Mechanisms of induction of apoptosis by anthraquinone anticancer drugs aclarubicin and mitoxantrone in comparison with doxorubicin: Relation to drug cytotoxicity and caspase-3 activation

We examined molecular events and morphological features associated with apoptosis induced by anthraquinone anticancer drugs aclarubicin, mitoxantrone and doxorubicin in two spontaneously immortalized cell lines (NIH 3T3 and B14) in relation to cytotoxicity of these drugs. The investigated cells showed similar sensitivity to aclarubicin but different sensitivity to doxorubicin and mitoxantrone: mitoxantrone was the most cytotoxic drug in both cell lines. All three drugs triggered both apoptosis and necrosis but none of these processes was positively correlated with their cytotoxicity. Apoptosis was the prevalent form of cell kill by aclarubicin, while doxorubicin and mitoxantrone induced mainly the necrotic mode of cell death. The extent and the timing of apoptosis were strongly dependent on the cell line, the type of the drug and its dose, and were mediated by caspase-3 activation. A significant increase in caspase-3 activity and the percentage of apoptotic cells, oligonucleosomal DNA fragmentation, chromatin condensation and formation of apoptotic bodies was observed predominantly in B14 cells. NIH 3T3 cells showed lesser changes and a lack of DNA fragmentation. Aclarubicin was the fastest acting drug, inducing DNA fragmentation 12 h earlier than doxorubicin, and 24 h earlier than mitoxantrone. Caspase-3 inhibitor Ac-DEVD-CHO did not show any significant effect on drug cytotoxicity and DNA nucleosomal fragmentation.     

Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis

We investigated the mode of cell death induced by the anthracyclines, aclarubicin, doxorubicin and daunorubicin in the human leukemia cell lines, HL60 and Jurkat. The cells were incubated with drug concentrations up to 500 nM for periods between 3 and 24 hours, followed by morphological and biochemical analyses. All three substances induced DNA fragmentation, evident as DNA laddering and appearance of cells with hypodiploid DNA content, externalisation of phosphatidyl serine, activation of caspases and degradation of the apoptosis-specific endonuclease inhibitor DFF45. However, concentrations and times necessary for these effects to occur were different, aclarubicin being the quickest acting drug with a lag phase of 3 h, followed by daunorubicin with 6 h and doxorubicin with 24 h. More importantly, aclarubicin induced these effects while the cell membrane was intact, whereas doxorubicin and daunorubicin led to immediate loss of membrane integrity. Programmed cell death is characterised by preservation of membrane integrity in order to allow removal of apoptotic bodies, whereas cell rupture is an early event in necrosis. We therefore suggest that, in our experimental settings, doxorubicin- and daunorubicin-induced cell death occurs by necrosis, while aclarubicin induces programmed cell death.     

Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death

Betanodavirus protein alpha induces cell apoptosis or secondary necrosis by a poorly understood process. In the present work, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed apoptotic cells at 24-72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium bromide dual dye staining from 48 to 72 h p.t. Protein alpha induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-xL on cell death, RNA2-transfected cells were co-transfected with zfBcl-x(L). Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein alpha induces apoptosis followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.     

Mechanism underlying oxidative stress-mediated lipotoxicity: exposure of J774.2 macrophages to triacylglycerols facilitates mitochondrial reactive oxygen species production and cellular necrosis

The aim of this study was to elucidate death pathways in macrophages resulting from exposure to triacylglycerols (TG), mechanisms which may be relevant to the development of atherosclerosis. A commercial TG emulsion (lipid emulsion, LE; 0.1-1.5 mg lipids/ml) was added to J774.2 cells in culture. Within the first 24 h after TG treatment, cellular reactive oxygen species (ROS) levels were strongly elevated and basal caspase-3 activity was attenuated. In contrast, after 48 h, ROS production was arrested. TG-mediated ROS production was demonstrated to be via mitochondrial complex 1 of the electron-transfer chain since the inhibitor of complex 1 rotenone significantly attenuated the cellular ROS levels in TG-treated cells. The TG effect culminated in cell death, with no caspase-3 activation. We therefore evaluated the effect of TG on apoptotic cells showing high caspase activity. TG induced elevated ROS levels and suppressed caspase-3 in apoptotic cells pretreated for 24 h with cycloheximide. Dual staining with propidium iodide and Annexin V followed by flow cytometric analysis showed that TG facilitated cell death with clear necrotic characteristics. To elucidate whether the necrotic cell death process is indeed oxidant dependent, antioxidant protection was studied. Treatment with N-acetylcysteine (NAC) (0.5 mM), ascorbic acid (0.5 mM), and resveratrol (0.2 mM) protected against the TG lipotoxic effect, while, surprisingly, lipophilic antioxidants did not. The combination of NAC, ascorbic acid, and resveratrol, each at much lower concentrations, had a synergistic protective effect. In conclusion, we show here for the first time that exposure to TG can directly regulate lipotoxicity in macrophages by inducing mitochondria-mediated prolonged oxidative stress; this, in turn, can inactivate the apoptotic caspase system, resulting in necrotic cell death which can be prevented by specific antioxidants.     

The induction of apoptosis by daunorubicin and idarubicin in human trisomic and diabetic fibroblasts

In this study, we investigated apoptosis induced in human trisomic and diabetic fibroblasts by daunorubicin (DNR) and its derivative, idarubicin (IDA). The cells were incubated with DNR or IDA for 2 h and then cultured in a drug-free medium for a further 2–48 h. The apoptosis in the cultured cell lines was assessed by biochemical analysis. We found that both drugs induced a timedependent loss of mitochondrial membrane potential, and a significant increase in intracellular calcium and caspase-3 activity. Mitochondrial polarization and changes in the level of intracellular calcium were observed during the first 2–6 h after drug treatment. Caspase-3 activation occurred in the late stages of the apoptotic pathway. Our findings also demonstrated that idarubicin was more cytotoxic and more effective than daunorubicin in inducing apoptosis in trisomic and diabetic fibroblasts.     

p53 regulates a non-apoptotic death induced by ROS

DNA damage induced by reactive oxygen species and several chemotherapeutic agents promotes both p53 and poly (ADP-ribose) polymerase (PARP) activation. p53 activation is well known to regulate apoptotic cell death, whereas robust activation of PARP-1 has been shown to promote a necrotic cell death associated with energetic collapse. Here we identify a novel role for p53 in modulating PARP enzymatic activity to regulate necrotic cell death. In mouse embryonic fibroblasts, human colorectal and human breast cancer cell lines, loss of p53 function promotes resistance to necrotic, PARP-mediated cell death. We therefore demonstrate that p53 can regulate both necrotic and apoptotic cell death, mutations or deletions in this tumor-suppressor protein may be selected by cancer cells to provide not only their resistance to apoptosis but also to necrosis, and explain resistance to chemotherapy and radiation even when it kills via non-apoptotic mechanisms.     

Changes in mitochondrial function induced by dequalinium precede oxidative stress and apoptosis in the human prostate-cancer cell line PC-3

Mitochondria play central roles in diverse physiological and pathological conditions associated with cell survival and death. Delocalized lipophilic cations, such as dequalinium (DQA), are accumulated in cancer cells attracted by the highly negative mitochondrial transmembrane potential of these cells. DQA showed a potent anticancer activity in cells from different malignancies. Here, we report the effect of DQA on PC-3 prostate cancer cells. Incubation with DQA at concentrations between 1.5 and 100 μM from 24 to 48 h decreases cell viability. The decrease in cell viability together with a loss of mitochondrial transmembrane potential induced an increase in reactive oxygen species production and cell death via caspase-3 dependent apoptotic pathway. DQA was shown to cause moderate to strong cell death in a time and concentration dependent manner, causing a most advantageous effect at a concentration of 10 μM applied for a long 48 h time period, which might be a consequence of the kinetics of intracellular DQA accumulation in mitochondria, but also of the mechanisms of DQA-induced cell death. This data shows DQA as a promising agent against the human prostate cancer PC-3 cell line, activating the caspase-3 dependent apoptotic pathway. This fact might be beneficial for possible future applications in cancer therapy.     

Mycoplasmal lipoproteins induce toll-like receptor 2- and caspases-mediated cell death in lymphocytes and monocytes

Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT-4 and Raji, and in one monocytic cell line, THP-1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL-60. These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA. The lipoproteins activated caspase-3 in both MOLT-4 and HL-60 cells, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase. The cytotoxicity to MOLT-4 and HL-60 cells was inhibited by various caspase inhibitors, Ac-DMQD-CHO, Ac-IETD-CHO, and Z-VAD-FMK. The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll-like receptor 2. Thus this study demonstrated that mycoplasmal lipoproteins induce caspases-dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2-mediated signaling.     

Rapid caspase-dependent cell death in cultured human breast cancer cells induced by the polyamine analogue N(1),N(11)-diethylnorspermine.

The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the cellular pools of putrescine, spermidine and spermine by down-regulating the activity of the polyamine biosynthetic enzymes and up-regulating the activity of the catabolic enzyme spermidine/ spermine N(1)-acetyltransferase (SSAT). In the breast cancer cell line L56Br-C1, treatment with 10 microm DENSPM induced SSAT activity 60 and 240-fold at 24 and 48 h after seeding, respectively, which resulted in polyamine depletion. Cell proliferation appeared to be totally inhibited and within 48 h of treatment, there was an extensive apoptotic response. Fifty percent of the cells were found in the sub-G(1) region, as determined by flow cytometry, and the presence of apoptotic nuclei was morphologically assessed by fluorescence microscopy. Caspase-3 and caspase-9 activities were significantly elevated 24 h after seeding. At 48 h after seeding, caspase-3 and caspase-9 activities were further elevated and at this time point a significant activation of caspase-8 was also found. The DENSPM-induced cell death was dependent on the activation of the caspases as it was inhibited by the general caspase inhibitor Z-Val-Ala-Asp fluoromethyl ketone. The results are discussed in the light of the L56Br-C1 cells containing mutated BRCA1 and p53, two genes involved in DNA repair.     

Aquatic birnavirus capsid protein,VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells

Aquatic birnavirus induces post-apoptotic necrotic cell death via a newly synthesized protein-dependent pathway. However, the involvement of viral genome-encoded protein(s) in this death process remains unknown. In the present study, we demonstrated that the submajor capsid protein, VP3, up-regulates the pro-apoptotic protein, Bad, in fish and mouse cells. Western blot analysis revealed that VP3 was expressed in CHSE-214 cells at 4 h post-infection (pi), indicating an early role during viral replication. We cloned the VP3 gene and tested its function in fish and mouse cells; VP3 overexpression induced apoptotic cell death by TUNEL assay. In addition, it up-regulated Bad gene expression in zebrafish ZLE cells by threefold at 12 h post-transfection (pt) and in mouse NIH3T3 cells by tenfold at 24 h pt. VP3 up-regulation of Bad expression altered mitochondria function, inducing mitochondrial membrane potential (MMP) loss and activating initiator caspase-9 and effector caspase-3. Furthermore, reduced Bad expression (65% reduction), MMP loss (up to 40%), and enhanced cell viability (up to 60%) upon expression of VP3 antisense RNA in CHSE-214 cells at 24 h post-IPNV infection was observed. Finally, overexpression of the anti-apoptotic gene, zfBcl-xL, reduced VP3-induced apoptotic cell death and caspase-3 activation at 24 h in fish cells. Taken together, these results suggest that aquatic birnavirus VP3 induces apoptosis via up-regulation of Bad expression and mitochondrial disruption, which activates a downstream caspase-3-mediated death pathway that is blocked by zfBcl-xL.     

Oxidative stress induces protein and DNA radical formation in follicular dendritic cells of the germinal center and modulates its cell death patterns in late sepsis

Profound depletion of follicular dendritic cells (FDCs) is a hallmark of sepsis-like syndrome, but the exact causes of the ensuing cell death are unknown. The cell death-driven depletion contributes to immunoparalysis and is responsible for most of the morbidity and mortality in sepsis. Here we have utilized immuno-spin trapping, a method for detection of free radical formation, to detect oxidative stress-induced protein and DNA radical adducts in FDCs isolated from the spleens of septic mice and from human tonsil-derived HK cells, a subtype of germinal center FDCs, to study their role in FDC depletion. At 24h post-lipopolysaccharide administration, protein radical formation and oxidation were significantly elevated in vivo and in HK cells as shown by ELISA and confocal microscopy. The xanthine oxidase inhibitor allopurinol and the iron chelator desferrioxamine significantly decreased the formation of protein radicals, suggesting the role of xanthine oxidase and Fenton-like chemistry in radical formation. Protein and DNA radical formation correlated mostly with apoptotic features at 24h and necrotic morphology of all the cell types studied at 48h with concomitant inhibition of caspase-3. The cytotoxicity of FDCs resulted in decreased CD45R/CD138-positive plasma cell numbers, indicating a possible defect in B cell differentiation. In one such mechanism, radical formation initiated by xanthine oxidase formed protein and DNA radicals, which may lead to cell death of germinal center FDCs.     

Se-methylselenocysteine induces apoptosis mediated by reactive oxygen species in HL-60 cells

Recent studies have implicated apoptosis as one of the most plausible mechanisms of the chemopreventive effects of selenium compounds, and reactive oxygen species (ROS) as important mediators in apoptosis induced by various stimuli. In the present study, we demonstrate that Se-methylselenocysteine (MSC), one of the most effective selenium compounds at chemoprevention, induced apoptosis in HL-60 cells and that ROS plays a crucial role in MSC-induced apoptosis. The uptake of MSC by HL-60 cells occurred quite early, reaching the maximum within 1 h. The dose-dependent decrease in cell viability was observed by MSC treatment and was coincident with increased DNA fragmentation and sub-G(1) population. 50 microM of MSC was able to induce apoptosis in 48% of cell population at a 24 h time point. Moreover, the release of cytochrome c from mitochondria and the activation of caspase-3 and caspase-9 were also observed. The measurement of ROS by dichlorofluorescein fluorescence revealed that dose- and time-dependent increase in ROS was induced by MSC. N-acetylcysteine, glutathione, and deferoxamine blocked cell death, DNA fragmentation, and ROS generation induced by MSC. Moreover, N-acetylcysteine effectively blocked caspase-3 activation and the increase of the sub-G(1) population induced by MSC. These results imply that ROS is a critical mediator of the MSC-induced apoptosis in HL-60 cells.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

Caspase-3 activity and carbonyl cyanide m-chlorophenylhydrazone-induced apoptosis in HL-60

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being "truly" necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as "truly" necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.     

Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation,enhanced ascorbate uptake,and upregulated Bcl-2 protein

Although it is well known that Bcl-2 can prevent apoptosis, the Bcl-2's anti-apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant-induced cell death and to investigate the role of Bcl-2 in the tert-butyl hydroperoxide (t-BuOOH)-induced oxidant injury in Rat-1 fibroblasts and their bcl-2 transfected counterparts, b5 cells. Treatment with t-BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat-1 cells than in b5 cells. The hydroperoxide t-BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 microM) for only 3 h induces death in Rat-1 cells, whereas their bcl-2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 microM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl-2 transfectants significantly suppressed t-BuOOH-induced increases in both lipid peroxidation and caspase-3 activation 3 and 1 h after t-BuOOH exposure, respectively, but failed to suppress either caspase-1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1-(14)C] ascorbic acid (Asc) into the bcl-2 transfectants was superior to that into the non-transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl-2 proteins was rapidly induced after t-BuOOH exposure in the transfectants, but not in non-transfectants, and restored till 24 h to the normal Bcl-2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase-3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl-2 on the t-BuOOH-induced cytotoxicity. And some of these events may correlate with Bcl-2 expression-induced partial enhanced anti-oxidant cellular ability including enrichment of intracellular Asc and oxidative stress-induced upregulation of Bcl-2 protein. On the other hand, ROS production and caspase-1 activation were not related to cytoprotection by Bcl-2.     

The lethal effect of bis-type azridinylnaphthoquinone derivative on oral cancer cells (OEC-M1) associated with anti-apoptotic protein bcl-2

Several drugs of aziridinylbenzoquinone analogs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill tumor cells preferentially within the hypoxic microenvironment. From our previous reported data, it was found that the synthesized 2-aziridin-1-yl-3-[(2-[2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio]ethoxy]ethyl)thio]naphthoquinone (AZ-1) is a bioreductive compound with potent lethal effect on oral cancer cell, OEC-M1. It was found in this study that the lethal effect of the oral cancer cell lines OEC-M1 induced by AZ-1 was mediated through the cell cycle arrest and apoptosis pathway. The LC50 values of OEC-M1 and KB cells induced by AZ-1 compound were 0.72 and 1.02 microM, respectively, which were much lower than that of normal fibroblast cells (SF with LC50 = 5.6 microM) with more than 90% of normal fibroblasts surviving as compared to control at a concentration of AZ-1 as high as 2 microM. It was interesting to note that the LC50 of monotype diaziridinylbenzoquinone compound, diaziquone (AZQ), was 50 microM on OEC-M1 cells. Comparing the cytotoxicity of AZ-1 and AZQ on OEC-M1 cells, AZ-1 is approximately 70 times more potent than AZQ. By using Western blot, both G2/M phase cell cycle arresting protein, cyclin B, and anti-apoptotic protein, bcl-2, were expressed in OEC-M1 cell when the concentrations of AZ-1 were increased from 0.125 to 0.5 microM and then decreased from 1 to 2 microM of AZ-1 treatment as compared with control for 24 h. Both proteins were expressed most abundantly at 0.5 microM AZ-1. However, the expression of bcl-2 protein in OEC-M1 was significantly decreasing in a dose-dependent manner and was only about 50% protein level at 2 microM AZ-1 for 48h as compared with control. The cell survival check protein p53 increased from 1.72- to 2.8-fold and 1.36- to 2.16-fold at concentrations of AZ-1 from 0.125 to 2.0 microM in a dose-dependently increasing manner on OEC-M1 as compared with control for 24 and48 h treatments, respectively. The apoptotic-related phenomena were observed, which included apoptotic body formation and the enzyme activity change of caspase-3. The apoptotic bodies and caspase-3 activity of OEC-M1 were induced only at 2 microM AZ-1 for a 24h treatment, yet apoptotic body formation was observed at as low as 0.5 microM AZ-1 and in a dose-dependently increasing manner for a 48 h treatment. The caspase-3 activity was increased 20.6%, 26.8%, and 84.2%, respectively, at 0.5, 1, and 2muM concentrations of AZ-1 for a 48 h treatment as compared with control. These results indicate that AZ-1 induced the cell death of OEC-M1 through the G2/M phase arrest of cell cycle and anti-apoptosis first and then apoptosis following a 48 h treatment. All of the pathway might be associated with bcl-2 and p53 protein expression. We propose that the AZ-1 could be used as anti-oral cancer drug for future studies with animal models.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.     

Aquatic birnavirus induces necrotic cell death via the mitochondria-mediated caspase pathway

Aquatic birnavirus induces necrotic cell death by an ill-understood process. Presently, we demonstrate that infectious pancreatic necrosis virus (IPNV) induces post-apoptotic necrotic cell death through loss of mitochondrial membrane potential (MMP) followed by caspase-3 activation in CHSE-214 cells. Progressive phosphatidylserine externalization was observed at 6 h post-infection (p.i.). This was followed by the development of bulb-like vesicles (bleb formation) at 8 h p.i. Progressive loss of MMP was also observed in IPNV-infected CHSE-214 cells beginning at 6 h p.i. At 8 h and 12 h p.i., IPNV-infected cells demonstrated a dramatic increase in MMP loss, rapid entry into necrotic cell death, and activation of caspase-9 and -3. Additionally, treatment with an inhibitor of MMP loss, bongkrekic acid, an adenine nucleotide translocase inhibitor, blocked IPNV-induced PS exposure and MMP loss, as well as reduced the activation of caspase-3. Taken together, our results suggest that IPNV induces apoptotic cell death via loss of MMP, thereby triggering secondary necrosis and caspases-3 activation. Furthermore, this death-signaling pathway is disrupted by bongkrekic acid in fish cells, indicating that this drug may serve to modulate IPNV-induced pathogenesis.     

Differential cytotoxic responses to low- and high-dose photodynamic therapy in human gastric and bladder cancer cells

Here, we present differential cytotoxic responses to two different doses of photodynamic therapies (PDTs; low-dose PDT [LDP] and high-dose PDT [HDP]) using a chlorin-based photosensitizer, DH-II-24, in human gastric and bladder cancer cells. Fluorescence-activated cell sorting analysis using Annexin V and propidium iodide (PI) showed that LDP induced apoptotic cell death, whereas HDP predominantly caused necrotic cell death. The differential cytotoxic responses to the two PDTs were further confirmed by a DiOC(6) and PI double-staining assay via confocal microscopy. LDP, but not HDP, activated caspase-3, which was inhibited by Z-VAD, Trolox, and BAPTA-AM. LDP and HDP demonstrated opposite effects on intracellular reactive oxygen species (ROS)/Ca(2+) signals; LDP stimulated intracellular ROS production, contributing to a transient increase of intracellular Ca(2+) , whereas HDP induced a massive and prolonged elevation of intracellular Ca(2+) responsible for the transient production of intracellular ROS. In addition, the two PDTs also increased in situ transglutaminase 2 (TG2) activity, with a higher stimulation by HDP, and this increase in activity was prevented by Trolox, BAPTA-AM, and TG2-siRNA. LDP-induced apoptotic cell death was strongly inhibited by Trolox and TG2-siRNA and moderately suppressed by BAPTA-AM. However, HDP-mediated necrotic cell death was partially inhibited by BAPTA-AM but not by TG2-siRNA. Thus, these results demonstrate that LDP and HDP induced apoptotic and necrotic cell death by differential signaling mechanisms involving intracellular Ca(2+) , ROS, and TG2.     

P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase-1, -3 and -8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP-induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP-induced cell death.