Mitochondrial reactive oxygen species affect sensitivity to curcumin-induced apoptosis

Curcumin exhibits anticancer activity in vivo and triggers tumor cell apoptosis in vivo and in vitro. Several in vitro studies suggest that curcumin-induced apoptosis is associated with reactive oxygen species (ROS) production and/or oxidative stress in transformed cells. This study compared and contrasted the effects of curcumin on human skin cancer cells and their respiration-deficient (rho0) clones to characterize the prospective oxidative stress signaling responsible for initiating apoptosis. Curcumin promoted a dose-and time-dependent G2/M cell cycle arrest and/or apoptosis in COLO 16 cells. Apoptosis induction in COLO 16 cells was associated with DNA fragmentation, cell shrinkage, the externalization of cell membrane phosphatidylserine, and mitochondrial disruption, which were preceded by an increase in intracellular ROS production. Pharmacologically lowering the mitochondrial bioenergetic capacity, as well as the constitutive ROS levels, in COLO 16 cells suppressed the cytotoxic effects of curcumin. Correspondingly, the rho0 counterparts of COLO 16 cells were markedly resistant to ROS production, mitochondrial disruption, and DNA fragmentation following curcumin exposure. These observations implied that the diminution of mitochondrial ROS production protected cells against the cytotoxic effects of curcumin, and support the notion that mitochondrial respiration and redox tone are pivotal determinants in apoptosis signaling by curcumin in human skin cancer cells.     

The novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid induces apoptosis of human myeloid leukemia cells by a caspase-8-dependent mechanism.

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces growth inhibition and differentiation of human myeloid leukemia cells. The present studies demonstrate that CDDO treatment results in apoptosis of U-937 and HL-60 myeloid leukemia cells. Similar to 1-beta-D-arabinofuranosylcytosine (ara-C), another agent that inhibits growth and induces apoptosis of these cells, CDDO induced the release of mitochondrial cytochrome c and activation of caspase-3. Overexpression of Bcl-X(L) blocked cytochrome c release, caspase-3 activation, and apoptosis in ara-C-treated cells. By contrast, CDDO-induced release of cytochrome c, and activation of caspase-3 were diminished only in part by Bcl-X(L). In concert with these findings, we demonstrate that CDDO, but not ara-C, activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism. The results also show that CDDO-induced cytochrome c release is mediated by caspase-8-dependent cleavage of Bid. These findings demonstrate that CDDO induces apoptosis of myeloid leukemia cells and that this novel agent activates an apoptotic signaling cascade distinct from that induced by the cytotoxic agent ara-C.     

Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain

The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.     

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.     

The role of Ca ions in restoration of the structure of interphase and mitotic chromosomes in PK living cells after hypotonic stress.

The dynamics of mitotic chromosome and interphase chromatin recondensation in living PK cells during their adaptation to hypotonic medium was studied. The recondensation process was found to be slowed down by the modification of plasma membrane with low concentrations of glutaraldehyde, while osmotic reactions of glutaraldehyde-treated cells remain unchanged. The effect of glutaraldehyde can be rapidly reversed by the addition of Ca(2+)-ionophore A23187. Intracellular Ca(2+)measurements show that the adaptation to hypotonic shock is accompanied by restoration of free Ca concentration, whereas the delay of chromatin condensation in glutaraldehyde-treated cells is paralleled by the decrease of Ca level. The mechanisms implying the role of low concentration of Ca(2+)in chromatin compactization in vivo are discussed.     

Ca2+ dysregulation induces mitochondrial depolarization and apoptosis: role of Na+/Ca2+ exchanger and AKT

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.     

Apoptotic cell death induced by hydrogen peroxide in NT2 parental and mitochondrial DNA depleted cells

Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death.     

Induction of apoptosis in human colon carcinoma COLO 205 cells by the recombinant α subunit of C-phycocyanin

The α-subunit of C-phycocyanin (CpcA) was expressed in Escherichia coli and purified. The recombinant CpcA inhibited the growth of human colon carcinoma COLO 205 cells. Typical apoptotic morphological characteristics, such as chromatin condensation and nuclear fragmentation, were observed in CpcA-treated COLO 205 cells by fluorescence microscopy and transmission electron microscopy. Moreover, the apoptotic process was associated with the Bax/Bcl-2 ratio up-regulation, mitochondrial membrane depolarization, cytochrome c release, and caspase-9 activation. These findings indicate that CpcA induced the death of COLO 205 cells through the intrinsic apoptotic pathway.     

Mitochondrial uncoupling protein-4 regulates calcium homeostasis and sensitivity to store depletion-induced apoptosis in neural cells

An increase in the cytoplasmic-free Ca(2+) concentration mediates cellular responses to environmental signals that influence a range of processes, including gene expression, motility, secretion of hormones and neurotransmitters, changes in energy metabolism, and apoptosis. Mitochondria play important roles in cellular Ca(2+) homeostasis and signaling, but the roles of specific mitochondrial proteins in these processes are unknown. Uncoupling proteins (UCPs) are a family of proteins located in the inner mitochondrial membrane that can dissociate oxidative phosphorylation from respiration, thereby promoting heat production and decreasing oxyradical production. Here we show that UCP4, a neuronal UCP, influences store-operated Ca(2+) entry, a process in which depletion of endoplasmic reticulum Ca(2+) stores triggers Ca(2+) influx through plasma membrane "store-operated" channels. PC12 neural cells expressing human UCP4 exhibit reduced Ca(2+) entry in response to thapsigargin-induced endoplasmic reticulum Ca(2+) store depletion. The elevations of cytoplasmic and intramitochondrial Ca(2+) concentrations and mitochondrial oxidative stress induced by thapsigargin were attenuated in cells expressing UCP4. The stabilization of Ca(2+) homeostasis and preservation of mitochondrial function by UCP4 was correlated with reduced mitochondrial reactive oxygen species generation, oxidative stress, and Gadd153 up-regulation and increased resistance of the cells to death. Reduced Ca(2+)-dependent cytosolic phospholipase A2 activation and oxidative metabolism of arachidonic acid also contributed to the stabilization of mitochondrial function in cells expressing human UCP4. These findings demonstrate that UCP4 can regulate cellular Ca(2+) homeostasis, suggesting that UCPs may play roles in modulating Ca(2+) signaling in physiological and pathological conditions.     

Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics

Deguelin exhibits chemopreventive properties in animal carcinogenesis models. The mechanism underpinning the chemopreventive effects of deguelin has not been fully elucidated. However, it has been suggested that this agent reduces ornithine decarboxylase activity, and perhaps the activity of other signaling intermediates associated with tumorigenesis, by inhibiting mitochondrial bioenergetics. We sought to determine if deguelin could trigger apoptosis by inhibiting mitochondrial bioenergetics. Therefore, we compared and contrasted the effects of deguelin on cells from two human cutaneous squamous cell carcinoma cell lines (parental cells) and their respiration-deficient clones lacking mitochondrial DNA (rho0). While deguelin promoted marked apoptosis in the parental cells in a dose- and time-dependent manner, it failed to do so in the rho0 clones. Furthermore, short-term exposure to deguelin diminished oxygen consumption by the parental cells and promoted mitochondrial permeability transition as evidenced by the dissipation of mitochondrial inner transmembrane potential, reactive oxygen species production, cardiolipin peroxidation, caspase activation, and mitochondrial swelling. Mitochondrial permeability transition was not observed in the rho0 clones exposed to deguelin. These results demonstrate that deguelin induces apoptosis in skin cancer cells by inhibiting mitochondrial bioenergetics and provide a novel mechanism for the putative anticancer activity of this agent.     

An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death

Apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity anchored to the mitochondrial inner membrane, is known to be involved in complex I maintenance. During apoptosis, AIF can be released from mitochondria and translocate to the nucleus, where it participates in chromatin condensation and large-scale DNA fragmentation. The mechanism of AIF release is not fully understood. Here, we show that a prolonged ( approximately 10 min) increase in intracellular Ca(2+) level is a prerequisite step for AIF processing and release during cell death. In contrast, a transient ATP-induced Ca(2+) increase, followed by rapid normalization of the Ca(2+) level, was not sufficient to trigger the proteolysis of AIF. Hence, import of extracellular Ca(2+) into staurosporine-treated cells caused the activation of a calpain, located in the intermembrane space of mitochondria. The activated calpain, in turn, cleaved membrane-bound AIF, and the soluble fragment was released from the mitochondria upon outer membrane permeabilization through Bax/Bak-mediated pores or by the induction of Ca(2+)-dependent mitochondrial permeability transition. Inhibition of calpain, or chelation of Ca(2+), but not the suppression of caspase activity, prevented processing and release of AIF. Combined, these results provide novel insights into the mechanism of AIF release during cell death.     

Extracellular superoxide released from mitochondria mediates mast cell death by advanced glycation end products

Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcepsilonRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (> or =0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcepsilonRI stimulation increased the cytosolic Ca(2+) levels to a similar extent, whereas GA, but not FcepsilonRI stimulation, caused mitochondrial Ca(2+) overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcepsilonRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca(2+) homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca(2+) overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions.     

Influence of a mitochondrial genetic defect on capacitative calcium entry and mitochondrial organization in the osteosarcoma cells

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.     

Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore.

We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade.     

Overexpressing GRP78 influences Ca2+ handling and function of mitochondria in astrocytes after ischemia-like stress

Ca(2+) transfer from endoplasmic reticulum (ER) to mitochondria at contact sites between the organelles can induce mitochondrial dysfunction and programmed cell death after stress. The ER-localized chaperone glucose-regulated protein 78kDa (GRP78/BiP) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects astrocytes against ischemic injury, reduces net flux of Ca(2+) from ER to mitochondria, increases Ca(2+) uptake capacity in isolated mitochondria, reduces free radical production, and preserves respiratory activity and mitochondrial membrane potential after stress. We conclude that GRP78 influences ER-mitochondrial Ca(2+) crosstalk to maintain mitochondrial function and protect astrocytes from ischemic injury.     

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis.

Apoptosis is a physiological counterbalance to mitosis and plays important roles in tissue development and homeostasis. Cytosolic Ca(2+) has been implicated as a proapoptotic second messenger involved in both triggering apoptosis and regulating cell death-specific enzymes. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and endoplasmic reticulum (ER) membranes; however, the molecular mechanism of Bax translocation and its relationship to Ca(2+) is largely unknown. Here we provide functional evidence for a synergistic interaction between the movements of intracellular Ca(2+) and cytosolic Bax in the induction of apoptosis. Overexpression of Bax in cultured cells causes a loss of ER Ca(2+) content. Depletion of ER Ca(2+) through activation of the ryanodine receptor enhances the participation of Bax into the mitochondrial membrane. Neither Bax translocation nor Bax-induced apoptosis is affected by buffering of cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting that depletion of ER Ca(2+) rather than elevation of cytosolic Ca(2+) is the signal for cell apoptosis. This dynamic interplay of Ca(2+) and Bax movements may serve as an amplifying factor in the initial signaling steps of apoptosis.     

Developmentally regulated ceramide synthase 6 increases mitochondrial Ca2+ loading capacity and promotes apoptosis

Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1-6/CerS1-6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C(16:0)-ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C(16:0)-ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca(2+)-loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca(2+) accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca(2+) uptake or decreasing Ca(2+)-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca(2+) homeostasis and calpain, which appears to be important in OL apoptosis during brain development.     

Ruthenium red-mediated suppression of Bcl-2 loss and Ca(2+) release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species

The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca(2+) was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca(2+) from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca(2+) levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.