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Results for “Apoptosis”

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PubMed2004

Soluble receptor (DcR3) and cellular inhibitor of apoptosis-2 (cIAP-2) protect human cytotrophoblast cells against LIGHT-mediated apoptosis.

LIGHT (tumor necrosis factor superfamily 14) is among the powerful apoptosis-inducing cytokines synthesized in human placentas. Here, we investigated mechanisms protecting cytotrophoblast (CTB) cells from LIGHT-mediated apoptosis. Viability assays and caspase-3 immunoblots using recombinant LIGHT were done to establish that CTB cells purified from term placentas resist LIGHT-induced apoptosis. Although the cells were also resistant to killing by another placental cytokine, interferon-gamma (IFN-gamma), a combination of the two induced apoptosis. Killing was prevented by DcR3-Fc fragment but not control human-Fc fragment, showing that apoptosis occurs via the LIGHT pathway and that soluble receptors provide protection. Next, two cellular inhibitors of apoptosis expressed in CTB cells, cellular inhibitor of apoptosis (cIAP)-1 and cIAP-2, were investigated for protection. Cellular IAP-1 was unchanged after stimulation with LIGHT whereas cIAP-2 mRNA and protein were elevated. The increase was abrogated by treating CTB cells with LIGHT + IFN-gamma, implying a central role for cIAP-2 in preventing LIGHT-mediated apoptosis and an ability of IFN-gamma to overcome cIAP-2 protection. Definitive evidence was provided in experiments that showed that cIAP-2 anti-sense morpholinos permit LIGHT to induce apoptosis in HT-29 cells. In summary, the data are consistent with the postulate that placental CTB cells are protected from LIGHT-mediated apoptosis by both soluble receptor, DcR3, and cIAP-2.

Apoptosis↗

PubMed2003

Modulation of macrophage apoptosis by antimycobacterial therapy: physiological role of apoptosis in the control of Mycobacterium tuberculosis.

Apoptosis is a form of cell death that avoids inflammatory responses. We had previously reported that Mycobacterium tuberculosis (Mtb) and Purified Protein Derivative (PPD) induce apoptosis in murine macrophages. The production of TNFalpha and IL-10 in response to Mtb infection modulates apoptosis by controlling nitric oxide production and caspase activation. Furthermore, Mtb triggers calcium influx responsible for mitochondrial alterations, an early pathway of apoptosis, independently of TNFalpha and IL-10. In tuberculosis patients apoptotic macrophages are found in granulomas and bronchoalveolar lavages, suggesting that apoptosis may participate in the control of Mtb. To further explore the role of macrophage apoptosis in tuberculosis, we studied the capacity of standard antimycobacterial drugs to modulate different events associated with the induction of apoptosis. The B10R murine macrophage line was infected or not with Mtb (5:1 bacteria to macrophage ratio) or exposed to PPD (10 microg/ml), in the presence or absence of varying concentrations (1-20 microg/ml) of anti mycobacterial drugs (isoniazid, rifampin, thiacetazone, streptomycin, and ethambutol). Inhibition of the intracellular growth of M. tuberculosis by all drugs studied/correlated with inhibition of permeability transition (PT) alterations; TNFalpha, IL-10, and nitric oxide production, and caspase-1 activation. However, these drugs did not affect PPD-induced apoptosis or its associated events, suggesting that the ability of antimycobacterial drugs to block macrophage apoptosis could be explained by their effects on the metabolic activities of Mtb. All drugs, except isoniazid, at higher concentrations, induced PT alterations in noninfected macrophages in a way that appears to be dependent of calcium, since a calcium chelator prevented it. The results presented herein suggest that the pharmacological manipulation of pathways associated with macrophage apoptosis may affect the intracellular growth of Mtb.

Animals↗

PubMed2002

Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis.

We investigated intracellular signalling pathways for apoptosis induced by epigallocatechin-3-gallate (EGCG) as compared with those induced by a toxic chemical substance (etoposide, VP16) or the death receptor ligand [tumour necrosis factor (TNF)]. EGCG as well as VP16 and TNF induced activation of two apoptosis-regulating mitogen-activated protein (MAP) kinases, namely c-Jun N-terminal kinase (JNK) and p38 MAP kinase, in both human leukaemic U937 and OCI-AML1a cells. In U937 cells, the apoptosis and activation of caspases-3 and -9 induced by EGCG but not VP16 and TNF were inhibited with SB203580, a specific inhibitor of p38, while those induced by EGCG and VP16 but not TNF were inhibited with SB202190, a rather broad inhibitor of JNK and p38. In contrast, the EGCG-induced apoptosis in OCI-AML1a cells was resistant to SB203580 but not to SB202190. Unlike TNF, EGCG did not induce the activation of nuclear factor-kappaB but rather induced the primary activation of caspase-9. N -Acetyl-L-cysteine (NAC) almost completely abolished apoptosis induced by EGCG under conditions in which the apoptosis induced by VP16 or TNF was not affected. The JNK/p38 activation by EGCG was also potently inhibited by NAC, whereas those by VP16 and TNF were either not or only minimally affected by NAC. In addition, dithiothreitol also suppressed both apoptosis and JNK/p38 activation by EGCG, and EGCG-induced activation of MAP kinase kinase (MKK) 3/6, MKK4 and apoptosis-regulating kinase 1 (ASK1) was suppressed by NAC. Dominant negative ASK1, MKK6, MKK4 and JNK1 potently inhibited EGCG-induced cell death. EGCG induced an intracellular increase in reactive oxygen species and GSSG, both of which were also inhibited by NAC, and the decreased synthesis of glutathione rendered the cell susceptible to EGCG-induced apoptosis. Taken together these results strongly suggest that EGCG executed apoptotic cell death via an ASK1, MKK and JNK/p38 cascade which is triggered by NAC-sensitive intracellular oxidative events in a manner distinct from chemically induced or receptor-mediated apoptosis.

Antioxidants↗

PubMed2001

Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis.

Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.

Apoptosis↗

PubMed1997

[Apoptosis and anti-apoptosis genes in the Bcl-2 family].

Apoptosis, or programmed cell death, is an active process of self-destruction, described a long time ago. However, the understanding of the molecular pathways which regulate programmed cell death is more recent and far from complete. Apoptosis occurs during embryonic and foetal development, and tissue remodeling, and its purpose is to assure homeostasis of cells and tissues. Apoptosis-defining morphological and biochemical changes are now well documented. Many physiological and non-physiological factors have been described as inducers of apoptosis. Several genes affecting various steps in programmed cell death must be expressed to trigger apoptosis. For example, ced-3 and ced-4 in the nematode C. elegans, and ICE, a gene found in mammals. In addition, the existence of genes suppressing apoptosis, like the human bcl-2 gene and a family of related bcl-2 genes was recently described. Several data dealing with these family of anti-apoptotic genes and some of their mechanisms of action are now currently available. It is clear that bcl-2 protects many cell lines from induced apoptosis. Other proteins, like bcl-xL, A1 or mcl-1 have the same anti-apoptotic function, but several molecules of the same family, like bcl-xS, bax-alpha or bak can trigger the opposite effect. It is known that bcl-2 can interact with other proteins. For example, bax, which can exist as a homodimer, is also able to form a heterodimer with bcl-2. A surexpression of bax in several cell lines allows to counteract the effect of bcl-2. R-ras p23 is another example, among others, of a protein interacting with bcl-2, and this results in an interruption of the apoptotic signal transduction pathway when bcl-2 is overexpressed. Some other explanations allowing a more detailed analysis of the molecular mechanisms of apoptosis and anti-apoptosis are discussed in this short review. Many interesting results suggest that bcl-2 is a death repressor molecule functioning in an anti-oxydant pathway, but other recent data seem to claim the contrary. Recently, the demonstration was made that apoptosis may require the activation of several classes of proteases. It seems now that bcl-2 has also a function of protease(s) inhibitor.

Animals↗

PubMed1997

Apoptosis in proliferative glomerulonephritis: decreased apoptosis expression in lupus nephritis.

BACKGROUND: Apoptosis is a mechanism of cellular death involved in the deletion of cells in hyperplasic processes which has been observed in proliferative glomerulonephritis. Patients with systemic lupus erythematosus (SLE) are known to have defective apoptosis but there is scarce information about apoptosis in the renal lesions of this disease. The present studies were done to evaluate apoptosis in relation to the intensity of proliferation in several glomerulonephritis and the possible association between this relationship and the chronic histologic changes in lupus nephritis. METHODS: Studies were done in renal biopsies from 19 patients with lupus nephritis (types, IV, Va, Vb) classified with respect to chronicity and activity indexes, five patients with acute poststreptococcal glomerulonephritis, five with idiopathic mesangioproliferative glomerulonephritis, four with membranous glomerulonephritis, four with minimal-change nephrotic syndrome, and three patients with focal segmental sclerosis. Seven normal kidneys which could not be used for transplantation were used as a control group. Apoptosis was determined by in situ DNA nick-end labelling techniques and proliferating cells were identified with a monoclonal antibody antiproliferating cell nuclear antigen (PCNA). RESULTS: Decreased number of apoptotic cells and a high ratio of PCNA: apoptosis were observed in glomerulus and tubulointerstitium of patients with lupus nephritis when compared to other proliferative glomerulonephritis and controls. In lupus nephritis, glomerular apoptosis had a negative correlation with the chronicity index (r = -0.606 P = 0.005). The number of apoptotic cells in the glomeruli was not correlated with the number of PCNA positive cells in lupus nephritis, in contrast with a striking linear correlation observed in acute poststreptococcal nephritis (r = 0.925, P = 0.024), a disease with excellent prognosis. CONCLUSIONS: Apoptosis is decreased in proliferative lupus nephritis. Intense proliferation without increment in apoptosis (a high PCNA: apoptosis ratio) is a characteristic of lupus nephritis associated with chronic renal histological changes.

Adolescent↗

PubMed1998

Epithelial cell proliferation, apoptosis, and apoptosis inhibition in inverted papillomas.

Inverted papillomas (IPs) are rare benign tumors of nasal epithelium with high recurrence rates and malignant transformation potential. Their etiology is still uncertain, and the mechanism of their growth has not yet been fully described. The purpose of this study was to detect, quantify, and compare cell proliferation, apoptosis, and apoptosis inhibition in hyperplastic epithelium from IPs and in inflammatory nasal polyps (NPs). IP samples were obtained after surgical removal of tumor in 13 patients, and NPs were sampled during endoscopic ethmoidectomy in 10 patients with nasal polyposis. Cell proliferation and apoptosis inhibition, respectively, were assessed by immunohistochemical identification of the proliferating cell nuclear antigen (PCNA) and the oncoprotein Bcl-2. Apoptosis was evaluated by analyzing the DNA fragmentation. Cell proliferation and apoptosis were significantly higher in IPs than in NPs (P = .0002 and P = .043, respectively), while apoptosis inhibition was significantly lower in IPs than in NPs (P = .001). Concerning IPs, cell proliferation was significantly higher than apoptosis (P = .0029) and apoptosis inhibition (P = .0015). The increase in epithelial cell proliferation seemed to be greater in IPs with dysplasia than in IPs without dysplasia. Increased epithelial cell proliferation, but not apoptosis and apoptosis inhibition, seems to be involved in the development of IP.

Adult↗

PubMed1996

Expression of apoptosis, proliferating cell nuclear antigen, and apoptosis-related antigens (bcl-2, c-myc, Fas, Lewis(y) and p53) in human cholangiocarcinomas and hepatocellular carcinomas.

In situ expression of apoptosis and its related antigens has rarely been evaluated in human liver tumors. Therefore, investigation using in situ nick end-labeling and immunohistochemical methods of the in situ expression of apoptosis, proliferating cells, and apoptosis-related antigens in 7 normal livers, 20 cholangiocarcinomas (CC) and 17 hepatocellular carcinomas (HCC) was done. Apoptotic cells as determined by the nick end-labeling method and proliferating cell nuclear antigen-positive cells were present in all specimens, and the percentage of them was significantly higher in CC than in HCC. Bcl-2 protein was present only in one CC and one HCC, but was occasionally noted in bile ducts in non-cancerous livers. C-myc and Fas antigens were not found in any of the cases. Lewisy antigen was expressed in 8 CC, but was absent in the other cases although bile ducts in non-cancerous livers frequently expressed Lewisy. p53 protein was present in 8 CC, but was absent in the other cases. Serial section observations showed that apoptotic cancer cells were consistently negative for proliferating cell nuclear antigen; bcl-2-positive cells did not show apoptosis; p53-positive cancer cells showed apoptosis. Some Lewisy-positive cancer cells showed apoptosis, while others did not. These data suggest that apoptosis and cell proliferation are involved in CC and HCC, and their degree is more severe in CC than in HCC. p53 protein (stimulative) may regulate apoptosis in some cases, whereas c-myc, Fas and Lewisy are not related to apoptosis in CC and HCC in vivo. Many other factors may regulate apoptosis in CC and HCC in vivo.

Apoptosis↗

PubMed2002

Avian reoviruses cause apoptosis in cultured cells: viral uncoating, but not viral gene expression, is required for apoptosis induction.

The cytopathic effect evidenced by cells infected with avian reovirus S1133 suggests that this virus may induce apoptosis in primary cultures of chicken embryo fibroblasts. In this report we present evidence that avian reovirus infection of cultured cells causes activation of the intracellular apoptotic program and that this activation takes place during an early stage of the viral life cycle. The ability of avian reoviruses to induce apoptosis is not restricted to a particular virus strain or to a specific cell type, since different avian reovirus isolates were able to induce apoptosis in several avian and mammalian cell lines. Apoptosis was also provoked in ribavirin-treated avian reovirus-infected cells and in cells infected with UV-irradiated reovirions, indicating that viral mRNA synthesis and subsequent steps in viral replication are not needed for apoptosis induction in avian reovirus-infected cells and that the number of inoculated virus particles, not their infectivity, is the critical factor for apoptosis induction by avian reovirus. Our finding that apoptosis is no longer induced when intracellular viral uncoating is blocked indicates that intraendosomal virion disassembly is required for apoptosis induction and that attachment and uptake of parental reovirions are not sufficient to cause apoptosis. Taken together, our results suggest that apoptosis is triggered from within the infected cell by viral products generated after intraendosomal uncoating of parental reovirions.

Animals↗

PubMed2000

Induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc: transrepression correlates with acceleration of apoptosis.

Analysis of amino-terminus mutants of c-Myc has allowed a systematic study of the interrelationship between Myc's ability to regulate transcription and its apoptotic, proliferative, and transforming functions. First, we have found that c-Myc-accelerated apoptosis does not directly correlate with its ability to transactivate transcription using the endogenous ornithine decarboxylase (ODC) gene as readout for transactivation. Furthermore, deletion of the conserved c-Myc box I domain implicated in transactivation does not inhibit apoptosis. Second, the ability of c-Myc to repress transcription, using the gadd45 gene as a readout, correlates with its ability to accelerate apoptosis. A conserved region of c-Myc implicated in mediating transrepression is absolutely required for c-Myc-accelerated apoptosis. Third, a lymphoma-derived Thr58Ala mutation diminishes c-Myc-accelerated apoptosis through a decreased ability to induce the release of cytochrome c from mitochondria. This mutation in a potential phosphorylation site does not affect cell cycle progression, providing genetic evidence that induction of cell cycle progression and acceleration of apoptosis are two separable functions of c-Myc. Finally, we show that the increased ability of Thr58Ala mutant to elicit cellular transformation correlates with its diminished ability to accelerate apoptosis. Bcl-2 overexpression blocked and the lymphoma-associated Thr58Ala mutation decreased c-Myc-accelerated apoptosis, and both led to a significant increase in the ability of Rat1a cells to form colonies in soft agar. This enhanced transformation was greater in soft agar containing a low concentration of serum, suggesting that protection from apoptosis is a mechanism contributing to the increased ability of these cells to proliferate in suspension. Thus, we show here for the first time that, in addition to mutations in complementary antiapoptotic genes, c-Myc itself can acquire mutations that potentiate neoplastic transformation by affecting apoptosis independently of cell cycle progression.

Animals↗

PubMed2005

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis.

AIM: To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma (HCC), the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS: Tree shrew HCC was induced with aflatoxin B1 (AFB1), and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30th-, 60th-, and 90th-wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique. RESULTS: The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bcl-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION: Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.

Aflatoxin B1↗

PubMed1999

Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis.

C-Jun amino terminal kinase/stress-activated protein kinases (JNK/SAPK) and p38 subgroups of mitogen-activated protein kinases have been suggested to play a critical role in apoptosis, cell growth, and/or differentiation. We found that a short exposure of SKT6 cells, which respond to erythropoietin (Epo) and induce erythroid differentiation, to osmotic or heat shock induced transient activation of JNK/SAPK and p38 and inactivation of ERK and resulted in erythroid differentiation without Epo, whereas long exposure of the cells to these stresses induced prolonged activation/inactivation of the same kinases and caused apoptosis. Inhibition of JNK/SAPK and p38 resulted in inhibition of stress-induced erythroid differentiation and apoptosis. Inhibition of ERK had no effect on stress-induced erythroid differentiation, but stimulated apoptosis. Activation of p38 and/or JNK/SAPK for a short time caused erythroid differentiation without Epo, although its prolonged activation induced apoptosis. Activation of ERK suppressed stress-induced apoptosis. These results indicate that short cellular stresses, inducing transient activation of JNK/SAPK and p38, lead to cell differentiation rather than apoptosis. Furthermore, activation of JNK/SAPK and p38 is required for both cell differentiation and apoptosis, and the duration of their activation may determine the cell fate, cell differentiation, and apoptosis. In contrast, inactivation of ERK is required for stress-induced apoptosis but not cell differentiation.

Animals↗

PubMed1997

Interleukin-1 beta converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection.

Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)-induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1 beta converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4(+) and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)- induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.

Antibodies, Monoclonal↗

PubMed2004

Hypoxia inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by blocking Bax translocation.

The hypoxic environment in solid tumors results from oxygen consumption by rapid proliferation of tumor cells. Hypoxia has been shown to facilitate the survival of tumor cells and to be a cause of malignant transformation. Hypoxia also is well known to attenuate the therapeutic activity of various therapies in cancer management. These observations indicate that hypoxia plays a critical role in tumor biology. However, little is known about the effects of hypoxia on apoptosis, especially on apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent apoptosis inducer that has been shown to specifically limit tumor growth without damaging normal cells and tissues in vivo. To address the effects of hypoxia on TRAIL-induced apoptosis, HCT116 human colon carcinoma cells were exposed to hypoxic or normoxic conditions and treated with soluble TRAIL protein. Hypoxia dramatically inhibited TRAIL-induced apoptosis in HCT116 cells, which are highly susceptible to TRAIL in normoxia. Hypoxia increased antiapoptotic Bcl-2 family member proteins and inhibitors of apoptosis proteins. Interestingly, these hypoxia-increased antiapoptotic molecules were decreased by TRAIL treatment to the levels lower than those of the untreated conditions, suggesting that hypoxia inhibits TRAIL-induced apoptosis via other mechanisms rather than up-regulation of these antiapoptotic molecules. Additional characterization revealed that hypoxia significantly inhibits TRAIL-induced translocation of Bax from the cytosol to the mitochondria in HCT116 and A549 cells, with the concomitant inhibition of cytochrome c release from the mitochondria. Bax-deficient HCT116 cells were completely resistant to TRAIL regardless of oxygen content, demonstrating a pivotal role of Bax in TRAIL-induced apoptotic signaling. Thus, our data indicate that hypoxia inhibits TRAIL-induced apoptosis by blocking Bax translocation to the mitochondria, thereby converting cells to a Bax-deficient state.

Antineoplastic Agents↗

PubMed2001

Signaling events triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): caspase-8 is required for TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a TNF family member and potent apoptosis inducer. In contrast to TNF-alpha or Fas ligand, relatively little is known about the signaling events activated by TRAIL. In particular, the initial caspase(s) required for TRAIL-induced apoptosis remains to be determined Caspase-3-like protease but not caspase-1-like protease (YVADase) activity rapidly increased in HeLa cells in response to TRAIL treatment. The increase in protease activity correlated with the profile of apoptotic cell death that was inhibited by the pan-caspase inhibitor Z-VAD-fmk. In response to TRAIL, caspase-8, an initiator caspase in death receptor-mediated apoptosis, was activated within 1 h in association with Bid cleavage, cytochrome c release, caspase-3 activation, and DNA fragmentation factor 45 cleavage. Z-IETD-fmk, a caspase-8 inhibitor, completely blocked caspase-8 activation and resulted in inhibition of caspase-3 (a caspase-3-like protease) activation and apoptotic cell death. Overexpression of a caspase-8 dominant negative mutant inhibited apoptosis induced by TRAIL. Caspase-8-deficient Jurkat cells were resistant to both TRAIL and Fas-induced apoptosis, whereas wild-type Jurkat cells were susceptible to both TRAIL- and Fas-induced apoptosis. The caspase-8-reintro duced caspase-8-deficient Jurkat cells acquired normal susceptibility to both TRAIL and agonistic Fas antibody. Reverse transcription-PCR and sequence analyses have revealed that these caspase-8-deficient Jurkat cell express wild-type caspase-10. Therefore, our data indicate that caspase-8 is required for TRAIL-induced apoptosis and suggest that caspase-10 may play a minor role, if any, in TRAIL-induced apoptosis.

Apoptosis↗

PubMed1998

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells.

We have previously shown that melanoma cells were resistant to apoptosis induced by TNF family members Fas ligand (FasL), TNF-alpha, and CD40L. FasL also was not involved in CD4 T cell-mediated killing of melanoma cells. In the present study, we have tested melanoma cells for their susceptibility to apoptosis induced by human TNF-related apoptosis-inducing ligand (TRAIL) and the ability of a mAb against TRAIL to inhibit apoptosis and CD4 CTL-mediated killing of melanoma and Jurkat target cells. The results show that TRAIL-induced apoptosis in cells from 7 of 10 melanoma cell lines tested as well as in Jurkat T cells. Susceptibility to apoptosis was increased in some of the cell lines by treatment with cyclohexamide or actinomycin D. The melanoma cells were resistant to apoptosis induced by FasL, TNF-alpha, and CD40L. mAb M180 against TRAIL inhibited apoptosis induced by TRAIL. It was also found to inhibit CD4 CTL-mediated killing of Jurkat T cells as well as autologous and allogeneic melanoma cells. The degree of inhibition produced by the mAb varied between different clones of CTL and according to the susceptibility of the target cells to TRAIL-induced apoptosis. These results suggest that TRAIL is an important mediator of cell death induced by CTL and may have an important therapeutic role against human melanoma.

Antibodies, Monoclonal↗

PubMed2005

A small molecule Smac-mimic compound induces apoptosis and sensitizes TRAIL- and etoposide-induced apoptosis in breast cancer cells.

Inhibitor of apoptosis protein (IAP) suppresses apoptosis through binding and inhibiting active caspases-3, -7 and -9 via its baculoviral IAP repeat (BIR) domains. During apoptosis the caspase inhibition by IAPs can be negatively regulated by a mitochondrial protein second mitochondrial-derived activator of caspase (Smac). Smac physically interacts with multiple IAPs and relieves their inhibitory effect on caspases-3, -7 and -9. Recently, a small molecule Smac-mimic compound (Smac-mimic), which potentiates TNF-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor (TNF)-alpha mediated cell death in glioblastoma T98G cells and HeLa cells, was identified and characterized. To determine the efficacy of this compound in breast cancer cells, we first measured protein expression of three IAPs: XIAP, cIAP-1, and cIAP-2 in nine independent breast cancer cell lines. Three cell lines were chosen: a high IAPs expressing line MDA-MB-231, and two low IAPs expressing lines, T47D and MDA-MB-453. The cell lines were tested for their sensitivity to Smac-mimic alone or in combination with TRAIL or etoposide. Acting alone, Smac-mimic was quite potent with a cytotoxic IC50 of 3.8 nM in high IAPs expressing MDA-MB-231 cells, but was inactive at a much higher concentration in low IAPs expressing T47D and MDA-MB-453 cells. In fact, as low as 2.5 nM of Smac-mimic alone was sufficient to activate caspase-3 and induce apoptosis in MDA-MB-231 cells. In combinational treatments with TRAIL or etoposide, Smac-mimic significantly sensitized cells to growth suppression in MDA-MB-231 cells, but to a lesser extent in T47D and MDA-MB-453 cells. Furthermore, it significantly synergized MDA-MB-231, but not T47D cells to apoptosis induced by either TRAIL or etoposide. Thus, in these cell lines, Smac-mimic acts in an apparent IAPs dependent manner to induce apoptosis alone as well as sensitizes breast cancer cells to TRAIL or etoposide induced apoptosis via caspase-3 activation.

Antineoplastic Agents, Phytogenic↗

PubMed2004

Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli and is aberrantly elevated in a variety of human cancers. Rhabdomyosarcoma tumors are the most common soft-tissue sarcoma in childhood. In this investigation, we demonstrate that CK2 is a key survival factor that protects tumor cells from TRAIL-induced apoptosis. We have demonstrated that inhibition of CK2 phosphorylation events by 5,6-dichlorobenzimidazole (DRB) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis. CK2 inhibition also induced rapid cleavage of caspase-8, -9, and -3, as well as the caspase substrate poly(ADP-ribose) polymerase after TRAIL treatment. Overexpression of Bcl-2 protected cells from TRAIL-induced apoptosis in the presence of the CK2 inhibitor. Death signaling by TRAIL in these cells was Fas-associated death domain and caspase dependent because dominant negative Fas-associated death domain or the cowpox interleukin 1beta-converting enzyme inhibitor protein cytokine response modifier A prevented apoptosis in the presence of DRB. Analysis of death-inducing signaling complex (DISC) formation demonstrated that inhibition of CK2 by DRB increased the level of recruitment of procaspase-8 to the DISC and enhanced caspase-8-mediated cleavage of Bid, thereby increasing the release of the proapoptotic factors cytochrome c, HtrA2/Omi, Smac/DIABLO, and apoptosis inducing factor (AIF) from the mitochondria, with subsequent degradation of X-linked inhibitor of apoptosis protein (XIAP). To further interfere with CK2 function, JR1 and Rh30 cells were transfected with either short hairpin RNA targeted to CK2alpha or kinase-inactive CK2alpha (K68M) or CK2alpha' (K69M). Data show that the CK2 kinase activity was abrogated and that TRAIL sensitivity in both cell lines was increased. Silencing of CK2alpha expression with short hairpin RNA was also associated with degradation of XIAP. These findings suggest that CK2 regulates TRAIL signaling in rhabdomyosarcoma by modulating TRAIL-induced DISC formation and XIAP expression.

Adaptor Proteins, Signal Transducing↗