Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells

Etoposide (VP-16) a topoisomerase II inhibitor induces apoptosis of tumor cells. The present study was designed to elucidate the mechanisms of etoposide-induced apoptosis in C6 glioma cells. Etoposide induced increased formation of ceramide from sphingomyelin and release of mitochondrial cytochrome c followed by activation of caspase-9 and caspase-3, but not caspase-1. In addition, exposure of cells to etoposide resulted in decreased expression of Bcl-2 with reciprocal increase in Bax protein. z-VAD.FMK, a broad spectrum caspase inhibitor, failed to suppress the etoposide-induced ceramide formation and change of the Bax/Bcl-2 ratio, although it did inhibit etoposide-induced death of C6 cells. Reduced glutathione or N-acetylcysteine, which could reduce ceramide formation by inhibiting sphingomyelinase activity, prevented C6 cells from etoposide-induced apoptosis through blockage of caspase-3 activation and change of the Bax/Bcl-2 ratio. In contrast, the increase in ceramide level by an inhibitor of ceramide glucosyltransferase-1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol caused elevation of the Bax/Bcl-2 ratio and potentiation of caspase-3 activation, thereby resulting in enhancement of etoposide-induced apoptosis. Furthermore, cell-permeable exogenous ceramides (C2- and C6-ceramide) induced downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio and subsequent activation of caspases-9 and -3. Taken together, these results suggest that ceramide may function as a mediator of etoposide-induced apoptosis of C6 glioma cells, which induces increase in the Bax/Bcl-2 ratio followed by release of cytochrome c leading to caspases-9 and -3 activation.     

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.     

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.     

Cordyceps sinensis mycelium extract induces human premyelocytic leukemia cell apoptosis through mitochondrion pathway

This study was to identify the signaling pathways for the induction of HL-60 cell apoptosis by Cordyceps sinensis mycelium extract (CSME). CSME at 25 mug/ml induced nuclear fragmentation and DNA degradation, two hallmark events of apoptosis, in the HL-60 cells within 12-24 hrs of treatment. Concomitantly, several major events in the mitochondrial signal pathway occurred, including the loss of MTP (DeltaPsi(m)), cytochrome c release into the cytoplasm, the decrease in Bcl-2 protein level, the translocation of Bax protein from cytoplasm into mitochondria, and the activation of caspase-2, -3, and -9, but caspase-8, the initiator caspase in the death receptor pathway, was not activated. These results suggest that CSME induces apoptosis in HL-60 cell through the mitochondrial pathway rather than the death receptor pathway.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

Caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells.

We reported previously that singlet oxygen, generated by irradiation of rose bengal with visible light, induced apoptosis in human promyelocytic leukemia HL-60 cells. However, the mechanism of apoptosis caused by this reactive oxygen species is unclear. In this study, we demonstrate that singlet oxygen induced caspase-3 activation and Z-DEVD-FMK, a caspase-3 inhibitor, blocked apoptosis induction, while caspase-1 activity was not detectable and the caspase-1 inhibitor Z-YVAD-FMK had a very limited effect on apoptosis. This suggests that the activation of caspase-3 by singlet oxygen is essential for the commitment of cells to undergo apoptosis. Further studies showed that singlet oxygen induced an increase in caspase-8 activity and a reduction in mitochondrial cytochrome c. Time course analysis indicated that the cleavage of caspase-8 precedes that of caspase-3. In addition, blockade of caspase-8 by Z-IETD-FMK inhibited cleavage of pro-caspase-3 and prevented loss of mitochondrial cytochrome c. These results suggest that caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis in HL-60 cells.     

Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells

We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca(2+) in the presence of inorganic phosphate (P(i)), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.     

Role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis

A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol. 77:11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome c release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome c release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.     

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.     

Effects of Chronic and Acute Zinc Supplementation on Myocardial Ischemia-Reperfusion Injury in Rats

A ginseng polysaccharide was extracted, purified, and modified by nitric acid-selenious acid (HNO₃-H₂SeO₃) method to yield one selenylation-modified polysaccharide (sGP). We reported for the first time the anticancer potential of sGP on the human promyelocytic leukemia HL-60 cell line and evaluated its relevant underlying mechanism. Our results showed that sGP markedly inhibited the growth of HL-60 cells via induction of apoptosis. The event of apoptosis was accompanied by the formation of apoptotic bodies; the release of cytochrome c; loss of mitochondrial membrane potential; and activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. In addition, western blot analysis showed that sGP inhibited antiapoptotic Bcl-2 protein expression and increased proapoptotic Bax protein expression in cells under identical conditions. Together, our study suggests that sGP induces apoptosis of HL-60 cells through the mitochondrial-dependent pathway.     

Mechanism of alpha-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of alpha-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (approximately 100 microM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.     

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis

Treatment of HL-60 cells with staurosporine (STS) induced mitochondrial cytochrome c efflux into the cytosol, which was followed by caspase-3 activation and apoptosis. Consistent with these observations, in vitro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required for caspase-3 activation. In contrast, treatment of HCW-2 cells (an apoptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, and apoptosis. In vitro assays strongly suggested that a lack of cytochrome c in the cytosol was the primary limiting factor for caspase-3 activation in HCW-2 cells. To explore the mechanism which regulates mitochondrial cytochrome c efflux, we developed an in vitro assay which showed that cytosolic extracts from STS-treated, but not untreated, HL-60 cells contained an activity, which we designated 'CIF' (cytochrome c-efflux inducing factor), which rapidly induced cytochrome c efflux from HL-60 mitochondria. In contrast, there was no detectable CIF activity in STS-treated HCW-2 cells although the mitochondria from HCW-2 cells were responsive to the CIF activity from STS-treated HL-60 cells. These experiments have identified a novel activity, CIF, which is required for cytochrome c efflux and they indicate that the absence of CIF is the biochemical explanation for the impaired ability of HCW-2 cells to activate caspase-3 and undergo apoptosis.     

LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.     

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells

The sphingomyelin-derived messenger ceramides provoke neuronal apoptosis through caspase-3 activation, while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase-3 activity. However, the mechanisms leading to the opposite regulation of caspase-3 by C2-ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2-ceramide-induced inhibition of mitochondrial potential and C2-ceramide-evoked cytochrome c release. C2-ceramide stimulated Bax expression, but had no effect on Bcl-2, while PACAP abrogated the action of C2-ceramide on Bax and stimulated Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked, respectively, by the JNK inhibitor L-JNKI1 and the MEK inhibitor U0126. L-JNKI1 prevented the alteration of mitochondria induced by C2-ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2-ceramide on mitochondrial potential. Moreover, L-JNKI1 inhibited the stimulatory effect of C2-ceramide on caspase-9 and -3 and prevented C2-ceramide-induced cell death. U0126 blocked PACAP-induced Bcl-2 expression, abrogated the inhibitory effect of PACAP on ceramide-induced caspase-9 activity, and promoted granule cell death. The present study reveals that C2-ceramide and PACAP exert opposite effects on Bax and Bcl-2 through, respectively, JNK- and ERK-dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro- and anti-apoptotic effects of C2-ceramide and PACAP.     

Mechanism of α-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of α-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca²⁺]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (～100 μM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.     

CWJ-081, a novel 3-arylisoquinoline derivative,induces apoptosis in human leukemia HL-60 cells partially involves reactive oxygen species through c-Jun NH2-terminal kinase pathway

In the present study, we investigated the effect of a novel 3-arylisoquinoline derivative 3-(6-ethyl-benzo[1,3]dioxol-5-yl)-7,8-dimethoxy-2-methyl-2H-isoquinolin-1-one (CWJ-081) on the induction of apoptosis and the putative molecular mechanism of its action in human leukemia cells. Treatment with CWJ-081 exhibited a characteristic feature of apoptosis including externalization of phosphatidylserine and formation of DNA fragmentation in human leukemia cell lines (HL-60, U-937, K-562). In addition, stimulation of HL-60 cells with CWJ-081 induced a series of intracellular events: (1) the activations of caspase-8, -9, and -3; (2) the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1); (3) the loss of mitochondrial membrane potential (ΔΨ_m); (4) the release of cytochrome c; and (5) the modulation of Bcl-2 family proteins. We further demonstrated that CWJ-081 induces reactive oxygen species (ROS) production and c-Jun NH₂-terminal kinase (JNK) activation. Pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) markedly inhibited the CWJ-081-induced JNK activation and apoptosis. Moreover, CWJ-081-induced apoptosis was suppressed in the presence of SP600125, a specific JNK inhibitor. Taken together, these data suggest that CWJ-081 induces apoptosis via the mitochondrial apoptotic pathway in HL-60 cells, and ROS-mediated JNK activation plays a key role in the CWJ-081-induced apoptosis.