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
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
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
[Objectives]To observe the effect of drug-containing serum from different extracts of Phyllanthus reticulatus leaf on the proliferation of human gastric cancer SGC-7901 cells and the effect of in vivo on the life exte...[Objectives]To observe the effect of drug-containing serum from different extracts of Phyllanthus reticulatus leaf on the proliferation of human gastric cancer SGC-7901 cells and the effect of in vivo on the life extension rate of mice with H22 ascites tumor,and to investigate the effects of chemical components such as Corilagin,ethyl gallate,ethyl brevifolincarboxylate,and gallic acid on the proliferation of human gastric cancer SGC-7901 cells.[Methods]MTT method was used to observe the inhibitory effect of drug-containing serum of different extracts of P.reticulatus leaf on human gastric cancer SGC-7901 cells in vitro to determine its active site.The active site was used as the research object to establish a Kunming mouse ascites tumor model,to investigate the effect on the life extension rate of mice with H22 ascites tumor,to further separate the monomer components from the effective fraction and investigate its effect on human gastric cancer SGC-7901 cells.[Results]Compared with the 10%blank serum control group,10%drug-containing serum of ethyl acetate and n-butanol in P.reticulatus leaf had significant inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells,and the inhibition rate was 34.99%and 28.68%,respectively(P<0.05).Compared with the blank group,the survival time of the high dose group of ethyl acetate in P.reticulatus leaf was significant(P<0.05).Gallic acid,Corilagin,and Brevifolincarboxylic acid ethyl ester had a significant inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells,with IC 50 of 26.52,70.45,and 158.86μg/mL,respectively.Ethyl gallate had no inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells,and its IC 50 was 251.96μg/mL.[Conclusions]The drug-containing serum of ethyl acetate and n-butanol extract of P.reticulatus can inhibit the proliferation of human gastric cancer SGC-7901 cells.Ethyl acetate of P.reticulatus leaves can increase the life extension rate of mice with H22 ascites tumor.Gallic acid,Corilagin,and Brevifolincarboxylic acid ethyl ester isolated from its active site are the material basis for inhibiting the proliferation of human gastric cancer SGC-7901 cells.展开更多
OBJECTIVE To investigate whether nimesulide can suppress tumor growth and induce apoptosis in SGC-7901 gastric cancer cells and to explore the molecular mechanism involved. METHODS SGC-7901 cells were cultured in RPMI...OBJECTIVE To investigate whether nimesulide can suppress tumor growth and induce apoptosis in SGC-7901 gastric cancer cells and to explore the molecular mechanism involved. METHODS SGC-7901 cells were cultured in RPMI 1640 medium con- taining different concentrations of nimesulide (0,12.5, 50, 100, 200, 400 Mmol/L). The MTT assay, morphological observation, electron microscopy (EM), immunohistochemical analysis and Western blot analysis were em- ployed to investigate the effects of nimesulide on the SGC-7901 cells and to explore possible related molecular mechanisms. RESULTS Nimesulide inhibited the growth of SGC-7901 cells and elicit- ed typical apoptotic morphologic changes. Nimesulide also decreased NF-κB and Bcl-2 expression, but increased the level of the Bax protein. The positive rate of Bcl -2 protein expression at 0, 50, 100 and 200 μmol/L of nimesulide was 58.3±14.0%, 50.2±9.9%, 32.8±5.0% and 22.7± 5.5% respectively based on immunohistochemical staining. The positive rate of Bax protein expression was 22.0 ±5.7%, 29.2 ±6.5%, 42.7 ±5.9% and 74.5±9.1% and the NF-κB expression was 74.2+10.9%, 61.8±7.6%, 36.7 ±10.9% and 17.5 ±12.3%, Significant differences were found be- tween 0 μmol/L and 100 μmoI/L and 200 μmol/L. Western blot analysis al- so showed that the expression of NF-κB was decreased. CONCLUSION Nimesulide suppresses tumor growth and induces apop- tosis by inhibiting NF-κB expression, which may be related to the over- expression of Bax relative to Bcl-2 expression.展开更多
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.
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 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 Natural Science Research Project of Guangxi University of Chinese Medicine in 2015"Study on Anti-tumor Effect of Phyllanthus reticulatus Leaf Extract"(p15028)Independent Research Program of Guangxi Key Laboratory of Traditional Chinese Medicine Pharmacology in 2015"Study on Phyllanthus reticulatus Leaf 7404 Nude Mice Xenografts"+1 种基金Young Teachers Enhancement Project of Guangxi Department of Education in 2018"Study on the Mechanism of Zhuang Medicine Phyllanthus reticulatus Leaf against Liver Cancer"(2018KY0300)2019-2021 Guangxi First-class Discipline Construction Open Project Fund for Young Scholars of Guangxi University of Chinese Medicine"Anti-hepatocarcinoma Mechanism of Autophagy Induced by Ethyl Acetate in Phyllanthus reticulatus Leaf Based on PI3K/AKT and STAT3/m TOR Signaling Pathway"(2019XK089)
文摘[Objectives]To observe the effect of drug-containing serum from different extracts of Phyllanthus reticulatus leaf on the proliferation of human gastric cancer SGC-7901 cells and the effect of in vivo on the life extension rate of mice with H22 ascites tumor,and to investigate the effects of chemical components such as Corilagin,ethyl gallate,ethyl brevifolincarboxylate,and gallic acid on the proliferation of human gastric cancer SGC-7901 cells.[Methods]MTT method was used to observe the inhibitory effect of drug-containing serum of different extracts of P.reticulatus leaf on human gastric cancer SGC-7901 cells in vitro to determine its active site.The active site was used as the research object to establish a Kunming mouse ascites tumor model,to investigate the effect on the life extension rate of mice with H22 ascites tumor,to further separate the monomer components from the effective fraction and investigate its effect on human gastric cancer SGC-7901 cells.[Results]Compared with the 10%blank serum control group,10%drug-containing serum of ethyl acetate and n-butanol in P.reticulatus leaf had significant inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells,and the inhibition rate was 34.99%and 28.68%,respectively(P<0.05).Compared with the blank group,the survival time of the high dose group of ethyl acetate in P.reticulatus leaf was significant(P<0.05).Gallic acid,Corilagin,and Brevifolincarboxylic acid ethyl ester had a significant inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells,with IC 50 of 26.52,70.45,and 158.86μg/mL,respectively.Ethyl gallate had no inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells,and its IC 50 was 251.96μg/mL.[Conclusions]The drug-containing serum of ethyl acetate and n-butanol extract of P.reticulatus can inhibit the proliferation of human gastric cancer SGC-7901 cells.Ethyl acetate of P.reticulatus leaves can increase the life extension rate of mice with H22 ascites tumor.Gallic acid,Corilagin,and Brevifolincarboxylic acid ethyl ester isolated from its active site are the material basis for inhibiting the proliferation of human gastric cancer SGC-7901 cells.
基金This work was supported by the grant form the Natural Science Foundation of the Education Department of Jiangsu Province of China (No. 05KJD320234 and 01KJB320011).
文摘OBJECTIVE To investigate whether nimesulide can suppress tumor growth and induce apoptosis in SGC-7901 gastric cancer cells and to explore the molecular mechanism involved. METHODS SGC-7901 cells were cultured in RPMI 1640 medium con- taining different concentrations of nimesulide (0,12.5, 50, 100, 200, 400 Mmol/L). The MTT assay, morphological observation, electron microscopy (EM), immunohistochemical analysis and Western blot analysis were em- ployed to investigate the effects of nimesulide on the SGC-7901 cells and to explore possible related molecular mechanisms. RESULTS Nimesulide inhibited the growth of SGC-7901 cells and elicit- ed typical apoptotic morphologic changes. Nimesulide also decreased NF-κB and Bcl-2 expression, but increased the level of the Bax protein. The positive rate of Bcl -2 protein expression at 0, 50, 100 and 200 μmol/L of nimesulide was 58.3±14.0%, 50.2±9.9%, 32.8±5.0% and 22.7± 5.5% respectively based on immunohistochemical staining. The positive rate of Bax protein expression was 22.0 ±5.7%, 29.2 ±6.5%, 42.7 ±5.9% and 74.5±9.1% and the NF-κB expression was 74.2+10.9%, 61.8±7.6%, 36.7 ±10.9% and 17.5 ±12.3%, Significant differences were found be- tween 0 μmol/L and 100 μmoI/L and 200 μmol/L. Western blot analysis al- so showed that the expression of NF-κB was decreased. CONCLUSION Nimesulide suppresses tumor growth and induces apop- tosis by inhibiting NF-κB expression, which may be related to the over- expression of Bax relative to Bcl-2 expression.
