Bid is cleaved upstream of caspase-8 activation during TRAIL-mediated apoptosis in human osteosarcoma cells

TRAIL induces apoptosis in many malignant cell types. In this study, we used the human papilloma virus (HPV) 16 E6 protein as a molecular tool to probe the TRAIL pathway in HCT116 colon carcinoma cells and U2OS osteosarcoma cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells expressing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAIL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8. Cathepsin B activity in U2OS cells was significantly activated shortly after exposure to TRAIL, and the cathepsin B inhibitor, CA074Me, inhibited both TRAIL- and anti-DR5-mediated apoptosis and delayed the cleavage of Bid. These findings suggest that TRAIL activates a pathway dependent on Bid, but largely independent of FADD and caspase-8, in U2OS cells.     

Fas death receptor signalling: roles of Bid and XIAP

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.     

Involvement of caspase-8 in chemotherapy-induced apoptosis of patient derived leukemia cell lines independent of the death receptor pathway and downstream from mitochondria

Resistance of leukemic cells to chemotherapy frequently occurs in patients with acute leukemia, which may be caused by alterations in common apoptotic pathways. Controversy exists whether cytostatic agents induce the mitochondrial or death receptor pathway of apoptosis. In the mitochondrial pathway cytochrome C release and caspase-9 activation play a central role in the induction of apoptosis, while formation of a Death Inducing Signaling Complex (DISC) and caspase-8 activation have been reported to be essential in death receptor-induced apoptosis. Here, we show in human derived myeloid and lymphoblastic leukemia cell lines that caspase-8 plays a more important role than previously expected in apoptosis mediated via the mitochondrial pathway. We demonstrated in these malignant cells chemotherapy-induced apoptosis independent of the death receptor pathway, since blocking this pathway using a retroviral construct encoding Flice inhibitory protein (FLIP) did not inhibit drug-induced apoptosis or caspase-8 activation, while overexpression of Bcl-2 completely inhibited both events. Furthermore, we showed that activation of caspase-8 by cytostatic agents occurred downstream from mitochondria. Since caspase-8 plays a central role in both death receptor- and chemotherapy-induced apoptosis of malignant cells from patients with acute leukemia, therapeutic strategies focusing at modulation and activation of caspase-8 may be successful in the treatment of drug-resistant malignancies. Supported by a grant of the Dutch Cancer Society/KWF Kankerbestrijding: 99-2122.     

沙门菌感染与Caspase­-8介导的宿主细胞调控性死亡研究进展

沙门菌主要通过食物传播,严重威胁了人类健康。肠道上皮细胞作为抵抗沙门菌入侵的重要屏障,可通过多种方式抵抗沙门菌的定植与入侵。同时,肠道固有层巨噬细胞可特异性识别正常菌群与沙门菌,激活炎性小体并分泌白细胞介素(interleukin,IL)-1β等炎症因子诱导炎症反应清除沙门菌。Caspase家族属于半胱氨酸蛋白酶,它们被激活后可执行各种细胞功能。Caspase-1是炎性小体的重要组成部分,可切割消皮素D(gasdermin D)诱导细胞焦亡,引发炎症反应。研究发现,Caspase-8同样参与炎性小体复合物的形成,但其功能尚不明确。新近研究发现,在沙门菌感染所诱导的细胞焦亡被抑制时,Caspase-8在炎性小体中被强烈激活,并在肠道上皮细胞和巨噬细胞中调控细胞死亡与炎症反应,以限制沙门菌感染。因此,Caspase-8在沙门菌感染期间也是调节宿主抗感染免疫的关键分子,研究其调控宿主细胞死亡以及炎症因子释放的机制对深入了解沙门菌感染与宿主抗感染免疫应答之间的关系具有重要意义。     

Synthesis, anticancer activities, interaction with DNA and mitochondria of manganese complexes

Two new complexes [(Etdpa)MnCl₂] and [(Adpa)Mn(Cl)(H₂O)] (Etdpa = ethyl bis(2-pyridylmethyl)amino-2-propionate; Adpa = bis(2-pyridylmethyl)amino-2-propionic acid) were synthesized and characterized by spectral methods. The crystal structure of [(Etdpa)MnCl₂] shows that the Mn(II) atom is coordinated by three N atoms (N1, N2, N3), one oxygen atom (O1) of the ligand (Etdpa) and two chloride atoms (Cl1, Cl2), forming a distorted octahedral geometry. The binding interaction between ct-DNA and the synthesized complexes was relatively weak, but they can inhibit the induced swelling of Ca²⁺-loaded mitochondria in a dose-dependent manner. The [(Adpa)Mn(Cl)(H₂O)] can cause the obvious decrease of mitochondria membrane potential. The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide) assay shows that the two Mn(II) complexes are more active against cancer cells. Especially [(Adpa)Mn(Cl)(H₂O)] can inhibit the proliferation of glioma cells with IC₅₀ 9.5 μM. Experimental results indicate that the [(Adpa)Mn(Cl)(H₂O)] could be a new potential antitumor complex to target the mitochondria.     

Signal transduction mediated by Bid,a pro—death Bcl—2 family proteins,connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells,the Fas/TNF-R1 death receptor pathway and the mitochondria pathway.The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis,mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events.However,death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly,bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins.Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals.Activated Bid is translocated to mitochondria and induces cytochrome c release,which in turn activates downstream caspases.Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.     

Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

In vitro analysis of Bcl-2 proteins in mitochondria and endoplasmic reticulum: similarities in anti-apoptotic functions and differences in regulation

Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.     

Caspase-8 dependent trail-induced apoptosis in cancer cell lines is inhibited by vitamin C and catalase

TNF-related apoptosis-inducing ligand (TRAIL/ Apo-2L) is a member of the TNF family of apoptosis-inducing proteins that initiates apoptosis in a variety of neoplastic cells while displaying minimal or absent cytotoxicity to most normal cells. Therefore, TRAIL is currently considered a promising target to develop anti-cancer therapies. TRAIL-receptor ligation recruits and activates pro-caspase-8, which in turn activates proteins that mediate disruption of the mitochondrial membranes. These events lead to the nuclear and cytosolic damage characteristic of apoptosis. Here we report that TRAIL-induced apoptosis is mediated by oxidative stress and that vitamin C (ascorbic acid), a potent nutritional antioxidant, protects cancer cell lines from apoptosis induced by TRAIL. Vitamin C impedes the elevation of reactive oxygen species (ROS) levels induced by TRAIL and impairs caspase-8 activation. We found that the removal of hydrogen peroxide by extracellular catalase during TRAIL-induced apoptosis also impairs caspase-8 activation. These data suggest that hydrogen peroxide is produced during TRAIL-receptor ligation, and that the increase of intracellular ROS regulates the activation of caspase-8 during apoptosis. Additionally we propose a mechanism by which cancer cells might resist apoptosis via TRAIL, by the intake of the nutritional antioxidant vitamin C. This work was supported by grants from the National Institutes of Health (CA 30388), the New York State Department of Health (M020113) and the Lebensfeld Foundation.     

Co-involvement of the mitochondria and endoplasmic reticulum in cell death induced by the novel ertargeted protein HAP

HAP (a homologue of the ASY/Nogo-B protein), a novel human apoptosis-inducing protein, was found to be identical to RTN3. In an earlier study, we demonstrated that HAP localized exclusively to the endoplasmic reticulum (ER) and that its overexpression could induce cell apoptosis via a depletion of endoplasmic reticulum (ER) Ca²⁺ stores. In this study, we show that overexpression of HAP causes the activation of caspase-12 and caspase-3. We still detected the collapse of mitochondrial membrane potential (Δωm) and the release of cytochrome c in HAP-overexpressing HeLa cells. All the results indicate that both the mitochondria and the ER are involved in apoptosis caused by HAP overexpression, and suggest that HAP overexpression may initiate an ER overload response (EOR) and bring about the downstream apoptotic events. Equal contribution to this paper     

Toll-like receptor 3 (TLR3) induces apoptosis via death receptors and mitochondria by up-regulating the transactivating p63 isoform alpha (TAP63alpha)

Toll-like receptor 3 (TLR3), a member of the pathogen recognition receptors, is widely expressed in various cells and has been shown to activate immune signaling pathways by recognizing viral double-stranded RNA. Recently, it was reported that the activation of TLR3 induced apoptosis in some cells, but the detailed molecular mechanism is not fully understood. In this study, we found that in endothelial cells polyinosinic-polycytidylic acid (poly(I-C)) induced dose- and time-dependent cell apoptosis, which was elicited by TLR3 activation, as TLR3 neutralization and down-regulation repressed the apoptosis. Poly(I-C) induced the activation of both caspases 8 and 9, indicating that TLR3 triggered the signaling of both the extrinsic and intrinsic apoptotic pathways. Poly(I-C) up-regulated tumor necrosis factor-related apoptosis-inducing ligand and its receptors, death receptors 4/5, resulting in initiating the extrinsic pathway. Furthermore, poly(I-C) down-regulated anti-apoptotic protein, B cell lymphoma 2 (Bcl-2), and up-regulated Noxa, a key Bcl-2 homology 3-only antagonist of Bcl-2, leading to the priming of the intrinsic pathway. A p53-related protein, the transactivating p63 isoform α (TAp63α), was induced by TLR3 activation and contributed to the activation of both the intrinsic and extrinsic apoptotic pathways. Both the cells deficient in p63 gene expression by RNA interference and cells that overexpressed the N-terminally truncated p63 isoform α (ΔNp63α), a dominant-negative variant of TAp63α, by gene transfection, survived TLR3 activation. Taken together, TAp63α is a crucial regulator downstream of TLR3 to induce cell death via death receptors and mitochondria.     

Catalytically active <Emphasis Type="Italic">Yersinia</Emphasis> outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIP_L degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55^−/− mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.     

Insight into the apoptosis-inducing action of alpha-bisabolol towards malignant tumor cells: involvement of lipid rafts and Bid

In a precedent report we showed that α-bisabolol, a sesquiterpene present widely in the plant kingdom, exerts a rapid and efficient apoptosis-inducing action selectively towards human and murine malignant glioblastoma cell lines through mitochondrial damage. The present study extends these data demonstrating the apoptosis-inducing action of α-bisabolol towards highly malignant human pancreatic carcinoma cell lines without affecting human fibroblast viability. The present study further shows the preferential incorporation of α-bisabolol to transformed cells through lipid rafts on plasma membranes and, thereafter, direct interaction between α-bisabolol and Bid protein, one of pro-apoptotic Bcl-2 family proteins, analyzed either by Surface Plasmon Resonance method or by intrinsic fluorescence measurement. Notions that lipid rafts are rich in plasma membranes of transformed cells and that Bid, richly present in lipid rafts, is deeply involved in lipid transport make highly credible the hypothesis that the molecular mechanism of α-bisabolol action may include its capacity to interact with Bid protein.     

Caspase-8及Caspase-9在肝细胞癌组织中的表达及意义

目的:观察凋亡相关因子半胱氨酸天冬氨酸蛋白酶8(caspase-8)和半胱氨酸天冬氨酸蛋白酶9(caspase-9)在肝细胞癌中的表达及临床病理意义。方法:选择我院自2012年7月~2013年4月的53例肝细胞肝癌患者作为观察组,选择同一时期在医院体检的50例正常肝组织患者作为对照组,采用原位分子杂交方法对两组患者的Caspase-8和Caspase-9mRNA进行检测。结果:观察组与对照组中caspase-8、caspase-9的阳性表达率分别为84.91%(45/53)、88.68%(47/53)和60.00%(30/50)、82.00%(41/50),观察组与对照组比较均有升高趋势,比值有显著性差异(P0.05)。结论:肝癌细胞中caspase-8、caspase-9的较高表达显示在肝癌的发生发展过程中起到重要作用,在肝癌组织细胞增殖凋亡中具有一定作用,肝癌中caspase-8和caspase-9蛋白的表达均对于判断肝癌预后具有一定的临床价值。     

Ⅱ型肺泡上皮细胞与特发性肺纤维化的关系研究进展

特发性肺纤维化（IPF）是一种严重影响肺通气与换气功能的下呼吸道慢性疾病,其发病机理目前尚不明确,表现为异常的间质炎症和纤维化,以及肺泡结构的破坏。而Ⅱ型肺泡上皮细胞（ATⅡ）作为维持肺结构和功能的关键细胞,在肺部纤维化的发生和发展中极其重要。在IPF中,各种原因所致的ATⅡ的受损和衰老凋亡,可能是纤维化发生的是始动因素。而在这之后,关于临时基质的形成、成纤维细胞的聚集、激活以及间质-上皮转化的过程,异常的ATⅡ也参与其中,并发挥着重要的作用。     

卵巢癌中Caspase-3 的表达及与化疗耐药相关性研究

目的:检测Caspase-3在卵巢癌中的表达并探讨其与卵巢癌化疗耐药相关性。方法:利用组织芯片技术结合免疫组化方法,对176例卵巢癌、70例良性卵巢肿瘤、50例正常卵巢组织中Caspase-3的表达进行检测,分析其表达与临床病理特征及化疗疗效的相关性。结果:卵巢癌中Caspase-3的表达显著低于正常卵巢及卵巢良性肿瘤,且卵巢癌FIGO临床分期越晚其表达越低(P<0.01),而与肿瘤的组织类型、病理分级及患者的年龄和月经状况无显著相关性(P>0.05)。Caspase-3在卵巢癌化疗耐药组的阳性率显著低于化疗敏感组(P<0.01)。结论:凋亡蛋白酶Caspase-3的缺表达可能参与了卵巢癌的发生发展过程,在卵巢癌化疗耐药的行程中发挥作用;Caspase-3的检测可能对指导卵巢癌临床化疗用药有帮助,可提高化疗效果和减少化疗耐药,并成为预测化疗疗效的有用指标。     

Caspase‐8: a key role in the pathogenesis of diabetic embryopathy

Maternal diabetes causes neural tube defects in embryos, which are associated with increased apoptosis in the neuroepithelium. Many factors, including effector caspases, have been shown to be involved in the events. However, the key regulators have not been identified and the underlying mechanisms remain to be addressed. Caspase‐8, an initiator caspase, has been shown to be altered in diabetic embryopathy, suggesting a role as an upstream apoptotic regulator. Using mouse embryos as a model system, this study demonstrates that caspase‐8 is required for the production of hyperglycemia‐associated embryonic malformations. Caspase‐8 was shown to be expressed in the developing neural tube. Its activity, as evidenced by enhanced cleavage, was increased by hyperglycemia. These changes were associated with increased formation of the active cleavage of Bid. Inhibition of caspase‐8 activity in high glucose–challenged embryos reduced the rate of embryonic malformation and this was associated with decreased apoptosis in the neuroepithelium of the neural tube. Inhibition of caspase‐8 activity also reduced hyperglycemia‐induced Bid activation and caspase‐9 cleavage. These data suggest that caspase‐8 may control diabetic embryopathy‐associated apoptosis via regulation of the Bid‐stimulated mitochondrion/caspase‐9 pathway. Birth Defects Res (Part B)86:72‐77, 2009. ©2009 Wiley‐Liss, Inc.     

Boron inhibits apoptosis in hyperapoptosis condition: Acts by stabilizing the mitochondrial membrane and inhibiting matrix remodeling

An abnormally high apoptosis has been associated with a number of clinical conditions including embryonal malformations and various pathologies such as neuronal degeneration and diabetes. In this study, boron is reported to inhibit apoptosis in hyperapoptosis conditions as demonstrated in a model of hyperapoptosis. Boron is a metalloid which is present in food in small amounts and is suggested here to inhibit apoptosis by stabilizing the mitochondrial membrane structure, thus preventing matrix remodeling and the release of cytochrome c, an apoptosis-inducer protein from the mitochondrion. The protective effect was assessed by measuring the changes in mitochondrial membrane potential, the levels of cytochrome c and downstream activation of caspase 3, besides phosphatidylserine exposure on the cell surface and DNA damage. The study has implication in clinical conditions characterized by hyperapoptosis as seen in certain embryonal malformations and various pathologies.     

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.