• Molecules and Cells
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• v.39 no.10
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• pp.742-749
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• 2016

The cancer chemo-preventive effects of equol have been demonstrated for a wide variety of experimental tumours. In a previous study, we found that equol inhibited proliferation and induced apoptotic death of human gastric cancer MGC-803 cells. However, the mechanisms underlying equol-mediated apoptosis have not been well understood. In the present study, the dual AO (acridine orange)/EB (ethidium bromide) fluorescent assay, the comet assay, MTS, western blotting and flow cytometric assays were performed to further investigate the pro-apoptotic effect of equol and its associated mechanisms in MGC-803 cells. The results demonstrated that equol induced an apoptotic nuclear morphology revealed by AO/EB staining, the presence of a comet tail, the cleavage of caspase-3 and PARP and the depletion of cIAP1, indicating its pro-apoptotic effect. In addition, equol-induced apoptosis involves the mitochondria-dependent cell-death pathway, evidenced by the depolarization of the mitochondrial membrane potential, the cleavage of caspase-9 and the depletion of Bcl-xL and full-length Bid. Moreover, treating MGC-803 cells with equol induced the sustained activation of extracellular signal-regulated kinase (ERK), and inhibiting ERK by U0126, a MEK/ERK pathway inhibitor, significantly attenuated the equol-induced cell apoptosis. These results suggest that equol induces mitochondria-dependent apoptosis in human gastric cancer MGC-803 cells via the sustained activation of the ERK1/2 pathway. Therefore, equol may be a novel candidate for the chemoprevention and therapy of gastric cancer.

• BMB Reports
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• v.55 no.7
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• pp.354-359
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• 2022

MitoNEET, a mitochondrial outer membrane protein containing the Asn-Glu-Glu-Thr (NEET) sequence, controls the formation of intermitochondrial junctions and confers autophagy resistance. Moreover, mitoNEET as a mitochondrial substrate undergoes ubiquitination by activated Parkin during the initiation of mitophagy. Therefore, mitoNEET is linked to the regulation of autophagy and mitophagy. Mitophagy is the selective removal of the damaged or unnecessary mitochondria, which is crucial to sustaining mitochondrial quality control. In numerous human diseases, the accumulation of damaged mitochondria by impaired mitophagy has been observed. However, the therapeutic strategy targeting of mitoNEET as a mitophagy-enhancing mediator requires further research. Herein, we confirmed that mitophagy is indeed activated by mitoNEET inhibition. CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which leads to mitochondrial depolarization, induces mitochondrial dysfunction and superoxide production. This, in turn, contributes to the induction of mitophagy; mitoNEET protein levels were initially increased before an increase in LC3-II protein following CCCP treatment. Pharmacological inhibition of mitoNEET using mitoNEET Ligand-1 (NL-1) promoted accumulation of Pink1 and Parkin, which are mitophagy-associated proteins, and activation of mitochondria-lysosome crosstalk, in comparison to CCCP alone. Inhibition of mitoNEET using NL-1, or mitoNEET shRNA transfected into RAW264.7 cells, abrogated CCCP-induced ROS and mitochondrial cell death; additionally, it activated the expression of PGC-1α and SOD2, regulators of oxidative metabolism. In particular, the increase in PGC-1α, which is a major regulator of mitochondrial biogenesis, promotes mitochondrial quality control. These results indicated that mitoNEET is a potential therapeutic target in numerous human diseases to enhance mitophagy and protect cells by maintaining a network of healthy mitochondria.

• The Korean Journal of Pharmacology
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• v.21 no.1
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• pp.12-26
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• 1985

The generation of $O^{-}_{2}{\cdot}$ and its toxic effects were studied with rat brain mitochondria. The production of $O^{-}_{2}{\cdot}$ from mitochondria in the presence of succinate and antimycin was demonstrated by SOD-inhibitable reduction of NBT. Although succinate can support the $O^{-}_{2}{\cdot}$ formation, the highest rate needs antimycin indicating that blockade of electron flow in the respiratory chain augments the univalent reduction of molecular oxygen. Under this condition, $H_2O_2$ was also observed to be produced. But its formation appears to be derived from the dismutation of the primary product, $O^{-}_{2}{\cdot}$ since the rate of $H_2O_2$ production was markedly decreased by NBT and ferricytochrome c. The $O^{-}_{2}{\cdot}$ and $H_2O_2$ produced were able to cause toxic actions to mitochondrial and extra-mitochondrial components as shown by lipid peroxidation of mitochondrial membrane, and inactivation and lysis of isocitrate dehydrogenase and erythrocytes added to the medium, respectively. In all the toxic actions observed, $Fe^{++}$ was required. It appears that in the toxic actions $OH{\cdot}$ generated from the iron-catalyzed Haber-Weiss reaction acts as a mediator. This was supported by the finding that mitochondria in the presence of succinate and antimycin produced ethylene from methional, and $Fe^{++}$ added increased the ethylene production. The observed toxic actions of mitochondrial $O^{-}_{2}{\cdot}$ may provide evidence supporting a potential role of mitochondria as a source of oxygen radicals to cause tissue damage.

• Journal of Life Science
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• v.29 no.12
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• pp.1393-1400
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• 2019

In the present study, we examined the effect of mitochondrial K⁺ channel opener diazoxide on the mitochondria-dependent apoptotic signaling in endothelial cells exposed to high glucose (HG) media. Endothelial cells derived from human umbilical veins were exposed to HG media containing 30 mM glucose, and the degree of apoptotic cell death associated with activation of the mitochondria-dependent apoptotic signaling pathway was determined. Exposure to HG media was seen to enhance apoptotic cell death in a time-dependent manner. In these cells, activation of caspases 3, 8, and 9 was observed, and while caspase-3 and -9 inhibitors suppressed the HG-induced apoptotic cell death, a caspase-8 inhibitor did not. The HG-treated cells exhibited disruption of mitochondrial membrane potential, formation of permeability transition pores, and cytosolic release of cytochrome c. Subsequently, diazoxide was seen to attenuate the HG-induced apoptotic cell death; caspase-9 activation was suppressed but caspase 8 was not. Diazoxide also suppressed the depolarization of mitochondrial membrane potential, the formation of mitochondrial permeability transition pores, and the release of cytochrome c. These effects were significantly inhibited by 5-hydroxydecanoate, a selective blocker of ATP-sensitive K⁺ channels (K_ATP). The present results demonstrate that diazoxide exhibits a beneficial effect to ameliorate HG-induced endothelial cell apoptosis. Opening the K_ATP could help preserve the functional integrity of mitochondria and provide an underlying mechanism to suppress HG-triggered apoptotic signaling.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.199-199
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• 2013

The distinctive cellular and mitochondrial dysfunctions of a human epithelial lung cancer cell line (H460) from a human lung fibroblastic normal cell line (MRC5) have been studied by dielectric barrier discharge (DBD) plasma treatment. The DBD plasma device have generated large amount of H2O2 and NOx in culture media which is dependent on plasma exposure time. It is found that the cell number of lung cancer cell H460 has been reduced more than the lung normal cell MRC5 as being increased exposure and incubation time. Also these both cell lines have showed mitochondria fragmentation under 5 minutes' plasma exposure, which is a clue of apoptosis. It is noted in this study that AnnexinV staining has showed not only early apoptosis, but also late apoptosis in lung cancer cell H460. Mitochondria enzyme activity and ATP generation have been also much reduced in lung cancer cell H460. Their mitochondrial membrane potential (${\Delta}{\psi}m$) has been found to be reduced in magnitude and shifted to the induced-potential level of cccp, while MRC5 mitochondrial membrane potential has been shifted slightly to that. These distinctively selective responses of lung cancer cell H460 from lung normal cell MRC5 gives us possibility of applying plasma to cancer therapy.

• Advances in Traditional Medicine
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• v.9 no.2
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• pp.106-114
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• 2009

Hepatocellular carcinoma is the world's most common primary malignant tumor of the liver. In-Jin-ho-Tang (IJHT) has been used as a traditional Chinese herbal medicine since ancient times, and today it is widely used as a medication for jaundice associated with inflammation of the liver. In-Jin-Ho-Tang is a drug preparation consisting of three herbs: Artemisiae Capillaris Herba (Artemisia capillaries $T_{HUNS}$, Injinho in Korean), Gardeniae Fructus (Gardenia jasminodes $E_{LLIS}$, Chija in Korean) and Rhei radix et rhizoma (Rheum palmatum L., Daehwang in Korean). This study investigated whether or not methanol extract of IJHT could induce HepG2 cancer cell death. Cytotoxic activity of IJHT on HepG2 cells was measured using an XTT assay, with an $IC_{50}$ value of $700{\mu}g/ml$ at 24 h Apoptosis induction by IJHT in HepG2 cells was verified by the cleavage of poly ADP-ribose polymerase, and a decrease in procaspase-3, -8, -9. Treatment of IJHT resulted in the release of cytochrome c into cytosol, loss of mitochondrial membrane potential (${\Delta}{\Psi}_m$), decrease in anti-apoptotic Bcl-2, and an increase in pro-apoptotic Bax expression. Thus, IJHT induced apoptosis in HepG2 cells via activation of caspase and mitochondria pathway. These results indicate that IJHT has potential as an anti-cancer agent.

• Journal of Microbiology
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• v.41 no.1
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• pp.41-45
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• 2003

N crassa mutant strain nap showed reduced growth rate, decreased electric membrane potential, and elevated intracellular ATP content in comparison to the wild type. Blue light induced a hyperpolarization of the membrane potential in both strains. The analysis of oxidative and phosphorylation activities of mitochondria isolated from the two strains has revealed that nap utilized more efficient oxidative pathways. The higher intracellular ATP content in the nap was presumably due to impaired transport systems of the plasma membrane, and to a lesser extent to the functioning of the fully competent respiratory chain. The excess ATP possibly accounts for carotenoid accumulation in the mutant.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.167.1-167.1
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• 2003

Here we show that panaxadiol, a ginseng saponin with a dammarane skeleton, induces acute apoptotic cell death in human hepatoma SK-HEP-1 cells as evidenced by analysis of DNA fragmentation, caspase activation, and changes in cell morphology. The kinetic study showed that panaxadiol-induced apoptosis is associated with depolarization of mitochondrial membrane potential and cytochrome c release. Sequential activations of caspases-depolarization of mitochondrial membrane potential and cytochrome c release. Sequential activations of caspases-9, and -3, or -7, but not of caspase 8 coincide well in a time dependent manner with mitochondrial membrane depolarization and cytochrome c release from mitochondria during apoptosis of SK-HEP-1 cells induced by treatment with panaxadiol. (omitted)

• Journal of Life Science
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• v.26 no.12
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• pp.1458-1465
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• 2016

We investigated the neuroprotective effect of an ethanol extract from Asparagus cochinchinensis (AC) against glutamate-induced toxicity in the HT22 hippocampal cell, which is an ideal in vitro model for oxidative stress. The neuroprotective effects of AC in HT22 cells were evaluated by analyzing cell viability, lactate dehydrogenase (LDH), flow cytometry for cell death types, reactive oxygen species (ROS), mitochondria membrane potential (MMP), and Western blot assays. In the cell death analysis, AC treatment resulted in significantly attenuated glutamate-induced loss of cell viability with a decrease in LDH release. AC treatment also reduced glutamate-induced apoptotic cell death. In the ROS and MMP analysis, AC treatment inhibited the elevation of intracellular ROS induced by glutamate exposure and the disruption of MMP. In oxidative stress-related proteins analysis, AC treatment inhibited the expression of poly ADP ribose polymerase and heme oxygenase-1 by glutamate. These results indicate that AC exerts a significant neuroprotective effect against glutamate-induced hippocampal damage by decreasing ROS production and stabilizing MMP. Thus, AC potentially provides a new strategy for the treatment of oxidative stress-related diseases.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.108-109
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• 2013

Mitochondria play key roles in the production of cell's energy. Their dominant function is the synthesis of adenosine 5'-triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (Pi) through the oxidative phosphorylation. Evaluation of drug-induced mitochondrial toxicity has become increasingly important since mitochondrial dysfunction has recently been implicated in numerous diseases including cancer and diabetes mellitus. Mitochondrial functions have been monitored via oxygen consumption, mitochondrial membrane potential, and more importantly via ATP synthesis since ATP synthesis is the most essential function of mitochondria. Various analytical methods have been employed to investigate ATP synthesis in mitochondria, including high performance liquid chromatography (HPLC), bioluminescence technique, and pH measurement. However, most of these methods are based on destructive analysis or indirect monitoring through the enzymatic reaction. Infrared absorption spectroscopy (IRAS) is one of the useful techniques for real-time, label-free, and direct monitoring of biological reactions [1,2]. However, the strong water absorption requires very short path length in the order of several micrometers. Transmission measurements with thin path length are not suitable for mitochondrial assays because solution handlings necessary for evaluating mitochondrial toxicity, such as rapid mixing of drugs and oxygen supply, are difficult in such a narrow space. On the other hand, IRAS in the multiple internal reflection (MIR) geometry provides an ideal optical configuration to combine solution handling and aqueous-phase measurement. We have recently reportedon a real-time monitoring of drug-induced necrotic and apoptotic cell death using MIR-IRAS [3,4]. Clear discrimination between viable and damaged cells has been demonstrated, showing a promise as a label-free and real-time detection for cell-based assays. In the present study, we have applied our MIR-IRAS system to mitochondria-based assays by monitoring ATP synthesis in isolated mitochondria from rat livers. Mitochondrial ATP synthesis and hydrolysis were in situ monitored with MIR-IRAS, while dissolved oxygen level and solution pH were simultaneously monitored with O2 and pH electrodes, respectively. It is demonstrated that ATP synthesis and hydrolysis can be monitored by the IR spectral changes in phosphate groups in adenine nucleotides and MIR-IRAS is useful for evaluating time-dependent drug effects of mitochondrial toxicants.