AIF与凋亡的关系及其相关的结直肠癌治疗研究

许多促凋亡信号转导分子作用于线粒体,引发线粒体外膜通透性的改变,从而引发潜在的促凋亡蛋白的释放.其中较为重要的一个为凋亡诱导因子(AIF).当收到致命信号后,由线粒体释放,并迅速从细胞质移位到细胞核里结合DNA并引起不依赖天冬半胱氨酸蛋白激酶(Caspase)的染色质凝集,从而导致细胞凋亡.AIF已逐步被发现在一些恶性癌细胞(如结直肠癌细胞)中具有重要作用,特别是它在细胞凋亡中的调控作用.最近一些研究成果表明,通过影响AIF释放或表达来诱导细胞凋亡已成为结直肠癌治疗的新的研究方向.     

丙泊酚对Ca2+诱导大鼠离体心肌线粒体损伤的影响

目的 探讨丙泊酚对Ca2+诱导大鼠离体心肌线粒体损伤的影响.方法 Wistar大鼠35只,断头处死迅速取出心脏制备线粒体悬液后,随机均分为五组:空白对照组(C组);CaCl2组(Ca2组),加入CaCl2100 nmol/mg prot;P25组,加入丙泊酚25 μp.mol/L)P50组,加入丙泊酚50 μmol/L;P100组,加入丙泊酚100 μmol/L.P25、P50、P100组也加入CaCl2 100 nmol/mg prot.20 min后采用Western blot法测定线粒体释放细胞色素C(Cyt C)和凋亡诱导因子(AIF)的水平.结果 与C组比较,其他四组Cyt C和AIF释放增加(P<0.01);与Ca2+组比较,P25组、P50组、P100组Cyt C、AIF释放减少(P<0.01);P50组、P100组又较P25组Cyt-C释放减少(P<0.01).结论 丙泊酚25、50、100μmol/L均可减轻Ca2+诱导大鼠离体心肌线粒体损伤,从而产生心肌保护作用.     

线粒体介导细胞凋亡和心肌保护新途径

在细胞凋亡过程中,线粒体起着主开关作用,线粒体通透性转换孔开放,线粒体跨膜电位耗损,细胞色素C和凋亡诱导因子释放到胞质中,通过两条途径分别导致细胞凋亡,Bcl－2家族蛋白以线粒体为靶位调控凋亡。线粒体K_(ATP)通道开放可以预防线粒体跨膜电位耗损而减少细胞凋亡,从而揭示了线粒体K_(ATP)通道开放产生心肌保护的新途径。     

羧甲基壳聚糖通过抑制线粒体途径保护氧化损伤诱导雪旺细胞凋亡

[目的]探讨线粒体途径在羧甲基壳聚糖(carboxymethylated chitosan,CMCS)保护过氧化氢(H_2O_2)诱导雪旺细胞(Schwann cells,SCs)凋亡中的作用及机制。[方法]体外培养SCs,S-100免疫荧光染色鉴定。将SCs分为空白对照组、H_2O_2诱导组、H_2O_2加CMCS处理组。流式细胞仪检测细胞凋亡比率,罗丹明(Rhodamine123)荧光染色检测细胞线粒体膜电位水平,Western blot检测SCs内细胞色素C(Cytochrome C)表达水平。[结果]本实验培养细胞经S-100荧光染色鉴定阳性率达95%以上,H_2O_2诱导SCs凋亡,降低线粒体膜电位,增加细胞色素C释放;而在加入CMCS后SCs凋亡比例降低,线粒体膜电位增加,细胞色素C释放减少。[结论]CMCS通过抑制线粒体细胞凋亡途径保护H_2O_2诱导SCs凋亡。     

线粒体途径在肿瘤坏死因子凋亡诱导配体诱导结肠癌细胞凋亡中的调节作用

目的探讨线粒体途径在肿瘤坏死因子凋亡诱导配体(TRAIL)诱导结肠癌细胞凋亡过程中的调节作用,为临床合理用药提供理论指导。方法采用流式细胞仪技术、荧光显色技术和Western印迹技术检测TRAIL处理结肠癌细胞SW1116后,在不同时间细胞凋亡情况、线粒体完整性改变(ΔΨm、cardiolipin情况)以及线粒体下游通路细胞色素C和Caspase-9的表达情况。结果TRAIL诱发结肠癌细胞凋亡,于4 h达凋亡高峰,凋亡指数为32．98%；在4 h出现线粒体ΔΨm下降和cardiolipin丢失增加,造成其内膜损伤；细胞色素C表达及Caspase-9酶活性随时间的延长而增加,24 h酶活性达到最大峰值为(48．12±2．21)μmol·L~(-1)·h~(-1)·mg~(-1)蛋白。TRAIL诱导的线粒体损伤可被Caspase抑制剂Z-VAD．fmk所抑制。结论线粒体途径参与TRAIL诱导结肠癌细胞的凋亡过程,以Caspase依赖方式引发线粒体ΔΨm和cardiolipin丢失,造成内膜损伤,导致细胞色素C释放和Caspase-9激活,诱发凋亡。     

线粒体介导细胞凋亡和心肌保护新途径

在细胞凋亡过程中，线粒体起着主开关作用，线粒体通透性转换孔开放，线粒体跨膜电位耗损，细胞色素C和凋亡诱导因子释放到胞质中，通过两条途径分别导致细胞凋亡，Bcl-2家族蛋白以线粒体为靶位调控凋亡。线粒体KATP通道开放可以预防线粒体跨膜电位耗损而减少细胞凋亡，从而揭示了线粒体KATP通道开放产生心肌保护的新途径。     

凋亡诱导因子和caspase-3在结直肠腺瘤-癌序列中的表达及相关性

目的探讨非caspase依赖凋亡途径的凋亡诱导因子（AIF）与caspase依赖凋亡途径的caspase-3在结直肠腺瘤．癌序列中的表达变化及两种蛋白之间的相关性。方法采用免疫组织化学染色检测18例正常黏膜、84例结直肠腺瘤和72例结直肠癌中AIF及caspase-3的表达情况。结果结直肠腺瘤组织中AIF及caspase-3的阳性表达率均明显高于正常黏膜组织（P〈0．05）;腺瘤组织中AIF阳性表达率与腺癌之间的差异无统计学意义（P〉0．05）,而caspase-3阳性表达率则明显高于腺癌组织（P〈0．05）;腺瘤组内绒毛状腺瘤AIF阳性表达率明显低于管状腺瘤（P〈0．05）,而caspase-3阳性表达率在两种腺瘤之间的差异无统计学意义（P〉0．05）。相关分析显示,AIF表达与caspase-3表达无显著相关性（P〉0．05）。结论AIF代表的非caspase依赖凋亡途径异常发生在腺瘤向癌突变早期．而caspase依赖途径的凋亡失控可能是更重要的促进结直肠癌发生的因素之一。非caspase依赖的凋亡途径与caspase依赖的凋亡途径是肿瘤发生中两条相对独立的细胞凋亡途径。     

聚酰胺树形分子递送Survivin反义寡核苷酸诱导结肠癌细胞凋亡的研究

目的 观察聚酰胺(PAMAM)树形分子高聚合物介导Survivin反义寡核苷酸(Sur-vivin-asODN)转染结直肠癌SW620细胞的可行性以及对结直肠癌SW620细胞凋亡的影响.方法 制备PAMAM反义基因复合物和阳离子脂质体反义基因复合物,透射电镜观察复合物的形态,激光散射粒径分析仪测定粒径,zeta电位分析仪测定zeta电位,离心法和紫外分光分度仪测定复合物的的包封率、载药率和体外DNA释放速度.将上述两种基因转染复合物转染结直肠癌细胞,测定其转染效率;检测转染后细胞中Survivin蛋白的表达和细胞的凋亡率.结果 PAMAM-Survivin-asODN复合物的粒径小于脂质体-Survivin-asODN复合物的粒径(P<0.01),但zeta电位高于后者(P<0.05);基因载药率、包封率两组差异无统计学意义;PAMAM对DNA持续释放14 d,但脂质体只持续5 d.PAMAM.Survivin-asODN转染结直肠癌细胞的效果强于脂质体-SurvivinasODN(P<0.05).转染后结直肠癌细胞Survivin蛋白的表达低于脂质体复合物(P<0.05),细胞的凋亡率高于脂质体复合物(P<0.05).结论 PAMAM能将Survivin-asODN高效递送到结直肠癌SW620细胞,降低Survivin蛋白表达并诱导结直肠癌细胞凋亡.     

南瓜蛋白诱导人胰腺癌细胞凋亡的线粒体机制

目的 探讨南瓜蛋白(CUS)诱导人胰腺癌细胞(PANC)-1凋亡的线粒体机制.方法 0、2.5、10.0、40.0 mg/L的CUS处理胰腺癌PANC-1 72 h,荧光显微镜和流式细胞仪观察线粒体膜电位(△Ψm)的变化,Western blot检测胞质细胞色素C(Cyt-C)的含量变化和细胞半胱氨酰天冬氨酸特异性蛋白酶-9(Caspase-9)和Caspase-3、B细胞淋巴瘤/白血病-2(bcl-2)和B细胞淋巴瘤/白血病-2相关X蛋白(bax)蛋白的表达水平.结果 0、2.5、10.0、40.0 mg/L CUS处理PANC-1 72 h后,△Ψm逐渐下降,分别为167.23±8.27、123.56±7.26、83.25±5.36和40.45±5.87,差异有统计学意义(P＜0.05);Western blot结果显示线粒体Cyt-C释放到细胞质的量逐渐增加,灰度值分别为0.29±0.05、1.69±0.35、1.87±0.40、2.47±0.32(P＜0.05);Caspase-9、Caspase-3和bax蛋白的表达逐渐增强,且其作用呈剂量依赖性(Caspase-9灰度值分别为0.15±0.03、0.19±0.05、0.49±0.09、0.89±0.08,P＜0.05;Caspase-3灰度值分别为0.88±0.08、1.81±0.19、2.36±0.38、2.92±0.24,P ＜0.05;bax灰度值分别为0.79±0.18、1.66±0.31、2.61±0.41、3.67±0.24,P＜0.05);而bcl-2蛋白表达无明显变化(bcl-2灰度值分别为0.88±0.08、0.82±0.18、0.84±0.04、0.82±0.13,P＞0.05).结论 线粒体途径在CUS诱导胰腺癌细胞凋亡中起重要作用,其机制可能是CUS通过激活促凋亡蛋白bax表达增加,降低△Ψm,促进线粒体Cyt-C的释放,从而激活Caspase系列酶,诱导胰腺癌细胞凋亡.     

GRIM-19的生物学活性及其与肿瘤关系的研究进展

干扰素/维甲酸联合应用诱导的细胞凋亡相关基因19(GRIM-19)是应用反义基因敲除法分离发现的一种新的细胞凋亡调节因子.GRIM-19存在于线粒体和细胞核中,在线粒体Ⅰ型呼吸过程中至关重要.GRIM-19参与细胞增生、凋亡调控过程,其表达水平降低或位点突变可以导致细胞的异常增生,同时,在病毒感染导致细胞癌变的过程中,GRIM-19可能是病毒癌基因结合的靶点.GRIM-19在肿瘤的形成和凋亡中发挥重要作用,可能成为一种新的肿瘤标记物,并可用于肿瘤早期筛选.     

Overexpression of SOD1 in transgenic rats attenuates nuclear translocation of endonuclease G and apoptosis after spinal cord injury

Spinal motor neurons are selectively vulnerable after spinal cord injury (SCI). Recent studies suggest they undergo apoptosis after caspase activation through a mitochondria-dependent apoptosis pathway, and that oxidative stress after SCI is likely to play a role. However, other signaling pathways of apoptosis that involve mitochondria have not been thoroughly studied after SCI. Apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are mitochondrial apoptogenic proteins that are capable of inducing neuronal apoptosis when translocated from mitochondria to nuclei through a caspase-independent pathway. In this study, we examined translocation of these proteins and apoptotic cell death of motor neurons. The role of oxidative stress was also studied using transgenic (Tg) rats that overexpress the intrinsic antioxidant copper/zinc-superoxide dismutase (SOD1). Western blots and an activity assay demonstrated that a greater amount of SOD1 and higher activity of SOD presented in mitochondria of Tg rats compared with wild-type (Wt) rats. Immunohistochemistry and Western blots showed translocation of EndoG and AIF from mitochondria to nuclei in motor neurons 1 day after SCI in both groups of rats. However, there was significantly less translocation of EndoG in the Tg rats compared with the Wt rats. Less apoptotic cell death was detected in the Tg rats than in the Wt rats 3 days after SCI. These results suggest that translocation of EndoG and AIF from mitochondria to nuclei may initiate a caspase-independent pathway of apoptosis. An increased level of SOD1 in mitochondria conceivably reduces oxidative stress, thereby attenuating EndoG translocation, and resulting in reduction of caspase-independent apoptosis.     

亚低温对大鼠脑缺血/再灌注海马神经元p-JNK,AIF的影响

目的 通过动态观察亚低温对SD大鼠全脑缺血海马神经元半胱氨酸蛋白激酶-3(Caspase-3),凋亡诱导因子(AIF),p-JNK表达的影响,探讨亚低温对脑保护的可能机制.方法 10～12周龄的成年雄性SD大鼠,参照Pulsinelli 等[1]的四血管阻断法制作全脑缺血模型,随机分为假手术组,全脑缺血再灌损伤组,亚低温再灌注组,每组根据再灌注不同时间点(2 h,1 d,3 d,7 d)制取海马标本,观察其组织形态学变化;免疫组化检测AIF,p-JNK;TUNEL染色检测海马区神经元凋亡;Caspase-3荧光活性检测.结果 假手术组各时间点无变化,缺血再灌注两组海马神经元凋亡在CA1区都从再灌注2 h开始,且在1 d达到高峰后回落,其中亚低温组在(1 d,3 d)明显降低了Caspase-3、AIF、p-JNK的活性,增加了存活细胞(P<0.05).结论 亚低温可显著减少SD大鼠缺血性海马神经元再灌注后凋亡的发生,其机制可能与抑制海马P-JNK的活化,减弱Caspase-3活性,同时通过非Caspase-3通路减少AIF的表达相关.     

姜黄素抑制人肾癌细胞769-P增殖及促凋亡的实验研究

【摘要】 目的 分析不同浓度姜黄素（curcumin，分子式C₂₁H₂₀O₆）对人肾癌细胞769-P的生物学作用及其调控机制。方法 体外培养人肾癌细胞769-P，用不同浓度姜黄素处理24 h后，采用MTT比色法测定姜黄素对细胞的增殖抑制作用，利用倒置显微镜观察细胞的显微形态结构，采用细胞划痕法观察细胞的迁移情况；Western Blot 法检测细胞内Caspase-3、 AIF蛋白质的表达情况；用反转录PCR法检测AIF、Bax mRNA的表达水平。结果 MTT法检测显示姜黄素能显著抑制人肾癌细胞系769-P的增殖，呈现浓度和时间效应关系；倒置显微镜结果显示姜黄素能够使肾癌细胞系的形态学发生改变，呈现出典型凋亡特征如核皱缩、核破裂、核肿胀等形态；细胞划痕实验显示，姜黄素抑制了肾癌细胞系的迁移；RT-PCR实验显示，姜黄素促进了BAX mRNA和AIF mRNA的表达；蛋白杂交实验显示，姜黄素促进了Caspase-3和AIF蛋白的活化，从而促进了凋亡。结论 姜黄素能显著抑制人肾癌细胞的增殖并促进其凋亡，这种生物学效应可能与激活Bax和AIF表达，活化Caspase-3的信号通路有关。     

Hydrogen peroxide induces programmed necrosis in rat nucleus pulposus cells through the RIP1/RIP3‐PARP‐AIF pathway

This study aimed to systematically investigate whether programmed necrosis contributes to H₂O₂‐induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H₂O₂ for various time periods. The cell viability was measured using a cell counting kit‐8, and the death rate was detected by Hoechst 33258/propidium iodide (PI) staining. The programmed necrosis‐related molecules receptor‐interacting protein 1 (RIP1), receptor‐interacting protein 3 (RIP3), poly (ADP‐ribose) polymerase (PARP), and apoptosis inducing factor (AIF) were determined by real‐time polymerase chain reaction and Western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy (TEM). In addition, the necroptosis inhibitor Necrostatin‐1 (Nec‐1), the PARP inhibitor diphenyl‐benzoquinone (DPQ) and small interfering RNA (siRNA) technology were used to indirectly evaluate programmed necrosis. Our results indicated that H₂O₂ induced necrotic morphologic and ultrastructural changes and an elevated PI positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec‐1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3‐mediated programmed necrosis, executed through the PARP‐AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1269–1282, 2018.

     

Suppressor of Cytokine Signaling 1 Inhibits Apoptosis of Islet Grafts Through Caspase 3 and Apoptosis-Inducing Factor Pathways in Rats

A significant portion of pancreatic islet grafts can be destroyed by apoptosis, failing to engraft in the early period after transplantation. Recently, we observed that overexpression of suppressor of cytokine signaling 1 (SOCS1) in islet grafts achieved an antiapoptotic effect, prolonging graft survival in a rat transplant model. Caspase 3 is the central executioner caspase that is activated by upstream cascades in a caspase-dependent apoptosis pathway. Apoptosis inducing factor (AIF) is a key protein that can be released from mitochondria, translocating to the nucleus in the caspase-independent apoptosis pathway. In this study, we investigated whether these two pathways were involved in cytoprotection afforded by SOCS1 on islet grafts. We used a chimeric adenovirus vector (Ad5F35-SOCS1) to enhance SOCS1 expression in isolated Sprague-Dawley rat islets, which were transplanted into recipients experiencing streptozotocin-induced diabetes. We analyzed the expressions of active (cleaved) caspase 3 and AIF on islets. The Ad5F35-SOCS1-infected islets with higher SOCS1 expression showed decreased levels of active caspase 3 and intranuclear AIF after treatment with tumor necrosis factor-α and cycloheximide in vitro. The diabetic recipients transplanted with Ad5F35-SOCS1-infected islets showed longer periods of normoglycemia versus recipients transplanted with mock-infected islets (P < .05) due to prolonged graft survival. A histological analysis indicated that the Ad5F35-SOCS1-infected islet grafts displayed decreased caspase 3 activation and AIF translocation (to nucleus) in the early posttransplant period. These results demonstrated that the expression of SOCS1 in islet grafts protected them from apoptosis through caspase 3 dependent and AIF caspase-independent-pathways.     

Apoptosis-inducing factor and colon cancer

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis, and tissue homeostasis. Since the vast majority of cytotoxic modalities exert their anti-tumor effects by induction of apoptosis, programmed cell death has emerged as a potential target for cancer treatment at various stages of tumor progression. Immuno-regulation and chemoradiosensitization are potential pathways where insight in apoptotic mechanisms may lead to improvement of chemoradiotherapeutic modalities. The central mediator of the intrinsic pathway of apoptosis is the mitochondrion, in which changes of the outer membrane's permeability cause an outflow of cytochrome c and more than 40 molecules involved in apoptosis. These include Smac/DIABLO, Omi/HTR A2, endonuclease G, and apoptosis inducing factor (AIF). AIF, a 57 kDa mitochondrial oxidoreductase, is released into the cytoplasm and translocates to the nucleus to induce cell death in response to poly-(ADP-ribose) polymerase-1 activation, resulting is DNA fragmentation independent of caspase activation. As a caspase-independent mechanism of apoptosis, AIF may be a potential target for chemoradiotherapeutic intervention in a number of malignancies. The aim of this review is to provide the available evidence of the role AIF in several malignancies with a particular emphasis in colon carcinogenesis.     

与基底细胞癌细胞凋亡相关的分子生物学研究--促凋亡分子Bad的克隆及其pEGFP-C3真核表达载体的构建

目的:克隆Bcl-2相关凋亡基因Bad并构建其真核表达载体,探讨其对肿瘤细胞凋亡的诱导作用。方法:采用RT-PCR的方法,扩增Bad基因全长片段。通过基因定向克隆,构建Bad基因的真核表达质粒载体。结果:经酶切鉴定、PCR及DNA序列测定鉴定,Bad基因表达质粒pEGFP-C3载体成功构建。结论:成功克隆Bcl-2相关促凋亡基因Bad并构建其真核表达载体pEGFP-C3-Bad,为进一步研究Bad在人肿瘤细胞系中的促凋亡作用奠定了实验基础。     

Modification of gene products involved in resistance to apoptosis in metastatic colon cancer cells: roles of Fas, Apaf-1, NFkappaB, IAPs, Smac/DIABLO, and AIF

BACKGROUND: Colon cancer becomes resistant to apoptosis as it acquires metastatic potential. SW480 and SW620 colon cancer cells were established from the same patient at different stages of tumor progression. The stage III colorectal cancer cell line (SW620) is more resistant to apoptosis. In the present report, we investigated the apoptotic gene products that might account for colon cancer evasion of immune attack and chemoradioresistance-induced apoptosis. METHODS: SW480 and SW620 cells were used for this experiment. Type 1 apoptosis was induced by CH-11. Type 2 apoptosis was induced by cisplatin and ionizing radiation. Apoptosis was determined by caspase-3 activity and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. Gene products Fas, TRAIL, c-FLIP, Bid, BAX, Bcl-2, Bcl-xL, Apaf-1, nuclear factor-kappa B, Smac/DIABLO, apoptosis inducing factor, and the inhibitors of apoptosis were investigated by immunocytochemistry and Western blot analyses. RESULTS: SW620 cell lines were more resistant to both Type 1 and Type 2 apoptosis induced by CH-11, cisplatin, and ionizing radiation, respectively. Examination of the extrinsic pathway demonstrated Fas receptor to be down-regulated in SW620. Apaf-1 was decreased in SW620 cells; while other members of the mitochondrial pathway including Bax, Bid, Bcl-xL, and Bcl-2 demonstrated minimal alterations of protein levels in both cell lines. Survivin and XIAP protein levels were increased in SW620 cells, which correlated with nuclear expression of nuclear factor-kappa B in SW620 cells but not SW480. Mitochondrial-released factors including Smac/DIABLO and apoptosis inducing factor were increased in SW480 cells. CONCLUSIONS: SW620 cells have acquired genetic defects both in the intrinsic and extrinsic pathways of apoptosis, which may explain in part the ability of colon cancer cells to escape the immune system and to become chemoradioresistant. These genes may be potential targets for chemoradiosensitization in advanced colorectal cancer.