Rapid and profound potentiation of Apo2L/TRAIL-mediated cytotoxicity and apoptosis in thoracic cancer cells by the histone deacetylase inhibitor Trichostatin A: the essential role of the mitochondria-mediated caspase activation cascade

Apo2L/TRAIL is actively investigated as a novel targeted agent to directly induce apoptosis of susceptible cancer cells. Apo2L/TRAIL-refractory cells can be sensitized to the cytotoxic effect of this ligand by cytotoxic chemotherapeutics. The aim of this study was to evaluate the in vitro tumoricidal activity of the Apo2L/TRAIL + Trichostatin A in cultured thoracic cancer cells and to elucidate the molecular basis of the synergistic cytotoxicity of this combination. Concurrent exposure of cultured cancer cells to sublethal concentrations of Apo2L/TRAIL and Trichostatin A resulted in profound enhancement of Apo2L/TRAIL-mediated cytotoxicity in all cell lines regardless of their intrinsic susceptibility to this ligand. This combination was not toxic to primary normal cells. While Apo2L/TRAIL alone or Trichostatin A alone mediated < 20% cell death, 60 to 90% of cancer cells were apoptotic following treatment with TSA + Apo2L/TRAIL combinations. Complete translocation of Bax from the cytosol to the mitochondria compartment was mainly observed in combination-treated cells and this was correlated with robust elevation of caspase 9 proteolytic activity indicative of activation of the mitochondria apoptogenic effect. Profound TSA + Apo2L/TRAIL–mediated cytotoxicity and apoptosis were completely abrogated by either Bcl2 over-expression or by the selective caspase 9 inhibitor, highlighting the essential role of mitochondria-dependent apoptosis signaling cascade in this process. Moreover, increased caspase 8 activity observed in cells treated with the TSA + Apo2L/TRAIL combination was completely suppressed by Bcl-2 over-expression or by the selective caspase 9 inhibitor indicating that the elevated caspase 8 activity in combination-treated cells was secondary to a mitochondria-mediated amplification feedback loop of caspase activation. These finding form the basis for further development of HDAC inhibitors + Apo2L/TRAIL combination as novel targeted therapy for thoracic malignancies. R.M. Reddy and W.-S. Yeow contributed equally to this work. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH.     

Arsenic trioxide and paclitaxel induce apoptosis by different mechanisms

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin colocalized with AIF. In contrast, paclitaxel-induced apoptosis MM depolarization, cytochrome C release and activation of caspase 9 were all blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.     

Arsenic Trioxide and Paclitaxel Induce Apoptosis by Different Mechanism

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin co-localized with AIF. In contrast, paclitaxel-induced apoptosis and MM depolarization, cytochrome C release and activation of caspase 9 was blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.     

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.     

PTHrP Overexpression Increases Sensitivity of Breast Cancer Cells to Apo2L/TRAIL

Parathyroid hormone-related protein (PTHrP) is a key component in breast development and breast tumour biology. PTHrP has been discovered as a causative agent of hypercalcaemia of malignancy and is also one of the main factors implicated in breast cancer mediated osteolysis. Clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis. Furthermore, PTHrP has been demonstrated to cause tumour cell death both in vitro and in vivo. Apo2L/TRAIL is a promising new anti-cancer agent, due to its ability to selectively induce apoptosis in cancer cells whilst sparing most normal cells. However, some cancer cells are resistant to Apo2L/TRAIL-induced apoptosis thus limiting its therapeutic efficacy. The effects of PTHrP on cell death signalling pathways initiated by Apo2L/TRAIL were investigated in breast cancer cells. Expression of PTHrP in Apo2L/TRAIL resistant cell line MCF-7 sensitised these cells to Apo2L/TRAIL-induced apoptosis. The actions of PTHrP resulted from intracellular effects, since exogenous treatment of PTHrP had no effect on Apo2L/TRAIL-induced apoptosis. Apo2L/TRAIL-induced apoptosis in PTHrP expressing cells occurred through the activation of caspase-10 resulting in caspase-9 activation and induction of apoptosis through the effector caspases, caspase-6 and -7. PTHrP increased cell surface expression of Apo2L/TRAIL death receptors, TRAIL-R1 and TRAIL-R2. Antagonistic antibodies against the death receptors demonstrated that Apo2L/TRAIL mediated its apoptotic signals through activation of the TRAIL-R2 in PTHrP expressing breast cancer cells. These studies reveal a novel role for PTHrP with Apo2L/TRAIL that maybe important for future diagnosis and treatment of breast cancer.     

MAPK/ERK overrides the apoptotic signaling from Fas, TNF, and TRAIL receptors

The tumor necrosis factor (TNF), Fas, and TNF-related apoptosis-inducing ligand (TRAIL) receptors (R) are highly specific physiological mediators of apoptotic signaling. We observed earlier that a number of FasR-insensitive cell lines could redirect the proapoptotic signal to an anti-apoptotic ERK1/2 signal resulting in inhibition of caspase activation. Here we determine that similar mechanisms are operational in regulating the apoptotic signaling of other death receptors. Activation of the FasR, TNF-R1, and TRAIL-R, respectively, rapidly induced subsequent ERK1/2 activation, an event independent from caspase activity. Whereas inhibition of the death receptor-mediated ERK1/2 activation was sufficient to sensitize the cells to apoptotic signaling from FasR and TRAIL-R, cells were still protected from apoptotic TNF-R1 signaling. The latter seemed to be due to the strong activation of the anti-apoptotic factor NF-kappaB, which remained inactive in FasR or TRAIL-R signaling. However, when the cells were sensitized with cycloheximide, which is sufficient to sensitize the cells also to apoptosis by TNF-R1 stimulation, we noticed that adenovirus-mediated expression of constitutively active MKK1 could rescue the cells from apoptosis induced by the respective receptors by preventing caspase-8 activation. Taken together, our results show that ERK1/2 has a dominant protecting effect over apoptotic signaling from the death receptors. This protection, which is independent of newly synthesized proteins, acts in all cases by suppressing activation of the caspase effector machinery.     

Switch in Fas-activated death signaling pathway as result of keratin 8/18-intermediate filament loss

Fas-induced apoptosis is initiated through the recruitment of FADD and procaspase 8 to form the death-inducing signaling complex (DISC). In some cells (type I cells) the initiator caspase 8 directly activates effector caspases such as procaspase 3, whereas in others (type II cells) the death signal is amplified through mitochondria. In epithelial cells, Fas-induced hierarchic caspase activation is also linked with DEDD, a member of the DED family that binds to keratin (K) intermediate filaments (IFs). Hepatocytes are type II cells and their IFs are made exclusively of K8/K18. We have shown previously that K8-null mouse hepatocytes, lacking K8/K18 IFs, are more sensitive than their wild-type counterparts to Fas-induced apoptosis. Here, by examining the cell-death kinetics and death-signaling ordering, we found that K8-null hepatocytes exhibited prominent DISC formation, higher procaspase 8 activation and direct procaspase 3 activation as reported for type I cells; however they experienced a reduced Bid cleavage and a stronger procaspase 9 activation. In addition, the K8/K18 loss altered the DEDD ubiquitination status and nuclear/cytoplasmic distribution. Together, the results suggest that the K8/K18 loss induces a switch in Fas-induced death signaling, likely through a DEDD involvement. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The ribonucleotide reductase R1 subunits of herpes simplex virus 1 and 2 protect cells against poly(I · C)-induced apoptosis

We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.     

A role for c-FLIPL in the regulation of apoptosis,autophagy, and necroptosis in T lymphocytes

Caspase 8 plays a dual role in the survival of T lymphocytes. Although active caspase 8 mediates apoptosis upon death receptor signaling, the loss of caspase 8 activity leads to receptor-interacting protein (RIP)-1/RIP-3-dependent necrotic cell death (necroptosis) upon TCR activation. The anti-apoptotic protein c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) suppresses death receptor-induced caspase 8 activation. Moreover, recent findings suggest that c-FLIP is also involved in inhibiting necroptosis and autophagy. It remains unclear whether c-FLIP protects primary T lymphocytes from necroptosis or regulates the threshold at which autophagy occurs. Here, we used a c-FLIP isoform-specific conditional deletion model to show that c-FLIP_L-deficient T cells underwent RIP-1-dependent necroptosis upon TCR stimulation. Interestingly, although previous studies have only described necroptosis in the absence of caspase 8 activity, we found that pro-apoptotic caspase 8 activity and apoptosis were also enhanced in c-FLIP_L-deficient T lymphocytes. Furthermore, c-FLIP_L-deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. Together, these findings indicate that c-FLIP_L plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes.     

Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA

Interferons enhance the cellular antiviral response by inducing expression of protective proteins. Many of these proteins are activated by dsRNA, a typical by-product of viral infection. Here we show that type-I and type-II interferons can sensitize cells to dsRNA-induced cytotoxicity. In caspase-8- or FADD-deficient Jurkat cells dsRNA induces necrosis, instead of apoptosis. In L929sA cells dsRNA-induced necrosis involves high reactive oxygen species production. The antioxidant butylated hydroxyanisole protects cells from necrosis, but shifts the response to apoptosis. Treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone or overexpression of Bcl-2 prevent this shift and promote necrosis. Our results suggest that a single stimulus can initiate different death-signaling pathways, leading to either necrotic or apoptotic cell death. Inhibition of key events in these signaling pathways, such as caspase activation, cytochrome c release or mitochondrial reactive oxygen species production, tips the balance between necrosis and apoptosis, leading to dominance of one of these death programs.     

CrmA Protects Against Apoptosis and Ceramide Formation in PC12 Cells

TNF- activated caspase 8 and caspase 3 in PC12 cells, leading to cell death by apoptosis (DNA fragmentation). TNF- caspase activation and cell killing were blocked by transfection and overexpression of the viral protein CrmA, which specifically inhibits caspase 8. CrmA was also able to block the TNF--induced increase in ceramide formation in PC12 cells. Conversely, if caspase 8 was activated by light-activated Rose Bengal, there was an increase in both ceramide and caspase 3–mediated apoptosis, which was blocked by CrmA overexpression. This suggested that caspase 8 increases ceramide either by increasing its synthesis or by activating sphingomyelinase. Since fumonisin B1 did not block and sphingomyelin decreased when ceramide increased, we concluded that activation of sphingomyelinase is the most likely mechanism. The Rose Bengal activation of caspase 8 and increased ceramide formation was blocked with IETD-CHO, to show that reactive oxygen species (also generated by Rose Bengal) were not responsible for the observed increase in ceramide. Thus in PC12 pheochromocytoma cells, ceramide appears to amplify the death signal and there appears to be a sequence of events: TNF; TRADD, pro-caspase 8, caspase 8, sphingomyelinase, ceramide, caspase 3, apoptosis.     

Expression of mitochondrial Apo2.7 molecules and caspase-3 activation in human lymphocytes treated with the ribosome-inhibiting mistletoe lectins and the cell membrane permeabilizing viscotoxins.

BACKGROUND: It is unclear whether expression of newly described mitochondrial Apo2.7 molecules (7A6 antigen) is specific for apoptosis or may also occur in necrosis. METHODS: We incubated human lymphocytes with the apoptosis-inducing mistletoe lectin (ML) I and the cell membrane-permeabilizing viscotoxins (VT), and measured cell death-associated changes by flow cytometry. RESULTS: In ML I-treated lymphocytes, Apo2.7 expression and caspase-3 activation was recognized within 24 h. In VT-treated cells, we observed an Apo2.7 expression with low fluorescence level, while active caspase-3 and DNA fragments (TUNEL) were not detected within 24 h. In these cells, caspase-3 activation was recognized 48 h later. As a major subset of ML-treated cells expressing Apo2.7 molecules did not activated caspase-3, while all caspase-3(+) cells did express Apo2.7, one may suggest that the caspase pathway is activated secondarily to mitochondrial events. CONCLUSIONS: Expression of Apo2.7 is sensitive marker of cell death but may not be specific for apoptosis alone as it can be detected also in cells treated with cell membrane-permeabilizing toxins. On the other hand, this expression may be the consequence of an induction of distinct "death signals" resulting in apoptosis later on.     

LY303511 amplifies TRAIL-induced apoptosis in tumor cells by enhancing DR5 oligomerization, DISC assembly, and mitochondrial permeabilization

Certain classes of tumor cells respond favorably to TRAIL due to the presence of cell surface death receptors DR4 and DR5. Despite this preferential sensitivity, resistance to TRAIL remains a clinical problem and therefore the heightened interest in identifying compounds to revert tumor sensitivity to TRAIL. We recently demonstrated that the phosphatidylinositide-3-kinase (PI3K) inhibitor, LY294002, and its inactive analog LY303511, sensitized tumor cells to vincristine-induced apoptosis, independent of PI3K/Akt pathway. Intrigued by these findings, we investigated the effect of LY303511 on TRAIL-induced apoptosis in HeLa cells. Preincubation of cells with LY30 significantly amplified TRAIL signaling as evidenced by enhanced DNA fragmentation, caspases 2, 3, 8, and 9 activation, and reduction in the tumor colony formation. This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the egress of cytochrome c and second mitochondrial activator of caspase/direct IAP-binding protein with low PI, cleavage of X-linked inhibitor of apoptosis protein, and activation of caspase 9. We link this execution signal to the ability of LY30 to downregulate cFLIP(S) and oligomerize DR5, thus facilitating the signaling of the death initiating signaling complex. The subsequent exposure to TRAIL resulted in processing/activation of caspase 8 and cleavage of its substrate, the BH3 protein Bid. These data provide a novel mechanism of action of this small molecule with the potential for use in TRAIL-resistant tumors.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8

Caspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results. The majority of 55 lung and breast carcinoma cell lines expressed mRNA for both caspase-8 and -10; however, immunoblot analysis revealed that caspase-10 protein expression was more frequently absent than that of caspase-8, suggesting a possible selective pressure against caspase-10 production in cancer cells. In nontransfected cells expressing both caspases, CD95L and Apo2L/TRAIL recruited endogenous caspase-10 as well as caspase-8 to their DISC, where both enzymes were proteolytically processed with similar kinetics. Caspase-10 recruitment required the adaptor FADD/Mort1, and caspase-10 cleavage in vitro required DISC assembly, consistent with the processing of an apoptosis initiator. Cells expressing only one of the caspases underwent ligand-induced apoptosis, indicating that each caspase can initiate apoptosis independently of the other. Thus, apoptosis signaling by death receptors involves not only caspase-8 but also caspase-10, and both caspases may have equally important roles in apoptosis initiation.     

c-Jun N-terminal kinase contributes to apoptotic synergy induced by tumor necrosis factor-related apoptosis-inducing ligand plus DNA damage in chemoresistant,p53 inactive mesothelioma cells

Apoptotic resistance of cancer cells may be overcome by the combination of treatments that activate the two major apoptotic pathways: (i) the death receptor pathway activated by death ligands and (ii) the DNA damage pathway activated by chemotherapy. We have previously shown that mesothelioma cells, resistant to most treatments, are sensitive to the combination of the death ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) plus chemotherapy. We investigated a possible role for c-Jun N-terminal kinase (JNK) in the synergistic effect, knowing that JNK can be activated separately by TRAIL and by DNA damage. We chose to study the M28 and REN human mesothelioma cell lines, which are p53-inactivated, to avoid an interaction between p53 and JNK. We showed that JNK was activated by TRAIL and by etoposide and that the activation was enhanced by the combination of the two treatments. We found this activation to be caspase-independent. To inhibit the JNK pathway, we used either dominant-negative constructs of JNK1 and JNK2 (compared with dominant-negative caspase 9) or a chemical inhibitor of the JNK pathway (SP600125). In cells treated with TRAIL plus etoposide, JNK inhibition increased cell survival and decreased apoptosis significantly. In transfected M28 cells, the effect of JNK inhibition was as great as that of the dominant-negative caspase 9 construct. We conclude that JNK contributes to the synergistic effect of TRAIL combined with DNA damage by mediating signals independent of p53 leading to apoptosis.     

Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis

Death receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP(S)) and a long, 55-kDa form (c-FLIP(L)). The role of c-FLIP(S) as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIP(L) remains controversial. Although overexpression of transfected c-FLIP(L) inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIP(L) on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIP(L) in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIP(L) augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIP(L) functions primarily as an inhibitor of death receptor-mediated apoptosis.     

The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis

To determine the apoptotic signaling pathway which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen-activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L-induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L-induced apoptosis is mediated by ROS-activated p38 MAP kinase followed by caspase activation in HeLa cells.