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1.
OBJECTIVE:To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus(C.orbiculatus) extract in human hepatocellular carcinoma cells.METHODS:Human hepatocellular carcinoma cells(HCCLM6) were treated with C.orbiculatus extract(COE) at different nontoxic concentrations(10,20,40,80,and 160 μg/mL).The effect of COE on HC-CLM6 viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assays.Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis.RESULTS:COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner.Apoptosis was accompanied by increased Bax expression and decreased Bcl-2 expression.In addition,COE treatment led to the release of cytochrome c,activation of caspase-3,and cleavage of poly(ADP-ribose) polymerase(PARP).Furthermore,activation of extracellular signal-regulated kinase(ERK),p38 kinase,and c-Jun N-terminal kinase(JNK) phosphorylation,and down-regulation of Akt phosphorylation was observed.CONCLUSION:COE induces mitochondrial-mediated,caspase-dependent apoptosis in HCCLM6 cells,which might be attributed to the activation of mitogen-activated protein kinase(MAPK) and inhibition of Akt signaling pathways.These data suggest that COE may be a potential treatment for human hepatocellular carcinoma.  相似文献   

2.
Background We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.Methods We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.Results The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors.The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2 -terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.Conclusion These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTDinduced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.  相似文献   

3.
Background Claudin-6 is a protein component of tight junctions and its expression could downregulate the malignant phenotype of breast carcinoma. Here we investigated the mechanisms of claudin-6 induced human MCF-7 breast cancer cells apoptosis, invasion, and migration. Methods Terminal deoxyribonucleotide transferase-mediated nick-end labeling assay and Annexin-V/PI double stain assay were carried out to evaluate apoptosis. Inhibitors of each pathway were used to inactivate the signaling pathways. The expression of claudin-6 and phosphate p38, Erk 1/2 and Akt protein levels was confirmed by Western blotting analysis. Invasive and migratory traits of claudin-6 expressing cells were determined by Boyden chamber invasion assay and monolayer wound-healing assay. Results Cells with high-level expression of claudin-6 had a higher rate of apoptosis than control cells. Western blotting assay showed that by contrast to control groups, p38 pathways were more activated in claudin-6 expressing cells. However, after inhibitor SB203580 treatment, the activation status could be significantly counteracted. Furthermore, by applying inhibitors to the apoptotic rate, invasive and migratory traits were also recovered in cells with claudin-6 expression. Conclusion Claudin-6 may function through p38 mitogen-activated protein kinase pathway, of which inhibition may reverse claudin-6-induced cell apoptosis, invasion, and migration.  相似文献   

4.
5.
The biological activity of adriamyein was investigated in humaq breast carcinoma (HBC) cells. Adriamycin inhibited the growth of a number of HBC cell lines and induced G1 arrest followed by apoptosis. In MCF-7 cells that harbor wild-type p53, adriamycin-induced G, arrest and apoptosis was accompanied by p53-independent regulation of WAF1/CIP1 as weal as bex mRNA levels. In MDA-MB-231 cells which possess a mutant p53, adriamycin-indueed G1 arrest and apoptosis was also associated with a concomitant up-regulation of WAF1/CIP1 mRNA while these cells did not express bex or bcl-2 messages. Thus, adriamycin induces G1 arrest and apoptosis via a unique pethway which appears to involve activation ot downstream effeetors of p53-independent manner.  相似文献   

6.
Objective: To study the mechanism of T-cell activation induced by non-lethal complement attack and the role of CD59 in this process. Methods: Human CD59 and its transmembrane counterpart CD59TM cDNA were transfected into murine thymoma EL-4 cells. Activation and proliferation of EL-4 transfectants were observed with MTr assay.Results: Both CD59 and CD59 TM cDNA expressed on EL-4 cells effectively inhibited complement-mediated membrane damage, Cross-linking of CD59 with antibody induced activation of CD59/EL-4 cells but not CD59TM/EL-4 cells, This effect was inhibited by Herbimycin A, a special protein tyrosine kinase (PTK) inhibitor, Non-lethal complement attack induced CD59/EL-4 but not CD59TM/EL-4 cell to proliferate, and this reaction was not blocked by Herbimycin A, Conclusion: CD59 takes part in T cell activation induced by non-lethal complement attack. The mechanisms of T cell activation induced by non-lethal complement attack are different from those by cross-linking of CD59.  相似文献   

7.
Objective:To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells.Methods:The cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry.The activities of caspase-3,-8,and-9 were detected by fluorescence resonance energy transfer technology and colorimetric assay,while the levels of Bcl-2,cytochrome c,and Bax were detected by Western blot method.Results:Induction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner.The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8.Further,the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor,a caspase-3 inhibitor,and a caspase-9 inhibitor but not by a caspase-8 inhibitor.Bcl-2 and Bax protein expressions were not changed by realgar.Conclusion:The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway,which sequentially activates caspase-9 and caspase-3.  相似文献   

8.
Background Docetaxel (DOC) therapy is well tolerated and shows high response rates in patients with hormone refractory prostate cancer (HRPC). There are many reports on the effect of rapamycin (RPM) on the treatment of carcinogenesis. The goal of this study was to test whether RPM could enhance the susceptibility of both androgen-dependent and -independent prostate carcinoma cells to DOC. Methods Prostate cancer (PC) cell lines (LNCap, PC3 and AILNCap) were cultured and treated with RPM and DOC alone or in combination. The effects of therapeutic agents on cells were determined by the WST-1 assay. Apoptosis induction was confirmed by flow cytometric analysis. The apopcyto caspase colorimetric assay kit was applied to measure the activities of caspases 3 and 9. The antitumor effects of RPM and DOC against PC cells were also assessed in nude mice using four randomized groups: control, RPM, DOC and combination drug therapy by measuring tumor size. All the animals tolerated both RPM and DOC without significant weight loss. Results RPM and DOC caused dosage-dependent growth suppression of PC cells. RPM could increase the susceptibility of PC cells to DOC significantly, and combined treatment with RPM and DOC caused synergistic growth suppression in all examined PC cell lines by isobolographic analysis. Both RPM and DOC significantly induced apoptosis in a dosage-dependent manner. RPM (10 nmol/L), DOC (1 nmol/L), and combined treatment induced apoptosis rate were 8%, 17% and 38%, respectively (the control was 2%). RPM could promote the apoptosis induced by DOC in PC cell lines. Both RPM and DOC significantly increased the caspase activity in a dosage-dependent manner. The relative activities of caspase 9 in control, RPM, DOC and RPM+DOC groups were 0.22±0.02, 0.36±0.06, 0.47±0.05 and 0.84±0.08, respectively. The relative activities of caspase 3 were 0.21±0.02, 0.24±0.05, 0.42±0.06 and 0.81±0.09, respectively. Either RPM or DOC alone significantly inhibited the growth of PC cells in nude mice compared to the control. The combination of RPM and DOC produced a significant reduction in tumor volume when compared to RPM or DOC alone. After 5-week treatment, the tumor sizes of LNCap in control, RPM, DOC and RPM+DOC groups were (570±56) mm3, (412±41) mm3, (425±46) mm3 and (221±26) mm3, respectively. Conclusions RPM could significantly increase the susceptibility of both androgen-dependent and -independent PC cells to DOC; the synergy of RPM and DOC was demonstrated. RPM enhanced the DOC-induced upregulation of caspase activity, resulting in an increasing number of cells in sub-G1 phases. The synergy of the combined treatment might be observed in both androgen-dependent and -independent PC cell lines.  相似文献   

9.
Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin. Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTr assay. Fluorescent microscopic analysis of apoptotic cells stained with 4‘ , 6‘-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis. Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK) , inhibitor PD 98059. Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.  相似文献   

10.
Objective Oxidative stress plays an important role in retinal pigmental epithelium (RPE) death during aging and the development of age-related macular degeneration.Although early reports indicate that reactive oxygen species (ROS) including H2O2 can trigger apoptosis at lower concentrations and necrosis at higher concentrations,the exact molecular mechanism of RPE death is still unclear.The purpose of this study was to investigate the molecular pathways involved in RPE death induced by exogenous ROS,especially at higher concentrations.Methods Cultured ARPE-19 cells were treated with H2O2 at different concentrations and cell viability was measured with the MTT assay.Cell death was morphologically studied by microscopy using APOPercentage assay and PI staining.Furthermore,the impact of oxidative stress on ARPE-19 cells was assessed by HO-1 and PARP-1 Western blotting and by the protection of antioxidant EGCG.Calcium influx was determined using the fura-2 calcium indicator and the role of intracellular calcium overload in ARPE-19 cell death was evaluated following cobalt treatment to block calcium effects.Results H2O2 reduced the viability of ARPE-19 cells in a concentration-dependent manner,which was presented as a typical s-shaped curve.Cell death caused by high concentrations of H2O2 was confirmed to be programmed necrosis.Morphologically,dying ARPE-19 cells were extremely swollen and lost the integrity of their plasma membrane,positively detected with APOPercentage assay and PI staining.24-hour treatment with 500 ?mol/L H2O2 induced remarkable up-regulation of HO-1 and PARP-1 in ARPE-19 cells.Moreover,antioxidant treatment using EGCG effectively protected cells from H2O2-induced injury,increasing cell viability from 14.17%±2.31% to 85.77%±4.58%.After H2O2 treatment,intracellular calcium levels were highly elevated with a maximum concentration of 1200nM.Significantly,the calcium channel inhibitor cobalt was able to blunt this calcium influx and blocked the necrotic pathway,rescuing the ARPE-19 cell from H2O2-induced death.Conclusions At high concentrations,H2O2 induces ARPE-19 cell death through a regulated necrotic pathway with calcium overload as a critical step in the cell death program.  相似文献   

11.
Role of p38 protein kinase in heat-induced Raw cells apoptosis   总被引:5,自引:1,他引:4  
Aftercellsareexposedtoastressingagent,celldeathcanbeobservedasapoptosis,necrosisandmitoticdeathApoptosisorphysiologiccelldeath,isundergeneticcontrolandischaracterizedmorphologicallybycellshrinkage,marginationofchromatinandnuclearfragmentationGenomicDNA…  相似文献   

12.
目的 探讨p38蛋白激酶信号传导的过程及其在细胞中的特异性作用机制。方法 应用间接荧光标记免疫探针技术及共聚焦激光扫描技术观察单核细胞中p38蛋白激酶的分布及LPS对其分布的影响。结果p38在未受刺激的静息单核细胞及内皮生长因子(EGF)刺激的单核细胞胞浆和胞核中荧光强度均呈弥散性分布;脂多糖(LPS)刺激使细胞核区的荧光强度明显增强,而胞浆区域的荧光强度降低。激酶动力学的研究显示,p38激活先于p38移位。LPS刺激后30 min,p38激酶活性即达到高峰,随后2 h内逐步下降,p38激酶活性呈一过性增高;LPS刺激45 min后,核区荧光强度达到峰值,且在2 h内维持在高水平。结论 单核细胞由LPS激活后,其p38蛋白激酶由胞浆转位到胞核,反应具有一定的特异性;p38移位入核依赖于p38磷酸化活化及由细胞浆移位到细胞核是一系列连续发生的事件。  相似文献   

13.
目的:探讨II型CD20抗体诱导靶细胞程序性死亡的机制,为制备具有较强肿瘤杀伤能力的新型CD20抗体提供理论依据。方法:将淋巴瘤细胞Raji分别与 I型CD20抗体利妥昔单抗(Rituximab)和II型CD20抗体托西莫单抗(Tositumomab)孵育后,采用磷脂酰丝氨酸V(annexin V)和碘化丙啶(propidium iodide,PI)双染,流式细胞仪比较两种抗体处理后程序性死亡细胞所占比例;在CD20抗体处理前,用工作浓度的caspase抑制剂Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone)或伏马毒素(fumonisin B1,FB1)预处理Raji细胞,并采用流式细胞仪比较预处理组与未预处理组淋巴瘤细胞程序性死亡所占的比例。收集CD20抗体处理过的Raji细胞,应用高效液相色谱法检测不同的CD20抗体处理组间淋巴瘤细胞内神经酰胺(ceramide)的水平,并用相同方法检测FB1对Tositumomab引起淋巴瘤细胞内ceramide水平变化的抑制作用。用不同浓度的C2-ceramide与淋巴瘤细胞进行孵育,16h后用annexin V & PI双染检测其诱导靶细胞程序性死亡的能力。结果:10 µg/mL的I型抗体Rituximab几乎不能诱导Raji细胞的程序性死亡,而相同浓度的II型CD20抗体Tositumomab能够诱导(28.6±4.2)%的Raji细胞产生程序性死亡,两者差异有统计学意义(t=26.48,P<0.01),这种程序性死亡不能被caspase抑制剂Z-VAD-FMK所抑制(F=3.01,P>0.05)。Tositumomab处理后能够引起Raji细胞内ceramide水平的升高(t=28.48,P<0.01),且5~40 µmol/L的C2-ceramide能够通过剂量依赖性的关系诱导Raji细胞的程序性死亡(F=2.71,P>0.05)。Ceramide合成的FB1可以显著抑制Tositumomab引起的Raji细胞内ceramide水平的升高(F=20.18,P<0.01),进而显著抑制Tositumomab介导的程序性死亡(F=17.02,P<0.01)。结论:II型CD20抗体Tositumomab而非I型CD20抗体Rituximab可以通过介导靶细胞内ceramide水平的升高,诱导靶细胞的程序性死亡,这种程序性死亡与经典的caspase通路无关。  相似文献   

14.
 【目的】 探讨番茄红素对脂多糖(LPS)所诱导的RAW264.7巨噬细胞炎症因子生成的影响及其作用的分子机制。【方法】 分别用1510 μmol/L 的番茄红素孵育细胞1 h,再用1 μg/mL LPS 处理细胞不同时间,分别用Griess法和ELISA法检测RAW264.7巨噬细胞培养基中NO及IL-6的含量,用Western-blot检测核因子-κB(NF-κB) p65磷酸化和非磷酸化I-κBα丝裂原活化蛋白激酶(MAPKs)的蛋白表达量。【结果】 番茄红素能有效地降低炎性因子NO和IL-6分泌,进一步研究显示番茄红素能够抑制LPS诱导I-κBα磷酸化和降解NF-κB核转移,阻断ERK1/2和p38 MAPK激活,而对JNK活化没有影响。【结论】 番茄红素能够通过抑制ERK1/2 和p38 MAPK信号通路的激活而抑制巨噬细胞NF-κB依赖的炎症因子NO和IL-6生成,这可能是番茄红素防治一些炎症相关性疾病的作用机制之一  相似文献   

15.
目的探讨神经酰胺对大鼠脑胶质瘤细胞C6自噬性死亡的影响及其作用机制。方法不同浓度神经酰胺作用于C6细胞后,用MTT法检测细胞的存活率,流式法检测细胞凋亡的改变;Western blotting及电镜的方法观察细胞自噬水平的改变,以及JNK及其下游靶分子c-Jun的磷酸化水平变化;最后进一步借助JNK特异性抑制剂SP600125抑制JNK的活性,观察其对神经酰胺诱导的细胞自噬情况的影响。结果与对照组相比,神经酰胺作用24 h后,C6细胞存活率明显降低,且具有剂量依赖性,差异有统计学意义(P<0.05);与对照组相比,神经酰胺作用24 h后,C6细胞死亡明显升高且具有剂量依赖性,差异有统计学意义(P<0.05),但其中凋亡性死亡比例较低;神经酰胺作用后细胞内自噬小体数目,LC3B/LC3A的比值,Beclin-1的表达水平,以及JNK和c-Jun磷酸化水平都显著升高,差异具有统计学意义(P<0.05);提前给予JNK特异性抑制剂SP600125抑制JNK的活性后,显著阻断神经酰胺诱导的细胞自噬。结论神经酰胺可诱导胶质瘤细胞C6发生自噬性死亡,其诱导胶质瘤细胞发生自噬的机制可能与激活JNK信号通路有关。  相似文献   

16.
p120连环蛋白表达变化对NF-κB信号通路的影响及有关机制   总被引:1,自引:1,他引:0  
目的 通过甲醛(FA)刺激建立体外气道炎症反应模型,探讨甲醛刺激后人气道上皮细胞株(16HBE)中p120连环蛋白(p120ctn)的表达变化及其对核转录因子-κB(NF-κB)信号通路的影响,从而进一步分析气道炎症发生发展的有关机制.方法 培养的16HBE细胞分为对照组和实验组,实验组用100 μmol/L的甲醛处理...  相似文献   

17.
Background Recent studies have demonstrated that dexamethasone (DEX) interferes with immune responses by targeting key functions of dendritic cells (DCs) at the earliest stage. However, the cellular and molecular mechanisms are still incompletely understood. This study aimed to explore the possible mechanisms by investigating the roles of DEX on differentiation, maturation & function of murine DCs and the effects of DEX on DCs via Toll-like receptor 4 (TLR4)-nuclear factor (NF)-KB mediated signal pathway. Methods Immature DCs (imDCs) were cultured from murine bone marrow (BM) cells. We added DEX into culture medium at different time. The expression of CD11c, CD86 and I-Ab (mouse MHC class II molecule) was determined by flow cytometry. We determined the expression of NF-κB and its inhibitory protein I-κBα by electrophoretic mobility shift assay (EMSA) and Western blotting, respectively. The productions of interleukin (IL)-12p70 and IL-10 in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Results DEX impaired differentiation of DCs from murine bone marrow progenitors, and inhibited lipopolysaccharide (LPS) induced maturation of DCs. DEX significantly inhibited NF-κB expression of normal DCs, the higher the DEX concentration or the longer the DEX treatment time, the more obvious the effect. However, DEX had little effect on LPS-induced NF-KB activation, and partially impaired LPS-induced I-κBα degradation. DEX significantly decreased LPS induced IL-12p70 production by DCs. Interestingly, our results showed a synergistic effect between DEX and LPS on the production of IL-10 by DCs. Conclusions DEX inhibits the differentiation and maturation of murine DCs involved in TLR4-I-κB-NF-κB pathway, and also indirectly impairs Thl development and interferes with the Thl-Th2 balance through IL-12 and/or IL-10 secretion by DCs.  相似文献   

18.
Li M  Liu J  Hu WL  Jia CH  Li HY  Wen ZH  Zou ZP  Bai XC  Luo SQ 《南方医科大学学报》2011,31(9):1504-1508
目的探讨二甲双胍对肾癌细胞凋亡的影响及其机制。方法通过Annexin V-FITC/PI染色镜下观察及流式细胞仪检测观察二甲双胍对786-O细胞凋亡的影响,通过免疫印迹检测AMPK信号及Caspase 9表达探讨可能分子机制。结果二甲双胍能够诱导786-O在低浓度血清环境凋亡,激活Caspase 9,二甲双胍能够激活786-O细胞中AMPK信号,但与其凋亡诱导效应无关。结论二甲双胍能够诱导肾癌细胞在低浓度血清环境凋亡,具有潜在治疗肾癌作用。  相似文献   

19.
目的:探讨H2型松弛素(Relaxin‐2)对血管平滑肌细胞(VSMCs)迁移的作用及分子机制。方法采用划痕实验和Transwell实验检测Relaxin‐2对VSMCs的迁移作用;采用蛋白质印迹法检测其对细胞信号蛋白丝氨酸苏氨酸蛋白激酶(Akt)、细胞外信号调节激酶(ERK)、核因子‐κB(NF‐κB) p65的影响。结果 Relaxin‐2可剂量依赖性促进VSMCs的迁移,应用NF‐κB抑制剂BAY 11‐7082可阻断Relaxin‐2诱导的基质金属蛋白酶‐9(MMP‐9)和基质金属蛋白酶‐2(MMP‐2)的表达,磷脂酰肌醇3激酶(PI3K)抑制剂LY294002和ERK抑制剂 U0126预处理可以降低Relaxin‐2引起的NF‐κB p65的激活。结论 Relaxin‐2可能通过活化PI3K/Akt和ERK信号通路激活NF‐κB p65,进而促进MMP‐9和MMP‐2的表达,从而诱导VSMCs迁移效应。  相似文献   

20.
脑溢安对脑出血大鼠脑内p38MAPK信号转导通路的影响   总被引:4,自引:3,他引:1  
目的研究脑溢安对脑出血大鼠脑内p38丝裂原活化蛋白激酶(Mitogen-activated protein kinase,p38MAPK)信号转导通路的影响,探讨中药脑溢安颗粒(简称脑溢安)对脑出血的保护作用机制.方法采用胶原酶注射建立大鼠脑出血模型,应用免疫共沉淀、激酶反应及Western blot技术检测脑出血大鼠脑内p38MApK活性变化及脑溢安对p38MAPK活性的影响.结果脑出血损伤后1 h p38MAPK活性增强,出血损伤后6 h达高峰,12 h后下降,至24 h p38MAPK活性消失,脑溢安治疗组(简称治疗组)在脑出血损伤后1,6和12h各时间点p38MAPK活性均较模型组减低.结论脑出血大鼠脑内p38MAPK活性增强,脑溢安能抑制脑出血损伤激活的p38MAPK信号转导通路.  相似文献   

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