Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-alpha-induced apoptosis. STS activated I kappa B kinase and nuclear factor-kappa B (NF-kappa B) nuclear translocation and DNA binding but inhibited transactivation of I kappa B-alpha, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-kappa B reporter in transfected Huh-7 cells.     

Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis

Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases.     

The PKC delta inhibitor, rottlerin, induces apoptosis of haematopoietic cell lines through mitochondrial membrane depolarization and caspases' cascade

Rottlerin is a widely selective protein kinase C delta (PKCdelta) inhibitor isolated from Mallotus philippinensis. It shown to be effective against several human tumor cell lines and in potentiating chemotherapy-induced cytotoxcicity. Using the trypan blue exclusion assay, we demonstrated that rottlerin reduced the viability in a dose- and time-dependent manner of human leukemia HL60 cells, human acute T cell leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. Rottlerin caused apoptosis and the apaptotic processing was inhibited by a caspase inhibitor, z-VAD-fmk, in these haematopoietic cells. The apoptosis-inducing activities were determined by nuclear condensation, sub-G1 appearance, DNA fragmentation, loss of mitochondrial membrane potential (Deltapsim), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Expression of PKCdelta and Bcl-2 protein inhibited Deltapsim change and repressed cell death. These studies suggest that the cytotoxic effects of rottlerin through inhibition of PKCdelta cause mitochondrial dysfunction, cytochrome c release from mitochondria into cytoplasm and the activation of caspases' cascade.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells.

BACKGROUND: There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C. MATERIALS AND METHODS: We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2. RESULTS: beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine. CONCLUSIONS: beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

Caspase-9 Activation by the Apoptosome Is Not Required for Fas-mediated Apoptosis in Type II Jurkat Cells

Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x_L were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x_L-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.     

Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells

Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x(L) had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C(2)-ceramide, induced cytochrome c release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x(L) overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

Apoptosis-resistant mitochondria in T cells selected for resistance to Fas signaling

Jurkat leukemic T cells are highly sensitive to the extrinsic pathways of apoptosis induced via the death receptor Fas or tumor necrosis factor-related apoptosis-inducing ligand as well as to the intrinsic/mitochondrial pathways of death induced by VP-16 or staurosporin. We report here that clonal Jurkat cell lines selected for resistance to Fas-induced apoptosis were cross-resistant to VP-16 or staurosporin. Each of the apoptotic pathways was blocked at an apical phase, where common regulators of apoptosis have not yet been defined. The Fas pathway was blocked at the level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in resistant cells in response to either Fas-cross-linking or VP-16 treatment. Also, no apoptosis-associated alterations in the mitochondrial inner membrane, outer membrane, or matrix were detected in resistant Jurkat cells treated with VP-16. Thus, no changes in permeability transition, loss in inner membrane cardiolipin, generation of reactive oxygen species, or release of cytochrome c were observed in resistant cells treated with VP-16. Further, unlike purified mitochondria from wild type cells, those obtained from resistant cells did not release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. These results identify a defect in mitochondria ability to release intermembrane proteins in response to Bid or Bax as a mechanism of resistance to chemotherapeuetic drugs. Further, the selection of VP-16-resistant mitochondria via elimination of Fas-susceptible cells may suggest the existence of a shared regulatory component between the extrinsic and intrinsic pathways of apoptosis.     

Retroactive pathway involving mitochondria in electroloaded cytochrome c-induced apoptosis. Protective properties of Bcl-2 and Bcl-XL

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Deltapsim) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Deltapsim drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.     

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose

Glucose uptakeand metabolism inhibit hypoxia-induced apoptosis in a varietyof cell types, but the underlying molecular mechanisms remain poorlyunderstood. In the present study, we explore hypoxia-mediated celldeath pathways in Jurkat cells in the presence and absence ofextracellular glucose. In the absence of extracellular glucose, hypoxiacaused cytochrome c release, caspase 3 andpoly(ADP-ribose)polymerase cleavage, and DNA fragmentation; thisapoptotic response was blocked by the caspase 9 inhibitorz-LEHD-FMK. The presence of extracellular glucose during hypoxiaprevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In theseconditions, overexpression of the caspase 8 inhibitor v-FLIP preventedhypoxia-mediated cell death. Thus hypoxia can stimulate twoapoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucoseuptake and metabolism, and the other independent of cytochromec release and resulting from activation of the deathreceptor pathway, which is accelerated by glucose uptake and metabolism.

     

Necrotic death pathway in Fas receptor signaling

A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.     

Pramanicin induces apoptosis in Jurkat leukemia cells: A role for JNK,p38 and caspase activation

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells.