AIM: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms. METHODS: Expression of multidrug resistance-associated...AIM: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms. METHODS: Expression of multidrug resistance-associated protein(MRP) was detected using reverse transcriptionpolymerase chain reaction(RT-PCR). Flow cytometry was used to assay the expression of P-glycoprotein(P-gp), Bcl-2,Bax, and the mean fluorescent intensity of intracellular rhodamine 123 in the cells. Meanwhile, the protein levels of Bcl-2 and Bax were also detected by Western blotting analysis. The sensitivity of cells to the anticancer agent, vincrimycin(VCR), and the intracellular [^3H]VCR accumulation were determined by tetrazolium blue (MTT) assay and a liquid scintillation counter, respectively. RESULTS: Expression of MRP and P-gp in SGC7901/VCR cells was 6.04-and 8.37-fold higher as compared with its parental SGC7901 cells, respectively. After treatment with 1, 5, 10, and 20 umol/L mifepristone, SGC7901/VCR cells showed a 1.34-, 2.29-, 3.11-, and 3.71-fold increase in the accumulation of intracellular VCR, a known substrate of MRP, and a 1.03-, 2.04-, 3.08-, and 3.68-fold increase in the retention of rhodamine 123, an indicator of P-gp function, respectively. MTT assay revealed that the resistance of SGC7901/VCR cells to VCR was 11.96-fold higher than that of its parental cells. The chemosensitivity of SGC7901/VCR cells to VCR was enhanced by 1.02-, 7.19-, 12.84-, and 21.17-fold after treatment with mifepristone at above-mentioned dose. After 96 h of incubation with mifepristone 10 umol/L, a concentration close to plasma concentrations achievable in human, the expression of Bcl-2 protein was decreased to (9.21+0.65)% from (25.32+1.44)%, whereas the expression of Bax protein was increased to (19.69+1.13)% from (1.24+0.78)% (P<0.01). Additionally, the effects of mifepristone on the expression of Bcl-2 and Bax proteins in SGC7901/VCR cells were further demonstrated by Western blotting analysis. CONCLUSION: Mifepristone has potent reversal effect on MDR in SGC7901/VCR via inhibiting the function of MRP and P-gp, modulating the expression of Bcl-2 and Bax proteins, and enhancing the sensitivity to anticancer agent VCR.展开更多
AIM:To explore whether antisense blocking of protein kinase C alpha(PKCα)would reverse multi-drug resistance(MDR)in the vincristine(VCR)-resistant human gastric cancer cell line SGC7901/VCR. METHODS:SGC7901/VCR cells...AIM:To explore whether antisense blocking of protein kinase C alpha(PKCα)would reverse multi-drug resistance(MDR)in the vincristine(VCR)-resistant human gastric cancer cell line SGC7901/VCR. METHODS:SGC7901/VCR cells expressing antisense PKCα,SGC7901/VCR/aPKC,were established by transfection with a recombinant plasmid reversely inserted with PKCαcDNA.Empty vector(PCI-neo)transfected cell clones,SGC7901/VCR/neo,served as the control.Western blot method was used to detect PKCαcontent in SGC7901,SGC7901/VCR,SGC7901/ VCR/neo and SGC7901/VCR/aPKC cells,using PKCα-specific antibody.The sensitivity of SGC7901,SGC7901/ VCR,SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin(DOX)in vitro was determined by MTT assay.The uptake of DOX in these cells was detected with fluorescence spectrophotometer.RESULTS:Western blot analysis showed that the PKCαprotein level was about 8.7-fold higher in SGC7901/ VCR cells than that in SGC7901 cells,whereas the protein expression of PKCαwas reduced by 78%in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells.SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity,accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells. CONCLUSION:PKCαpositively regulates MDR in SGC7901 cells,and inhibition of PKCαcan partially attenuate MDR in human gastric cancer cells.展开更多
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 troglita...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 Western 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.展开更多
基金Supported by the National Key Research Project Fundation of China,No.96-905-02-01,and the National Natural Science Fundation of China,No.39630340
文摘AIM: To explore the reversal effect of mifepristone on multidrug resistance (MDR) in drug-resistant human gastric cancer cell line SGC7901/VCR and its mechanisms. METHODS: Expression of multidrug resistance-associated protein(MRP) was detected using reverse transcriptionpolymerase chain reaction(RT-PCR). Flow cytometry was used to assay the expression of P-glycoprotein(P-gp), Bcl-2,Bax, and the mean fluorescent intensity of intracellular rhodamine 123 in the cells. Meanwhile, the protein levels of Bcl-2 and Bax were also detected by Western blotting analysis. The sensitivity of cells to the anticancer agent, vincrimycin(VCR), and the intracellular [^3H]VCR accumulation were determined by tetrazolium blue (MTT) assay and a liquid scintillation counter, respectively. RESULTS: Expression of MRP and P-gp in SGC7901/VCR cells was 6.04-and 8.37-fold higher as compared with its parental SGC7901 cells, respectively. After treatment with 1, 5, 10, and 20 umol/L mifepristone, SGC7901/VCR cells showed a 1.34-, 2.29-, 3.11-, and 3.71-fold increase in the accumulation of intracellular VCR, a known substrate of MRP, and a 1.03-, 2.04-, 3.08-, and 3.68-fold increase in the retention of rhodamine 123, an indicator of P-gp function, respectively. MTT assay revealed that the resistance of SGC7901/VCR cells to VCR was 11.96-fold higher than that of its parental cells. The chemosensitivity of SGC7901/VCR cells to VCR was enhanced by 1.02-, 7.19-, 12.84-, and 21.17-fold after treatment with mifepristone at above-mentioned dose. After 96 h of incubation with mifepristone 10 umol/L, a concentration close to plasma concentrations achievable in human, the expression of Bcl-2 protein was decreased to (9.21+0.65)% from (25.32+1.44)%, whereas the expression of Bax protein was increased to (19.69+1.13)% from (1.24+0.78)% (P<0.01). Additionally, the effects of mifepristone on the expression of Bcl-2 and Bax proteins in SGC7901/VCR cells were further demonstrated by Western blotting analysis. CONCLUSION: Mifepristone has potent reversal effect on MDR in SGC7901/VCR via inhibiting the function of MRP and P-gp, modulating the expression of Bcl-2 and Bax proteins, and enhancing the sensitivity to anticancer agent VCR.
基金Supported by The Research Fund of the Educational Departmentof Zhejiang Provincial Government,No.20070609the Research Fund of Jiaxing Science and Technology Bureau,No.2007AY2033
文摘AIM:To explore whether antisense blocking of protein kinase C alpha(PKCα)would reverse multi-drug resistance(MDR)in the vincristine(VCR)-resistant human gastric cancer cell line SGC7901/VCR. METHODS:SGC7901/VCR cells expressing antisense PKCα,SGC7901/VCR/aPKC,were established by transfection with a recombinant plasmid reversely inserted with PKCαcDNA.Empty vector(PCI-neo)transfected cell clones,SGC7901/VCR/neo,served as the control.Western blot method was used to detect PKCαcontent in SGC7901,SGC7901/VCR,SGC7901/ VCR/neo and SGC7901/VCR/aPKC cells,using PKCα-specific antibody.The sensitivity of SGC7901,SGC7901/ VCR,SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin(DOX)in vitro was determined by MTT assay.The uptake of DOX in these cells was detected with fluorescence spectrophotometer.RESULTS:Western blot analysis showed that the PKCαprotein level was about 8.7-fold higher in SGC7901/ VCR cells than that in SGC7901 cells,whereas the protein expression of PKCαwas reduced by 78%in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells.SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity,accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells. CONCLUSION:PKCαpositively regulates MDR in SGC7901 cells,and inhibition of PKCαcan partially attenuate MDR in human gastric cancer cells.
基金supported by grants from Natural Sciences Foundation of Hubei Province (No.2007ABA065)Science and Technology Key Project of Health Bureau of Hubei Province (No.JX1B006)
文摘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 Western 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.
