Objective Previous investigations have shown that N-acetylcysteine （NAC） could regulate diverse cell type＇s apoptosis. The purpose of this study was to evaluate the mechanism of NAC reversed apoptosis of cardiomyoc...Objective Previous investigations have shown that N-acetylcysteine （NAC） could regulate diverse cell type＇s apoptosis. The purpose of this study was to evaluate the mechanism of NAC reversed apoptosis of cardiomyocytes induced by hypoxia-reoxygenation （H/R）. Methods Cardiomyocytes were treated with hypoxia 6 h and reoxygenation 72 h in the absence and presence of NAC （100/2mol/ L）. The ROS was assayed by using Image-iTTM LIVE green reactive oxygen species detection kit. The viability of cell was assayed with trypan blue. Early stages ofapoptosis were assessed by flow cytometry using Annexin V, and late stages of apoptosis were assessed using TUNEL system. Bcl2 and bax mRNA levels were determined by real-time quantitative PCR. Bcl2, bax, p38 and pp38 protein levels were determined by western blot. Results We found that H/R could markedly increase ROS generation and induce the apoptosis of cardiomyocytes （P〈0.01）. NAC （10012 mol/L） significantly reduced the generation of ROS and apoptosis （P all 〈0.01）. NAC also significantly reduced the protein ratio of pp38 and p38 and increased the RNA and protein ratio of bcl2 and bax （P all 〈0.01）. Conclusion The results showed that NAC significantly reduced apoptosis through inhibiting the phosphorylation of p38 signal pathway, which has potential value for clinical cardiac diseases （J Geriatr Cardio12009; 6：168-172）.展开更多
A novel marine active polypeptide （PCF）, isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether ...A novel marine active polypeptide （PCF）, isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCE Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42-5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.展开更多
AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-...AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.展开更多
Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK- positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mecha- nism of resis...Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK- positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mecha- nism of resistance was studied. Methods Cell viability was determined using the MTT assay. Crizotinib-induced apoptosis in H2228 and H2228 crizotinib-resistant cells treated with the indicated doses of crizotinib was measured at different times （24 h, 48 h, 72 h） using flow cytometry. The levels of p-ALK, ALK, p-STAT3, STAT3, and survivin after treatment of cells with 0, 0.3, and 1 pM crizotinib for 72 h were determined using Western blot analysis. DNA sequencing was used to identify mutations in H2228 crizotinib-resistant cells. Results The crizotinib IC50 values in H2228 and H2228 crizotinib-resistant cells at 72 h were 334.5 nM and 3418 nM, respectively. The resistance index of 1-12228 crizotinib-resistant cells was 10.20. Crizotinib induced apoptosis in H2228 cells and reduced the levels of p-ALK, p-STAT3, and survivin. In contrast, no changes in the levels of p-ALK, p-STAT3, and survivin were observed in H2228 crizotinib-resistant cells. The mutations 2067G--,A and 2182G--,C in EML4-ALK were present in the H2228 crizotinib-resistant cells. Conclusion Crizotinib decreased the viability of H2228 cells in a dose- and time-dependent manner. In the STAT3/survivin pathway, downregulation of p-ALK, p-STAT3, and survivin might contribute to crizo- tinib-induced apoptosis in H2228 ceils. However, the STAT3/survivin pathway in H2228 crizotinib-resistant cells was unaffected by crizotinib treatment. Acquired resistance in H2228 cells might be related to ALK mutations.展开更多
基金This work was supported by National Natural Science Foundation of China （No 30800464）.
文摘Objective Previous investigations have shown that N-acetylcysteine （NAC） could regulate diverse cell type＇s apoptosis. The purpose of this study was to evaluate the mechanism of NAC reversed apoptosis of cardiomyocytes induced by hypoxia-reoxygenation （H/R）. Methods Cardiomyocytes were treated with hypoxia 6 h and reoxygenation 72 h in the absence and presence of NAC （100/2mol/ L）. The ROS was assayed by using Image-iTTM LIVE green reactive oxygen species detection kit. The viability of cell was assayed with trypan blue. Early stages ofapoptosis were assessed by flow cytometry using Annexin V, and late stages of apoptosis were assessed using TUNEL system. Bcl2 and bax mRNA levels were determined by real-time quantitative PCR. Bcl2, bax, p38 and pp38 protein levels were determined by western blot. Results We found that H/R could markedly increase ROS generation and induce the apoptosis of cardiomyocytes （P〈0.01）. NAC （10012 mol/L） significantly reduced the generation of ROS and apoptosis （P all 〈0.01）. NAC also significantly reduced the protein ratio of pp38 and p38 and increased the RNA and protein ratio of bcl2 and bax （P all 〈0.01）. Conclusion The results showed that NAC significantly reduced apoptosis through inhibiting the phosphorylation of p38 signal pathway, which has potential value for clinical cardiac diseases （J Geriatr Cardio12009; 6：168-172）.
基金Supported by the National Natural Science Foundation of China (No 30471458)the Natural Science Foundation of Shandong Province,China (No Z2007c09)
文摘A novel marine active polypeptide （PCF）, isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCE Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42-5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.
基金Supported by Medical and Health Research Foundation of Zhejiang Province,No. 2009B019
文摘AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.
基金Supported by grants from the Bureau of Science and Technology,Guangxi Zhuang Autonomous Zone,China(No.201017)National Natural Science Foundation of China(No.81060188 and 81260357)
文摘Objective This study investigated the role of the STAT3/survivin signaling pathway in the EML4-ALK- positive lung adenocarcinoma cell line H2228 before and after crizotinib-induced resistance. The mecha- nism of resistance was studied. Methods Cell viability was determined using the MTT assay. Crizotinib-induced apoptosis in H2228 and H2228 crizotinib-resistant cells treated with the indicated doses of crizotinib was measured at different times （24 h, 48 h, 72 h） using flow cytometry. The levels of p-ALK, ALK, p-STAT3, STAT3, and survivin after treatment of cells with 0, 0.3, and 1 pM crizotinib for 72 h were determined using Western blot analysis. DNA sequencing was used to identify mutations in H2228 crizotinib-resistant cells. Results The crizotinib IC50 values in H2228 and H2228 crizotinib-resistant cells at 72 h were 334.5 nM and 3418 nM, respectively. The resistance index of 1-12228 crizotinib-resistant cells was 10.20. Crizotinib induced apoptosis in H2228 cells and reduced the levels of p-ALK, p-STAT3, and survivin. In contrast, no changes in the levels of p-ALK, p-STAT3, and survivin were observed in H2228 crizotinib-resistant cells. The mutations 2067G--,A and 2182G--,C in EML4-ALK were present in the H2228 crizotinib-resistant cells. Conclusion Crizotinib decreased the viability of H2228 cells in a dose- and time-dependent manner. In the STAT3/survivin pathway, downregulation of p-ALK, p-STAT3, and survivin might contribute to crizo- tinib-induced apoptosis in H2228 ceils. However, the STAT3/survivin pathway in H2228 crizotinib-resistant cells was unaffected by crizotinib treatment. Acquired resistance in H2228 cells might be related to ALK mutations.
