Photodynamic therapy-induced death of MCF-7 human breast cancer cells: a role for caspase-3 in the late steps of apoptosis but not for the critical lethal event

Photodynamic therapy (PDT) causes mitochondrial damage and induces apoptosis through release of cytochrome c and activation of caspase-3. To test whether caspase 3 is the sole executioner of apoptosis and its role in overall cell lethality, we compared the response of MCF-7c3 cells that express a stably transfected CASP-3 gene to that of parental MCF-7:SW8 cells transfected with vector alone (MCF-7v). Following photosensitization with the phthalocyanine Pc 4 and red light, cytochrome c was released from the mitochondria to equivalent extents in the two cell lines. However, the appearance of apoptotic indicators, such as active caspase-3 (DEVDase), cleavage of poly(ADP-ribose) polymerase, and oligonucleosomal DNA fragmentation, was observed only in MCF-7c3 cells during the first 6 h after photosensitization. Although production of 50-kb DNA fragments and chromatin condensation were found in PDT-treated MCF-7v cells by 20-24 h posttreatment, the rate and extent of apoptosis were much less than in MCF-7c3 cells. MCF-7c3 cells were more sensitive to photosensitization than were MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to photodynamic killing when evaluated by a clonogenic assay. These results show (a) the importance of assessing overall cell death by clonogenic assay; (b) that the critical lethal event is independent of caspase-3, perhaps at or near the release of cytochrome c from mitochondria; and (c) that the caspase-3-mediated events appear to be irrelevant in determining overall killing of cells.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Hepatocyte growth factor/scatter factor blocks the mitochondrial pathway of apoptosis signaling in breast cancer cells

The cytokine hepatocyte growth factor/scatter factor (HGF/SF) has been found to protect a variety of epithelial and cancer cell types against cytotoxicity and apoptosis induced by DNA damage, but the specific apoptotic signaling events and the levels at which they are blocked by HGF/SF have not been identified. We found that treatment of MDA-MB-453 human breast cancer cells with adriamycin (also known as doxorubicin, a DNA topoisomerase IIalpha inhibitor) induced a series of time-dependent events, including the mitochondrial release of cytochrome c and apoptosis-inducing factor, mitochondrial membrane depolarization, activation of a set of caspases (caspase-9, -3, -7, -2, and -8), cleavage of poly(ADP-ribose) polymerase (PARP), and up-regulation of expression of the Fas ligand. All of these events were blocked by preincubation of the cells with HGF/SF. In contrast, the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone blocked some of these events (e.g. caspase-3 activation and PARP cleavage) but did not block cytochrome c release or mitochondrial depolarization. These findings suggest that HGF/SF functions, in part, upstream of the mitochondria to block mitochondrial apoptosis signaling, prevent activation of multiple caspases, and protect breast cancer cells against apoptosis.     

Cytochrome c release upon Fas receptor activation depends on translocation of full-length bid and the induction of the mitochondrial permeability transition.

In Jurkat cells Bid was cleaved upon activation of the Fas receptor with an anti-Fas antibody. The caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-CH(2)F (IETD) prevented the cleavage of Bid and the loss of viability. The nuclear enzyme poly(ADP-ribose)polymerase (PARP) was also cleaved upon the activation of caspases, and IETD similarly prevented PARP cleavage. The PARP inhibitor 3-aminobenzamide (3-AB) restored the cell killing in the presence of IETD, an effect that occurred without restoration of the cleavage of Bid or PARP. In the presence of 3-AB and IETD, translocation occurred of full-length Bid to the mitochondria. The induction of the mitochondrial permeability transition (MPT) was documented by the cyclosporin A (CyA) sensitivity of the release of cytochrome c, the release of malate dehydrogenase from the mitochondrial matrix, the loss of the mitochondrial membrane potential, and the pronounced swelling of these organelles, as assessed by electron microscopy. In addition to preventing all evidence of the MPT, CyA prevented the loss of cell viability, without effect on the cleavage of either Bid or PARP. The prevention of PARP cleavage by inhibition of caspase-3 resulted in a 10-fold activation of the enzyme and a resultant depletion of NAD and ATP. The PARP inhibitor 3-AB prevented the loss of NAD and ATP. Depletion of ATP by metabolic inhibitors similarly prevented the cell killing. It is concluded that the cleaving of PARP in Fas-mediated apoptosis allowed expression of an energy-dependent cell death program that included the translocation of full-length Bid to the mitochondria with induction of the MPT.     

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

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

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.     

Myrtucommulone from <Emphasis Type="Italic">Myrtus communis</Emphasis> induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9

Myrtucommulone (MC) is a unique, nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis). Here, we addressed the potential of MC to induce apoptosis of cancer cells. MC potently induced cell death of different cancer cell lines (EC₅₀ 3–8 μM) with characteristics of apoptosis, visualized by the activation of caspase-3, -8 and -9, cleavage of poly(ADP-ribose)polymerase (PARP), release of nucleosomes into the cytosol, and DNA fragmentation. MC was much less cytotoxic for non-transformed human peripheral blood mononuclear cells (PBMC) or foreskin fibroblasts (EC₅₀ cell death = 20–50 μM), and MC up to 30 μM hardly caused processing of PARP, caspase-3, -8 and -9 in human PBMC. MC-induced apoptosis was mediated by the intrinsic rather than the extrinsic death pathway. Thus, MC caused loss of the mitochondrial membrane potential in MM6 cells and evoked release of cytochrome c from mitochondria. Interestingly, Jurkat cells deficient in caspase-9 were resistant to MC-induced cell death and no processing of PARP or caspase-8 was evident. In cell lines deficient in either CD95 (Fas, APO-1) signalling, FADD or caspase-8, MC was still able to potently induce cell death and PARP cleavage. Conclusively, MC induces apoptosis in cancer cell lines, with marginal cytotoxicity for non-transformed cells, via the mitochondrial cytochrome c/Apaf-1/caspase-9 pathway. I. Tretiakova and D. Blaesius contributed equally to this work.     

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.     

Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia

It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3.     

Bcl-xL blocks transforming growth factor-beta 1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizing Apaf-1-dependent caspase activation in prostate epithelial cells

The mechanism by which transforming growth factor-beta1 (TGF-beta1) induces apoptosis of prostate epithelial cells was studied in the NRP-154 rat prostate epithelial cell line. TGF-beta 1 down-regulates expression of Bcl-xL and poly(ADP-ribosyl)polymerase (PARP), promotes cytochrome c release, up-regulates expression of latent caspase-3, and activates caspases 3 and 9. We tested the role of Bcl-xL in this cascade by stably overexpressing Bcl-xL to prevent loss by TGF-beta 1. Clones overexpressing Bcl-xL are resistant to TGF-beta 1 with respect to induction of apoptosis, cytochrome c release, activation of caspases 9 and 3, and cleavage of PARP; yet they remain sensitive to TGF-beta 1 by cell cycle arrest, induction of both fibronectin and latent caspase-3 expression, and loss of PARP expression. We show that Bcl-xL associates with Apaf-1 in NRP-154 cells; but this association does not inhibit the activation of caspases 9 and 3 by cytochrome c. Together, our data suggest that TGF-beta1 induces apoptosis through loss of Bcl-xL, leading to cytochrome c release and the subsequent activation of caspases 9 and 3. Moreover, our data demonstrate that the antiapoptotic effect of Bcl-xL occurs by inhibition of mitochondrial cytochrome c release and not through antagonizing Apaf-1-dependent processing of caspases 9 and 3.     

Mitochondrial cytochrome c release and caspase-3-like protease activation during indomethacin-induced apoptosis in rat gastric mucosal cells

Indomethacin (IND), a nonsteroidal anti-inflammatory drug, has been known to cause gastric mucosal injury as a side effect. Using a rat gastric mucosal cell line, RGM1, we determined whether apoptosis is involved in IND-mediated gastropathy, and whether caspase activation and mitochondrial cytochrome c release play an important role in producing apoptosis of IND-treated RGM1 cells in the presence of serum. IND caused caspase-3-like protease activation followed by apoptosis in a dose- and time-dependent manner. Caspase-1-like protease activity did not change during IND-induced apoptosis. IND also increased mitochondrial cytochrome c release in a time-dependent fashion. Mitochondrial cytochrome c efflux occurred just before or at the same time as caspase-3-like protease activation, and preceded the increase in apoptotic cell numbers. Z-VAD-FMK, a caspase inhibitor, inhibited both the increase in caspase-3-like protease activity and apoptosis in IND-treated RGM1 cells but did not affect caspase-1-like protease activity or mitochondrial cytochrome c release. These observations suggest that the apoptosis of gastric mucosal cells could be involved in IND-induced gastropathy, that cytochrome c is released from mitochondria into the cytosol during the early phase of IND-mediated apoptosis, and that subsequent activation of caspase-3-like protease, but not caspase-1-like protease, is required for the execution of apoptosis.     

Synergistic induction of apoptosis in murine hepatoma Hepa1-6 cells by IFN-gamma and TNF-alpha

In this study, we examined the susceptibility of murine hepatoma Hepa1-6 cells to undergo IFN-gamma- and/or TNF-alpha-induced apoptosis. IFN-gamma or TNF-alpha alone had no demonstrable cytotoxic effects, whereas IFN-gamma and TNF-alpha in combination induced apoptosis drastically in Hepa1-6 cells. During this apoptosis, an increase in caspase-3- and -8-like protease activities and activation of caspase-3, identified by the appearance of its p17 fragment, were observed. Moreover, the cytotoxic induction and caspase-3 activation were effectively inhibited by Z-Asp-CH(2)-DCB (Z-Asp), a caspase inhibitor. Further, an elevation of cytochrome c in the cytosol, in a parallel to activation of caspase-3, was observed in a time-dependent manner. Concurrently, up-regulation of caspase-11 gene expression and processing of procaspase-11 were detected during this apoptosis. These results suggest that the caspase-3 activation, the release of cytochrome c from mitochondria, and increased caspase-11 gene expression involve in synergistic induction of apoptosis in Hepa1-6 cells by IFN-gamma and TNF-alpha.     

Characterization of Cholix toxin-induced apoptosis in HeLa cells

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.     

Resistance to CD95/Fas-induced and ceramide-mediated apoptosis of human melanoma cells is caused by a defective mitochondrial cytochrome c release

Intracellular CD95/Fas-signaling pathways have not been investigated in melanoma yet. Two different CD95 receptor-induced apoptotic pathways are presently known in other cell types: (i) direct activation of caspase-8 and (ii) induction of ceramide-mediated mitochondrial activation, both leading to subsequent caspase-3 activation. In the present study, five of 11 melanoma cell populations were shown to release cytochrome c from mitochondria, which activates caspase-3 and finally results in DNA fragmentation upon treatment with the agonistic monoclonal antibody CH-11. In contrast, this apoptotic pathway was not activated in the remaining six melanoma cell populations. Interestingly, the susceptibility of melanoma cells to CD95L/FasL-triggered cell death was clearly correlated with N-acetylsphingosine-mediated apoptosis. Our results are in line with a defect upstream of mitochondrial cytochrome c release in resistant cells.     

Mitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.     

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.     

Cationic liposomes induce macrophage apoptosis through mitochondrial pathway

To clarify the mechanism of apoptosis of the macrophage-like cell line RAW264.7 induced by cationic liposomes, we focused on the mitochondria and investigated the changes in mitochondrial membrane potential and the release of cytochrome c following treatment of cationic liposomes composed of stearylamine (SA-liposomes). SA-liposomes induced mitochondrial membrane depolarization and also the release of cytochrome c from mitochondria. Caspase-3 was also activated by SA-liposome treatment. Pretreatment of cells with N-acetylcysteine, a scavenger of reactive oxygen species (ROS), conferred resistance to the induction of the membrane depolarization, cytochrome c release, and caspase-3 activation by SA-liposomes. These results indicated that SA-liposomes caused the apoptosis in RAW264.7 cells through the mitochondrial pathway, and ROS generation was required for this phenomenon.     

Activation of protein kinase C inhibits TRAIL-induced caspases activation, mitochondrial events and apoptosis in a human leukemic T cell line

TRAIL causes apoptosis in numerous types of tumor cells. However, the mechanisms regulating TRAIL-induced apoptosis remain to be elucidated. We have investigated the role of PKC in regulating TRAIL-induced mitochondrial events and apoptosis in the Jurkat T cell line. We found a caspase-dependent decline in mitochondrial membrane potential and translocation of cytochrome c from mitochondria into the cytosol in response to TRAIL. Both these events were prevented by PKC activation. Moreover, PKC activation considerably reduced the activation of caspases, PARP cleavage and apoptosis when induced upon TRAIL treatment. MAPK activation was involved in the mechanism of PKC-mediated inhibition of TRAIL-induced cytochrome c release from mitochondria. Furthermore, inhibition of the MAPK pathway partially reversed the PKC-mediated inhibition of TRAIL-induced apoptosis. Besides, PKC activation may also inhibit the TRAIL-induced apoptosis through a MAPK-independent mechanism. Altogether, these results indicate a negative role of PKC in the regulation of apoptotic signals generated upon TRAIL receptor activation.