Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by...Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.展开更多
目的探讨过表达SARI(suppressor of AP-1 and regulated by IFN)对宫颈癌细胞增殖的影响及可能的分子机制。方法在He La细胞和Ca Ski细胞转染可表达SARI的质粒后,使用Real-time PCR和Western blot检测细胞中SARI表达水平的变化;CCK-8法...目的探讨过表达SARI(suppressor of AP-1 and regulated by IFN)对宫颈癌细胞增殖的影响及可能的分子机制。方法在He La细胞和Ca Ski细胞转染可表达SARI的质粒后,使用Real-time PCR和Western blot检测细胞中SARI表达水平的变化;CCK-8法检测上述宫颈癌细胞模型的增殖;流式细胞术分析上述细胞模型的周期及凋亡;报告基因检测转录因子AP-1的转录活性;Western blot检测抗凋亡分子Mcl-1及Bcl-2的表达。结果成功构建过表达SARI的细胞模型。SARI表达水平上调后,细胞增殖受到抑制(P<0.01);早期凋亡细胞增加但细胞周期无明显变化。转录因子AP-1的转录活性及Mcl-1的表达明显下调(P<0.05),但Bcl-2的表达无明显变化。结论宫颈癌细胞中过表达SARI诱导宫颈癌细胞发生早期凋亡,可能是通过抑制AP-1的转录并下调抗凋亡分子Mcl-1的表达来实现的。展开更多
AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-...AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. Hep G2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.RESULTS: As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant(P < 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partiallyinhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant(P > 0.05). The effect on inducing apoptosis in Hep G2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant(P < 0.展开更多
AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resis...AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.展开更多
文摘Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.
文摘目的探讨过表达SARI(suppressor of AP-1 and regulated by IFN)对宫颈癌细胞增殖的影响及可能的分子机制。方法在He La细胞和Ca Ski细胞转染可表达SARI的质粒后,使用Real-time PCR和Western blot检测细胞中SARI表达水平的变化;CCK-8法检测上述宫颈癌细胞模型的增殖;流式细胞术分析上述细胞模型的周期及凋亡;报告基因检测转录因子AP-1的转录活性;Western blot检测抗凋亡分子Mcl-1及Bcl-2的表达。结果成功构建过表达SARI的细胞模型。SARI表达水平上调后,细胞增殖受到抑制(P<0.01);早期凋亡细胞增加但细胞周期无明显变化。转录因子AP-1的转录活性及Mcl-1的表达明显下调(P<0.05),但Bcl-2的表达无明显变化。结论宫颈癌细胞中过表达SARI诱导宫颈癌细胞发生早期凋亡,可能是通过抑制AP-1的转录并下调抗凋亡分子Mcl-1的表达来实现的。
文摘AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. Hep G2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.RESULTS: As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant(P < 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partiallyinhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant(P > 0.05). The effect on inducing apoptosis in Hep G2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant(P < 0.
基金Supported by Research grants from Merck KGaA,Darmstadt,Germany,to Schulze-Bergkamen H
文摘AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.
