三氧化二砷对肝癌细胞系SMMC-7721凋亡及 Smac caspase-9、caspase-3 蛋白表达的影响

目的：研究三氧化二砷（As203）对人肝癌细胞SMMC-7721的促凋亡作用及对Smac、caspase-9、caspase-3表达的影响。方法：人肝癌细胞SMMC-7721经As20,处理,共分为四组,分别为空白对照组、低剂量组、中等剂量组、高剂量组。分别采用MTT、Hoechst33258染色法、Annexin V-FITC／PI双染法观察其对SMMC．7721细胞增殖的抑制,凋亡细胞核的形态学变化,以及诱导凋亡作用;采用Westemblot法检测凋亡相关蛋白Smac、caspase-9、caspase-3表达的变化。结果：MTT显示：As203在体外能明显抑制SMMC-7721的生长,具有时间剂量依赖关系,与空白对照组相比,其余三组细胞生存率明显下降,差异均有统计学意义（P〈0．05）;Hoechst33258显示细胞呈明显的凋亡细胞形态学特征,具有剂量依赖性;AnnexinV-FITC／PI双染法显示：As203作用24小时可诱导SMMC-7721细胞凋亡,且呈剂量依赖性,与空白对照组相比（2．69±0．58）,其余三组（4．01±0．58）、（5．99±1．69）、（9．26±2．34）差异均有统计学意义（P〈0．05）;Westernblot显示：As2O3作用SMMC-7721细胞24小时,Smac、caspase-9、caspase-3表达上升,呈剂量依赖性,与空白对照组相比,其余三组蛋白表达量明显增加,差异均有统计学意义（P〈0．05）。结论：-定量的As203能抑制SMMC-7721细胞增殖,促进其凋亡,其机制可能与调控Smac、caspase-9、caspase-3表达有关。     

嵌合重组caspase-3诱导表达促进肿瘤细胞凋亡

通过稳定转染人宫颈癌HeLa细胞,建立了野生型caspase-3（wt-casp3）,大小亚基序列颠倒的重组caspase-3 (r-casp3),和N端融合绿脓杆菌外毒素（PE）转膜肽段的嵌合重组caspase-3 (cr-casp3)的诱导表达细胞系.蜕皮素诱导后细胞中检测到目的基因的表达,MTT检测和细胞计数结果表明,r-casp3和cr-casp3诱导表达后有效地导致HeLa细胞死亡,通过测定细胞中caspase-3活性,以及细胞周期检测、DNA梯状电泳条带检测(DNA ladder)、电镜观察等证实r-casp3和cr-casp3诱导表达后细胞发生了凋亡,且二者的促凋亡活性相当,而wt-casp3诱导表达细胞并未出现上述效应.结果表明,与野生型caspase-3活化需要上游分子的切割不同,重组caspase-3具有自发的促凋亡活性,而N端PE肽段的融合不影响这种活性,因此PE转膜结构域和重组caspase-3有望参与构建能转膜进入细胞内部,并杀伤细胞的新型肿瘤治疗分子.     

中国地鼠口腔颊囊黏膜癌变过程凋亡相关基因caspase-3、caspase-9、Bax、Bcl-2的表达

目的探讨凋亡相关基因caspase-3、caspase-9、Bax、Bcl-2在中国地鼠口腔正常黏膜、上皮单纯增生、上皮异常增生和鳞状细胞癌组织中的表达及其意义。方法采用免疫组织化学法、RT-PCR检测正常中国地鼠口腔黏膜上皮、单纯增生上皮、异常增生上皮和鳞癌组织中caspase-3、caspase-9、Bax、Bcl-2蛋白及mRNA的表达。结果在口腔黏膜癌变过程中,鳞癌组织中抑制凋亡蛋白Bcl-2表达明显高于口腔正常黏膜、上皮单纯增生、上皮异常增生(P0.05);异常增生上皮caspase-3、caspase-9、Bax表达均明显高于正常组(P0.05),但随着增生程度的加重,caspase-3、caspase-9、Bax表达明显降低(P0.05)。相关性分析显示,鳞癌组织中Bcl-2与Bax、caspase-3、caspase-9呈负相关(P0.05)。RT-PCR结果表明,与正常组织相比,鳞癌组织中Bcl-2高表达,而caspase-3、caspase-9、Bax的mRNA表达显著下调(P0.05)。结论本实验在中国地鼠鳞癌组织中caspase-3、caspase-9、Bax表达降低而Bcl-2表达上升,揭示了caspase-3、caspase-9、Bax、Bcl-2表达与口腔鳞癌的发生、发展密切相关,可以为口腔颊囊黏膜癌的基因治疗提供一些线索或并为评价OSCC的生物学特征及预后提供参考。     

消癌平诱导caspase-3活化动态过程的实时监测

消癌平是从萝摩科(Asclepiadaceae)植物通关藤(Marsdenia tenacissima)根部提取的药物,已经广泛应用于癌症和多种炎症的临床治疗,并且具有较好的疗效。将消癌平注射液与细胞培养液按照1∶1的比例混合后共同培养人肺腺癌(ASTC-a-1)细胞,通过CCK-8方法检测发现消癌平能够显著抑制细胞的增长。然后利用荧光共聚焦扫描显微镜和荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术在稳定表达了SCAT3质粒的单个ASTC-a-1细胞中实时动态监测消癌平处理后SCAT3的空间分布以及SCAT3被caspase-3切割的动态过程。实验结果表明:消癌平处理后20 min中SCAT3开始向细胞核以及细胞膜部位转移,约在100min左右SCAT3被迅速切割。     

Changes in FADD levels,distribution, and phosphorylation in TNFα-induced apoptosis in hepatocytes is caspase-3, caspase-8 and BID dependent

FADD/MORT1 (The adaptor protein of Fas Associate Death Domain/Mediator of Receptor Induced Toxicity) is essential for signal transduction of death receptor signaling. We have previously shown that FADD is significantly up-regulated in TNFα/ActD induced apoptosis. Over-expression of FADD also induces death of lung cancer cells and primary hepatocytes. We hypothesize that the increase in detectable FADD levels require the proximal steps in apoptotic signaling and speculated that FADD would be redistributed in cells destined to undergo apoptosis. We show that monomeric non-phosphorylated FADD is up-regulated in hepatocytes treated with TNFα/ActD and that it accumulates in the cytoplasm. Nuclear phosphorylated FADD decreases with TNFα/ActD treatment. Dimeric FADD in the cytoplasm remains constant with TNFα/ActD. The change in FADD levels and distribution was dependent on caspase-3, caspase-8 activity and the presence of BID. Thus, changes in FADD levels and distribution are downstream of caspase activation and mitochondria changes that are initiated by the formation of the DISC complex. Changes in FADD levels and distribution may represent a novel feed-forward mechanism to propagate apoptosis signaling in hepatocytes. Xiaoying Zhang and Raghuveer Vallabhaneni contributed equally to the work.     

晚期糖基化白蛋白通过激活caspase-12途径诱导巨噬细胞凋亡

本文旨在研究晚期糖基化白蛋白(advanced glycated albumin,AGE-alb)对巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)凋亡途径关键分子caspase-12的影响,以阐明AGE-alb对巨噬细胞凋亡的诱导作用及机制。体外培养RAW264.7巨噬细胞,分别给予AGE-alb(2、4和6 g/L)、正常对照白蛋白(control albumin,C-alb,4 g/L)、ERS诱导剂衣霉素(tunicamycin,TM,4 mg/L)处理,或以ERS抑制剂4-苯丁酸(4-phenylbutyric acid,PBA,5 mmol/L)预处理细胞1 h,然后与AGE-alb(4 g/L)共孵育24 h。采用MTT法检测细胞活力,TUNEL法检测细胞凋亡情况,试剂盒测定培养基乳酸脱氢酶(lactate dehydrogenase,LDH)活性,免疫印迹法检测caspase-12表达变化。结果显示:与TM相似,AGE-alb显著诱导RAW264.7巨噬细胞损伤,表现为细胞活力降低,LDH漏出和细胞凋亡率明显增加,且呈浓度依赖性。AGE-alb明显上调caspase-12活性,尤其在4和6 g/L浓度时更为显著(P0.01)。然而,PBA可抑制AGE-alb所致的巨噬细胞活力降低以及LDH漏出和凋亡增加,且减轻AGE-alb诱导的caspase-12活化(P0.05)。上述结果提示,AGE-alb可诱导RAW264.7巨噬细胞凋亡,其机制可能与激活caspase-12介导的ERS凋亡途径有关。     

斜纹夜蛾spli caspase-1部分基因的克隆与分析

紫外线、芹菜夜蛾核型多角体病毒(sfMNPV)和苜蓿银纹夜蛾核型多角体病毒(AcMNPV)都可诱导斜纹夜蛾(Spodoptera littoralis)SL-1细胞凋亡,这说明在SL-1细胞中Caspase表达活性高,本实验从莲纹夜蛾(Spodoptera littoralis)和草地贪夜蛾(Spodoptera frugiperda)序列出发,根据其中保守性高的序列设计引物,提取SL-1细胞中的总RNA,并进行逆转录和PCR扩增,获得了一条419bp的cDNA。     

腺性膀胱炎及膀胱癌中livin 和caspase-9 的表达

目的:研究在腺行膀胱炎(cystitis glandularis,CG)以及膀胱癌(bladder carcinoma,BC)中livin和caspase-9的表达,探讨腺性膀胱炎与膀胱癌可能存在的关系。方法:收集哈尔滨医科大学附属第三医院2014年1月~2014年10月住院患者60例组织石蜡标本,应用SP(streptavidin-perosidase)法检测livin和caspase-9在腺性膀胱炎组织(30例)、膀胱癌组织(30例)和正常组织(30例)中的表达。结果:正常膀胱、CG和BC组织中livin阳性表达率分别为0/30,9/30(30%)和14/30(46.67%),差异有统计学意义(P0.05);caspase-9在正常膀胱、CG和BC组织中的阳性表达率分别20/30(66.67%),15/30(50%),3/30(10%),差异有统计学意义(P0.05)。结论:腺性膀胱炎可能是正常膀胱组织向膀胱腺癌发展的一个中间阶段,在CG向BC发展过程中livin和caspase-9的异常表达具有普遍性,临床上应积极治疗并密切随访,并可将livin和caspase-9作为诊断早期膀胱癌的检测指标。     

重构型人caspase-8基因的表达诱导HeLa细胞凋亡

以两种大、小亚基基因次序颠倒的重构型人caspase-8基因的pIRES2-EGFP真核表达载体转染HeLa细胞,用间接免疫荧光染色、免疫细胞化学染色和电镜观察等方法,研究重构型人caspase-8基因在HeLa细胞中的表达及其促凋亡活性.结果显示,两种重构型人caspase-8基因在HeLa细胞中的表达可以有效地诱导HeLa细胞凋亡.     

Anesthetic propofol attenuates the isoflurane-induced caspase-3 activation and Aβ oligomerization

Accumulation and deposition of β-amyloid protein (Aβ) are the hallmark features of Alzheimer''s disease. The inhalation anesthetic isoflurane has been shown to induce caspase activation and increase Aβ accumulation. In addition, recent studies suggest that isoflurane may directly promote the formation of cytotoxic soluble Aβ oligomers, which are thought to be the key pathological species in AD. In contrast, propofol, the most commonly used intravenous anesthetic, has been reported to have neuroprotective effects. We therefore set out to compare the effects of isoflurane and propofol alone and in combination on caspase-3 activation and Aβ oligomerization in vitro and in vivo. Naïve and stably-transfected H4 human neuroglioma cells that express human amyloid precursor protein, the precursor for Aβ; neonatal mice; and conditioned cell culture media containing secreted human Aβ40 or Aβ42 were treated with isoflurane and/or propofol. Here we show for the first time that propofol can attenuate isoflurane-induced caspase-3 activation in cultured cells and in the brain tissues of neonatal mice. Furthermore, propofol-mediated caspase inhibition occurred when there were elevated levels of Aβ. Finally, isoflurane alone induces Aβ42, but not Aβ40, oligomerization, and propofol can inhibit the isoflurane-mediated oligomerization of Aβ42. These data suggest that propofol may mitigate the caspase-3 activation by attenuating the isoflurane-induced Aβ42 oligomerization. Our findings provide novel insights into the possible mechanisms of isoflurane-induced neurotoxicity that may aid in the development of strategies to minimize potential adverse effects associated with the administration of anesthetics to patients.     

灵芝孢子粉对戊四氮活化海马神经细胞caspase-9 表达的研究

目的:本实验研究灵芝孢子粉对戊四氮活化大鼠海马神经细胞caspase-9表达变化的影响,进一步探索灵芝孢子粉对癫痫大鼠脑保护的作用机制和癫痫与海马神经细胞凋亡调控基因之间的关系。方法:通过制备癫痫模型,RT-PCR和Western blotting检测正常对照组、癫痫模型组和灵芝孢子粉用药组caspase-9的表达。结果:癫痫模型组和灵芝孢子粉用药组caspase-9的表达较正常对照组均升高;其中癫痫模型组caspase-9的表达水平与对照组比较明显升高,灵芝孢子粉用药组caspase-9的表达水平与癫痫模型组比较明显降低,差异有统计学意义。结论:本研究结果证实,Caspase-9介导了癫痫大鼠神经细胞凋亡机制,说明灵芝孢子粉有效成份能充分作用于脑组织,可以调控caspase-9的表达,发挥抗凋亡的神经保护作用。     

多奈哌齐对血管性痴呆小鼠海马caspase-3表达的影响

目的探讨盐酸多奈哌齐对血管性痴呆(VD)小鼠海马半胱氨酸蛋白酶-3(cysteinyl aspirate specific-proteinase3,caspase-3)表达的调节作用。方法采用双侧颈总动脉反复缺血-再灌注法制备小鼠VD模型。90只雄性昆明小鼠随机分为3组,假手术组(30只)、模型组(30只)及盐酸多奈哌齐治疗组(30只)。在术后第29天、第30天,经跳台试验和水迷宫试验进行行为学测试,用免疫组化和western blot方法检测各组小鼠海马caspase-3的表达变化。结果盐酸多奈哌齐治疗组小鼠的学习和记忆成绩明显高于VD模型组小鼠(P<0.05);免疫组化和Western blot结果发现,盐酸多奈哌齐治疗组小鼠海马CAl区caspase-3蛋白阳性表达的平均光密度值显著低于VD模型组(P<0.05)。结论盐酸多奈哌齐能够降低VD小鼠海马caspase-3的表达。     

β-淀粉样蛋白通过激活caspase-3诱导PC12细胞凋亡

目的探讨β-淀粉样蛋白25-35片段（Aβ25-35）对体外培养的大鼠嗜铬瘤细胞PC12细胞促凋亡机制。方法采用四甲基偶氮唑蓝（MTT）法观察不同浓度的Aβ25-35干预PC12细胞24h后的细胞活性;将细胞分为对照组、实验组（即20 mmol/L Aβ25-35组）,流式细胞技术观察两组PC12细胞凋亡率;免疫细胞化学染色法观察PC12细胞凋亡基因caspase-3的表达。结果PC12细胞活性呈Aβ25-35剂量依赖性降低,且浓度为20 mmol/L时降低最显著;PC12细胞实验组的凋亡率为23.03%±1.22%,对照组为2.42%±0.87%（P〈0.01）;caspase-3实验组的阳性表达较对照组明显增加（P〈0.01）。结论Aβ可通过激活促凋亡基因caspase-3诱导PC12细胞凋亡。     

Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ～200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.     

大鼠脑缺血再灌注后Cathepsin D和caspase-9表达的动态变化

目的:探讨大鼠缺血再灌注后溶酶体组织蛋白酶D(Cathepsin D CD)、caspase-9在不同时段蛋白质及mRNA表达变化。方法:将60只S D大鼠随机分为三组:正常组(10只),假手术组(10只),脑缺血再灌注组(40只),线栓法制备大脑中动脉梗死模型(MCAO),免疫组化及RT-PCR法分别检测Cathepsin D、caspase-9的蛋白和mRNA表达。结果:与正常组和假手术组比较,模型组大鼠脑缺血再灌注损伤后6h Cathepsin D的蛋白和mRNA表达明显增强(P<0.05),24h达高峰,48h仍保存高水平。caspase-9蛋白和mRNA6h开始明显升高,12h达高峰,此后缓慢下降,但48h组仍显著高于对照组(P<0.05),结论:Cathepsin D、caspase-9在大鼠脑缺血再灌注后表达增强,溶酶体可能参与了脑缺血再灌注损伤后神经细胞凋亡的过程。     

Important role of β1-integrin in fucoidan-induced apoptosis via caspase-8 activation

Fucoidan induces apoptosis by activating caspase-8 in human MCF-7 breast cancer cells, but the detailed mechanism for this is not understood. We demonstrate here that fucoidan interacted with the cell surface, and silencing the β1-integrin gene expression inhibited fucoidan-induced apoptosis accompanied by caspase-8 activation. Fucoidan induced formation of the β1-integrin-caspase-8 complex. These data indicate that β1-integrin is an important factor for the cell-surface binding of fucoidan and plays an important role in fucoidan-induced apoptosis. Fucoidan also induced recruitment of caspase-8 to the β1-integrin intracellular domain, cleaved it into the activated protein by direct combination with β1-integrin, and induced apoptosis via the caspase cascade in MCF-7 cells.