Tomoko Kanno

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca2+ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca2+ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca2+ increased the generation of H2O2 by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca2+ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca2+-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca2+ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.

Mitochondrial Dysfunction in the Senescence Accelerated Mouse (SAM)

Oxidative damage to DNA, proteins, and lipids in mitochondria caused by free radicals may be one factor in aging. Oxidative phosphorylation was estimated in liver mitochondria from senescence accelerated mice (SAMP8) and a senescence resistant substrain (SAMR1). The respiratory control ratio decreased in liver mitochondria of SAMP8 during aging, and it was estimated that at 18 months of age this respiratory control value suggested that it might be insufficient to provide ATP synthesis necessary for normal cell metabolism. In addition, the ADP/O, an index of efficiency of ATP synthesis, was depressed at 18 months of age. Dinitrophenol-dependent uncoupled respiration in liver mitochondria of SAMP8 mice was markedly decreased with aging, suggesting a dysfunctional energy transfer mechanism in mitochondria of aged SAMP8 mice. Active uptake of calcium in liver mitochondria was markedly dysfunctional in SAMP8 mice with aging, and uncoupling of respiration was induced more easily in aged mitochondria. Milder effects on these functional parameters were observed in SAMR1 mice. A similar dysfunction was also observed in heart mitochondria of SAMP8 mice at 12 months of age. The amount of Bcl-x in liver mitochondria was slightly decreased in SAMP8. We suggest that these changes in mitochondrial function may be related to the shorter life span of the senescence accelerated mouse.

Activation of caspase‐3‐like protease by digitonin‐treated lysosomes

Apoptosis, a naturally occurring programmed cell death or cell ‘suicide’, has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria‐mediated pathway has been postulated to be one of the activation mechanism of caspase‐3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin‐treated lysosomes strongly activates Ac‐DEVD‐CHO inhibitable caspase‐3‐like protease. Activation of caspase‐3‐like protease by digitonin‐treated lysosomal fractions was specifically suppressed by leupeptin and E‐64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase‐3‐like protease.

Mechanism of α-tocopheryl succinate-induced apoptosis of promyelocytic leukemia cells

Selective induction of apoptosis in tumor cells is important for treating patients with cancer. Because oxidative stress plays an important role in the process of apoptosis, we studied the effect of α-tocopheryl succinate (VES) on the fate of cultured human promyelocytic leukemia cells (HL-60). The presence of fairly low concentrations of VES inhibited the growth and DNA synthesis of HL-60 cells, and also induced their apoptosis via a mechanism that was inhibited by z-VAD-fluoromethylketone (z-VAD-fmk), an inhibitor of pan-caspases. VES activated various types of caspases, including caspase-3, 6, 8, and 9, but not caspase-1. VES triggered the reaction leading to the cleavage of Bid, a member of the death agonist Bcl-2 family, and released cytochrome c (Cyt.c) from the mitochondria into the cytosol by a z-VAD-fmk-inhibitable mechanism. VES transiently increased the intracellular calcium level [Ca2+]i and stimulated the release of Cyt.c in the presence of inorganic phosphate (Pi). However, high concentrations of VES (∼100 μM) hardly induced swelling of isolated mitochondria but depolarized the mitochondrial membrane potential by a cyclosporin A (CsA)-insensitive mechanism. These results indicate that VES-induced apoptosis of HL-60 cells might be caused by activation of the caspase cascade coupled with modulation of mitochondrial membrane function.

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.

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

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

Inhibition of stimulus-specific neutrophil superoxide generation by alpha-tocopherol.

Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of alpha-tocopherol, inhibited the superoxide (O2-.) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myrisate 13-acetate (PMA). ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1microM. This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 microM). The alpha-tocopherol sensitive O2-. generation was also observed in neutrophils induced by dioctanoylglycerol (diC8) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS). The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC. The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O2-. generation. These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O2-. generation by neutrophils.

INHIBITION OF NEUTROPHIL-SUPEROXIDE GENERATION BY ALPHA -TOCOPHEROL AND COENZYME Q

Effects of various derivatives of alpha-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2.-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8 inhibited O2.- generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only with alpha-tocopherol, but also with beta-, gamma-, delta-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By contrast, no inhibition was observed with 2-carboxy-2, 5, 7, 8-tetramethyl-6-chromanol (CTMC, trolox) or 2, 2, 5, 7, 8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2.- generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16, 24 and 32 carbon isoprenoid chains corresponding to CoQ4, 6, and 8 inhibited O2.- generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2.- generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50 for O2.- generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2.- generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

Tributyltin interacts with mitochondria and induces cytochrome c release.

Although triorganotins are potent inducers of apoptosis in various cell types, the critical targets of these compounds and the mechanisms by which they lead to cell death remain to be elucidated. There are two major pathways by which apoptotic cell death occurs: one is triggered by a cytokine mediator and the other is by a mitochondrion-dependent mechanism. To elucidate the mechanism of triorganotin-induced apoptosis, we studied the effect of tributyltin on mitochondrial function. We found that moderately low doses of tributyltin decrease mitochondrial membrane potential and induce cytochrome c release by a mechanism inhibited by cyclosporine A and bongkrekic acid. Tributyltin-induced cytochrome c release is also prevented by dithiols such as dithiothreitol and 2,3-dimercaptopropanol but not by monothiols such as GSH, N-acetyl-L-cysteine, L-cysteine and 2-mercaptoethanol. Further studies with phenylarsine oxide agarose revealed that tributyltin interacts with the adenine nucleotide translocator, a functional constituent of the mitochondrial permeability transition pore, which is selectively inhibited by dithiothreitol. These results suggest that, at low doses, tributyltin interacts selectively with critical thiol residues in the adenine nucleotide translocator and opens the permeability transition pore, thereby decreasing membrane potential and releasing cytochrome c from mitochondria, a series of events consistent with established mechanistic models of apoptosis.

Suppression of T3- and fatty acid-induced membrane permeability transition by l-carnitine

Cytochrome c (Cyt. c) is known to be released from the mitochondria into the cytosol by means of the membrane permeability transition (MPT) mechanism, thereby activating caspase cascade activity, and inducing cell apoptosis. Recently we reported that L-carnitine suppressed palmitoyl-CoA-induced MPT as well as apoptosis in some cell types (Biochem. Pharmacol, in press). In the present study T(3) was found to induce MPT and Cyt. c release, while cyclosporin A (CsA), bovine serum albumin (BSA) and L-carnitine were found to inhibit this action in a concentration-dependent manner. Similarly, long chain fatty acid (LCFA) also induced MPT and Cyt. c release, which was then inhibited by CsA, BSA and L-carnitine. From these results the authors postulate that T(3)-induced MPT is in part regulated by fatty acid metabolism through a dynamic balance between LCFAs and L-carnitine.