Terfenadine induces thymocyte apoptosis via mitochondrial pathway

The treatment of rat thymocytes with 10 microM terfenadine resulted in a significant increase in DNA fragmentation. The DNA fragmentation induced by terfenadine was dependent on its concentration and incubation time. In terfenadine-treated cells, the translocation of phosphatidylserine from the inside of plasma membrane to the outside, an early event of the apoptotic process, and chromatin condensation, the morphological characterization of apoptotic cell death, were observed. Terfenadine stimulated caspase-8, -9 and -3-like activities in an incubation time-dependent manner in thymocytes. The active forms of caspase-3 and -9 were detected in the extract from terfenadine-treated cells by immunoblotting analysis using specific antibodies to caspases, but active caspase-8 was not found in this fraction. Decrease in mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol were observed in terfenadine-treated thymocytes. These results suggest that terfenadine induces apoptosis in rat thymocytes via mitochondrial pathway.     

Pyranocoumarin(+/-)-4'-O-acetyl-3'-O-angeloyl-cis-khellactone induces mitochondrial-dependent apoptosis in HL-60 cells

The pyranocoumarin (+)-4'-O-acetyl-3 'O-angeloyl-cis-khellactone (PC) isolated from Radix Peucedani (root of Peucedanum praeruptorum Dunn) showed a dose-dependent effect at 10 -30 pg/mL on causing apoptotic DNA and nuclear fragmentations in HL-60 cells. After 24 h of PC treatment there were losses of mitochondrial membrane potential and cytochrome c. PC also increased total cellular and mitochondrial Bax protein, stimulated an increase in caspase-dependent Bcl-2 cleavage but showed no effect on Bcl-Xv. These observations strongly suggest activation of the mitochondria apoptotic pathway. The pan-specific caspase inhibitor, ZVAD-fmk, abolished the PC-induced apoptosis,whereas the caspase-8 inhibitor IETD-fmk showed no effect, implying the involvement of the caspase 9 pathway. PC caused a 2 to 12 hour transient increase in phospho-ERK, and a 72 h-long activation of JNK. Pre-treatment with the MEK inhibitor PD98059, which suppresses ERK activation, paradoxically promoted PC-induced mitochondrial cytochrome c release, procaspase-3 and -8 cleavage, and enhanced apoptosis. Our results show that PC triggers mitochondria-mediated apoptosis in HL-60 cells, and the involvement of ERK and JNK signal pathways in the process.     

Quinacrine induces cytochrome c-dependent apoptotic signaling in human cervical carcinoma cells

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of DiOC6 demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (delta pai(m)). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector, caspase-3. Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDGA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDGA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.     

Methamphetamine induces apoptosis in an immortalized rat striatal cell line by activating the mitochondrial cell death pathway

Methamphetamine is a neurotoxic drug of abuse known to cause cell death both in vitro and in vivo. Nevertheless, the molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that methamphetamine-induced apoptosis is associated with early (2 h) overexpression of bax, decreases of mitochondrial membrane potential and oxygen consumption as well as release of cytochrome c from mitochondria. In addition, activated caspase-9 was detected at 4 h post-METH exposure. Cell death was detectable by annexin V and propidium iodide staining after 8 h of methamphetamine exposure. At that time, the majority of the cells were stained by annexin V alone, with some cells being stained for both annexin V and propidium iodide. Moreover, cleavage of caspase-3, poly (ADP-ribose) polymerase and DNA fragmentation-related factor 45 was detected at 8 h post drug treatment. These results indicate that methamphetamine-induced apoptotic cell death results from early overexpression of bax, reduction of mitochondrial respiration and membrane potential and release of mitochondrial cytochrome c with subsequent activation of the caspase cascade.     

Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and -9 in HL-60 cells by a mechanism which does not involve Fas signaling

Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin (DAU) in vitro, but have the advantage of blocking nucleoside transport, inhibiting both DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these antitumor drugs were tested for their ability to trigger the release of mitochondrial cytochrome c (Cyt c) and the caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type, drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector caspase-3 is responsible for PARP-1 cleavage, 4 microM concentrations of DAU and these TT bisquinones all maximally induce caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since caspase-3 may be activated by initiator caspase-9 and -8, it is significant to show that such caspase activation cascade is induced by 4 microM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce caspase-3, -8 and -9 activities may be linked to their quinone functionality and cytotoxicity. Interestingly, 4 microM concentrations of TT bisquinones retain their ability to induce caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 microM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 microM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Caspase-2 and -8 may both act upstream of mitochondria to promote Cyt c release, but caspase-2 is already maximally activated 6 h after 4 microM DAU or TT13 treatments, whereas DAU- or TT-induced caspase-8 and -9 activities peak at 9 h. Pre-treatments with 15 microM of the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally block DAU- and TT13-induced caspase-2, -8 and -9 activities, whereas pre-treatments with 15 microM of the caspase-8 inhibitor z-Ile-Glu-Thr-Asp (IETD)-fmk prevent DAU and TT13 from inducing caspase-8 activities without affecting their caspase-2- and -9-inducing activities, suggesting that the induction of apical caspase-2 activity by these drugs may be a critical upstream event required for the activation of other downstream caspases, including caspase-9 and the mitochondrial amplification loop through caspase-8. However, the mechanisms by which DAU and TT13 induce the release of mitochondrial Cyt c appear to be caspase-independent since they are both insensitive to similar pre-treatments with 100 microM of these specific caspase-2 and -8 inhibitors. Moreover, pre-treatments with 10 microg/ml of the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb are all unable to prevent DAU and TT13 from inducing Cyt c release and caspase-2, -8 and -9 activities, suggesting that the Fas-FasL signaling pathway is not involved in the mechanism by which these quinone antitumor drugs trigger apoptosis in HL-60 cells.     

Teratogen-induced activation of caspase-9 and the mitochondrial apoptotic pathway in early postimplantation mouse embryos

Previously we showed that teratogen-induced cell death in mouse embryos is apoptotic in nature, i.e., involves the release of cytochrome c from mitochondria and the subsequent activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation. Herein we show that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine also activate caspase-9, the apical caspase in the mitochondrial apoptotic pathway. Activation of procaspase-9 is associated with the cleavage of this proenzyme and the generation of two forms of the large subunit, primarily a 39-kDa subunit (p39) but also a lesser amount of a 37-kDa subunit (p37). We also present data that support the idea that the teratogen-induced formation of the p37 subunit in vivo occurs by the cytochrome c-mediated processing of procaspase-9, whereas the p39 subunit is formed by an amplification loop involving caspase-3. We also previously showed that the release of cytochrome c, activation of caspase-3, cleavage of PARP, and DNA fragmentation are blocked in cells of the developing heart, which are resistant to teratogen-induced cell death. We now show that this block in the mitochondrial apoptotic pathway in heart cells extends to the activation of procaspase-9. Thus, our cumulative data indicate that hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine induce cell death in Day 9 mouse embryos by activating the mitochondrial apoptotic pathway. In addition, our data suggest that cells of the Day 9 mouse embryo that are resistant to teratogen-induced cell death possess multiple mechanisms for inhibiting the mitochondrial apoptotic pathway after a teratogenic exposure.     

The heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis in cocultures of rat parenchymal and nonparenchymal liver cells.

In this study, we investigated the mechanism of apoptosis by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in cocultures of parenchymal and nonparenchymal liver cells, since the liver consists of various cell types and they cooperatively respond to chemicals. It was found that cocultures were more susceptible to cell death by Trp-P-1 than culture of each cell type alone. In cocultures, Trp-P-1 induced DNA fragmentation accompanied by the activation of 18-kDa endonuclease. Trp-P-1 (30 microM) caused a rapid increase in Bid protein level in mitochondria and the leakage of cytochrome c from mitochondria into the cytosol 15 min after treatment. On the other hand, an increase in Bax protein and a decrease in Bcl-2 protein were detected in the mitochondrial fraction 2 h after treatment following the increases in p53 protein level and DNA binding activity of NF-kappa B. Caspase-8 was activated within 30 min followed by the activation of downstream caspases as measured using the corresponding peptide substrates. The activation of caspases was also confirmed by cleavage of caspase-3, poly(ADP-ribose)polymerase, and protein kinase C-delta as analyzed by Western blotting. A peptide inhibitor of caspase-8 diminished DNA ladder formation and the activation of downstream caspases, but a caspase-9 inhibitor and pyrrolidinedithiocarbamate as an inhibitor of NF-kappa B showed only partial inhibition, suggesting that caspase-8 is the apical caspase in the cascade. These results led to the conclusion that Trp-P-1 mainly drives the caspase-8-mediated pathway that involves Bid, accompanied by a delay in the p53/NF-kappa B-mediated side pathway that involves Bax, Bcl-2, and caspase-9.     

A key role for caspase-2 and caspase-3 in the apoptosis induced by 2-chloro-2'-deoxy-adenosine (cladribine) and 2-chloro-adenosine in human astrocytoma cells

Both the anticancer agent 2-chloro-2'-deoxy-adenosine (Cladribine) and its derivative 2-chloro-adenosine induce apoptosis of human astrocytoma cells (J Neurosci Res 60:388-400, 2000). In this study, we have analyzed the involvement of caspases in these effects. Both compounds produced a gradual and time-dependent activation of "effector" caspase-3, which preceded the appearance of the nuclear signs of apoptosis, suggesting a temporal correlation between these two events. Moreover, the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (fmk) suppressed both caspase-3 activation and apoptosis induction. "Initiator" caspase-9 and caspase-8 were only marginally activated at later times in the apoptotic process. Accordingly, at concentrations that selectively inhibit these caspases, neither N-benzyloxycarbonyl-Leu-Glu-His-Asp-fmk nor N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fmk could prevent adenosine analog-induced cell death. To definitively rule out a role for the caspase-9/cytochrome c-dependent mitochondrial pathway of cell death, neither adenosine analog had any effect on mitochondrial membrane potential, which was instead markedly reduced by other apoptotic stimuli (e.g., deoxyribose, NaCN, and betulinic acid). Consistently, although the latter triggered translocation of mitochondrial cytochrome c to the cytoplasm, no cytosolic accumulation of cytochrome c was detected with adenosine analogs. Conversely, 1 to 7 h after addition of either adenosine analog (i.e., before the appearance of caspase-3 activation), caspase-2 activity was surprisingly and markedly increased. The selective caspase-2 inhibitor N-benzyloxy carbonyl-Val-Asp-Val-Ala-Asp-fmk significantly reduced both adenosine analogs-induced caspase-2 activation and the associated cell death. We conclude that adenosine analogs induce the apoptosis of human astrocytoma cells by activating an atypical apoptotic cascade involving caspase-2 as an initiator caspase, and effector caspase-3. Therefore, these compounds could be effectively used in the pharmacological manipulation of tumors characterized by resistance to cell death via either the mitochondrial or caspase-8/death receptor pathways.     

Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS

Diclofenac is a non-steroidal anti-inflammatory drug that is widely used clinically but side effects associated with the administration of the drug have been reported. The apoptotic effect of the drug has been evaluated in human and rat hepatocytes. Apoptosis was observed after exposure to sub-cytotoxic concentrations of the drug, without overlapping with cell necrosis. Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 8 and 9 mediate the cell-receptor and the mitochondria-initiated apoptotic pathways, respectively. Inhibition of both caspases prevented activation of downstream caspases, thus indicating that diclofenac at least activates caspase 3 and both effector caspases 8 and 9. The hierarchy of caspase activation by diclofenac was investigated. Analysis of kinetics revealed a simultaneous activation of these caspases that was maximal after 12 hr of exposure to the drug. Inhibitors of MPT, prevented the downstream activation of the caspase cascade, thus showing that diclofenac opened the mitochondrial pore. On the other hand, antioxidants were able to prevent caspase activation by diclofenac, revealing that oxidative stress at the mitochondrial level is in the root of MPT induction and caspase cascade activation. Caspase activation is not mediated by Bid cleavage, suggesting that the cell-receptor pathway seems not to be involved. However, a dose-dependent release of caspase 8 from the mitochondria was observed, indicating that caspase 8 can be processed independently of cell death receptors. Caspases 8 and 9 are very likely the apical caspases in diclofenac-induced apoptosis. In addition, an early dose-dependent increase of bclX(L) expression parallel to the generation of reactive oxygen species in the mitochondria was found. In conclusion, the mitochondrial pathway is very likely the only pathway involved in diclofenac-induced apoptosis, which was related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac.     

Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways

The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.     

Cadmium-induced apoptosis through the mitochondrial pathway in rainbow trout hepatocytes: involvement of oxidative stress

Cadmium (Cd) induces oxidative stress and apoptosis in trout hepatocytes. We therefore investigated the involvement of the mitochondrial pathway in the initiation of apoptosis and the possible role of oxidative stress in that process. This study demonstrates that hepatocyte exposure to Cd (2, 5 and 10 microM) triggers significant caspase-3, but also caspase-8 and -9 activation in a dose-dependent manner. Western-blot analysis of hepatocyte mitochondrial and cytosolic fractions revealed that cytochrome c (Cyt c) was released in the cytosol in a dose-dependent manner, whereas the pro-apoptotic protein Bax was redistributed to mitochondria after 24 and 48 h exposure. We also found that the expression of anti-apoptotic protein Bcl-xL, known to be regulated under mild oxidative stress to protect cells from apoptosis, did not change after 3 and 6 h exposure to Cd, then increased after 24 and 48 h exposure to 10 microM Cd. In the second part of this work, two antioxidant agents, 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) (100 microM) and N-acetylcysteine (NAC, 100 microM) were used to determine the involvement of reactive oxygen species (ROS) in Cd-induced apoptosis. Simultaneously exposing trout hepatocytes to Cd and TEMPO or NAC significantly reduced caspase-3 activation after 48 h and had a suppressive effect on caspase-8 and -9 also, mostly after 24 h. Lastly, the presence of either one of these antioxidants in the treatment medium also attenuated Cd-induced Cyt c release in cytosol and the level of Bax in the mitochondria after 24 and 48 h, while high Bcl-xL expression was observed. Taken together, these data clearly evidenced the key role of mitochondria in the cascade of events leading to trout hepatocyte apoptosis in response to Cd and the relationship that exists between oxidative stress and cell death.     

Immunocytochemical detection of members of the caspase cascade of apoptosis in high-grade astrocytomas

During the physiological process of PCD, the cell initiates a sequence of events culminating in the disintegration of the cell into small, membrane-bound apoptotic bodies. The intrinsic part of the PCD program arises from the mitochondria when it releases cytochrome c from the mitochondrial intermembrane space into the cytosol, forming the caspase-activating complex or apoptosome. The family of caspases is involved in the execution of genetically controlled PCD. Caspase-3 is expressed in normal and neoplastically transformed human cells and, like other caspases, is synthesized as an inactive, 32kDa proenzyme. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of cell nuclei. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis, while caspase-9 is the initiator caspase in the caspase cascade in apoptotic normal and neoplastically transformed cells. During our immunocytochemical study, a sensitive, four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results did in fact demonstrate the presence of high apoptotic activity within the cellular microenvironment of high-grade astrocytomas and glioblastomas. The observations identified cytoplasmic expression of caspase-3 and caspase-6 in more than 50 per cent of tumor cells, caspase-8 and caspase-9 in more than 10 per cent of tumor cells in high-grade anaplastic ASTR and glioblastoma. The immunocytochemical expression pattern in about 10 per cent of the tumor cells for caspase-3 and caspase-6 and about 1 to 5 per cent of the tumor cells for caspase-8 and caspase-9 demonstrated a translocation tendency from the cytoplasm to the cell nuclei in the apoptotic cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. We suggest that caspase-3, -6, -8 and -9 immunocytochemistry could have prognostic and immunotherapeutic significance in the treatment of these highly malignant glial tumors.     

Mechanism of dibucaine-induced apoptosis in promyelocytic leukemia cells (HL-60)

Dibucaine, a local anesthetic, inhibited the growth of promyelocytic leukemia cells (HL-60) without inducing arrest of the cell cycle and differentiation to granulocytes. Typical DNA fragmentation and DNA ladder formation were induced in a concentration- and time-dependent manner. The half-maximal concentration of dibucaine required to induce apoptosis was 100 microM. These effects were prevented completely by the pan-caspase inhibitor z-Val-Ala-Asp-(OMe)-fluoromethylketone (z-VAD-fmk), thereby implicating the cysteine aspartase (caspase) cascade in the process. Dibucaine activated various caspases, such as caspase-3, -6, -8, and -9 (-like) activities, but not caspase-1 (-like) activity, and induced mitochondrial membrane depolarization and the release of cytochrome c (Cyt.c) from mitochondria into the cytosol. Processing of pro-caspase-3, -8, and -9 by dibucaine was confirmed by western blot analysis. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to dibucaine. However, 100 microM dibucaine scarcely inhibited oxidative phosphorylation, but it induced membrane permeability transition in isolated rat liver mitochondria. Taken together, these data suggest that dibucaine induced apoptosis of HL-60 cells through activation of the caspase cascade in conjunction with Cyt.c release induced by a processed product of Bid and depolarization of the mitochondrial membrane potential.     

Kalopanaxsaponin A induces apoptosis in human leukemia U937 cells through extracellular Ca influx and caspase-8 dependent pathways

In the present study, we investigated the effect of KPS-A on the apoptotic activity and the molecular mechanism of the action in human leukemia. Treatment with KPS-A significantly increased apoptotic DNA fragmentation in human histiocytic lymphoma U937 cells as shown by DAPI staining, flow cytometry, and agarose gel electrophoresis. In addition, stimulation of U937 cell with KPS-A induced a series of intracellular events: (1) the activations of caspase-8, caspase-9, and caspase-3; (2) the translocations of Bid and Bax proteins to mitochondria; (3) the loss of mitochondrial membrane potential; and (4) the increased release of cytochrome c to the cytosol. Pretreatment with a specific caspases-8, -9 or -3 inhibitor, neutralized the pro-apoptotic activity of KPS-A in U937 cells. We further demonstrated that KPS-A markedly induced an increase in intracellular Ca²⁺ level, which was reversed by EGTA, a general calcium chelator, but not by TMB-8 and dantrolene, intracellular Ca²⁺ release blockers. Moreover, KPS-A-induced DNA fragmentation and caspase activation were substantially reduced in the presence of EGTA. Taken together, these results suggest that KPS-A may play therapeutic role for leukemia via the potent apoptotic activity through Ca²⁺/caspases-8/MPT/caspases-9/caspases-3 signaling pathway.     

Saucernetin-7 isolated from Saururus chinensis induces caspase-dependent apoptosis in human promyelocytic leukemia HL-60 cells

In the present study, we investigated the effect of saucernetin-7 (a biologically active compound isolated from the underground parts of Saururus chinensi) on the induction of apoptosis and the putative pathways of its action in HL-60 human promyelocytic leukemia cells. Saucernetin-7-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, DNA laddering by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed saucernetin-7-induced DNA ladder formation, thereby implicating the caspase cascade in the apoptotic process. We also observed that saucernetin-7 caused the activations of caspase-3, -8 and -9, and that it induced Bid cleavage, the mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria, but it had no effect on Bcl-2 and Bcl-xL levels. Taken together, the present study demonstrates that saucernetin-7 is a potent inducer of apoptosis and that its activity is facilitated by caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, release of cytochrome c into cytoplasm, and subsequently caspase-3 activation, which offers a potential mechanism for the apoptosis-inducing activity of saucernetin-7.     

4-hydroxynonenal induces apoptosis via caspase-3 activation and cytochrome c release

We investigated the mechanism by which 4-hydroxynonenal (HNE), a major aldehydic product of lipid peroxidation, induces apoptosis in tumor cells. Treatment of human colorectal carcinoma (RKO) cells with HNE-induced poly-ADP-ribose-polymerase (PARP) cleavage and DNA fragmentation in a dose- and time-dependent manner. The induction of PARP cleavage and DNA fragmentation paralleled caspase-2, -3, -8, and -9 activation. Pretreatment of cells with an inhibitor of caspase-3, z-DEVD-fmk, or a broad spectrum caspase inhibitor, z-VAD-fmk, abolished caspase activation and subsequent PARP cleavage. Constitutive expression of high levels of Bcl-2 protected cells from HNE-mediated apoptosis. In addition, Bcl-2 overexpression inhibited cytochrome c release from mitochondria and subsequent caspase-2, -3, and -9 activation. These findings demonstrate that HNE triggers apoptotic cell death through a mitochondrion-dependent pathway involving cytochrome c release and caspase activation. Bcl-2 overexpression protected cells from HNE-induced apoptosis through inhibition of cytochrome c release.     

Activation of protein kinase C delta by proteolytic cleavage contributes to manganese-induced apoptosis in dopaminergic cells: protective role of Bcl-2

Chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. Apoptosis has been shown to occur in manganese-induced neurotoxicity; however, the down-stream cellular target of caspase-3 that contributes to DNA fragmentation is not established. Herein, we demonstrate that proteolytic activation of protein kinase Cdelta (PKCdelta) by caspase-3 plays a critical role in manganese-induced apoptotic cell death. Treatment of PC12 cells with manganese caused a sequential activation of mitochondrial-dependent pro-apoptotic events, including mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, and DNA fragmentation. Overexpression of Bcl-2 in PC12 cells remarkably attenuated each of these events, indicating that the mitochondrial-dependent apoptotic cascade contributes to manganese-induced apoptosis. Furthermore, PKCdelta was proteolytically cleaved by caspase-3, causing a persistent activation of the kinase. The manganese-induced proteolytic cleavage of PKCdelta was significantly blocked by Bcl-2-overexpression. Administration of active recombinant PKCdelta induced DNA fragmentation in PC12 cells, suggesting a pro-apoptotic role of PKCdelta. Furthermore, expression of catalytically inactive mutant PKCdelta(K376R) via a lentiviral gene delivery system effectively attenuated manganese-induced apoptosis. Together, these results suggest that the mitochondrial-dependent caspase cascade mediates apoptosis via proteolytic activation of PKCdelta in manganese-induced neurotoxicity.     

A decisive role of mitochondria in defining rate and intensity of apoptosis induction by different alkaloids

Sanguinarine, chelerythrine and chelidonine possess prominent apoptotic effects towards cancer cells. In this study, we found that sanguinarine and chelerythrine induce apoptosis in human CEM T-leukemia cells, and that is accompanied by an early increase in cytosolic cytochrome c that precedes caspases-8, -9 and -3 processing. During apoptosis induction by sanguinarine and chelerythrine, reactive oxygen species (ROS) was rapidly generated and DeltaPsi(mt) dissipated, while Bax, Bcl-2 and Bcl-X((L/S)) proteins' content in the mitochondrial fraction did not change significantly. Caspase-3 activation and DNA fragmentation were considerably inhibited by N-acetyl-cysteine (NAC). Chelidonine induced only a slight release of cytochrome c (12h), parallel to caspase-3 activation. Effect of sanguinarine or chelerythrine towards mitochondria was confirmed by marked changes in morphology of this organelle (3h), while chelidonine did not affect mitochondria intactness. Sanguinarine or chelerythrine also caused an intensive DNA damage in cells in 1h, however a massive increase in number of such impaired cells occurred in 6h, while chelidonine induced intensive DNA damage in 15-20% cells only in 24h. Thus, our results demonstrated that rapid cytochrome c release in CEM T-leukemia cells exposed to sanguinarine or chelerythrine was not accompanied by changes in Bax, Bcl-2 and Bcl-X((L/S)) proteins in the mitochondrial fraction, and preceded activation of the initiator caspase-8.     

Imperatorin,a furanocoumarin from Angelica dahurica (Umbelliferae), induces cytochrome c-dependent apoptosis in human promyelocytic leukaemia,HL-60 Cells

Imperatorin, a biologically active furanocoumarin from the roots of Angelica dahurica (Umbelliferae), was found to induce apoptosis in human promyelocytic leukaemia, HL-60 cells. DNA fragmentation assay, morphology-based evaluation, and flow cytometric analysis demonstrated that imperatorin at micromolar concentrations was able to trigger apoptosis of HL-60 cells. Neither necrosis nor differentiation was observed at cytotoxic micromolar concentrations of imperatorin. Further studies showed that the cytochrome c/caspase-9 pathway was responsible for imperatorin-induced apoptosis; i.e., mitochondrial membrane was depolarized, Bcl-2 was down-regulated, cytochrome c was released from mitochondria, caspase-9 and caspase-3 were activated, and poly(ADP-ribose) polymerase was cleaved. Furthermore, imperatorin-induced apoptosis was significantly blocked by Z-VAD-FMK (a broad spectrum caspase inhibitor), Z-LEHD-FMK (a caspase-9 inhibitor) and Ac-DMQD-CHO (a caspase-3 inhibitor), but not by Z-IEDT-FMK (a caspase-8 inhibitor).     

Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling

Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system. AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound. Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9. In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines. Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr. The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective. Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively. During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8. However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation. Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells.