HSP60, Bax, apoptosis and the heart

HSP60 has primarily been known as a mitochondrial protein that is important for folding key proteins after import into the mitochondria. It is now clear that a significant amount of HSP60 is also present in the extra-mitochondrial cytosol of many cells. In the heart, this cytosolic HSP60 complexes with Bax, Bak and Bcl-XL, but not with Bcl-2. Reduction in HSP60 expression precipitates apoptosis, but does not alter mitochondrial function. During hypoxia, HSP60 cellular distribution changes, with HSP60 leaving the cytosol, and translocating to the plasma membrane. Total cellular HSP60 does not change until 10 h of reoxygenation; however, release of cytochrome c from the mitochondria occurs prior to reoxygenation, coinciding with the redistribution of HSP60. The changes in HSP60, Bax and cytochrome c during hypoxia can be replicated by ATP depletion. HSP60 has also been shown to accelerate the cleavage of pro-caspase3. Thus, HSP60 has a complex role in apoptosis in the cell. Its binding to Bax under normal conditions suggests a key regulatory role in apoptosis.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.     

c-Myc and caspase-2 are involved in activating Bax during cytotoxic drug-induced apoptosis

Activation of Bax following diverse cytotoxic stress has been shown to be an essential gateway to mitochondrial dysfunction and activation of the intrinsic apoptotic pathway characterized by cytochrome c release with caspase-9/-3 activation. Interestingly, c-Myc has been reported to promote apoptosis by destabilizing mitochondrial integrity in a Bax-dependent manner. Stress-induced activation of caspase-2 may also induce permeabilization of mitochondria with activation of the intrinsic death pathway. To test whether c-Myc and caspase-2 cooperate to activate Bax and thereby mediate intrinsic apoptosis, small interfering RNA was used to efficiently knock down the expression of c-Myc, caspase-2, and Apaf-1, an activating component in the apoptosome, in two human cancer cell lines, lung adenocarcinoma A-549 and osteosarcoma U2-OS cells. Under conditions when the expression of endogenous c-Myc, caspase-2, or Apaf-1 is reduced 80-90%, cisplatin (or etoposide)-induced apoptosis is significantly decreased. Biochemical studies reveal that the expression of c-Myc and caspase-2 is crucial for cytochrome c release from mitochondria during cytotoxic stress and that Apaf-1 is only required following cytochrome c release to activate caspases-9/-3. Although knockdown of c-Myc or caspase-2 does not affect Bax expression, caspase-2 is important for cytosolic Bax to integrate into the outer mitochondrial membrane, and c-Myc is critical for oligomerization of Bax once integrated into the membrane.     

Heat shock protein 60 or 70 activates nitric-oxide synthase (NOS) I- and inhibits NOS II-associated signaling and depresses the mitochondrial apoptotic cascade during brain stem death

The cellular and molecular basis of brain stem death remains an enigma. As the origin of a "life-and-death" signal that reflects the progression toward brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this phenomenon. Here, we evaluated the hypothesis that heat shock proteins (HSPs) play a neuroprotective role in the RVLM during brain stem death and delineated the underlying mechanisms, using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult. In Sprague-Dawley rats, proteomic, Western blot, and real-time PCR analyses demonstrated that Mev induced de novo synthesis of HSP60 or HSP70 in the RVLM without affecting HSP90 level. Loss-of-function manipulations of HSP60 or HSP70 in the RVLM using anti-serum or antisense oligonucleotide potentiated Mev-elicited cardiovascular depression alongside reduced nitric-oxide synthase (NOS) I/protein kinase G signaling, enhanced NOS II/peroxynitrite cascade, intensified nucleosomal DNA fragmentation, elevated cytoplasmic histone-associated DNA fragments or activated caspase-3, and augmented the cytochrome c/caspase-3 cascade of apoptotic signaling in the RVLM. Co-immunoprecipitation experiments further revealed a progressive increase in the complex formed between HSP60 and mitochondrial or cytosolic Bax or mitochondrial Bcl-2 during Mev intoxication, alongside a dissociation of the cytosolic HSP60-Bcl-2 complex. We conclude that HSP60 and HSP70 confer neuroprotection against Mev intoxication by ameliorating cardiovascular depression via an anti-apoptotic action in the RVLM. The possible underlying intracellular processes include enhancing NOS I/protein kinase G signaling and inhibiting the NOS II/peroxynitrite cascade. In addition, HSP60 exerts its effects against apoptosis by blunting Mev-induced activation of the Bax/cytochrome c/caspase-3 cascade.     

Lethal toxin from Clostridium sordellii induces apoptotic cell death by disruption of mitochondrial homeostasis in HL-60 cells

Lethal toxin (LT) from Clostridium sordellii (strain IP82) inactivates in glucosylating the small GTPases Ras, Rap, Ral and Rac. In the present study we show that LT-IP82 induces cell death via an intrinsic apoptotic pathway in the myeloid cell-line HL-60. LT-IP82 was found to disrupt mitochondrial homeostasis as characterized by a decrease in mitochondrial transmembrane potential and cardiolipin alterations, associated with the release of cytochrome c in the cytosol. Time-course studies of caspase activation revealed that caspase-9 and caspase-3 were activated before caspase-8. Moreover, although LT-IP82-induced cell death was abrogated by caspase-inhibitors, these inhibitors did not suppress mitochondrial alterations, indicating that caspase activation occurs downstream of mitochondria. Protection of mitochondria by Bcl-2 overexpression prevented mitochondrial changes as well as apoptosis induction. Furthermore, evidence is provided that LT-IP82-induced apoptosis is not a consequence of cortical actin disorganization, suggesting that Rac inactivation does not initiate the apoptotic process. Cell exposure to LT-IP82 leads to a co-localization of the toxin with a mitochondrial marker within 2 h. Therefore, we suggest that LT-IP82 could act at the mitochondrion level independently of its enzymatic effect on small GTPases.     

A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release

FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.     

Prostaglandin A2 activates intrinsic apoptotic pathway by direct interaction with mitochondria in HL-60 cells

HL-60 cells treated by prostaglandin (PG) A₂ showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA₂-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA₂-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA₂, mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA₂. Finally, it was shown that PGA₂-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA₂ activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA₂ plays a pivotal role.     

Immunosuppressant FTY720 induces apoptosis by direct induction of permeability transition and release of cytochrome c from mitochondria

FTY720 has immunosuppressive activity in experimental organ transplantation and shows a prompt and protracted decrease of blood T lymphocytes upon oral administration. The blood lymphocyte decrease in vivo was mainly a result of FTY720-induced apoptosis. However, this apoptotic mechanism is not well understood. We examined the mechanism of FTY720-induced apoptosis in lymphoma. Western blotting and fluorescent caspase-specific substrate revealed that caspase-3 is involved in FTY720-induced apoptosis, whereas caspase-1 is not. Apoptotic cell death was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, suggesting that caspase activation is essential for FTY720-induced apoptosis. FTY720 reduced mitochondrial transmembrane potential and released cytochrome c from the mitochondria of intact cells as well as in a cell-free system even in the presence of Z-VAD-FMK. As these mitochondrial reactions occurred before caspase activation, we concluded that FTY720 directly influences mitochondrial functions. The inhibition of mitochondrial permeability transition by Bcl-2 overexpression or by chemical inhibitors prevented all apoptotic events occurring in intact cells and in a cell-free system. Moreover, using a cell-free system, FTY720 did not directly affect isolated nuclei or cytosol. These results indicate that FTY720 directly affects mitochondria and triggers permeability transition to induce further apoptotic events.     

Molecular regulation of autophagy and apoptosis during ischemic and non-ischemic cardiomyopathy

A significant understanding of the genetic signaling pathways governing the extrinsic and intrinsic apoptotic pathways has been established. In recent years, the role of apoptosis in the heart during ischemic and non-ischemic cardiomyopathies has been under investigation and reported to contribute to ventricular remodeling and heart failure. Autophagy has been recently characterized as an essential cellular process in the heart, but whether autophagy functions as a pro-death or pro-survival program during disease conditions is still not completely understood. The mitochondrial death protein Bnip3 has been implicated in both apoptosis and autophagy, and its role in both processes is also discussed.     

Mitochondrially localized active caspase-9 and caspase-3 result mostly from translocation from the cytosol and partly from caspase-mediated activation in the organelle. Lack of evidence for Apaf-1-mediated procaspase-9 activation in the mitochondria

Active caspase-9 and caspase-3 have been observed in the mitochondria, but their origins are unclear. Theoretically, procaspase-9 might be activated in the mitochondria in a cytochrome c/Apaf-1-dependent manner, or activated caspase-9 and -3 may translocate to the mitochondria, or the mitochondrially localized procaspases may be activated by the translocated active caspases. Here we present evidence that the mitochondrially localized active caspase-9 and -3 result mostly from translocation from the cytosol (into the intermembrane space) and partly from caspase-mediated activation in the organelle rather than from the Apaf-1-mediated activation. Apaf-1 localizes exclusively in the cytosol and, upon apoptotic stimulation, translocates to the perinuclear area but not to the mitochondria. In most cases, the mitochondrially localized procaspase-9 and -3 are released early during apoptosis and translocate to the cytosol and/or perinuclear area. Cytochrome c and the mitochondrial matrix protein Hsp60 are also rapidly released to the cytosol early during apoptosis. Both the early release of proteins like cytochrome c and Hsp60 from the mitochondria as well as the later translocation of the active caspase-9/-3 are partially inhibited by cyclosporin A, an inhibitor of mitochondrial membrane permeabilization. The mitochondrial active caspases may function as a positive feedback mechanism to further activate other or residual mitochondrial procaspases, degrade mitochondrial constituents, and disintegrate mitochondrial functions.     

Molecular regulation of autophagy and apoptosis during ischemic and non-ischemic cardiomyopathy

A significant understanding of the genetic signaling pathways governing the extrinsic and intrinsic apoptotic pathways has been established. In recent years, the role of apoptosis in the heart during ischemic and non-ischemic cardiomyopathies has been under investigation and reported to contribute to ventricular remodeling and heart failure. Autophagy has been recently characterized as an essential cellular process in the heart, but whether autophagy functions as a pro-death or pro-survival program during disease conditions is still not completely understood. The mitochondrial death protein Bnip3 has been implicated in both apoptosis and autophagy, and its role in both processes is also discussed.     

Salidroside inhibits H2O2-induced apoptosis in PC12 cells by preventing cytochrome c release and inactivating of caspase cascade

We used a rat pheochromocytoma (PC12) cell line to study the effects of salidroside on hydrogen peroxide (H(2)O(2))-induced apoptosis. In PC12 cells, H(2)O(2)-induced apoptosis was accompanied by the down-regulation of Bcl-2, the up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol, and the activation of caspase-3, -8 and -9. However, salidroside suppressed the down-regulation of Bcl-2, the up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol. Moreover, salidroside attenuated caspase-3, -8 and -9 activation, and eventually protected cells against H(2)O(2)-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with salidroside can block H(2)O(2)-induced apoptosis by regulating Bcl-2 family members and by suppressing cytochrome c release and caspase cascade activation.     

Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.     

PS-341 (bortezomib) induces lysosomal cathepsin B release and a caspase-2-dependent mitochondrial permeabilization and apoptosis in human pancreatic cancer cells

PS-341 (bortezomib) is a potent and reversible proteosome inhibitor that functions to degrade intracellular polyubiquitinated proteins. PS-341 induces apoptosis and has shown broad antitumor activity with selectivity for transformed cells. We studied the effect of PS-341 on lysosomal and mitochondrial permeabilization, including the role of caspase-2 activation in apoptosis induction in the BxPC-3 human pancreatic carcinoma cell line. PS-341 induced a dose-dependent apoptosis in association with reactive oxygen species generation and cleavage of caspase-2 to its 33- and 14-kDa fragments. PS-341 disrupted lysosomes with redistribution of cathepsin B to the cytosol, as shown using fluorescence confocal microscopy, that was blocked by the free radical scavenger tiron but not by a caspase-2 inhibitor (benzyloxycarbonyl (Z)-VDVAD-fluoromethyl ketone (FMK)). PS-341-induced caspase-2 activation was attenuated by a selective pharmacological inhibitor of cathepsin B (R-3032), suggesting that cathepsin B release occurs upstream of caspase-2. PS-341-induced mitochondrial depolarization was attenuated by Z-VDVAD-FMK, tiron, and an inhibitor of the mitochondrial permeability transition pore (bongkrekic acid). Regulation of mitochondrial permeability by caspase-2 was confirmed using caspase-2 small interfering RNA. PS-341-induced cytochrome c release and phosphatidylserine externalization were attenuated by Z-VDVAD-FMK and partially by R-3032. PS-341 activated the BH3-only proteins Bik and Bim and down-regulated Bcl-2 and Bcl-xL mRNA and protein expression. Taken together, PS-341 induces lysosomal cathepsin B redistribution upstream of caspase-2. Caspase-2 activation regulates PS-341-induced mitochondrial depolarization and apoptosis, suggesting that caspase-2 can serve as a link between lysosomal and mitochondrial permeabilization.     

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.     

Transforming growth factor-beta 1 induces apoptosis through Fas ligand-independent activation of the Fas death pathway in human gastric SNU-620 carcinoma cells

To date, two major apoptotic pathways, the death receptor and the mitochondrial pathway, have been well documented in mammalian cells. However, the involvement of these two apoptotic pathways, particularly the death receptor pathway, in transforming growth factor-beta 1 (TGF-beta 1)-induced apoptosis is not well understood. Herein, we report that apoptosis of human gastric SNU-620 carcinoma cells induced by TGF-beta 1 is caused by the Fas death pathway in a Fas ligand-independent manner, and that the Fas death pathway activated by TGF-beta 1 is linked to the mitochondrial apoptotic pathway via Bid mediation. We showed that TGF-beta 1 induced the expression and activation of Fas and the subsequent caspase-8-mediated Bid cleavage. Interestingly, expression of dominant negative FADD and treatment with caspase-8 inhibitor efficiently prevented TGF-beta 1-induced apoptosis, whereas the treatment with an activating CH11 or a neutralizing ZB4 anti-Fas antibody, recombinant Fas ligand, or Fas-Fc chimera did not affect activation of Fas and the subsequent induction of apoptosis by TGF-beta 1. We further demonstrated that TGF-beta 1 also activates the mitochondrial pathway showing Bid-mediated loss of mitochondrial membrane potential and subsequent cytochrome c release associated with the activations of caspase-9 and the effector caspases. Moreover, all these apoptotic events induced by TGF-beta 1 were found to be effectively inhibited by Smad3 knockdown and also completely abrogated by Smad7 expression, suggesting the involvement of the Smad3 pathway upstream of the Fas death pathway by TGF-beta 1.     

Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria

Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway.     

Haplophytin-A induces caspase-8-mediated apoptosis via the formation of death-inducing signaling complex in human promyelocytic leukemia HL-60 cells

Haplophytin-A (10-methoxy-2,2-dimethyl-2,6-dihydro-pyrano[3,2-c]quinolin-5-one), a novel quinoline alkaloid, was isolated from the Haplophyllum acutifolium. In this study, we investigated the effect of haplophytin-A on the apoptotic activity and the molecular mechanism of action in human promyelocytic leukemia HL-60 cells. Treatment with haplophytin-A (50 μM) induced classical features of apoptosis, such as, DNA fragmentation, DNA ladder formation, and the externalization of annexin-V-targeted phosphatidylserine residues in HL-60 cells. In addition, haplophytin-A triggered the activations of caspase-8, -9, and -3, and the cleavage of poly (ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, haplophytin-A caused the loss of mitochondrial membrane potential (ΔΨ_m) and the release of cytochrome c and Smac/DIABLO to the cytosol, and modulated the expression levels of Bcl-2 family proteins. We further demonstrated that knockdown of caspase-8 using its siRNA inhibited the mitochondrial translocation of tBid, the activations of caspase-9 and caspase-3, and subsequent DNA fragmentation by haplophytin-A. Furthermore, haplophytin-A-induced the formation of death-inducing signaling complex (DISC) and then activated caspase-8 in HL-60 cells. During haplophytin-A-induced apoptosis, caspase-8-stimulated tBid provide a link between the death receptor-mediated extrinsic pathway and the mitochondria- mediated intrinsic pathway. Taken together, these results suggest that the novel compound haplophytin-A play therapeutical role for leukemia via the potent apoptotic activity through the extrinsic pathway, involving the intrinsic pathway.