• International Journal of Oral Biology
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• v.40 no.2
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• pp.103-109
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• 2015

Growing evidence suggests that mitochondrial reactive oxygen species (ROS) are involved in various pain states. This study was performed to investigate whether ROS-induced changes in neuronal excitability in trigeminal subnucleus caudalis are related to ROS generation in mitochondria. Confocal scanning laser microscopy was used to measure ROS-induced fluorescence intensity in live rat trigeminal caudalis slices. The ROS level increased during the perfusion of malate, a mitochondrial substrate, after loading of 2',7'-dichlorofluorescin diacetate ($H_2DCF-DA$), an indicator of the intracellular ROS; the ROS level recovered to the control condition after washout. When pre-treated with phenyl N-tert-butylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), malate-induced increase of ROS level was suppressed. To identify the direct relation between elevated ROS levels and mitochondria, we applied the malate after double-loading of $H_2DCF-DA$ and chloromethyl-X-rosamine (CMXRos; MitoTracker Red), which is a mitochondria-specific fluorescent probe. As a result, increase of both intracellular ROS and mitochondrial ROS were observed simultaneously. This study demonstrated that elevated ROS in trigeminal subnucleus caudalis neuron can be induced through mitochondrial-ROS pathway, primarily by the leakage of ROS from the mitochondrial electron transport chain.

• BMB Reports
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• v.55 no.10
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• pp.494-499
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• 2022

PTEN-induced putative kinase 1 (PINK1) is a serine/threonine kinase that phosphorylates several substrates and exerts neuroprotective effects against stress-induced apoptotic cell death. Mutations in PINK1 have been linked to autosomal recessive forms of Parkinson's disease (PD). Mitophagy is a type of autophagy that selectively promotes mitochondrial turnover and prevents the accumulation of dysfunctional mitochondria to maintain cellular homeostasis. Toll-interacting protein (Tollip) was initially identified as a negative regulator of IL-1β receptor signaling, suppressing inflammatory TLR signaling cascades. Recently, Tollip has been reported to play a role in autophagy and is implicated in neurodegeneration. In this study, we determined whether Tollip was functionally linked to PINK1-mediated mitophagy. Our results demonstrated that Tollip promoted the mitochondrial processing of PINK1 and altered the localization of PINK1, predominantly to the cytosol. This action was attributed to increased binding of PINK1 to mitochondrial processing peptidase β (MPPβ) and the subsequent increase in MPPβ-mediated mitochondrial PINK1 cleavage. Furthermore, Tollip suppressed mitophagy following carbonyl cyanide m-chlorophenylhydrazone-induced mitochondrial dysfunction. These findings suggest that Tollip inhibits mitophagy via the PINK1/parkin pathway upon mitochondrial damage, leading to the blockade of PINK1-mediated neuroprotection.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.5
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• pp.299-304
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• 2005

Cardiac hypertrophy contributes an increased risk to major cerebrovascular events. However, the molecular mechanisms underlying cerebrovascular dysfunction during cardiac hypertrophy have not yet been characterized. In the present study, we examined the molecular mechanism of isoproterenol (ISO)-evoked activation of Ras/Raf/MAPK pathways as well as PKA activity in cerebral artery of rabbits, and we also studied whether the activations of these signaling pathways were altered in cerebral artery, during ISO-induced cardiac hypertrophy compared to heart itself. The results show that the mRNA level of c-fos (not c-jun and c-myc) in heart and these genes in cerebral artery were considerably increased during cardiac hypertrophy. These results that the PKA activity and activations of Ras/Raf/ERK cascade as well as c-fos expression in rabbit heart during cardiac hypertrophy were consistent with previous reports. Interestingly, however, we also showed a novel finding that the decreased PKA activity might have differential effects on Ras and Raf expression in cerebral artery during cardiac hypertrophy. In conclusion, there are differences in molecular mechanisms between heart and cerebral artery during cardiac hypertrophy when stimulated with β2 adrenoreceptor (AR), suggesting a possible mechanism underlying cerebrovascular dysfunction during cardiac hypertrophy.

• Journal of Physiology & Pathology in Korean Medicine
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• v.23 no.4
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• pp.888-894
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• 2009

It has been reported that silibinin, a natural polyphenolic flavonoid, induces cell death in various cancer cell types. However, the underlying mechanisms by which silibinin induces apoptosis in human glioma cells are poorly understood. The present study was therefore undertaken to examine the effect of silibinin on glioma cell apoptosis and to determine its underlying mechanism in human glioma cells. Apoptosis was estimated by FACS analysis. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (${\Psi}m$) were measured using fluorescence dyes DCFH-DA and $DiOC_6$(3), respectively. Cytochrome c release from mitochondria and caspase-3 activation were estimated by Western blot analysis using specific antibodies. Exposure of cells to 30 mM silibinin induced apoptosis starting at 6 h, with increasing effects after 12-48h in a time-dependent manner. Silibinin caused ROS generation and disruption of ym, which were associated with the silibinin-induced apoptosis. The silibinin-induced ROS generation and disruption in ym were prevented by inhibitors of mitochondrial electron transport chain. The hydrogen peroxide scavenger catalase blocked ROS generation and apoptosis induced by silibinin. Silibinin induced cytochrome c release into cytosolic fraction and its effect was prevented by catalase and cyclosporine A. Silibinin treatment caused caspase-3 activation, which was inhibited by DVED-CHO and cyclosporine A. Pretreatment of caspase inhibitors also protected against the silibinin-induced apoptosis. These findings indicate that ROS generation plays a critical role in the initiation of the silibinin-induced apoptotic cascade by mediation of the mitochondrial apoptotic pathway including the disruption of ${\Psi}m$, cytochrome c release, and caspase-3 activation.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.5
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• pp.1322-1329
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• 2008

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruits on cell viability in human glioma cells. The present study was undertaken to examine the effect of mulberry fruit (Mar; Fructus) on cell viability and to determine its underlying mechanism in human glioma cells. Cell viability and cell death were estimated by MTT assay and trypanblue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with $DiOC_6$(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Mori Fructus resulted in apoptotic cell death in a dose- and time-dependent manner. Mori Fructus increased ROS generation and the Mori Fructus-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in Mari Fructus-induced cell death. Western blot analysis showed that Mori Fructus treatment caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). Mori Fructus induced depolarization of mitochondrial membrane potential and its effect was inhibited by the antioxidants NAC and catalase. Mori Fructus induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by Mori Fructus and caspase inhibitors prevented the Mori Fructus-induced cell death. These findings suggest that Mori Fructus results in human glioma cell death through ROS-dependent mitochondrial pathway in human glioma cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10495-10500
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• 2015

Background: To evaluate the effects of bufalin in A549 human lung adenocarcinoma epithelial cells in vitro and assess the underlying mechanisms. Materials and Methods: Human A549 non-small cell lung cancer (NSCLC) cells were treated with various concentrations of bufalin. Cell proliferation was measured by CCK-8 assay, apoptotic cell percentage was calculated by flow cytometry and morphological change was observed by inverted phase contrast microscopy/transmission electron microscopy. In addition, the membrane potential of mitochondria was detected by JC-1 fluorescence microscopy assay, and the related protein expression of cytochrome C and caspase-3 was analyzed by Western blotting. Results: Bufalin could inhibit the proliferation of A549 cells via induction of apoptosis, with the evidence of characteristic morphological changes in the nucleus and mitochondria. Furthermore, bufalin decreased the mitochondrial membrane potential with up-regulation of cytochrome C in the cytosol, and activation of caspase-3. Conclusions: Bufalin inhibits the proliferation of A549 cells and triggers mitochondria-dependent apoptosis, pointing to therapeutic application for NSCLC.

• Korean Journal of Clinical Laboratory Science
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• v.42 no.1
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• pp.46-54
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• 2010

Ceramide induces cell death in a variety of cell types however, the underlying molecular mechanisms related to renal epithelial cells remain unclear. The present study was undertaken to determine the role of extracellular signal-regulated protein kinase (ERK) in ceramide-induced cell death in renal epithelial cells. An established renal proximal tubular cell line of opossum kidney (OK) cells was used for this research. Ceramide induced apoptotic cell death in these cells. Western blot analysis showed that ceramide induced activation of ERK. The ERK activation and cell death induced by ceramide were prevented by the ERK inhibitor PD98059. Ceramide caused cytochrome C release from mitochondria into the cytosol as well as activation of caspase-3. Both effects were prevented by PD98059. The ceramide-induced cell death was also prevented by a caspase inhibitor. These results suggest that ceramide induces cell death through an ERK-dependent mitochondrial apoptotic pathway in OK cells.

• BMB Reports
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• v.45 no.1
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• pp.7-13
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• 2012

Phospholipase D (PLD), a superfamily of signaling enzymes that most commonly generate the lipid second messenger Phosphatidic Acid (PA), is found in diverse organisms from bacteria to man and functions in multiple cellular pathways. A fascinating member of the family, MitoPLD, is anchored to the mitochondrial surface and has two reported roles. In the first role, MitoPLD-generated PA regulates mitochondrial shape through facilitating mitochondrial fusion. In the second role, MitoPLD performs a critical function in a pathway that creates a specialized form of RNAi required by developing spermatocytes to suppress transposon mobilization during meiosis. This spermatocyte-specific RNAi, known as piRNA, is generated in the nuage, an electron-dense accumulation of RNA templates and processing proteins that localize adjacent to mitochondria in a structure also called intermitochondrial cement. In this review, we summarize recent findings on these roles for MitoPLD functions, highlighting directions that need to be pursued to define the underlying mechanisms.

• Parasites, Hosts and Diseases
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• v.60 no.5
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• pp.371-371
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• 2022

• Molecules and Cells
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• v.42 no.12
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• pp.893-905
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• 2019

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion processes that closely related to their function. Disruption of mitochondrial dynamics has been demonstrated in acute kidney injury (AKI), which could eventually result in cell injury and death. Previously, we reported that augmenter of liver regeneration (ALR) alleviates renal tubular epithelial cell injury. Here, we gained further insights into whether the renoprotective roles of ALR are associated with mitochondrial dynamics. Changes in mitochondrial dynamics were examined in experimental models of renal ischemia-reperfusion (IR). In a model of hypoxia-reoxygenation (HR) injury in vitro, dynamin-related protein 1 (Drp1) and mitochondrial fission process protein 1 (MTFP1), two key proteins of mitochondrial fission, were downregulated in the Lv-ALR + HR group. ALR overexpression additionally had an impact on phosphorylation of Drp1 Ser637 during AKI. The inner membrane fusion protein, Optic Atrophy 1 (OPA1), was significantly increased whereas levels of outer membrane fusion proteins Mitofusin-1 and -2 (Mfn1, Mfn2) were not affected in the Lv-ALR + HR group, compared with the control group. Furthermore, the mTOR/4E-BP1 signaling pathway was highly activated in the Lv-ALR + HR group. ALR overexpression led to suppression of HR-induced apoptosis. Our collective findings indicate that ALR gene transfection alleviates mitochondrial injury, possibly through inhibiting fission and promoting fusion of the mitochondrial inner membrane, both of which contribute to reduction of HK-2 cell apoptosis. Additionally, fission processes are potentially mediated by promoting tubular cell survival through activating the mTOR/4E-BP1 signaling pathway.