Eun Sook Han

18β-Glycyrrhetinic acid induces apoptotic cell death in SiHa cells and exhibits a synergistic effect against antibiotic anti-cancer drug toxicity

Defects in mitochondrial function have been shown to participate in the induction of cell death in cancer cells. The present study was designed to assess the toxic effect of 18beta-glycyrrhetinic acid against human cervix and uterus tumor cell line SiHa cells in relation to the mitochondria-mediated cell-death process and evaluate the combined toxic effect of 18beta-glycyrrhetinic acid and anti-cancer drugs. 18beta-Glycyrrhetinic acid induced the nuclear damage, changes in the mitochondrial membrane permeability, formation of reactive oxygen species and depletion of glutathione in SiHa cells. It caused cell death by inducing the increase in the pro-apoptotic Bax protein and cytochrome c levels, reduction in anti-apoptotic Bcl-2 level, subsequent caspase-3 activation and loss of the mitochondrial transmembrane potential. Unlike 18beta-glycyrrhetinic acid, a pro-compound glycyrrhizin up to 100 microM did not induce cell death and depletion of glutathione. Combined treatment of mitomycin c (or doxorubicin) and 18beta-glycyrrhetinic acid revealed a synergistic toxic effect. Meanwhile, combination of camptothecin and 18beta-glycyrrhetinic acid exhibited an additive cytotoxic effect. Results suggest that 18beta-glycyrrhetinic acid may cause cell death in SiHa cells by inducing the mitochondrial membrane permeability change, leading to cytochrome c release and caspase-3 activation. The effect may be associated with increased formation of reactive oxygen species and depletion of glutathione. Combined treatment of antibiotic anti-cancer drug and 18beta-glycyrrhetinic acid seems to exhibit a synergistic toxic effect.

Differential effect of catecholamines and MPP+ on membrane permeability in brain mitochondria and cell viability in PC12 cells

The present study examined the effect of dopamine, 6-hydroxydopamine (6-OHDA), and MPP(+) on the membrane permeability transition in brain mitochondria and on viability in PC12 cells. Dopamine and 6-hydroxydopamine induced the swelling and membrane potential change in mitochondria, which was inhibited by addition of antioxidant enzymes, SOD and catalase. In contrast, antioxidant enzymes did not reduce the effect of MPP(+) on mitochondrial swelling and membrane potential. Catecholamines enhanced the Ca(2+) uptake and release by mitochondria, and the addition of MPP(+) induced Ca(2+) release. Catecholamines induced a thiol oxidation in mitochondria that was decreased by antioxidant enzymes. MPP(+) showed a little effect on the cytochrome c release from mitochondria and did not induce thiol oxidation. Catecholamines and MPP(+) induced a cell death, including apoptosis, in PC12 cells that was inhibited by addition of antioxidant enzymes. The result suggests that the oxidation of dopamine and 6-hydroxydopamine could modulate the membrane permeability in brain mitochondria and induce PC12 cell death, which may be ascribed to oxidative stress. MPP(+) appears to exert a toxic effect on neuronal cells by the action, which is different from catecholamines.

Protective effect of harmalol and harmaline on MPTP neurotoxicity in the mouse and dopamine-induced damage of brain mitochondria and PC12 cells.

The present study elucidated the protective effect of β‐carbolines (harmaline, harmalol, and harmine) on oxidative neuronal damage. MPTP treatment increased activities of total superoxide dismutase, catalase, and glutathione peroxidase and levels of malondialdehyde and carbonyls in the basal ganglia, diencephalon plus midbrain of brain compared with control mouse brain. Coadministration of harmalol (48 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. Harmaline, harmalol, and harmine attenuated both the 500 μM MPP+‐induced inhibition of electron flow and membrane potential formation and the 100 μM dopamine‐induced thiol oxidation and carbonyl formation in mitochondria. The scavenging action of β‐carbolines on hydroxyl radicals was represented by inhibition of 2‐deoxy‐d‐ribose degradation. Harmaline and harmalol (100 μM) attenuated 200 μM dopamine‐induced viability loss in PC12 cells. The β‐carbolines (50 μM) attenuated 50 μM dopamine‐induced apoptosis in PC12 cells. The compounds alone did not exhibit significant cytotoxic effects. The results indicate that β‐carbolines attenuate brain damage in mice treated with MPTP and MPP+‐induced mitochondrial damage. The compounds may prevent dopamine‐induced mitochondrial damage and PC12 cell death through a scavenging action on reactive oxygen species and inhibition of monoamine oxidase and thiol oxidation.

Protective effect of harmaline and harmalol against dopamine‐ and 6‐hydroxydopamine‐induced oxidative damage of brain mitochondria and synaptosomes, and viability loss of PC12 cells

The present study elucidated the protective effect of β‐carbolines (harmaline, harmalol and harmine) against oxidative damage of brain mitochondria, synaptosomes and PC12 cells induced by either dopamine or 6‐hydroxydopamine. Harmaline, harmalol and antioxidant enzymes (superoxide dismutase/SOD and catalase) decreased the alteration of mitochondrial swelling and membrane potential induced by 200 µm dopamine or 100 µm 6‐hydroxydopamine. Deprenyl attenuated the dopamine‐induced mitochondrial dysfunction but did not reduce the effect of 6‐hydroxydopamine. While β‐carbolines inhibited the electron flow in mitochondria, they did not enhance the depressant effect of catecholamines. β‐Carbolines and antioxidant enzymes reversed the depression of synaptosomal Ca2+ uptake induced by 10 µm catecholamines. The compounds inhibited the catecholamine‐induced thioredoxin reductase inhibition, thiol oxidation and carbonyl formation in mitochondria and synaptosomes. β‐Carbolines decreased the reactive species‐induced deoxyribose degradation. Harmaline and harmalol reduced the catecholamine‐induced loss of the transmembrane potential and of cell viability in PC12 cells.β‐Carbolines alone did not show a significant cytotoxic effect on PC12 cells. The results suggest that β‐carbolines may attenuate the dopamine‐ or 6‐hydroxydopamine‐induced alteration of brain mitochondrial and synaptosomal functions, and viability loss in PC12 cells, by a scavenging action on reactive oxygen species and inhibition of thiol oxidation.

Protective effect of serotonin on 6-hydroxydopamine- and dopamine-induced oxidative damage of brain mitochondria and synaptosomes and PC12 cells.

The present study elucidated the effects of indoleamines (serotonin, melatonin, and tryptophan) on oxidative damage of brain mitochondria and synaptosomes induced either by 6-hydroxydopamine (6-OHDA) or by iron plus ascorbate and on viability loss in dopamine-treated PC12 cells. Serotonin (1-100 microM), melatonin (100 microM), and antioxidant enzymes attenuated the effects of 6-OHDA, iron plus ascorbate, or 1-methyl-4-phenylpyridinium on mitochondrial swelling and membrane potential formation. Serotonin and melatonin decreased the attenuation of synaptosomal Ca(2+) uptake induced by either 6-OHDA alone or iron plus ascorbate. Serotonin and melatonin inhibited the production of reactive oxygen species, formation of malondialdehyde and carbonyls, and thiol oxidation in mitochondria and synaptosomes and decreased degradation of 2-deoxy-D-ribose. Unlike serotonin, melatonin did not reduce the iron plus ascorbate-induced thiol oxidation. Tryptophan decreased thiol oxidation and 2-deoxy-D-ribose degradation but did not inhibit the production of reactive oxygen species and formation of oxidation products in the brain tissues. Serotonin and melatonin attenuated the dopamine-induced viability loss, including apoptosis, in PC12 cells. The results suggest that serotonin may attenuate the oxidative damage of mitochondria and synaptosomes and the dopamine-induced viability loss in PC12 cells by a decomposing action on reactive oxygen species and inhibition of thiol oxidation and shows the effect comparable to melatonin. Serotonin may show a prominent protective effect on the iron-mediated neuronal damage.

Inhibition of MPP+-induced mitochondrial damage and cell death by trifluoperazine and W-7 in PC12 cells.

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of trifluoperazine and W-7 on the MPP+-induced mitochondrial damage and cell death in undifferentiated PC12 cells. Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) at 0.5-1 microM significantly reduced the loss of cell viability in PC12 cells treated with 500 microM MPP+. Trifluoperazine and W-7 (0.5-1 microM) inhibited the nuclear damage, the loss of the mitochondrial transmembrane potential followed by cytochrome c release, and the elevation of intracellular Ca2+ levels due to MPP+ in PC12 cells and attenuated the formation of reactive oxygen species and the depletion of GSH. Calmodulin antagonists at 5-10 microM exhibited a cytotoxic effect on PC12 cells, and compounds at 10 microM did not attenuate cytotoxicity of MPP+. Calmodulin antagonists (0.5-1 microM) significantly reduced rotenone-induced mitochondrial damage and cell death, whereas they did not attenuate cell death and elevation of intracellular Ca2+ levels due to H2O2 or ionomycin. The results show that trifluoperazine and W-7 exhibit a differential inhibitory effect against cytotoxicity of MPP+ depending on concentration. Both compounds at the concentrations less than 5 microM may attenuate the MPP+-induced viability loss in PC12 cells by suppressing change in the mitochondrial membrane permeability and by lowering the intracellular Ca2+ levels.

Lamotrigine inhibition of rotenone- or 1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death.

Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the effect of antiepileptic lamotrigine against the cytotoxicity of mitochondrial respiratory complex I inhibitors rotenone and 1-methyl-4-phenylpyridinium (MPP+) in relation to the mitochondria-mediated cell death process and oxidative stress. Both rotenone and MPP+ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in differentiated PC12 cells. Lamotrigine significantly attenuated the rotenone- or MPP+-induced mitochondrial damage leading to caspase-3 activation, increased oxidative stress and cell death. The preventive effect of lamotrigine against the toxicity of rotenone was greater than its effect on that of MPP+. The results show that lamotrigine seems to reduce the cytotoxicity of rotenone and MPP+ by suppressing the mitochondrial permeability transition formation, leading to cytochrome c release and subsequent activation of caspase-3. The preventive effect may be ascribed to its inhibitory action on the formation of reactive oxygen species and depletion of GSH. Lamotrigine seems to exert a protective effect against the neuronal cell injury due to the mitochondrial respiratory complex I inhibition.

Protective effect of boldine on dopamine-induced membrane permeability transition in brain mitochondria and viability loss in PC12 cells.

Boldine ([S]-2,9-dihydroxy-1,10-dimethoxyaporphine) has been shown to exert antioxidant and anti-inflammatory effects. The present study elucidated the protective effect of boldine on catecholamine-induced membrane permeability transition in brain mitochondria and viability loss in PC12 cells. Dopamine (200 microM) and 6-hydroxydopamine (6-OHDA, 100 microM) attenuated Ca(2+) and succinate-induced mitochondrial swelling and membrane potential formation. Boldine (10-100 microM) and 10 microg/mL of superoxide dismutase (SOD) or catalase reduced the effect of catecholamine oxidation on brain mitochondria. Boldine, SOD, and catalase decreased catecholamine-induced mitochondrial cytochrome c release. Antioxidant enzymes attenuated the depressant effect of catecholamines on mitochondrial electron flow, whereas boldine did not reduce it. Boldine inhibited the catecholamine-induced decrease in thioredoxin reductase activity and the increase in thiol oxidation in mitochondria. It also showed a scavenging action on hydrogen peroxide and hydroxyl radicals and decreased the formation of melanin from dopamine. Boldine and antioxidant enzymes decreased the dopamine-induced cell death, including apoptosis, in PC12 cells. The results suggest that boldine may attenuate the catecholamine oxidation-induced brain mitochondrial dysfunction and decrease the dopamine-induced death of PC12 cells through a scavenging action on reactive oxygen species and inhibition of melanin formation and thiol oxidation.

Prevention of 7-ketocholesterol-induced mitochondrial damage and cell death by calmodulin inhibition

Oxysterols such as 7-ketocholesterol and 25-hydroxycholesterol formed under enhanced oxidative stress in the brain are suggested to induce neuronal cell death. The present study investigated the effect of calmodulin antagonists (trifluoperazine, W-7 and calmidazolium) against the cytotoxicity of 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress. PC12 cells exposed to 7-ketocholesterol revealed nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. N-Acetylcysteine, trolox, carboxy-PTIO and Mn-TBAP reduced the cytotoxic effect of 7-ketocholesterol. Calmodulin antagonists attenuated the 7-ketocholesterol-induced nuclear damage, formation of the mitochondrial permeability transition and cell viability loss in PC12 cells. The results suggest that calmodulin antagonists may prevent the 7-ketocholesterol-induced viability loss in PC12 cells by suppressing formation of the mitochondrial permeability transition, leading to the release of cytochrome c and subsequent activation of caspase-3. The effects seem to be ascribed to their depressant action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that calmodulin inhibition may exhibit a protective effect against the neurotoxicity of 7-ketocholesterol.

N-methylated β-carbolines protect PC12 cells from cytotoxic effect of MPP+ by attenuation of mitochondrial membrane permeability change

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of beta-carbolines (harmaline and harmalol) on the MPP(+)-induced change in the mitochondrial membrane permeability and cell death in differentiated PC12 cells. beta-Carbolines and antioxidants (superoxide dismutase, catalase, ascorbate or rutin) prevented the loss of cell viability in PC12 cells treated with 250 microM MPP(+), while the effects of N-acetylcysteine and dithiothreitol were not observed. beta-Carbolines reduced the condensation and fragmentation of nuclei caused by MPP(+) in PC12 cells. beta-Carbolines alone did not exhibit a significant cytotoxic effect on PC12 cells. beta-Carbolines (50 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species (ROS) and depletion of GSH caused by MPP(+) in PC12 cells. beta-Carbolines reduced the hydrogen peroxide- or SIN-1-induced cell death in PC12 cells. The results suggest that beta-carbolines may attenuate the MPP(+)-induced viability loss in PC12 cells by inhibition of change in the mitochondrial membrane permeability and by antioxidant effect.