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PubMed1996

Calcium-related damage in ischemia.

The objective of this hypothesis article is to review evidence supporting a role for calcium in mediating ischemic brain damage, and to present data which puts mitochondrial dysfunction in the center of interest. The assumptions/postulates put forward, relating to global/forebrain and to focal ischemia, are as follows. (1) In brief ischemia of the global/forebrain type neuronal necrosis, particularly in the CA1 sector of the hippocampus, is conspicuously delayed. It is postulated that the initial events during ischemia, and in the immediate recirculation period, lead to a perturbation of cell calcium homeostasis, with a gradual postischemic rise in the free cytosolic calcium concentration (Ca2+i). When the latter reaches a certain limiting value mitochondria start accumulating calcium. It is hypothesized that intramitochondrial calcium accumulation triggers a permeability transition of the inner mitochondrial membrane (MPT), leading to production of reactive oxygen species, release of calcium, and an increase in the cytosol calcium concentration of a potentially adverse nature. (2) If ischemia of this "cardiac arrest" type is prolonged, or complicated by preischemic hyperglycemia, neuronal necrosis is enhanced and pan-necrotic lesions appear. Such insults are known to cause rapidly developing mitochondrial failure, but the involvement of calcium has not yet been demonstrated. (3) In focal ischemia, core tissues probably suffer a metabolic insult similar to that affecting brain tissues in global/forebrain ischemia. Thus, calcium influx and calcium overload of mitochondria are predictable, but available data only demonstrate rapidly developing, secondary energy failure, mitochondrial dysfunction, and enhanced influx of 45Ca. Thus, although secondary mitochondrial failure has been proved, a causative link between calcium influx and bioenergetic failure remains to be proved. Perifocal, penumbral tissues are exposed to spontaneously occurring depolarisation waves, leading to cellular efflux of K+ and influx of Ca2+. The latter may lead to gradual mitochondrial calcium overload triggering a MPT, and cell death. Although conclusive evidence has not yet been presented available results suggest a link between calcium influx, mitochondrial overload, and cell death.

Animals↗
PubMed2002

Stress-induced mitochondrial depolarization and oxidative damage in PSP cybrids.

Increased oxidative damage and mitochondrial dysfunction have been suggested to play critical roles in the pathogenesis of progressive supranuclear palsy (PSP) yet the specific intracellular defects which cause and can result from these oxidative and bioenergetic defects remain unclear. To extend our previous PSP cybrid findings, we measured electron transport chain (ETC) activities in cell lines expressing mitochondrial genes from patients with PSP. Further, we measured changes in mitochondrial membrane potential as well as lipid peroxidation in PSP and control cybrids in response to mitochondrial toxins. We observed significant decreases in complex I+III activity in PSP cybrids as well as significant increases in markers of lipid oxidative damage as compared to control cybrids. These results coupled with previous reports from this and other laboratories strongly suggest contributory roles of mitochondrial dysfunction and oxidative damage in PSP, possibly due to genetic abnormalities and/or damage of mitochondrial DNA.

Brain↗
PubMed2005

Regulation of inward rectifier K+ channels by shift of intracellular pH dependence.

The mechanistic link between mitochondrial metabolism and inward rectifier K+ channel activity was investigated by studying the effects of a mitochondrial inhibitor, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on inward rectifiers of the Kir2 subfamily expressed in Xenopus oocytes, using two-electrode voltage-clamp, patch-clamp, and intracellular pH recording. FCCP inhibited Kir2.2 and Kir2.3 currents and decreased intracellular pH, but the pH change was too small to account for the inhibitory effect by itself. However, pre-incubation of oocytes with imidazole prevented both the pH decrease and the inhibition of Kir2.2 and Kir2.3 currents by FCCP. The pH dependence of Kir2.2 was shifted to higher pH in membrane patches from FCCP-treated oocytes compared to control oocytes. Therefore, the inhibition of Kir2.2 by FCCP may involve a combination of intracellular acidification and a shift in the intracellular pH dependence of these channels. To investigate the sensitivity of heteromeric channels to FCCP, we studied its effect on currents expressed by heteromeric tandem dimer constructs. While Kir2.1 homomeric channels were insensitive to FCCP, both Kir2.1-Kir2.2 and Kir2.1-Kir2.3 heterotetrameric channels were inhibited. These data support the notion that mitochondrial dysfunction causes inhibition of heteromeric inward rectifier K+ channels. The reduction of inward rectifier K+ channel activity observed in heart failure and ischemia may result from the mitochondrial dysfunction that occurs in these conditions.

Animals↗
PubMed1999

Purine catabolism: links to mitochondrial respiration and antioxidant defenses?

Type I diabetes in rodents is associated with a spectrum of liver mitochondrial abnormalities ranging from evidence of oxidative stress and altered antioxidant defenses to frank defects in respiration rates and respiratory control ratios. To better address the myriad changes in redox metabolism in these mitochondria, we have applied new chromatographic techniques that enable simultaneous analysis of multiple components of pathways of interest (e.g., purine catabolites and oxidation by-products). We report here a portion of these results, which, in conjunction with other reported data, suggest that purine catabolism may contribute to mitochondrial antioxidant defenses by producing the antioxidant urate. In liver mitochondria from diabetic rats, increases in uric acid (threefold) and its direct precursor xanthine (sixfold) were observed in moderate diabetes, but levels fell essentially to normal in severe disease. Failure to maintain elevated xanthine and uric acid occurred contemporaneously with progressive mitochondrial dysfunction. Regression analysis revealed altered precursor-product relationships between xanthine, its precursors, and uric acid. An independent set of studies in isolated rat liver mitochondria showed that mitochondrial respiration was associated with essentially uniform decreases (approximately 30%) in all purine catabolites measured (urate, xanthine, hypoxanthine, guanine, guanosine, and xanthosine). That result suggests the potential for steady production of urate. Taken together, the two studies raise the possibility that purine catabolism may be a previously unappreciated component of the homeostatic response of mitochondria to oxidant stress and may play a critical role in slowing progressive mitochondrial dysfunction in certain disease states.

Animals↗
PubMed2000

Cytoplasm mediates both development and oxidation-induced apoptotic cell death in mouse zygotes.

Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175 microM H(2)O(2) that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P<0.01) in zygotes reconstituted from untreated pronuclei and H(2)O(2)-stressed cytoplasts than those of nuclear transfer controls. Instead, the arrested, reconstituted zygotes displayed TUNEL staining at a similar rate to that of H(2)O(2)-treated controls, suggesting that apoptotic potential could be transferred cytoplasmically. On the other hand, rates of cleavage and development to morula and blastocyst of the reconstituted zygotes, derived from stressed pronuclei and untreated cytoplasm, were significantly increased (P<0.05), compared to those of H(2)O(2)-treated, control zygotes, indicating that healthy cytoplasm could partly rescue pronuclei from oxidative stress. Although oxidation stressed both nuclei and cytoplasm, cytoplasm was more sensitive than nuclei to oxidative stress. It is suggested that cytoplasm, most likely mitochondria, plays a central role in mediating both development and apoptotic cell death induced by oxidative stress in mouse zygotes.

Animals↗
PubMed2004

Oocyte mitochondria: strategies to improve embryogenesis.

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.

Animals↗
PubMed2002

Thioltransferase (glutaredoxin) mediates recovery of motor neurons from excitotoxic mitochondrial injury.

Mitochondrial dysfunction involving electron transport components is implicated in the pathogenesis of several neurodegenerative disorders and is a critical event in excitotoxicity. Excitatory amino acid L-beta-N-oxalylamino-L-alanine (L-BOAA), causes progressive corticospinal neurodegeneration in humans. In mice, L-BOAA triggers glutathione loss and protein thiol oxidation that disrupts mitochondrial complex I selectively in motor cortex and lumbosacral cord, the regions affected in humans. We examined the factors regulating postinjury recovery of complex I in CNS regions after a single dose of L-BOAA. The expression of thioltransferase (glutaredoxin), a protein disulfide oxidoreductase regulated through AP1 transcription factor was upregulated within 30 min of L-BOAA administration, providing the first evidence for functional regulation of thioltransferase during restoration of mitochondrial function. Regeneration of complex I activity in motor cortex was concurrent with increase in thioltransferase protein and activity, 1 hr after the excitotoxic insult. Pretreatment with alpha-lipoic acid, a thiol delivery agent that protects motor neurons from L-BOAA-mediated toxicity prevented the upregulation of thioltransferase and AP1 activation, presumably by maintaining thiol homeostasis. Downregulation of thioltransferase using antisense oligonucleotides prevented the recovery of complex I in motor cortex and exacerbated the mitochondrial dysfunction in lumbosacral cord, providing support for the critical role for thioltransferase in maintenance of mitochondrial function in the CNS.

Animals↗
PubMed1999

Effect of N-acetylcysteine on mitochondrial function following traumatic brain injury in rats.

Efficacy of N-acetylcysteine (NAC) in traumatic brain injury (TBI)-induced mitochondrial dysfunction was evaluated following controlled cortical impact injury in rats. Respiratory function and calcium transport of rat forebrain mitochondria from injured and uninjured hemispheres were examined. NAC significantly restored mitochondrial electron transfer, energy coupling capacity, calcium uptake activity and reduced calcium content absorbed to brain mitochondrial membranes when examined 12 h post-TBI if NAC was administered i.p. 5 min before injury or 30 min or 1 h postinjury. Glutathione (reduced form, GSH) levels in brain tissues were decreased at all time points examined over a 14-day observation period, while mitochondrial GSH levels significantly decreased only at 3 days and 14 days following TBI. NAC treatment given within 1 h greatly restored brain GSH levels from 1 h to 14 days and mitochondrial GSH levels from 12 h to 14 days post-TBI. NAC did not show protective effects when given 2 h postinjury. Our data indicate that NAC administered postinjury at an early stage can effectively restore TBI-induced mitochondrial dysfunction and the protective effect of NAC may be related to its restoration of GSH levels in the brain.

Acetylcysteine↗
PubMed1982

Encephalopathy of Reye's syndrome: a review of pathogenetic hypotheses.

The pathogenesis of Reye's syndrome encephalopathy was analyzed in terms of uniform criteria designed to clarify and assist evaluation of the leading hypotheses. Three of these hypotheses derive from known metabolic sequelae of hepatic mitochondrial dysfunction and the severe systemic catabolism of protein, fats, and carbohydrates that characterize the syndrome biochemically: hyperammonemia, hyperfattyacidemia, and hyperlactatemia. In addition, there is a fourth hypothesis of generalized mitochondrial insult affecting brain, muscle, and other organs as well as liver. The weight of evidence favors hyperammononemia as a sufficient factor while recognizing important interrelationships with the other observed biochemical derangements. How the catabolism and hepatic mitochondrial dysfunction are produced by the triggering viral infection remains unknown. Therapeutic efforts have thus far not succeeded in definitive metabolic intervention. Such reversal of the clinical syndrome would lead to confirmation of the necessary pathogenetic factors; this type of intervention remains the chief goal of metabolic research in Reye's syndrome.

Acidosis↗
PubMed2006

Autophagy as a protective response to Bnip3-mediated apoptotic signaling in the heart.

Bnip3 is a member of the 'BH3-only' Bcl-2 subfamily which has been implicated in apoptotic,(1) necrotic(2) and autophagic cell death.(3,4) We recently reported that Bnip3 is a key mediator of mitochondrial dysfunction and cell death in the ex vivo heart following ischemia/reperfusion (I/R).(5) Moreover, we found that Bnip3 was involved in upregulation of autophagy in I/R and that Bnip3-mediated mitochondrial dysfunction correlated with upregulation of autophagy. Using a model of simulated I/R and overexpression of Bnip3 in HL-1 cardiac myocytes, we determined that Bnip3-mediated upregulation of autophagic activity constituted a protective response against Bnip3 death signaling. Here we present additional evidence that enhanced autophagic activity functions as a cytoprotective pathway to oppose ischemia/reperfusion-related apoptosis.

Animals↗
PubMed2000

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis.

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.

Apoptosis↗
PubMed2002

Pk11195, a mitochondrial benzodiazepine receptor antagonist, reduces apoptosis threshold in Bcl-X(L) and Mcl-1 expressing human cholangiocarcinoma cells.

BACKGROUND: Cholangiocarcinoma cells express high levels of the antiapoptotic proteins Bcl-X(L) and Mcl-1 and are markedly chemo- and radioresistant. Mitochondria have emerged as central players in apoptosis. Antiapoptotic members of the Bcl-2 protein family localise to the outer mitochondrial membrane and regulate mitochondrial release of apoptogenic proteins. Mitochondrial benzodiazepine receptor (mBzR) ligands have been shown to reverse Bcl-2 action and facilitate apoptosis. AIM: We evaluated the ability of the mBzR antagonist Pk11195 to overcome preapoptotic mitochondrial dysfunction in Egi-1 and Tfk-1, two human cholangiocarcinoma cell lines expressing high levels of Bcl-X(L) and Mcl-1. MATERIALS AND METHODS: Cells growing in culture were used to perform in vitro experiments over 48-96 hours following treatment. The cytotoxic agents used were 5 fluorouracil 10 microM and etoposide (Vp16) 10 microM, together with ultraviolet and 0.5-1 Gy x ray irradiation with or without 75 microM Pk11195. Apoptosis and mitochondrial dysfunction were measured at single cell resolution by flow cytometry using the mitochondrial fluorochrome DiOC6(3). Severe combined immunodeficient non-obese diabetic (SCID-NOD) mice with subcutaneous xenografts using the Egi-1 and Tfk-1 cell lines were treated with etoposide with or without addition of Pk11195 over a 72 hour period during which time the xenograft growth patterns were monitored. RESULTS: In vitro, the effect of Pk11195 on induction of apoptosis in cholangiocarcinoma cells following stimulation by chemotherapy or radiotherapy was found to be both time and dose dependent, with Pk11195 increasing rates of apoptosis by 50-95%. Intraperitoneal administration of Pk11195 in combination with Vp16 was found to increase the growth inhibiting effects of Vp16 on xenografts during the treatment phase. PK11195 75 microM on its own had no intrinsic cytotoxic efficacy. CONCLUSION: This is the first study to demonstrate that functional antagonism of coexpressed Bcl-X(L) and Mcl-1 proteins using the mBzR antagonist Pk11195 can facilitate apoptosis in cholangiocarcinoma following chemotherapy and radiotherapy.

Antineoplastic Agents↗
PubMed2004

Mechanisms of non-alcoholic steatohepatitis.

In 1980, the term non-alcoholic steatohepatitis was coined to describe a new syndrome occurring in patients who usually were obese (often diabetic) females who had a liver biopsy picture consistent with alcoholic hepatitis, but who denied alcohol use. The causes of this syndrome were unknown, and there was no defined therapy. More than two decades later, this clinical syndrome is only somewhat better understood, and still there is no Food and Drug Administration-approved or even generally accepted drug therapy. Patients with primary non-alcoholic steatohepatitis typically have the insulin resistance syndrome (synonymous with the metabolic syndrome, syndrome X, and so forth), which is characterized by obesity, diabetes, hyperlipidemia, hypertension, and, in some instances, other metabolic abnormalities such as polycystic ovary disease. Secondary non-alcoholic steatohepatitis may be caused by drugs such as tamoxifen, certain industrial toxins, rapid weight loss, and so forth. The cause of non-alcoholic steatohepatitis remains elusive, but most investigators agree that a baseline of steatosis requires a second hit capable of inducing inflammation, fibrosis, or necrosis for non-alcoholic steatohepatitis to develop. Our research group has focused its efforts on the interactions of nutritional abnormalities, cytokines, oxidative stress with lipid peroxidation, and mitochondrial dysfunction in the induction of steatohepatitis, both alcoholic and non-alcoholic in origin. Research findings from other laboratories also support the role of increased cytokine activity, oxidative stress, and mitochondrial dysfunction in the pathogenesis of non-alcoholic steatohepatitis. The objectives of this article are to review the (1) definition and clinical features of non-alcoholic steatohepatitis, (2) potential mechanisms of non-alcoholic steatohepatitis, and (3) potential therapeutic interventions in non-alcoholic steatohepatitis.

Alcoholism↗
PubMed1999

Local mitochondrial function following traumatic brain injury in rats.

The effect of lateral fluid percussion injury on mitochondrial function in the rat brain was investigated by quantitative imaging of changes in the regional activity of succinate dehydrogenase (SDH), a mitochondrial enzyme of the tricarboxylic acid cycle for adenosine triphosphate production. Regional SDH was measured in the frontal, parietal, temporal, and occipital cortices, CA1 and CA2-3 of the hippocampus, thalamus, corpus callosum, caudate/putamen, and cerebellum 1 hour and 72 hours after low, medium, and high pressure injury. No regional difference between the hemispheres in the activity of SDH was observed in the sham group. The hippocampus showed high SDH activity. The CA2-3 regions showed the highest activity among the regions examined. The corpus callosum, which is white matter, showed the lowest. One hour after low pressure fluid percussion injury, only the frontal lobe showed significantly lower SDH activity than the sham control in the ipsilateral hemisphere, whereas after 72 hours SDH activity was significantly lower in the frontal, parietal, and temporal lobes. SDH activity was significantly lower in the frontal, parietal, and temporal lobes in the medium and high pressure injury groups than in the sham control 1 hour after injury, and SDH activity in the CA1 and CA2-3 of the hippocampus was significantly decreased 72 hours after injury. No decrease in SDH activity was observed in any region of the contralateral hemisphere either 1 hour or 72 hours after injury. Mitochondrial dysfunction of the ipsilateral cortex and hippocampus following fluid percussion injury is correlated with the severity of injury and advances with time after injury. The results suggest that progression of mitochondrial dysfunction is associated with secondary bioenergetic deterioration.

Animals↗
PubMed2002

Animal models of anti-HIV drugs exposure during pregnancy: effects on neurobehavioral development.

In the last 10 years, zidovudine (AZT) has become the main prophylactic therapy against vertical HIV-1 transmission. AIDS Clinical Trials Group (ACTG) 076 have demonstrated that the administration of AZT to HIV-infected women during their third trimester of pregnancy, trough labor and given orally to babies for 6 weeks, reduced by two-thirds the rate of vertical infection. Although the rapid diffusion of this regimen into clinical practice together with the implementation of HIV counseling and testing practices have dramatically reduced the vertical transmission rate in the US and Western Europe, there is a growing concern on the adverse effects of antiretroviral therapy on the fetus and the newborn. In fact, even though shorter regimen therapies that are less complex and expensive to implement in poor countries have been demonstrated as effective as ACTG 076 regimen, the distribution of the risk of vertical transmission in the developing countries is still very high. Consequently, a large number of unborns will be a candidate to developmental exposure to antiretroviral agents. To date, data on the transplacental mutagenicity, carcinogenicity and mitochondrial dysfunction induced by developmental exposure to AZT have been reported in several animal models. Furthermore, one study reported severe yet few human cases of cardiomyopathy and neurological disease likely associated with mitochondrial dysfunction in uninfected infants of seropositive mothers perinatally exposed to AZT. For all of these reasons, many investigations have been focusing on the assessment of the potential adverse effects of nucleoside reverse transcriptase (RT) inhibitors (NRTI) administration during development. A survey of the main results derived from clinical and animal studies is reported here, focusing on those neurobehavioral studies that have been looking for specific and/or aspecific changes in the nervous system induced by NRTI exposure in utero.

Animals↗
PubMed2004

Mitochondrial signaling: the retrograde response.

Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus that influences many cellular and organismal activities under both normal and pathophysiological conditions. In yeast it is used as a sensor of mitochondrial dysfunction that initiates readjustments of carbohydrate and nitrogen metabolism. In both yeast and animal cells, retrograde signaling is linked to TOR signaling, but the precise connections are unclear. In mammalian cells, mitochondrial dysfunction sets off signaling cascades through altered Ca(2+) dynamics, which activate factors such as NFkappaB, NFAT, and ATF. Retrograde signaling also induces invasive behavior in otherwise nontumorigenic cells implying a role in tumor progression.

Aging↗
PubMed2002

Calpain and mitochondria in ischemia/reperfusion injury.

Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4-23 of Bcl-x(L) conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca2+-induced deltaPsi(m) loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.

Animals↗
PubMed2004

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells.

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.

Apoptosis↗