Mechanism of apoptosis induced by apigenin in hepg2 human hepatoma cells: involvement of reactive oxygen species generated by NADPH oxidase

Although plant-derived flavonoids have been reported to have anti-cancer activities, the exact mechanism of these actions is not completely understood. In this study we investigated the role for reactive oxygen species (ROS) as a mediator of the apoptosis induced by apigenin, a widespread flavonoid in plant, in HepG2 human hepatoma cells. Apigenin reduced cell viability, and induced apoptotic cell death in a dose-dependent manner. In addition, it evoked a dose-related elevation of intracellular ROS level. Treatment with various inhibitors of the NADPH oxidase (diphenylene iodonium, apocynin, neopterine) significantly blunted both the generation of ROS and induction of apoptosis induced by apigenin. These results suggest that ROS generated through the activation of the NADPH oxidase may play an essential role in the apoptosis induced by apigenin in HepG2 cells. These results further suggest that apigenin may be valuable for the therapeutic management of human hepatomas.     

First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics

A decline in energy is common in aging, and the restoration of mitochondrial bioenergetics may offer a common approach for the treatment of numerous age-associated diseases. Cardiolipin is a unique phospholipid that is exclusively expressed on the inner mitochondrial membrane where it plays an important structural role in cristae formation and the organization of the respiratory complexes into supercomplexes for optimal oxidative phosphorylation. The interaction between cardiolipin and cytochrome c determines whether cytochrome c acts as an electron carrier or peroxidase. Cardiolipin peroxidation and depletion have been reported in a variety of pathological conditions associated with energy deficiency, and cardiolipin has been identified as a target for drug development. This review focuses on the discovery and development of the first cardiolipin-protective compound as a therapeutic agent. SS-31 is a member of the Szeto-Schiller (SS) peptides known to selectively target the inner mitochondrial membrane. SS-31 binds selectively to cardiolipin via electrostatic and hydrophobic interactions. By interacting with cardiolipin, SS-31 prevents cardiolipin from converting cytochrome c into a peroxidase while protecting its electron carrying function. As a result, SS-31 protects the structure of mitochondrial cristae and promotes oxidative phosphorylation. SS-31 represents a new class of compounds that can recharge the cellular powerhouse and restore bioenergetics. Extensive animal studies have shown that targeting such a fundamental mechanism can benefit highly complex diseases that share a common pathogenesis of bioenergetics failure. This review summarizes the mechanisms of action and therapeutic potential of SS-31 and provides an update of its clinical development programme.

LINKED ARTICLES

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Effects of polychlorinated biphenyls on the neutrophil NADPH oxidase system

Polychlorinated biphenyls (PCBs) are reported to induce the formation of reactive oxygen species (ROS) in human neutrophil granulocytes through the activation of the NADPH oxidase. The purpose of the present study is to elucidate the cellular mechanisms responsible for the activation of the NADPH oxidase after exposure to PCB. We have previously shown that PCB activates human neutrophil granulocytes through a calcium dependent activation of phospholipase D and/or phospholipase C, followed by the activation of protein kinase C. In the present study, pharmacological characterization of Aroclor (A) 1242-induced respiratory burst in human neutrophils was conducted by the use of enzymatic inhibitors. Pre-incubation with U0126, SB203580, SP600125, cyclosporin A and FK506 attenuated the A 1242-induced respiratory burst, measured by DCF-fluorescence, and luminol-amplified chemiluminescence. Our results show that the Erk1/2 kinases and p38MAPK/JNK are involved in ROS formation in neutrophils exposed to A 1242.     

In vitro effects of acetylcholinesterase reactivators on monoamine oxidase activity

Administration of acetylcholinesterase (AChE) reactivators (oximes) is usually used in order to counteract the poisoning effects of nerve agents. The possibility was suggested that oximes may show some therapeutic and/or adverse effects through their action in central nervous system. There are no sufficient data about interaction of oximes with monoaminergic neurotransmitter's systems in the brain. Oxime-type AChE reactivators pralidoxime, obidoxime, trimedoxime, methoxime and HI-6 were tested for their potential to affect the activity of monoamine oxidase of type A (MAO-A) and type B (MAO-B) in crude mitochondrial fraction of pig brains. The compounds were found to inhibit fully MAO-A with half maximal inhibitory concentration (IC₅₀) of 0.375 mmol/l (pralidoxime), 1.53 mmol/l (HI-6), 2.31 mmol/l (methoxime), 2.42 mmol/l (obidoxime) and 4.98 mmol/l (trimedoxime). Activity of MAO-B was fully inhibited by HI-6 and pralidoxime only with IC₅₀ 4.81 mmol/l and 11.01 mmol/l, respectively. Methoxime, obidoxime and trimedoxime displayed non-monotonic concentration dependent effect on MAO-B activity. Because oximes concentrations effective for MAO inhibition could not be achieved in vivo at the cerebral level, we suppose that oximes investigated do not interfere with brain MAO at therapeutically relevant concentrations.     

rBTI induces apoptosis in human solid tumor cell lines by loss in mitochondrial transmembrane potential and caspase activation

The molecular mechanisms and the possible effects of a recombinant buckwheat trypsin inhibitor (rBTI) on the induction of apoptosis in the human solid tumor cells (EC9706, HepG2 and HeLa) were investigated. An MTT assay showed that rBTI could specifically inhibit the growth of solid tumor cells in a dose- and time-dependent manner. Analysis by flow cytometry indicated that the apoptosis of several tumor cells increased after treatment with rBTI in range of 6.25–50 μg/ml. DNA electrophoresis analysis showed the ‘DNA ladder’, typical of apoptosis. rBTI-induced apoptosis was shown to involve Bax and Bak up-regulation, Bcl-2 and Bcl-xl down-regulation, release of cytochrome c from the mitochondria to the cytosol, activation of caspase-3 and -9 and disruption of the mitochondrial transmembrane potential (Δψ_m). The z-DEVD-fmk caspase-3 inhibitor significantly inhibited rBTI-induced apoptosis. We concluded that rBTI can induce the apoptosis in several types of human solid tumor cells and promotes apoptosis through the mitochondrial apoptotic pathway.     

Dehydromonocrotaline induces cyclosporine A-insensitive mitochondrial permeability transition/cytochrome c release

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in plants of the genus Crotalaria that causes cytotoxicity and genotoxicity in animals and humans. It is well established that the toxicity of MCT results from its hepatic bioactivation to dehydromonocrotaline (DHM), an alkylating agent, but the exact mechanism of action remains unknown. In a previous study, we demonstrated DHM's inhibition of mitochondrial NADH-dehydrogenase activity at micromolar concentrations, which is an effect associated with a significant reduction in ATP synthesis. As a follow-up study, we have evaluated the ability of DHM to induce mitochondrial permeability transition (MPT) and its associated processes in isolated rat liver mitochondria. In the presence of 10 μM Ca²⁺, DHM (50-250 μM) elicited MPT in a concentration-dependent, but cyclosporine A-independent manner, as assessed by mitochondrial swelling, which is associated with mitochondrial Ca²⁺ efflux and cytochrome c release. DHM (50-250 μM) did not cause hydrogen peroxide accumulation but did deplete endogenous glutathione and NAD(P)H, while oxidizing protein thiol groups. These results potentially indicate the involvement of mitochondria, via apoptosis, in the well-documented cytotoxicity of monocrotaline.     

Monochloramine produces reactive oxygen species in liver by converting xanthine dehydrogenase into xanthine oxidase

In the present study, we assessed the influence of monochloramine (NH₂Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH₂Cl (2.5-20 μM) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH₂Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH₂Cl (5 μM)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH₂Cl methionine (25 μM). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH₂Cl (5 μM)-induced XD/XO interconversion. These imply that NH₂Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate, it was found that NH₂Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH₂Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.     

Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Calpha in C2C12 myocytes

Protein kinase C (PKC) agonists including phorbol 12-myristate 13-acetate (PMA) not only induce the redistribution of cytosolic PKC to various subcellular compartments but also activate the kinase domain of the protein. In the present study we have investigated the nature of mitochondrial PKC pool and its effects on mitochondrial function in cells treated with PMA. Treatment of C2C12 myoblasts, C6 glioma and COS7 cells with PMA resulted in a dramatic redistribution of intracellular PKCalpha pool, with large fraction of the protein pool sequestered in the mitochondrial compartment. We also observed mitochondrial PKCdelta accumulation in a cell restricted manner. The intramitochondrial localization was ascertained by using a combination of protection against protease treatment of isolated mitochondria and immunofluorescence microscopy. PMA-induced mitochondrial localization of PKCalpha was accompanied by increased mitochondrial PKC activity, altered cell morphology, disruption of mitochondrial membrane potential, decreased complex I and pyruvate dehydrogenase activities, and increased mitochondrial ROS production. All of these changes could be retarded by treatment with PKC inhibitors. These results show a direct role for PMA-mediated PKCalpha translocation to mitochondria in inducing mitochondrial toxicity.     

Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway

Quinolones (QNs)-induced arthropathy is an important toxic effect in immature animals leading to restriction of their therapeutic use in pediatrics. However, the exact mechanism still remains unclear. Recently, we have demonstrated that ofloxacin, a typical QN, induces apoptosis of alginate microencapsulated juvenile rabbit joint chondrocytes by disturbing the beta 1 integrin functions and inactivating the ERK/MAPK signaling pathway. In this study, we extend our initial observations to further elucidate the mechanism(s) of ofloxacin-induced apoptosis by utilizing specific caspase inhibitors. Pretreatment with both caspase-9-specific inhibitor zLEHD-fmk and caspase-8 inhibitor zIETD-fmk attenuated ofloxacin-induced apoptosis and activation of caspase-3 of chondrocyte in a concentration-dependent manner, as determined by fluorescent dye staining, enzyme activity assay and immunoblotting. Furthermore, the activation of caspase-9, -8 and -3 stimulated by ofloxacin was significantly inhibited in the presence of zIETD-fmk while pretreatment with zLEHD-fmk only blocked the activation of caspase-9 and -3. Ofloxacin also stimulated a concentration-dependent translocation of cytochrome c from mitochondria into the cytosol and a decrease of mitochondrial transmembrane potential, which was completely inhibited by zIETD-fmk. In addition, ofloxacin was found to increase the level of Bax, tBid, p53 in a concentration- and time-dependent manner. Taken together, The current results indicate that the caspase-8-dependent mitochondrial pathway is primarily involved in the ofloxacin-induced apoptosis of microencapsulated juvenile rabbit joint chondrocytes.     

Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes

We have previously shown that, in trout hepatocytes, exposure to a high dose of copper (Cu) leads to disruption of Ca(2+) homeostasis and elevated formation of reactive oxygen species (ROS), with the latter ultimately causing cell death. In the present study, we aimed at identifying, using a lower Cu concentration, the role of mitochondria in this scenario, the potential involvement of the mitochondrial permeability transition (MPT), and the mode of cell death induced by the metal. Incubation with 10 muM Cu resulted in a strong stimulation of ROS formation, and after 2 h of exposure a significant increase of both apoptotic and necrotic cells was seen. Co-incubation of Cu-treated hepatocytes with the iron-chelator deferoxamine significantly inhibited ROS production and completely prevented cell death. The origin of the radicals generated was at least partly mitochondrial, as visualized by confocal laser scanning microscopy. Furthermore, ROS production was diminished by inhibition of mitochondrial respiration, but since this also aggravated the elevation of intracellular Ca(2+) induced by Cu, it did not preserve cell viability. In a sub-population of cells, Cu induced a decrease of mitochondrial membrane potential and occurrence of the MPT. Cyclosporin A, which did not inhibit ROS formation, prevented the onset of the MPT and inhibited apoptotic, but not necrotic, cell death. Cu-induced apoptosis therefore appears to be dependent on induction of the MPT, but the prominent contribution of mitochondria to ROS generation also suggests an important role of mitochondria in necrotic cell death.     

Linking energy metabolism to dysfunctions in mitochondrial respiration--a metabolomics in vitro approach

The study presented here describes the application of metabolite profiling of highly polar, intracellular metabolites after incubation of a mammalian fibroblast cell line with inhibitors of mitochondrial function. A metabolomics approach was used to assess the complex response of the cellular energy metabolism. Metabolic profiles of phosphorylated and carboxylated intracellular metabolites were assessed by UPLC-MS/MS and used to predict the mode of mitochondrial toxicity. Based on distinct metabolic patterns, multivariate data analysis allowed for the discrimination of two groups of toxins: inhibitors of the electron transport in mitochondrial membranes (complex IV inhibitors) and uncouplers of oxidative phosphorylation. Beyond these known interferences, metabolic profiling was able to reveal additional inhibitory effects on the cellular energy metabolism. Most prominently, for three of the toxins, metabolic patterns also disclosed an enhanced activity of the glycerol phosphate shuttle inferring the inhibition of NADH dehydrogenase at complex I. Secondly, inhibition of the electron transport was accompanied by a limiting availability of citric acid cycle intermediates and aspartate. Concomitantly, specific perturbations of the purine nucleotide cycle were observed. We have shown here that metabolomic approaches may assist to predict complex modes of action of toxic compounds on cellular level as well as to unravel specific dysfunctions in the energy metabolism.     

Impaired mitochondrial energy metabolism and kinetic properties of cytochrome oxidase following acute aluminium phosphide exposure in rat liver

The present study was designed with an aim to analyze the effect of acute aluminium phosphide (ALP) exposure (10 mg/kg b.wt, intragastrically) on the kinetic characteristics of cytochrome oxidase and energy metabolism in male Wistar rat liver mitochondria. Liver mitochondrial preparations from ALP-treated rats exhibited significant decrease (66%) in the activity of cytochrome oxidase suggesting that there was a decrease in the catalytic efficiency of the active oxidase molecules on ALP treatment. The decreased activity of cytochrome oxidase with altered NADH and succinic dehydrogenase activities might have contributed towards a significant decline in state 3 and state 4 respiration as observed. These alterations in the electron transport chain complexes in turn adversely affected the ATP synthesis rate as well as ATP levels in the mitochondria isolated from treated rats. The alterations in the respiratory chain, was followed by enhanced lipid peroxidation in rat liver mitochondria which might have further contributed to change in the fluidity of membrane as depicted by decreased fatty acid content of liver mitochondria. However, no significant change was observed in cholesterol and phospholipids content in our study. The present study thus highlights the significance of altered mitochondrial respiratory chain functions and membrane integrity after acute ALP exposure.     

Inhibition of phosphate transport in rat heart mitochondria by 3'-azido-3'-deoxythymidine due to stimulation of superoxide anion mitochondrial production

In order to gain some insight into the mechanism by which 3'-azido-3'-deoxythymidine (AZT) damages mitochondria, we investigated whether externally added AZT can stimulate reactive oxygen species (ROS) production by rat heart mitochondria (RHM). An increase in superoxide anion ((O(2)(.-)) production was measured in RHM added with AZT, by using a photometrically method which allows an early O(2)(.-) detection by following the absorbance increase at 550 nm due to the ferricytochrome c reduction. Such an increase was found to be prevented from externally added superoxide dismutase. The stimulation of O(2)(.-) mitochondrial production induced by AZT was found to occur under conditions in which mitochondrial oxygen consumption was prevented by both inhibitors of electron flow and ATP synthesis. Since ROS can cause mitochondrial carrier impairment, we investigated whether AZT can affect mitochondrial permeability in virtue of its capability to stimulate ROS production. In this regard, we studied the transport of phosphate (P(i)), by measuring the mitochondrial shrinkage that takes place as a result of P(i) uptake by RHM previously swollen in a calcium acetate medium. As a result of the AZT-dependent O(2)(.-) production, uncompetitive inhibition of the rate of P(i) transport in RHM was found (K(i) of about 10 microM), consistently, such an inhibition was found to prevent by certain known ROS scavengers, i.e. superoxide dismutase, the antioxidant Vitamin C and reduced gluthatione.     

依达拉奉治疗老年糖尿病周围神经病变的研究

目的观察依达拉奉治疗老年糖尿病周围神经病变（DPN）的临床疗效并探讨其机制。方法老年DPN患者61例,随机分为治疗组33例和常规组28例．两组均予基础治疗,治疗组加用依达拉奉联合前列地尔,常规组予弥可保及前列地尔,疗程均为2周。治疗前后分别判定症状。检查神经传导速度。测定血清MDA、LPO浓度及SOD活力。结果与常规组（53．6％）相比,治疗组症状显著改善（81．8％,P〈0．05）;神经传导速度增快（P〈0．05）;常规组血清MDA（6．13±1．46VS6．25±1．56μmol·L^-1,P〉0．05）、LPO（14．82±2．25VS15．24±2．49μmol·L^-1,P〉0．05）、SOD活力（57．14±9．28VS55．75±11．21U·mL^-1,P〉0．05）无明显变化,而治疗组MDA（4．32±1．09VS6．15±1．34μmol·L^-1,P〈0．05）、LPO（10．80±2．37vs14．55±2．25μmol·L^-1 P〈0．05）浓度明显降低。SOD活力（76．85±10．76VS61．27±12．98U·mL^-1 P〈0．05）显著升高。结论依达拉奉可有效改善DPN患者的临床症状,其机制可能与抗氧化作用有关。     

Comparative pharmacology of chemically distinct NADPH oxidase inhibitors

BACKGROUND AND PURPOSE

Oxidative stress [i.e. increased levels of reactive oxygen species (ROS)] has been suggested as a pathomechanism of different diseases, although the disease-relevant sources of ROS remain to be identified. One of these sources may be NADPH oxidases. However, due to increasing concerns about the specificity of the compounds commonly used as NADPH oxidase inhibitors, data obtained with these compounds may have to be re-interpreted.

EXPERIMENTAL APPROACH

We compared the pharmacological profiles of the commonly used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and 4-(2-amino-ethyl)-benzolsulphonyl-fluoride (AEBSF), as well as the novel triazolo pyrimidine VAS3947. We used several assays for detecting cellular and tissue ROS, as none of them is specific and artefact free.

KEY RESULTS

DPI abolished NADPH oxidase-mediated ROS formation, but also inhibited other flavo-enzymes such as NO synthase (NOS) and xanthine oxidase (XOD). Apocynin interfered with ROS detection and varied considerably in efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low micromolar concentrations, and interfered neither with ROS detection nor with XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without effect.

CONCLUSIONS AND IMPLICATIONS

Our data suggest that triazolo pyrimidines such as VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to be a major source of ROS in aortas of SHRs.     

Inhibitory activity of monoamine oxidase by coumarins fromPeucedanum japonicum

Four coumarins were isolated from chloroform extract of the root of Peucedanum japonicum and identified as praeruptorin A(1), xanthotoxin (2), psoralen (3) and bergapten (4) on the basis of spectroscopic methods. The inhibitory activities of these coumarins on monoamine oxidase prepared by mouse brain were tested. The IC50 values of them were shown to be 27.4 microM (1), 40.7 microM (2), 35.8 microM (3), and 13.8 microM (4), in vitro.