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 tha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
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
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 tha
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.
