Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

Upstream regulatory role for XIAP in receptor-mediated apoptosis

X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of cell death that functions by suppressing caspases 3, 7, and 9. Here we describe the establishment of Jurkat-derived cell lines stably overexpressing either full-length XIAP or a truncation mutant of XIAP that can only inhibit caspase 9. Characterization of these cell lines revealed that following CD95 activation full-length XIAP supported both short- and long-term survival as well as proliferative capacity, in contrast to the truncation mutant but similar to Bcl-x(L). Full-length XIAP was also able to inhibit CD95-mediated caspase 3 processing and activation, the mitochondrial release of cytochrome c and Smac/DIABLO, and the loss of mitochondrial membrane potential, whereas the XIAP truncation mutant failed to prevent any of these cell death events. Finally, suppression of XIAP levels by RNA interference sensitized Bcl-x(L)-overexpressing cells to death receptor-induced apoptosis. These data demonstrate for the first time that full-length XIAP inhibits caspase activation required for mitochondrial amplification of death receptor signals and that, by acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation.     

Overexpression of Par‐4 sensitizes TRAIL‐induced apoptosis via inactivation of NF‐κB and Akt signaling pathways in renal cancer cells

The prostate‐apoptosis‐response‐gene‐4 (Par‐4) is up‐regulated in prostate cells undergoing programmed cell death. Furthermore, Par‐4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor‐mediated cell death pathways. In this study, we investigated how Par‐4 modulates TRAIL‐mediated apoptosis in TRAIL‐resistant Caki cells. Par‐4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par‐4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase‐8 and the effector caspase‐3, together with an enforced cleavage of XIAP and c‐FLIP. TRAIL‐induced reduction of XIAP and c‐FLIP protein levels in Par‐4 overexpressing cells was prevented by z‐VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL‐treated Par‐4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par‐4 recovered Bcl‐2 level to basal level induced by wild type Par‐4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par‐4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par‐4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par‐4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl‐2, Akt, and NF‐κB. J. Cell. Biochem. 109: 885–895, 2010. © 2010 Wiley‐Liss, Inc.     

cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis

Sensitivity to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and the lysosomal pathway of cell death are features of cancer cells. However, it is unknown if TRAIL cytotoxic signaling engages the lysosomal pathway of cell death. Our aim, therefore, was to ascertain if TRAIL killing involves lysosomal permeabilization. TRAIL-induced apoptosis of hepatocellular carcinoma cells (HuH-7, Hep3B) was associated with lysosomal permeabilization, as demonstrated by redistribution of the lysosomal protease cathepsin B into the cytosol. Pharmacological and short hairpin RNA-targeted inhibition of cathepsin B reduced apoptosis. Because cellular FLICE-inhibitory protein (cFLIP) inhibits TRAIL-induced cell death and is frequently overexpressed by human cancers, the ability of cFLIP to prevent lysosomal permeabilization during TRAIL treatment was examined. Enforced long-form cFLIP (cFLIP(L)) expression reduced release of cathepsin B from lysosomes and attenuated apoptosis. cFLIP(L) overexpression was also associated with robust p42/44 MAPK activation following exposure to TRAIL. In contrast, cFLIP(L) overexpression attenuated p38 MAPK activation and had no significant effect on JNK and NF-kappaB activation. Inhibition of p42/44 MAPK by PD98059 restored TRAIL-mediated lysosomal permeabilization and apoptosis in cFLIP-overexpressing cells. In conclusion, these results demonstrate that lysosomal permeabilization contributes to TRAIL-induced apoptosis of hepatocellular carcinoma cells and suggest that cFLIP(L) cytoprotection is, in part, due to p42/44 MAPK-dependent inhibition of lysosomal breakdown.     

Anti-apoptotic protein BRE/BRCC45 attenuates apoptosis through maintaining the expression of caspase inhibitor XIAP in mouse Lewis lung carcinoma D122 cells

Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.     

Sanguinarine-induced apoptosis: generation of ROS, down-regulation of Bcl-2, c-FLIP, and synergy with TRAIL

Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis and other poppy-fumaria species, possessing potent antibacterial, antifungal, and anti-inflammatory activities. In this study, we investigated the underling mechanisms by which sanguinarine induce apoptosis in human breast cancer MDA-231 cells. Treatment of MDA-231 cells with sanguinarine induced remarkable apoptosis accompanying the generation of ROS. Consistently, sanguinarine-induced apoptosis was mediated by the increased reproductive cell death. Pretreatment with NAC or GSH attenuated sanguinarine-induced apoptosis, suggesting the involvement of ROS in this cell death. During sanguinarin-induced apoptosis, protein levels of pro-caspase-3, Bcl-2, cIAP2, XIAP, and c-FLIPs were reduced. Sanguinarine-mediated apoptosis was substantially blocked by ectopic expression of Bcl-2 and cFLIPs. Additionally, we found that sub-lethal doses of sanguinarine remarkably sensitized breast cancer cells to TRAIL-mediated apoptosis, but the cell death induced by sanguinarine and TRAIL in combination was not blocked by overexpression of Bcl-2 or Akt. Therefore, combinatory treatment of sanguinarine and TRAIL may overcome the resistance of breast cancer cells due to overexpression of Akt or Bcl-2.     

Cutting edge: cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein protects germinal center B cells from apoptosis during germinal center reactions

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIP(L)), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIP(L) decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIP(L) degradation and blocks all signs of apoptosis, even in the presence of anti-Fas ABS: cFLIP(L) expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.     

Sodium butyrate sensitizes human colon adenocarcinoma COLO 205 cells to both intrinsic and TNF-α-dependent extrinsic apoptosis

Overexpression of cFLIP protein seems to be critical in the antiapoptotic mechanism of immune escape of human COLO 205 colon adenocarcinoma cells. Actually, cFLIP appears to inhibit the death receptor ligand-mediated cell death. Application of the metabolic inhibitor sodium butyrate (NaBt), short-chain volatile fatty acid, sensitized COLO 205 cells to TNF-α-mediated apoptosis. Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP and simultaneous up-regulation of TNF-R1 protein levels. Additionally, the combined TNF-α and NaBt treatment caused cleavage of Bid and caspase-9 activation, as well as cytochrome c release from mitochondria. Thus, the evidence of this study indicates that NaBt facilitates the death receptor signal evoked by TNF-α. Moreover, NaBt alone initiated intrinsic apoptosis, that in turn was abolished by intracellular BCL-2 delivery. It confirms the involvement of mitochondria in the proapoptotic activity of NaBt. The activation of mitochondrial pathway was substantiated by up-regulated expression of BAK with concomitant reduction of antiapoptotic BCL-x_L, XIAP and survivin proteins. These findings suggest that NaBt could represent a good candidate for the new therapeutic strategy aimed to improve chemo- and immunotherapy of colon cancer.     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

Novel Polypyridyl chelators deplete cellular zinc and destabilize the X-linked inhibitor of apoptosis protein (XIAP) prior to induction of apoptosis in human prostate and breast cancer cells

X-linked inhibitor of apoptosis protein (XIAP), inhibits the initiation and execution phases of the apoptotic pathway. XIAP is the most potent member of the inhibitor of apoptosis protein (IAP) family of the endogenous caspase inhibitors. Therefore, targeting XIAP may be a promising strategy for the treatment of apoptosis-resistant malignancies. In this study, we systematically studied the relationships of chemical structures of several novel ligands to their zinc (Zn)-binding ability, molecular target XIAP, and tumor cell death-inducing activity. We show that treatment of PC-3 prostate cancer and MDA-MB-231 breast cancer cells with these membrane-permeable Zn-chelators with different Zn affinities results in varying degrees of XIAP depletion. Following decreased level of XIAP expression, we also show apoptosis-related caspase activation and cellular morphological changes upon treatment with strong Zn-chelators N4Py and BnTPEN. Addition of Zn has a full protective effect on the cells treated with these chelators, while iron (Fe) addition has only partial protection that, however, can be further increased to a comparable level of protection as Zn by inhibition of ROS generation, indicating that cell death effects mediated by Fe- but not Zn-complexes involve redox cycling. These findings suggest that strong Zn-chelating agents may be useful in the treatment of apoptosis-resistant human cancers.     

Changes in expression of anti-apoptotic protein, cFLIP, in granulosa cells during follicular atresia in porcine ovaries

Follicular selection is performed in mammalian ovaries, as most follicles undergo atresia during follicular development and growth. Follicular regression is indicated to begin with granulosa cell apoptosis. To reveal the molecular mechanisms of the selection, we examined the changes in the levels of cellular-Flice like inhibitory protein (cFLIP) expression in porcine granulosa cells. cFLIP is the homologue of intracellular apoptosis inducer (procaspase-8/Flice), and has two alternative splicing isoforms: cFLIP short form (cFLIP(S)) and long form (cFLIP(L)). By competing with caspase-8, cFLIP inhibits apoptosis initiated by death receptors. The changes in the levels of cFLIP(S) and cFLIP(L) mRNA and protein expression in granulosa cells were determined by RT-PCR and Western blotting, respectively. cFLIP(L) mRNA and protein were highly expressed in granulosa cells of healthy follicles and decreased during atresia. cFLIP(S) mRNA levels in granulosa cells were low and showed no change among the stages of follicular development, and its protein level was extremely low. We examined the changes in the localization of cFLIP mRNAs in pig ovaries by in situ hybridization and found that cFLIP(L) is abundant in granulosa cells of healthy follicles in comparison with those of atretic follicles. Immunohistochemical analyses demonstrated that the cFLIP protein is highly expressed in the granulosa cell of healthy follicles but weakly expressed in that of atretic follicles. We presumed that cFLIP, especially cFLIP(L), plays an anti-apoptotic role in the granulosa cells of healthy follicles of pig ovaries, and that cFLIP could be a major survival factor that determines whether growth or atresia occurs in porcine follicles.     

Lack of X-linked inhibitor of apoptosis protein leads to increased apoptosis and tissue loss following neonatal brain injury

Neurological deficits caused by H-I (hypoxia-ischaemia) to the perinatal brain are often severely debilitating and lead to motor impairment, intellectual disability and seizures. Perinatal brain injury is distinct from adult brain injury in that the developing brain is undergoing the normal process of neuronal elimination by apoptotic cell death and thus the apoptotic machinery is more easily engaged and activated in response to injury. Thus cell death in response to neonatal H-I brain injury is partially due to mitochondrial dysfunction and activation of the apoptosome and caspase 3. An important regulator of the apoptotic response following mitochondrial dysfunction is XIAP (X-linked inhibitor of apoptosis protein). XIAP inhibits apoptosis at the level of caspase 9 and caspase 3 activation, and lack of XIAP in vitro has been shown to lead to increased apoptotic cell death. In the present study we show that mice lacking the gene encoding the XIAP protein have an exacerbated response to neonatal H-I injury as measured by tissue loss at 7 days following the injury. In addition, when the XIAP-deficient mice were studied at 24 h post-H-I we found that the increase in injury correlates with an increased apoptotic response in the XIAP-deficient mice and also with brain imaging changes in T2-weighted magnetic resonance imaging and apparent diffusion coefficient that correspond to the location of apoptotic cell death. These results identify a critical role of XIAP in regulating neuronal apoptosis in vivo and demonstrate the enhanced vulnerability of neurons to injury in the absence of XIAP in the developing brain.     

Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection

Cell death pathways are likely regulated in specialized subcellular microdomains, but how this occurs is not understood. Here, we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates the inhibitor of apoptosis (IAP) protein survivin on Ser20 in the cytosol, but not in mitochondria. This phosphorylation event disrupts the binding interface between survivin and its antiapoptotic cofactor, XIAP. Conversely, mitochondrial survivin or a non-PKA phosphorylatable survivin mutant binds XIAP avidly, enhances XIAP stability, synergistically inhibits apoptosis, and accelerates tumor growth, in vivo. Therefore, differential phosphorylation of survivin by PKA in subcellular microdomains regulates tumor cell apoptosis via its interaction with XIAP.     

cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms

The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. This 2 MDa intracellular complex that we designate "Ripoptosome" is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIP(L) prevents Ripoptosome formation, whereas, intriguingly, cFLIP(S) promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the "rheostat" that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the?Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.     

Bcl-2 attenuates anticancer agents-induced apoptosis by sustained activation of Akt/protein kinase B in U937 cells

Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family contributes to resistance to anticancer therapeutic drugs. Thus, this protein represent attractive target for novel anticancer agents. In the present study, we determined the effect of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-γ1 degradation and Akt activation during the various anticancer agents-induced apoptosis. Treatment with chrysin for 12 h produced morphological features of apoptosis in U937 cells, which was associated with caspase-3 activation and PLC-γ1 degradation. Induction of apoptosis was also accompanied by down-regulation of XIAP and inactivation of Akt. Chrysin-induced caspase-3 activation, PLC-γ1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit ceramide-, and Akt specific inhibitor (SH-6)-induced apoptosis by sustained Akt activation. Thus, our findings imply that some of the biological functions of Bcl-2 may be attributed to their ability to inhibit anticancer agents-induced apoptosis through the sustained Akt activation.     

Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP(L) and XIAP protein levels and ERK activation

We established TRAIL-resistant MDA-231/TR cells from MDA-231 parent cells to understand the mechanism of TRAIL resistance in breast cancer cells. The selected TRAIL-resistant cells were cross-resistant to TNF-alpha/cycloheximide but remained sensitive to DNA-damage drugs such as oxaliplatin and etoposide. The expression levels of death receptors (DR4 and DR5), FADD, cIAP1, cIAP2, and Bcl-2 family were not changed in TRAIL-treated both cells. Significant down-regulation of XIAP and cFLIP was occurred after TRAIL treatment in MDA-231 cells whereas their levels were sustained in MDA-231/TR cells. TRAIL-mediated activation of ERK and JNK were also observed in parent MDA-231 cells but not in MDA-231/TR cells. However, TRAIL-resistant cells showed constitutive activation state after treatment with TRAIL. Pretreatment with PD98059 or transfection of MKK1-DN (dominant negative) expression vector attenuated TRAIL resistance in MDA-231/TR cells. Our findings provide the evidence that the sustained expression level of cFLIP(L) and XIAP protein and constitutive ERK activation may lead to acquired TRAIL resistance in breast cancer cells.