Effect of Aged Garlic Extract on Caspase-3 Activity,In Vitro

Abstract

In neurodegeneration, such as Alzheimer's disease (AD), apoptosis results in the loss of valuable neurons. A key mechanism in apoptosis is the activation of caspase-3. Caspase-3 activity first becomes detectable early in apoptosis, continues to increase as cells undergo apoptosis, and rapidly declines in late stages of apoptosis. Its activity is an early marker of cells undergoing apoptosis. Caspase-3 catalyzes the formation of β-amyloid peptide, a hallmark of AD. The purpose of the study was to determine whether dietary aged garlic extract (AGE), with known antioxidant properties and neuroprotection against Alzheimer's β-amyloid cytotoxicity, inhibits the caspase-3 activity in vitro. Caspase-3 activity was assayed according to the prescribed protocol and incubated overnight at ambient temperature. We report that AGE inhibits caspase-3 in dose dependent manner. Caspase-8 was not inhibited by AGE. As a caspase-3 inhibitor, AGE may be effective in reducing apoptotic death of neurons since caspase inhibitors have been shown to inhibit neuronal cell death. We propose a scheme for the ameliorative effect of AGE on deleterious effects of β-amyloid and possibly uncontrolled caspase-3 activity.     

Different molecular events account for butyrate-induced apoptosis in two human colon cancer cell lines

We studied the molecular events underlying butyrate-induced apoptosis in two different colon cancer cell lines: Caco-2, a well defined cancer cell and RSB, a cell line obtained from a colonic tumor of an ulcerative colitis patient. Caco-2 and RSB cells were exposed to 2, 5 and 10 mmol/L butyrate for 48 h. Caspase-1 was cleaved in Caco-2-cells at all butyrate concentrations, whereas in RSB-cells caspase-1 expression was undetectable. In RSB cells, butyrate dose-dependently induced caspase-3 cleavage, whereas in Caco-2-cells, butyrate up-regulated expression of the caspase-3 active subunit. Caspase-3-specific activity, cytoplasmic nucleosome concentration and growth were directly correlated with butyrate doses in both cell lines; however, the response was more pronounced in Caco-2 than in RSB cells. Expression of the cleaved poly(ADP-ribose) polymerase (PARP) product was elevated in both cell lines at the highest butyrate concentration. Bak expression gradually increased as a function of butyrate concentrations in both cell lines. At 10 mmol/L butyrate, expression increased by fivefold and sevenfold in Caco-2 and RSB cells, respectively. The highest expression of Bcl-2 was observed in control Caco-2 cells, and expression decreased with increasing butyrate concentration. This effect was not observed in RSB cells. Inactivation of caspase-1 with Z-YVAD-FMK abrogated butyrate-induced apoptosis in Caco-2 but not in RSB cells. Inactivation of caspase-3 with Z-DVED-FMK completely inhibited butyrate-induced apoptosis in RSB cells whereas this effect was less pronounced in Caco-2 cells. Our data demonstrate that butyrate-induced apoptosis is activated via different apoptotic pathways in diversely stratified colon cancers.     

诱导胃癌细胞凋亡中天冬氨酸特异性半胱氨酸蛋白酶的表达

目的　研究天冬氨酸特异性半胱氨酸蛋白酶 (caspase 8)在维生素E琥珀酸酯 (VES)诱导胃癌细胞凋亡过程中的作用。方法　以人胃癌SGC 790 1细胞作为靶细胞 ,采用WesternBlot法、荧光分光光度法、DAPI染色法探讨caspase 8在VES诱导SGC 790 1细胞凋亡过程中的表达和活性。结果　经 5、10、2 0mg/LVES处理后 ,细胞中caspase 8蛋白表达增加 ,caspase 8活性分别为 ( 17 83±1 82 )U和 ( 33 74± 2 6 5 )U、( 5 2 34± 3 10 )U ,均呈剂量 效应关系 ;用caspase 8抑制剂z 异亮氨酸 谷氨酸 苏氨酸 天冬氨酸 (IETD fmk)处理SGC 790 1细胞后 ,降低了VES诱导的细胞凋亡率及caspase 8的活性。结论　caspase 8是VES诱导SGC 790 1细胞凋亡的重要参与者。     

Intracellular mechanisms mediating tocotrienol-induced apoptosis in neoplastic mammary epithelial cells

Tocotrienols and tocopherols represent the two subgroups that make up the vitamin E family of compounds. However, tocotrienols display significantly more potent apoptotic activity in neoplastic mammary epithelial cells than tocopherols. Studies were conducted to determine the intracellular mechanism(s) mediating tocotrienol-induced apoptosis in neoplastic +SA mouse mammary epithelial cells in vitro. An initial step in apoptosis is the activation of 'initiator' caspases (caspase-8 or -9) that subsequently activate 'effector' caspases (caspase-3, -6 and -7) and induce apoptosis. Treatment with cytotoxic doses of alpha-tocotrienol (20 microM) resulted in a time-dependent increase in caspase-8 and caspase-3 activity. Combined treatment with specific caspase-8 or caspase-3 inhibitors completely blocked alpha-tocotrienol-induced apoptosis and caspase-8 or caspase-3 activity, respectively. In contrast, alpha-tocotrienol treatment had no effect on caspase-9 activation, and combined treatment with a specific caspase-9 inhibitor did not block alpha-tocotrienol-induced apoptosis in (+)SA cells. Since caspase-8 activation is associated with the activation of death receptors, such as Fas, tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL) receptors, studies were conducted to determine the exact death receptor(s) and ligand(s) involved in mediating tocotrienol-induced caspase-8 activation and apoptosis. Treatment with Fas-ligand (FasL), Fas-activating antibody, or TRAIL failed to induce cell death in (+)SA neoplastic mammary epithelial cells, suggesting that these cells are resistant to death receptor-induced apoptosis. Moreover, treatment with cytotoxic doses of alpha-tocotrienol did not alter the intracellular levels of Fas, FasL, or Fas-associated death domain (FADD) in these cells. Western blot analysis also showed that alpha-tocotrienol did not induce FasL or FADD translocation from the cytosolic to membrane fraction in these cells. Finally, treatment with Fas-blocking antibody did not reverse the tocotrienol-induced apoptosis in (+)SA cells. These data demonstrate that tocotrienol-induced caspase-8 activation and apoptosis is not mediated through death receptor activation in malignant (+)SA mammary epithelial cells. Resistance to death receptor-induced apoptosis has been shown to be associated with increased expression of apoptosis-inhibitory proteins, such as FLICE-inhibitory protein (FLIP), and enhanced signalling of the phosphatidylinositol 3-kinase (PI3K)/PI3K-dependent kinase (PDK)/Akt mitogenic pathway. Additional studies showed that treatment with cytotoxic doses of alpha-tocotrienol decreased total, membrane, and cytosolic levels of FLIP, and reduced phosphorylated PDK-1 (active) and phosphorylated-Akt (active) levels in these cells. In summary, these findings demonstrate that tocotrienol-induced caspase-8 activation and apoptosis in malignant (+)SA mammary epithelial cells is not mediated through the activation of death receptors, but appears to result from the suppression of the PI3K/PDK/Akt mitogenic signalling pathway, and subsequent reduction in intracellular FLIP expression.     

Risk factors for Alzheimer's disease: role of multiple antioxidants,non-steroidal anti-inflammatory and cholinergic agents alone or in combination in prevention and treatment

The etiology of Alzheimer's disease (AD) is not well understood. Etiologic factors, chronic inflammatory reactions, oxidative and nitrosylative stresses and high cholesterol levels are thought to be important for initiating and promoting neurodegenerative changes commonly found in AD brains. Even in familial AD, oxidative stress plays an important role in the early onset of the disease. Mitochondrial damage and proteasome inhibition represent early events in the pathogenesis of AD, whereas increased processing of amyloid precursor protein (APP) to beta-amyloid (Abeta) fragments (Abeta(40) and Abeta(42)) and formation of senile plaques and neurofibrillary tangles (NFTs) represent late events. We propose a hypothesis that in idiopathic AD, epigenetic components of neurons such as mitochondria, proteasomes and post-translation protein modifications (processing of amyloid precursor protein to beta-amyloid and hyperphosphorylation of tau), rather than nuclear genes, are the primary targets for the action of diverse groups of neurotoxins. Based on epidemiologic, laboratory and limited clinical studies, we propose that a combination of non steroidal anti-inflammatory drugs (NSAIDs) and appropriate levels and types of multiple micronutrients, including antioxidants, may be more effective than the individual agents in the prevention, and they, in combination with a cholinergic agent, may be more effective in the treatment of AD than the individual agents alone. In addition, agents, which can prevent formation of plaques or dissolve these plaques may further enhance the efficacy of our proposed treatment strategy.     

2-甲氧基雌二醇诱导卵巢癌细胞凋亡的研究

目的探讨2-甲氧基雌二醇（2-ME）诱导卵巢癌细胞系OVCAR-3细胞和卵巢癌原代培养细胞凋亡的作用及其机制。方法以2-ME作用于卵巢癌OVCAR-3细胞和原代培养细胞,采用MTT法检测细胞存活率、Annexin V—FITC／PI双染流式细胞术检测细胞凋亡,JC-1染色流式细胞术检测细胞线粒体膜电位（△ψm）,比色测定试剂盒检测caspase-3和caspase-8活性。结果与对照组相比,OVCAR-3细胞2-ME组存活率显著下降（100％vs70．5％）,凋亡率明显增加（5．3％VS40．5％）;卵巢癌原代培养细胞2-ME组存活率显著下降（100％vs61．8％）,凋亡率明显增加（5．4％VS36．9％）;此外,2-ME明显降低卵巢癌OVCAR-3细胞和原代培养细胞的AtOm,提高caspase-3和caspase-8活性。而预先以Z—VAD阻断caspase能逆转2-ME的效应。结论2-ME通过凋亡信号通路的内源性和外源性途径,诱导卵巢癌细胞凋亡。     

Caspase-3和p38MAPK在MMT诱导的PC-3M细胞凋亡中的作用

目的确定Caspase-3和p38MAPK在大气污染物甲基环戊二烯羰基锰（MMT）诱导人前列腺细胞系PC-3M凋亡过程中的作用。方法1mmol／LMMT诱导PC-3M细胞后，化学发光法检测细胞Caspase-3活力的变化，MTS法检测Caspase-3特异性多肽抑制剂Z-DEVD-FMK对细胞存活率的影响，Western blot法检测p38MAPK在细胞凋亡过程中的活性变化，MTS及化学发光法检测p38MAPK抑制剂SB203580对细胞凋亡率和Caspase-3活力的影响。结果在MMT诱导的PC-3M细胞凋亡中，Caspase-3被明显激活差异有统计学意义（P〈0．01）；Z-DEVD-FMK可明显提高PC-3M细胞存活率差异有统计学意义（P〈0．01）；p38MAPK磷酸化程度明显升高；SB203580可抑制细胞凋亡差异有统计学意义（P〈0．01），同时使Caspase-3活力下降差异有统计学意义（P〈0．01）。结论Caspase-3及p38MAPK参与了MMT诱导的PC-3M细胞凋亡。     

Caspase-3和p38 MAPK在MMT诱导的PC-3M细胞凋亡中的作用

目的确定Caspase-3和p38 MAPK在大气污染物甲基环戊二烯羰基锰(MMT)诱导人前列腺细胞系PC-3M凋亡过程中的作用。方法1 mmol/L MMT诱导PC-3M细胞后,化学发光法检测细胞Caspase-3活力的变化,MTS法检测Caspase-3特异性多肽抑制剂Z-DEVD-FMK对细胞存活率的影响,Western blot法检测p38 MAPK在细胞凋亡过程中的活性变化,MTS及化学发光法检测p38 MAPK抑制剂SB203580对细胞凋亡率和Caspase-3活力的影响。结果在MMT诱导的PC-3M细胞凋亡中, Caspase-3被明显激活差异有统计学意义(P<0.01);Z-DEVD-FMK可明显提高PC-3M细胞存活率差异有统计学意义(P<0.01);p38 MAPK磷酸化程度明显升高;SB203580可抑制细胞凋亡差异有统计学意义(P<0.01),同时使Caspase-3活力下降差异有统计学意义(P<0.01)。结论Caspase-3及p38MAPK参与了MMT诱导的PC-3M细胞凋亡。     

醋酸铅对人股动脉内皮细胞凋亡及天冬氨酸特异性半胱氨酸蛋白酶-3表达的影响

目的探讨醋酸铅对人股动脉内皮细胞凋亡和天冬氨酸特异性半胱氨酸蛋白酶-3（caspase-3）活性的影响。方法醋酸铅染毒人股动脉内皮细胞后,用Hochest 33258染色法检测细胞形态学变化,用细胞免疫化学法检测caspase-3、P53、Bax、Bc l-2的表达;用流式细胞仪检测细胞凋亡率。结果经10μmol/L醋酸铅处理24、48 h后,Hochest 33258-PI染色显示醋酸铅组细胞呈凋亡形态学改变。动脉内皮细胞的细胞免疫化学结果都显示醋酸铅组的caspase-3、P53和Bax阳性表达升高,而Bc l-2的阳性表达下降。其流式细胞仪检测结果表明醋酸铅组细胞凋亡率较对照组显著升高（P〈0.05）。结论醋酸铅可促进动脉内皮细胞凋亡,其机制可能与capase-3的活化有关。     

乙醇对胎鼠脑新皮质神经干细胞凋亡的影响

目的探讨乙醇对胎鼠神经干细胞凋亡的影响。方法将妊娠14 d胎鼠脑新皮质来源的神经干细胞暴露于终浓度分别为0(对照)、25、50、100 mmol/L的乙醇培养基溶液3 d。采用Annexin V/PI法检测细胞早期和晚期凋亡情况,采用JC-1探针法检测线粒体膜电位的改变,并检测凋亡相关基因Bcl-2、Bax、Caspase-3 mRNA和蛋白的表达。结果与对照组比较,50、100 mmol/L乙醇染毒组神经干细胞的早期凋亡细胞率明显升高,差异有统计学意义(P0.05);而各浓度乙醇染毒组神经干细胞的晚期凋亡率无明显改变。且随着乙醇染毒浓度的升高,神经干细胞的早期凋亡细胞率呈上升趋势。仅100 mmol/L乙醇染毒组神经干细胞线粒体膜电位低于对照组,差异有统计学意义(P0.05)。与对照组比较,各浓度乙醇染毒组神经干细胞Bax mRNA的表达水平均上升,差异有统计学意义(P0.05);而Bcl-2和Caspase-3 mRNA的表达水平均无明显改变。且随着乙醇染毒浓度的升高,Bax mRNA表达量呈上升趋势。与对照组比较,50、100 mmol/L乙醇染毒组神经干细胞Bax蛋白的表达水平均升高,而Bcl-2/Bax蛋白的比值均降低,差异有统计学意义(P0.05);各浓度乙醇染毒组神经干细胞Bcl-2、Caspase-3蛋白的表达水平均无明显改变。结论乙醇可引起神经干细胞早期凋亡,其机制可能与线粒体损伤和Bax表达上调有关。     

酒精对小鼠NIT-1细胞氧化损伤及凋亡的影响

目的　研究酒精能否诱导小鼠胰岛素瘤细胞(NIT -1)凋亡,初步探讨凋亡发生机制。方法　体外培养的小鼠NIT- 1细胞用不同浓度酒精处理后,采用DNA琼脂糖凝胶电泳法观察酒精对小鼠NIT- 1细胞凋亡的影响;测定细胞丙二醛(MDA)和谷胱甘肽(GSH)的含量以及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH- Px ,GPX)的活性以判断细胞氧化程度;采用比色法测定Caspase 3酶活性。结果　酒精导致NIT 1细胞琼脂糖凝胶电泳显示典型的梯状凋亡条带。与对照组比,剂量组MDA生成增多,SOD、GSH -Px活性下降,GSH含量下降。酒精作用后NIT 1细胞caspase- 3活性升高,升高程度与作用时间及剂量有关。结论　提示高浓度酒精可导致体外培养的小鼠NIT- 1胰岛β细胞氧化抗氧化失衡,氧化应激诱导细胞发生凋亡,凋亡的发生很可能与Caspase- 3激活有关。     

醋酸铅对人肾小管上皮细胞凋亡及Caspase-3的表达影响

目的探讨醋酸铅对人肾小管上皮细胞(HK-2)凋亡作用及Caspase-3活性的影响。方法醋酸铅(5、10、20μmol/L)染毒HK-26、12、24、48h后,四甲基偶氮唑蓝(MTT)检测细胞增殖。经铅(5、10、20μmol/L)染毒24h后细胞免疫化学法、RT-PCR和Western Blotting检测Caspase-3的转录与表达。结果与空白对照组相比,不同浓度的铅均不同程度的抑制了HK-2的增殖(P﹤0.01)。细胞免疫化学法、RT-PCR和Western Blotting结果显示醋酸铅组的Caspase-3转录与表达升高。结论醋酸铅可促进人肾小管上皮细胞的凋亡,Caspase-3参与了凋亡过程。     

Involvement of caspase 3 mediated apoptosis in hematopoietic cytotoxicity of metabolites of ethylene glycol monomethyl ether

The hematopoietic toxicity of ethylene glycol monomethyl ether (EGME) and its metabolites, methoxy acetaldehyde (MALD) and methoxyacetic acid (MAA), was analyzed using human bone marrow cells from a lymphoma patient without bone marrow involvement and a human leukemia cell line, HL60. After 24-hour incubation, the concentrations of 50 percent inhibition (IC50) of human hematopoietic progenitor cells with MALD or MAA were 3 mM and 3.9 mM, respectively, and EGME (10 mM or more) did not show any cytotoxicity. IC50 (after 48-hour exposure) of MALD and MAA on HL60 cells were 2.45 mM and 5.6 mM, respectively, suggesting that both hematopoietic progenitor cells and HL60 have a similar sensitivity. DNA ladder formation, a characteristics of apoptosis, was observed in MALD- or MAA-treated HL60 cells, but not in EGME-treated samples. Caspase-3 enzyme activity, the effector of the apoptotic process, was greatly enhanced with MALD treatment. The inhibitor of caspase-3 repressed cell death induced with MALD as well as MAA.     

Depletion of intracellular zinc induced apoptosis in cultured hippocampal neurons through Raf/MEK/ERK pathways

An experiment was performed to observe the changes in Raf-1 kinase/mitogen-activated protein kinase ERK (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways in cultured hippocampal neurons and its correlation with neurons apoptosis induced by intracellular zinc depletion. Cultured hippocampal neurons were exposed to a cell membrane-permeant zinc chelator TPEN (2 μM), and to TPEN plus zinc sulfate (5 μM) for 24 h. Cultures were then processed to detect neuronal viability by the methyl thiazolyl tetrazolium assay, while apoptosis rate was simultaneously observed by the flow cytometric analysis. Caspase-3, Raf-1, pMEK, pERK1/2, and pCREB protein levels were examined by Western blot assays. The viability in TPEN-incubated neurons was notably decreased, apoptosis rate and expression of caspase-3 significantly increased compared to untreated controls. The significant down-regulation of Raf/MEK/ERK signaling pathway and expression of pCREB were decreased in TPEN-treated neurons. Co-addition of zinc almost completely reversed TPEN-induced alterations described. The results demonstrated zinc-modulated apoptosis and the expression of Raf/MEK/ERK at the protein level in hippocampal neurons. It is possible that zinc depletion-induced apoptosis in cultured hippocampal neurons may be relevant to the changes of Raf/MEK/ERK signaling pathway.     

Generation of anti-beta-amyloid antibodies via phage display technology towards Alzheimer's disease vaccination

The pathology of Alzheimer's disease (AD) shows a significant correlation between beta-amyloid peptide (betaAP) deposition and the clinical severity of dementia. The ability of site-directed antibodies towards the N-terminal region of beta-amyloid peptide to suppress in vitro formation of toxic beta-amyloid serves as a factual basis for in vivo investigations. We localized the epitope of these anti-aggregating antibodies, and injection of phage displaying this epitope induced antibodies against the whole anti-beta-amyloid peptide. In Alzheimer's diseased transgenic mice, these antibodies are delivered from the periphery to the CNS preventing beta-amyloid formation and/or dissolving such aggregates. Performance of such antigens opens up possibilities for development of an efficient, long-lasting immunization procedure for treatment of Alzheimer's disease.     

Induction of apoptosis by the aqueous extract of Rubus coreanum in HT-29 human colon cancer cells

OBJECTIVES: The incompletely ripened fruit of Rubus coreanum (IRFRC) has been used in traditional herbal medicine to manage various diseases. To explore the possibility that IRFRC has chemopreventive effects, we examined whether or not extracts of IRFRC inhibits HT-29 cell growth and explored the mechanism for this effect. METHODS: We cultured HT-29 cells in the presence of the aqueous or ethanol extract of IRFRC. DNA synthesis was estimated by 5-bromo-2'-deoxyuridine incorporation. We measured apoptosis using a DNA fragmentation assay and Annexin V staining. We used western blot analyses to determine the cleavage of caspases and poly (adenosine diphosphate-ribose) polymerase. RESULTS: Aqueous extract of IRFRC substantially inhibited viable HT-29 cell number in a dose-dependent manner, whereas ethanol extract had only a minimal effect. Aqueous extract inhibited DNA synthesis and induced apoptosis of HT-29 cells in a dose-dependent manner. Aqueous extract induced cleavage of caspase-3, -7, and -9 and induced the activity of caspase-3 and cleavage of poly (adenosine diphosphate-ribose) polymerase. CONCLUSIONS: We have shown that aqueous extract of IRFRC inhibits cell proliferation and stimulates apoptosis in HT-29 cells, and that this may be mediated by its ability to activate the caspase-3 pathway. It remains to be determined whether the aqueous extract of IRFRC has chemopreventive activities in animal models.     

Curcumin-Induced Apoptosis in PC3 Prostate Carcinoma Cells Is Caspase-Independent and Involves Cellular Ceramide Accumulation and Damage to Mitochondria

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 μ M curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.     

中性内肽酶高表达对β-淀粉样肽诱导的PC12细胞凋亡的保护作用

目的 确定中性内肽酶（NEP）高表达对β-淀粉样肽（AB）诱导的PC12细胞凋亡保护作用的分子机制。方法 利用脂质体转染法建立可稳定表达NEP和失活NEP的PC12细胞系。逆转录-聚合酶链反应（RT-PCR）法和免疫印迹法（Western blot法）检测基因在细胞中的表达。利用AB25-35诱导PC12细胞，CellTiter96AQueous单溶液检测系统（MTS）检测细胞存活率；分光光度法检测乳酸脱氢酶（LDH）含量、活性氧（ROS）生成量水平变化；化学发光法检测细胞三磷酸腺苷（ATP）生成量和Caspase-3表达量的变化。结果 在AB25-35诱导PC12细胞凋亡中，NEP高表达可明显提高细胞存活率，降低培养基中LDH含量和ROS生成量；提高细胞ATP释放量；抑制Caspase-3在细胞凋亡中的激活，与正常PC12细胞相比，差异均有统计学意义（P〈0．05）。稳定表达失活NEP的PC12细胞则对细胞凋亡没有影响。结论 NEP通过降低氧化应激反应对细胞线粒体的损伤，抑制凋亡关键蛋白Caspase-3的激活，从而提高AB25-35诱导的神经细胞的存活率。     

Activation of neutral sphingomyelinase participates in ethanol-induced apoptosis in Hep G2 cells

The mechanism underlying ethanol-induced apoptosis in liver cells is not clear. Sphingomyelin (SM) metabolism is a novel signal transduction pathway that has an impact on apoptosis in many cell types. We investigated whether the SM pathway is involved in ethanol-induced apoptosis in the liver. Hep G2 cells were treated with ethanol followed by assaying apoptosis, sphingomyelinase (SMase) activity, caspase-3 activity, and the changes of SM content in the cells. We found that ethanol dose-dependently increased apoptosis and the effect was accompanied by increases of caspase-3 activity and neutral SMase activity. At concentrations of 80 and 160 mM, ethanol significantly increased caspase-3 activity by 120% and neutral SMase activity by 24%. The activity of acid SMase was only slightly increased without statistical significance. C(2)-ceramide, the exogenous SM metabolite, mimicked the effects of ethanol on apoptosis and caspase-3 activation. When the SM content was determined 24 h after treatment with ethanol, its level was 15% lower than that of controls. The results indicate that metabolism of SM triggered by neutral SMase participates in ethanol-induced apoptosis in Hep G2 cells and activation of caspase-3 is involved in the apoptotic pathway.     

曲古抑菌素A阻断Stat3信号通路诱导前列腺癌细胞凋亡的实验研究

目的本实验研究组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)对前列腺癌细胞DU145的抑制作用及其对信号传导与转录激活因子3(Stat3)信号通路的影响。方法采用不同浓度的TSA处理DU145不同时间后,四氮甲基唑蓝比色法测定TSA对细胞活力的抑制效应;流式细胞仪分析细胞周期的改变;蛋白印迹实验检测细胞凋亡相关蛋白半胱氨酸蛋白酶(Caspase)家族的Caspase-8、Caspase-9、Caspase-3、二磷酸腺苷核糖多聚酶(PARP)及Stat3信号蛋白活性(phospho-Stat3)的变化。结果 TSA时间和剂量依赖性地抑制DU145细胞的增殖,TSA处理细胞24 h和48 h后,细胞生存率分别是85.7%和68.7%;细胞经TSA处理后,细胞形态和细胞周期均发生明显的变化,细胞周期被阻断在G0/G1期,其细胞百分比从55.6%增加到68.5%;Western blot检测结果显示,TSA作用DU145细胞后,Stat3信号蛋白的磷酸化水平下降,同时IL-6对Stat3的刺激诱导作用也被TSA所阻断;Caspase-8、Caspase-9、Caspase-3、PARP等凋亡蛋白被TSA诱导活化,并发生显著剪切。结论 TSA能够通过抑制Stat3信号通路的活性来诱导DU145细胞凋亡。