methods Two siRNAs(mdr1si2631 and mdr1si3071) specifically targeting mdr1 gene were designed and synthesized by transcription in vitro.The siRNA duplexes were used to transfect into the gastric cancer SGC7901/VCR cells.The expression levels of mdr1 mRNA and P-gp were detected by RT-PCR and immunohistochemistry respectively.The accumulation of intracellular adriamycin(ADR)was examined by flow cytometry and the cell sensitivity to ADR was demonstrated by MTT.Results The expression level of mdr1 mRNA treated by siRNAs for 48?hours was decreased in the SGC7901/VCR cells.The mdr1 RT-PCR product in the transfected mdr1si2631 SGC7901/VCR cells could hardly been found,similar to its parental SGC7901 cells,the ratio of mdr1 and ?-actin in the control SGC7901/VCR group was 1.05?0.10,the transfected mdr1si3071 group was 0.16?0.03(P0.05).The RT-PCR results showed that the mdr1 mRNA expression level in the mdr1 si2631 group decline more obviously than that in the mdr1si307l group,near by the level in its parental SGC7901 cells.The P-gp immunoreactivity(IR)in brownish-colored granules was located on the cell membrane.The P-gp IR became weaker in the SGC7901/VCR cells treated by siRNAs for 48 hours and the P-gp expression level in both transfected siRNA groups was decreased.The values of adriamycin-specific fluorescence intensity and the positive rates of intracellular ADR in both transfected siRNA groups were increased.The relative reversal efficiency of the SGC7901/VCR cells to ADR detected by MTT was 79.59%in mdr1 si2631 group and 59.98%in mdr1si3071 group respectively.Conclusion siRNA could reverse mdr1 gene mediated multidrug resistance in gastric cancer SGC790l/VCR cells.