Objectives This study examined the protective effect of salubrinal and the mechanism underlying this protection against tunicamycin （TM）- and hypoxia-induced apoptosis in rat cardiomyocytes. Methods Neonatal rat car...Objectives This study examined the protective effect of salubrinal and the mechanism underlying this protection against tunicamycin （TM）- and hypoxia-induced apoptosis in rat cardiomyocytes. Methods Neonatal rat cardiomyocytes were cultured from the ventricles of l-day-old Wistar rats. Cells were exposed to different concentrations of salubrinal （10, 20, and 40 gmol/L） for 30 min followed by TM treatment or hypoxia for 36 h. Apoptosis was measured by a multiparameter HCS （high content screening） apoptosis assay, TUNEL assay and flow cytometry. The phosphorylation of eukaryotic translation initiation factor 2 subunit alpha （eIF2c0 and the expression of cleaved caspase-12 were determined by Western blotting. C/EBP homologous protein （CHOP） was detected by immunocytochemistry. Results HCS, TUNEL assays and flow cytometry showed that salubrinal protected cardiomyocytes against apoptosis induced by TM or hypoxia. Western blotting showed that salubrinal protected cardiomyocytes against apoptosis by inducing eIF2ct phosphorylation and down-regulating the expression of the endoplasmic reticulum stress-mediated apoptotic proteins, CHOP and cleaved caspase-12. Conclusions Our study suggests that salubrinal protects rat cardiomyocytes against TM- or hypoxia-associated apoptosis via a mechanism involving the inhibition of ER stress-mediated apoptosis.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
We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA f...We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.展开更多
Objective To assess the effects of Yi Qi Jie Du Formula(YQJDF)combined with salinomycin(SAL)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying molecular mechanisms.Methods Cell counting meth...Objective To assess the effects of Yi Qi Jie Du Formula(YQJDF)combined with salinomycin(SAL)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying molecular mechanisms.Methods Cell counting methods,the CCK-8 assay,transwell migration assay and JC-1 staining,were used to observe the effects of the combination on the proliferation,migration and apoptosis of NPC stem cells,respectively.Western blot was used to detect the levels of protein in NPC-SCs.Results YQJDF combined with SAL had a synergistic effect on the inhibition of proliferation and migration and induction of NPCSC apoptosis.Mechanistically,YQJDF combined with SAL synergistically upregulated the levels of apoptotic proteins,including cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9.Moreover,YQJDF combined with SAL synergistically decreased the levels of CD44,p-c-Src,Ras,p-PKCδ,p-MEK,p-c-Raf,p-ERK1/2 and p-AKT proteins.Conclusions The combination of YQJDF and SAL has a synergistic effect on the inhibition of NPC-SC proliferation and migration and induction of apoptosis,which may be closely related to the downregulation of the CD44/Ras signaling pathway.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
Efficient functioning of the endoplasmic reticulum(ER) is very important for most cellular activities, such as protein folding and modification. The ER closely interacts with other organelles, including the Golgi body...Efficient functioning of the endoplasmic reticulum(ER) is very important for most cellular activities, such as protein folding and modification. The ER closely interacts with other organelles, including the Golgi body, endosome, membrane, and mitochondria, providing lipids and proteins for the repair of these organelles. ER stress can be induced by various abnormal materials in the cell. ER stress is a compensatory intracellular environment disorder that occurs during areaction. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system, but excessive ER activation can cause cell death. The Pubmed and Web of Science databases were searched for full-text articles, and the terms "endoplasmic reticulum stress/unfolded protein response/gynecologic tumor cell apoptosis" were used as key words. Thirty-five studies of ER stress and unfolded protein response published from 2000 to 2016 were analyzed. Stress triggers apoptosis through a variety of signaling pathways. Increasing evidence has shown that the ER plays an important role in tumor cell diseases. The present review discusses the molecular mechanisms underlying unfolded protein response and its ability to promote survival and proliferation in gynecologic tumor cells.展开更多
Objective This study aimed to analyze the mechanism of action of the Pingyang Jiangya Formula(平阳降压方,PYJYF)in treating hypertension,based on network pharmacology,and to verify the subsequent predictions through an...Objective This study aimed to analyze the mechanism of action of the Pingyang Jiangya Formula(平阳降压方,PYJYF)in treating hypertension,based on network pharmacology,and to verify the subsequent predictions through animal experiments.Methods The active components and related target genes of PYJYF were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM),Encyclopedia of Traditional Chinese Medicine(ETCM),and Drug Bank databases and available literature.The hypertension target genes were screened based on Therapeutic Target Database(TTD),GeneCards,Online Mendelian Inheritance in Man(OMIM),UniProt,and relevant literature.The component-disease-target network intersection target genes were inputted into the STRING database,and the key target genes were selected according to the degree algorithm.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed to explore the multitarget mechanism of action and molecular regulatory network of PYJYF in the treatment of hypertension.To verify this prediction,we used PYJYF to intervene in spontaneously hypertensive rats(SHRs)and Wistar–Kyoto rats(WKY)as normal control,and the noninvasive tail artery manometry method was used to measure systolic blood pressure(SBP)in the rat tail before PYJYF intervention.After drug intervention,the SBP of each group rats were measured and compared every week.Enzyme-linked immunosorbent assay(ELISA)was used to test plasma renin,angiotensin II(Ang II),and aldosterone(Ald)levels,and hematoxylin-eosin(HE)staining was used to observe pathological damage to the renal vessels in each group of rats.Western blot and reverse transcription real-time quantitative PCR(RT-PCR)were used to detect the protein and mRNA expression levels of PI3 K,AKT1,BAX,and Bcl-2,respectively.Results A total of 4123 hypertension targets were obtained from related databases.From the TCMSP and chemical databases,78 active components of PYJYF and the corresponding 401 drug targets were retrieved.Data analysis revealed that 208 drug targets directly interacted with the hypertension targets in PYJYF.The 10 targets most closely related to hypertension target proteins in PYJYF were directly retrieved from relevant databases.GO analysis revealed that 10 direct target proteins were involved in all aspects of the antihypertensive effects of PYJYF,as well as molecular biological processes,such as the regulation of blood pressure,renin-angiotensin-aldosterone system(RAAS),angiotensin-mediated ligand reactions,and biological stimulation of cardiomyocyte apoptosis.KEGG pathway enrichment analysis revealed that PYJYF directly affected 20 signaling pathways associated with hypertension.In animal experiments,PYJYF reduced the protein and m RNA levels of PI3 K,Akt,and Bax and upregulated the expression of the protein and m RNA levels of Bcl-2,reduced plasma renin,Ang II,and Ald levels,improved the hyperactivity of RAAS,and significantly reduced SBP in SHRs.Conclusion PYJYF is effective for hypertension therapy that acts through multiple compounds and targets.The possible underlying molecular mechanism includes regulating the PI3 K/Akt signaling pathway to suppress RAAS,increasing the ratio of Bcl-2/Bax proteins,and inhibiting apoptosis,thereby mediating the repair of renal and renal vascular damage caused by hypertension.These findings warrant further research for use in clinical settings.展开更多
Objective This study aimed to study the role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis of various lung adenocarcinoma cell lines and xenograft tumor models.Methods In vitro, H2228, H1993, and ...Objective This study aimed to study the role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis of various lung adenocarcinoma cell lines and xenograft tumor models.Methods In vitro, H2228, H1993, and A549 cells were treated with crizotinib. The inhibition of proliferation was quantitated by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. Apoptosis was quantified by flow cytometry. Expression of key proteins of the HGF/c-Met signaling pathway was examined by western blotting. In vivo, H1993 and A549 tumor cell xenograft models were established. Immunohistochemical analysis was used to determine protein expression of HGF and c-MET and the amount of phospho-c-MET(p-c-Met). Real-time quantitative polymerase chain reaction(PCR) was applied to examine the messenger RNA(m RNA) expression of c-MET and serine/threonine protein kinase(AKT). The expression and activation of the key proteins were evaluated by western blotting.Results In vitro, the growth of H1993, H2228, and A549 cells was inhibited after crizotinib treatment for 72 h. Apoptotic rates of H1993 and H2228 cells increased with the crizotinib concentration and exposure time. In vivo, the growth-inhibitory rate of crizotinib for H1993 xenografts was 72.3%. Positive expression rates of HGF and c-MET in H1993 xenografts were higher than those in A549 xenografts; the p-c-MET amount was the largest in H1993 xenograft control but the lowest in the H1993 xenograft with crizotinib treatment. The m RNA expression levels of c-MET and AKT in H1993 xenografts were higher than those of A549 xenografts. The protein levels of c-MET, AKT, and extracellular regulated protein kinases(ERK) in H1993 xenografts were higher than those in A549 xenografts; the p-AKT amount was higher in H1993 xenograft control than in A549 xenografts; the largest amount of p-c-MET was detected in H1993 xenograft control; the amount of p-ERK was the lowest in the H1993 xenograft with crizotinib treatment.Conclusion The HGF/c-Met signaling pathway may mediate crizotinib-induced apoptosis and inhibition of proliferation of lung adenocarcinoma cells.展开更多
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
OBJECTIVE: To examine whether electroacupuncture(EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis(AF) via tumor necrosis factor-α(TNF-α)-tumor necrosis factor receptor 1(TNFR1)-caspase-8 and...OBJECTIVE: To examine whether electroacupuncture(EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis(AF) via tumor necrosis factor-α(TNF-α)-tumor necrosis factor receptor 1(TNFR1)-caspase-8 and integrin β1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces.METHODS: Thirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui(GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) staining. The m RNA and protein expression levels of integrin β1 andAkt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively.RESULTS: Treatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin β1and Akt m RNA and protein levels compared with controls.CONCLUSION: Treatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin β1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.展开更多
OBJECTIVE： To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. METHODS： EC9706 cells were treated in the follow- ing six groups： blank group （n...OBJECTIVE： To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. METHODS： EC9706 cells were treated in the follow- ing six groups： blank group （no transfection）, nega- tive control group （transfection no-carrier）, trans- fection group （transfected with pSilencer-siK-ras）, taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and west- ern blotting were used to analyze the expression of MAPK1 （mitogen-activated protein kinases 1） and cyclin D1 in response to siRNA （small interfering RNA） transfection and taxol treatment. RESULTS： K-Ras （K-Ras gene） siRNA transfection of EC9706 esophageal squamous carcinoma cells de- creased the expression of K-Ras, MAPK1 and cyclinD1 at the mRNA and protein level. Reverse tran- scription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group （P〈0.05）. Western blotting showed that 72 h after EC9706 cell trans- fection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the ex- pression levels in the transfection group were sig- nificantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as com- pared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe. CONCLUSION： Apoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-me- diated inhibition of Ras signaling, with the most ob- vious increase observed in the transfection plus tax- ol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeu- tic agent, taxol. Ras knockdown also down-regulat- ed the expression of the downstream genes, MAPKI and cyclin DI, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.展开更多
Proliferation suppression and apoptosis are the prominent characteristics induced by heat stress（HS） in cells, whereas the effects of HS on cell growth（mass accumulation） are unknown. In this study, Lantang swine...Proliferation suppression and apoptosis are the prominent characteristics induced by heat stress（HS） in cells, whereas the effects of HS on cell growth（mass accumulation） are unknown. In this study, Lantang swine（an indigenous breed of China） skeletal muscle satellite cells（SCs） were pre-cultured at 37 °C for 24 h. The HS group was subjected to HS at 41 °C, while the control group was maintained at 37 °C. Heat shock protein 70（HSP70） expression and SC size are significantly increased（P〈0.05） by HS, but cell proliferation is suppressed（P〈0.05） and apoptosis is induced（P〈0.05）. HS led to a lower percentage of SCs in the G0/G1 phase（P〈0.05） together with a higher percentage of SCs in the S phase（P〈0.05）. However, the percentage of SCs in the G2/M phase was decreased（P〈0.05） at 48 h but then increased（P〈0.05） at 72 h with HS. In addition, the phosphorylation ratios of protein kinase b（Akt）, ribosomal protein S6 kinase（S6K）, and ribosomal protein S6 were increased（P〈0.05） by HS. Nevertheless, the phosphorylation ratios of the 4E binding protein 1 and the eukaryotic initiation factor-4E were indistinguishable（P〉0.05） from those of the control group. The phosphorylation ratio of the mammalian target of rapamycin（m TOR）（Ser^2448） increased（P〈0.05） within 48 h, and apparent differences were abrogated at 72 h（P〉0.05）. Moreover, cleaved caspase-3 expression was increased at 72 h（P〈0.05）. These findings indicate that HS induces apoptosis and disrupts cell cycle distribution to decrease the number of cells. Additionally, HS can promote SC growth via an activated Akt/m TOR/S6 K signaling pathway.展开更多
基金This study was supported by the Ministry Science Foundation of the Chinese People's Liberation Army during the 12th Five-Year Plan Period
文摘Objectives This study examined the protective effect of salubrinal and the mechanism underlying this protection against tunicamycin （TM）- and hypoxia-induced apoptosis in rat cardiomyocytes. Methods Neonatal rat cardiomyocytes were cultured from the ventricles of l-day-old Wistar rats. Cells were exposed to different concentrations of salubrinal （10, 20, and 40 gmol/L） for 30 min followed by TM treatment or hypoxia for 36 h. Apoptosis was measured by a multiparameter HCS （high content screening） apoptosis assay, TUNEL assay and flow cytometry. The phosphorylation of eukaryotic translation initiation factor 2 subunit alpha （eIF2c0 and the expression of cleaved caspase-12 were determined by Western blotting. C/EBP homologous protein （CHOP） was detected by immunocytochemistry. Results HCS, TUNEL assays and flow cytometry showed that salubrinal protected cardiomyocytes against apoptosis induced by TM or hypoxia. Western blotting showed that salubrinal protected cardiomyocytes against apoptosis by inducing eIF2ct phosphorylation and down-regulating the expression of the endoplasmic reticulum stress-mediated apoptotic proteins, CHOP and cleaved caspase-12. Conclusions Our study suggests that salubrinal protects rat cardiomyocytes against TM- or hypoxia-associated apoptosis via a mechanism involving the inhibition of ER stress-mediated apoptosis.
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.
文摘We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.
基金We thank for the funding support from the National Natural Science Foundation of China(No.81874408,No.81973914 and No.81573721)the Natural Science Foundation of Hunan Province,China(No.2019JJ40216)Study Fundation of Hunan Provincial Education Department(No.19B439).
文摘Objective To assess the effects of Yi Qi Jie Du Formula(YQJDF)combined with salinomycin(SAL)on nasopharyngeal carcinoma stem cells(NPC-SCs)and investigate the underlying molecular mechanisms.Methods Cell counting methods,the CCK-8 assay,transwell migration assay and JC-1 staining,were used to observe the effects of the combination on the proliferation,migration and apoptosis of NPC stem cells,respectively.Western blot was used to detect the levels of protein in NPC-SCs.Results YQJDF combined with SAL had a synergistic effect on the inhibition of proliferation and migration and induction of NPCSC apoptosis.Mechanistically,YQJDF combined with SAL synergistically upregulated the levels of apoptotic proteins,including cleaved Caspase-3,cleaved Caspase-7 and cleaved Caspase-9.Moreover,YQJDF combined with SAL synergistically decreased the levels of CD44,p-c-Src,Ras,p-PKCδ,p-MEK,p-c-Raf,p-ERK1/2 and p-AKT proteins.Conclusions The combination of YQJDF and SAL has a synergistic effect on the inhibition of NPC-SC proliferation and migration and induction of apoptosis,which may be closely related to the downregulation of the CD44/Ras signaling pathway.
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.
文摘Efficient functioning of the endoplasmic reticulum(ER) is very important for most cellular activities, such as protein folding and modification. The ER closely interacts with other organelles, including the Golgi body, endosome, membrane, and mitochondria, providing lipids and proteins for the repair of these organelles. ER stress can be induced by various abnormal materials in the cell. ER stress is a compensatory intracellular environment disorder that occurs during areaction. ER can sense the stress and respond to it through translational attenuation, upregulation of the genes for ER chaperones and related proteins, and degradation of unfolded proteins by a quality-control system, but excessive ER activation can cause cell death. The Pubmed and Web of Science databases were searched for full-text articles, and the terms "endoplasmic reticulum stress/unfolded protein response/gynecologic tumor cell apoptosis" were used as key words. Thirty-five studies of ER stress and unfolded protein response published from 2000 to 2016 were analyzed. Stress triggers apoptosis through a variety of signaling pathways. Increasing evidence has shown that the ER plays an important role in tumor cell diseases. The present review discusses the molecular mechanisms underlying unfolded protein response and its ability to promote survival and proliferation in gynecologic tumor cells.
基金funding support from the National Natural Science Foundation of China(No.81874464)National Major New Drug Development Project(No.2019ZX09301-103)Provincial Department of Graduate Research Innovation Project of Hunan(No.CX20190565)。
文摘Objective This study aimed to analyze the mechanism of action of the Pingyang Jiangya Formula(平阳降压方,PYJYF)in treating hypertension,based on network pharmacology,and to verify the subsequent predictions through animal experiments.Methods The active components and related target genes of PYJYF were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM),Encyclopedia of Traditional Chinese Medicine(ETCM),and Drug Bank databases and available literature.The hypertension target genes were screened based on Therapeutic Target Database(TTD),GeneCards,Online Mendelian Inheritance in Man(OMIM),UniProt,and relevant literature.The component-disease-target network intersection target genes were inputted into the STRING database,and the key target genes were selected according to the degree algorithm.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed to explore the multitarget mechanism of action and molecular regulatory network of PYJYF in the treatment of hypertension.To verify this prediction,we used PYJYF to intervene in spontaneously hypertensive rats(SHRs)and Wistar–Kyoto rats(WKY)as normal control,and the noninvasive tail artery manometry method was used to measure systolic blood pressure(SBP)in the rat tail before PYJYF intervention.After drug intervention,the SBP of each group rats were measured and compared every week.Enzyme-linked immunosorbent assay(ELISA)was used to test plasma renin,angiotensin II(Ang II),and aldosterone(Ald)levels,and hematoxylin-eosin(HE)staining was used to observe pathological damage to the renal vessels in each group of rats.Western blot and reverse transcription real-time quantitative PCR(RT-PCR)were used to detect the protein and mRNA expression levels of PI3 K,AKT1,BAX,and Bcl-2,respectively.Results A total of 4123 hypertension targets were obtained from related databases.From the TCMSP and chemical databases,78 active components of PYJYF and the corresponding 401 drug targets were retrieved.Data analysis revealed that 208 drug targets directly interacted with the hypertension targets in PYJYF.The 10 targets most closely related to hypertension target proteins in PYJYF were directly retrieved from relevant databases.GO analysis revealed that 10 direct target proteins were involved in all aspects of the antihypertensive effects of PYJYF,as well as molecular biological processes,such as the regulation of blood pressure,renin-angiotensin-aldosterone system(RAAS),angiotensin-mediated ligand reactions,and biological stimulation of cardiomyocyte apoptosis.KEGG pathway enrichment analysis revealed that PYJYF directly affected 20 signaling pathways associated with hypertension.In animal experiments,PYJYF reduced the protein and m RNA levels of PI3 K,Akt,and Bax and upregulated the expression of the protein and m RNA levels of Bcl-2,reduced plasma renin,Ang II,and Ald levels,improved the hyperactivity of RAAS,and significantly reduced SBP in SHRs.Conclusion PYJYF is effective for hypertension therapy that acts through multiple compounds and targets.The possible underlying molecular mechanism includes regulating the PI3 K/Akt signaling pathway to suppress RAAS,increasing the ratio of Bcl-2/Bax proteins,and inhibiting apoptosis,thereby mediating the repair of renal and renal vascular damage caused by hypertension.These findings warrant further research for use in clinical settings.
基金Supported by grants from the National Natural Sciences Foundation of China(No.81060188 and No.81260351)Guangxi Sciense&Technology Development Funds(No.2015139 and No.201017)
文摘Objective This study aimed to study the role of the HGF/c-Met signaling pathway in crizotinib-induced apoptosis of various lung adenocarcinoma cell lines and xenograft tumor models.Methods In vitro, H2228, H1993, and A549 cells were treated with crizotinib. The inhibition of proliferation was quantitated by a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay. Apoptosis was quantified by flow cytometry. Expression of key proteins of the HGF/c-Met signaling pathway was examined by western blotting. In vivo, H1993 and A549 tumor cell xenograft models were established. Immunohistochemical analysis was used to determine protein expression of HGF and c-MET and the amount of phospho-c-MET(p-c-Met). Real-time quantitative polymerase chain reaction(PCR) was applied to examine the messenger RNA(m RNA) expression of c-MET and serine/threonine protein kinase(AKT). The expression and activation of the key proteins were evaluated by western blotting.Results In vitro, the growth of H1993, H2228, and A549 cells was inhibited after crizotinib treatment for 72 h. Apoptotic rates of H1993 and H2228 cells increased with the crizotinib concentration and exposure time. In vivo, the growth-inhibitory rate of crizotinib for H1993 xenografts was 72.3%. Positive expression rates of HGF and c-MET in H1993 xenografts were higher than those in A549 xenografts; the p-c-MET amount was the largest in H1993 xenograft control but the lowest in the H1993 xenograft with crizotinib treatment. The m RNA expression levels of c-MET and AKT in H1993 xenografts were higher than those of A549 xenografts. The protein levels of c-MET, AKT, and extracellular regulated protein kinases(ERK) in H1993 xenografts were higher than those in A549 xenografts; the p-AKT amount was higher in H1993 xenograft control than in A549 xenografts; the largest amount of p-c-MET was detected in H1993 xenograft control; the amount of p-ERK was the lowest in the H1993 xenograft with crizotinib treatment.Conclusion The HGF/c-Met signaling pathway may mediate crizotinib-induced apoptosis and inhibition of proliferation of lung adenocarcinoma cells.
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the National Natural Science Foundation of China(No.81273836,Based on Ineegrin/FAK signaling pathways research the effects of electroacupuncture on the contents of apoptosisNo.81001554,Based on Wnt/β-catenin signaling pathways research the effects of electroacupuncture on the annulus fibrosis cells)
文摘OBJECTIVE: To examine whether electroacupuncture(EA) treatment inhibited cell apoptosis of intervertebral annulus fibrosis(AF) via tumor necrosis factor-α(TNF-α)-tumor necrosis factor receptor 1(TNFR1)-caspase-8 and integrin β1/Akt signaling pathways in a rat model of cervical intervertebral disc degeneration caused by unbalanced dynamic and static forces.METHODS: Thirty-two Sprague-Dawley rats were included in this study, of which 24 rats underwent surgery to induce cervical intervertebral disc degeneration, while eight rats received EA treatment at Dazhui(GV 14). Immunohistochemical staining was used to detect TNF-α, TNFR1, and caspase-8Apoptosis of AF cells was examined with terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) staining. The m RNA and protein expression levels of integrin β1 andAkt were evaluated with real-time polymerase chain reaction and western blot analysis, respectively.RESULTS: Treatment with EA decreased TUNEL-positive AF cells and lowered TNF-α, TNFR1 and caspase-8 positive cells compared with control groups. EA treatment also increased integrin β1and Akt m RNA and protein levels compared with controls.CONCLUSION: Treatment with EA inhibits AF cell apoptosis through suppression of the TNF-α-TNFR1-caspase-8 signal pathway and increases the expression of integrin β1 and Akt. EA may be a good alternative therapy for treating cervical spondylosis.
基金Supported by the Natural Science Research Program of the Education Department of Henan Province(No.2011B320012)
文摘OBJECTIVE： To study the effect of RNAi silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. METHODS： EC9706 cells were treated in the follow- ing six groups： blank group （no transfection）, nega- tive control group （transfection no-carrier）, trans- fection group （transfected with pSilencer-siK-ras）, taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and west- ern blotting were used to analyze the expression of MAPK1 （mitogen-activated protein kinases 1） and cyclin D1 in response to siRNA （small interfering RNA） transfection and taxol treatment. RESULTS： K-Ras （K-Ras gene） siRNA transfection of EC9706 esophageal squamous carcinoma cells de- creased the expression of K-Ras, MAPK1 and cyclinD1 at the mRNA and protein level. Reverse tran- scription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mRNAs were significantly lower in the transfection group than in the blank group （P〈0.05）. Western blotting showed that 72 h after EC9706 cell trans- fection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the ex- pression levels in the transfection group were sig- nificantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as com- pared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe. CONCLUSION： Apoptosis increased significantly in EC9706 esophageal carcinoma cells after siRNA-me- diated inhibition of Ras signaling, with the most ob- vious increase observed in the transfection plus tax- ol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeu- tic agent, taxol. Ras knockdown also down-regulat- ed the expression of the downstream genes, MAPKI and cyclin DI, thus inhibiting the growth, proliferation and metabolism of esophageal cancer cells.
基金Project supported by the National 948 Program of China(No.2011-G35)the National Basic Research Program(973)of China(No.2012CB124704)the Science and Technology Planning Project of Guangzhou,Guangdong Province,China(Nos.201510010020 and 201300000035)
文摘Proliferation suppression and apoptosis are the prominent characteristics induced by heat stress（HS） in cells, whereas the effects of HS on cell growth（mass accumulation） are unknown. In this study, Lantang swine（an indigenous breed of China） skeletal muscle satellite cells（SCs） were pre-cultured at 37 °C for 24 h. The HS group was subjected to HS at 41 °C, while the control group was maintained at 37 °C. Heat shock protein 70（HSP70） expression and SC size are significantly increased（P〈0.05） by HS, but cell proliferation is suppressed（P〈0.05） and apoptosis is induced（P〈0.05）. HS led to a lower percentage of SCs in the G0/G1 phase（P〈0.05） together with a higher percentage of SCs in the S phase（P〈0.05）. However, the percentage of SCs in the G2/M phase was decreased（P〈0.05） at 48 h but then increased（P〈0.05） at 72 h with HS. In addition, the phosphorylation ratios of protein kinase b（Akt）, ribosomal protein S6 kinase（S6K）, and ribosomal protein S6 were increased（P〈0.05） by HS. Nevertheless, the phosphorylation ratios of the 4E binding protein 1 and the eukaryotic initiation factor-4E were indistinguishable（P〉0.05） from those of the control group. The phosphorylation ratio of the mammalian target of rapamycin（m TOR）（Ser^2448） increased（P〈0.05） within 48 h, and apparent differences were abrogated at 72 h（P〉0.05）. Moreover, cleaved caspase-3 expression was increased at 72 h（P〈0.05）. These findings indicate that HS induces apoptosis and disrupts cell cycle distribution to decrease the number of cells. Additionally, HS can promote SC growth via an activated Akt/m TOR/S6 K signaling pathway.
