• Journal of Microbiology and Biotechnology
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• v.30 no.12
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• pp.1885-1895
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• 2020

Rumex japonicus Houtt (RJH) is a valuable plant used in traditional medicine to treat several diseases, such as scabies and jaundice. In this study, Jurkat cell growth inhibitory extracts of R. japonicus roots were subjected to bioassay-guided fractionation, resulting in the isolation of three naphthalene derivatives (3-5) along with one anthraquinone (6) and two phenolic compounds (1 and 2). Among these compounds, 2-methoxystypandrone (5) exhibited potent anti-proliferative effects on Jurkat cells. Analysis by flow cytometry confirmed that 2-methoxystypandrone (5) could significantly reduce mitochondrial membrane potential and promote increased levels of mitochondrial reactive oxygen species (ROS), suggesting a strong mitochondrial depolarization effect. Real-time quantitative polymerase chain reaction (qPCR) analysis was also performed, and the results revealed that the accumulation of ROS was caused by reduced mRNA expression levels of heme oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In addition, 2-methoxystypandrone (5) triggered strong apoptosis that was mediated by the arrest of the G0/G1 phase of the cell cycle. Furthermore, 2-methoxystypandrone (5) downregulated p-IκB-α, p-NF-κB p65, Bcl2, and Bcl-xl and upregulated BAX proteins. Taken together, these findings revealed that 2-methoxystypandrone (5) isolated from RJH could potentially serve as an early lead compound for leukemia treatment involving intracellular signaling by increasing mitochondrial ROS and exerting anti-proliferative effects.

• Journal of Ginseng Research
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• v.46 no.5
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• pp.646-656
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• 2022

Background: In addition to its use as a health food, ginseng is used in cosmetics and shampoo because of its extensive health benefits. The ginsenoside, Rh2, is a component of ginseng that inhibits tumor cell proliferation and differentiation, promotes insulin secretion, improves insulin sensitivity, and shows antioxidant effects. Methods: The effects of Rh2 on cell survival, extracellular matrix (ECM) protein expression, and cell differentiation were examined. The antioxidant effects of Rh2 in UV-irradiated normal human dermal fibroblast (NHDF) cells were also examined. The effects of Rh2 on mitochondrial function, morphology, and mitophagy were investigated in UV-irradiated NHDF cells. Results: Rh2 treatment promoted the proliferation of NHDF cells. Additionally, Rh2 increased the expression levels of ECM proteins and growth-associated immediate-early genes in ultraviolet (UV)-irradiated NHDF cells. Rh2 also affected antioxidant protein expression and increased total antioxidant capacity. Furthermore, treatment with Rh2 ameliorated the changes in mitochondrial morphology, induced the recovery of mitochondrial function, and inhibited the initiation of mitophagy in UV-irradiated NHDF cells. Conclusion: Rh2 inhibits mitophagy and reinstates mitochondrial ATP production and membrane potential in NHDF cells damaged by UV exposure, leading to the recovery of ECM, cell proliferation, and antioxidant capacity.

• Food Science of Animal Resources
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• v.24 no.2
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• pp.151-155
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• 2004

This study was performed to develop the method of differentiation fresh and frozen meat by using the measurement of mitochondrial malate dehydrogenase. The principle of this experiment is based on the fact the enzyme proteins associated with mitochondria membrane could be released by freezing. The methods were studied by measurements of protein concentration of meat press juice, WHC (water-holding capacity), drip loss and mitochondrial malate dehydrogenase enzyme activity. Samples were stored at 4$^{\circ}C$ and -18$^{\circ}C$ during storage period, respectively. Protein concentration of meat press juice was ranged from 8.5 mg/mL to 12.7 mg/mL and increased by freezing below at -18$^{\circ}C$(p<0.05). The WHC was not significantly different between fresh meat and frozen chicken meat (p＞0.05). The amount of drip loss of fresh and frozen chicken meat at 4$^{\circ}C$ and -18$^{\circ}C$ was not significantly different (p＞0.05). Mitochondrial malate dehydrogenase activity of frozen meat (-18$^{\circ}C$) was significantly higher (p＜0.05) than that of fresh meat. Also, enzyme activity of frozen meat was maintained at the same level after 3 minutes reaction. But fresh meat had not this reaction. From these results, it suggests that mitochondrial malate dehydrogenase can be used as a promising enzyme to differentiate between fresh and frozen meat.

• 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.

• Applied Microscopy
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• v.40 no.4
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• pp.261-266
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• 2010

ATP is the energy source synthesized at the electron transferase that consist of complex I, II, III, IV and V in mitochondrial cristae. The complex V functions as ATPase which composed of sub-complex $F_0$ and $F_1$. Porin or VDAC (voltagedependent anion-selective channel), is a family of small pore-forming proteins of the mitochondrial outer membrane, and play important roles in the regulated flux of anion, proton and metabolites between the cytosolic and mitochondrial compartments. The channel allows the diffusion of negatively charged solutes such as succinate, malate, and ATP in the fully open state, but of positively charged ions in subconducting state. In this study, in order to investigate the relationship of the function and localization between porin and ATPase we observed the distribution of porin and ATPase in the mitochondria of the bovine heart. Monoclonal antibodies against porin and ATPase ${\beta}$-subunit were used to detect porin and ATPase using light microscope with immunohistochemistry and immunofluorescence, and using electron microscope with immunogold-labeling. ATPase were stained in longitudinal section region in cardiac muscle, porin were stained in longitudinal section region in cardiac muscle. We viewed more specific pattern of localization and distribution of these proteins using immunofluorescence method. There were some region which were labeled with porin or ATPase respectively, and others which were labeled both proteins in cardiac muscle. The electron microscope results showed that immunogold labeled porin were labeled locally at mitochondrial outer membrane and ATPase were labeled evenly at mitochondrial cristae. But ATPase was not labeled at mitochondria cristae. These results confirmed the subcellular localizations of porin and ATPase in mitochondrial outer membrane and cristae. Also, we assumed that ATP synthesis always does not activation in all mitochondria exist in the bovine cardiac muscle.

• Molecules and Cells
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• v.27 no.3
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• pp.321-327
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• 2009

Voltage-dependent anion channels (VDACs) are reported to be porin-type, ${\beta}$-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.

• CELLMED
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• v.3 no.1
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• pp.9.1-9.10
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• 2013

In traditional systems of medicine including homeopathy, the Condurango extract (Con) is often used to cure stomach cancer mainly, without having any scientific validation of its anti-cancer ability. Con has therefore been tested against non-small-cell lung cancer cells (NSCLC) A549 and NCI-H522 (H522) known to contain the KRAS mutation, making them resistant to most chemotherapeutic agents. As cancer cells generally defy cytotoxicity developed by chemopreventive agents and escape cell death, any drug showing the capability of preferentially killing cancer cells through apoptosis is worth consideration for judicious application. A549 and H522 cells were exposed to $0.35{\mu}g/{\mu}l$ and $0.25{\mu}g/{\mu}l$ of Con, respectively, for 48 h and analysed based on various protocols associated with apoptosis and DNA damage, such as MTT assay to determine cell viability, LDH assay, DNA fragmentation assay, comet assay, and microscopical examinations of DNA binding fluorescence stains like DAPI, Hoechst 33258 and acridine orange/ethidium bromide to determine the extent of DNA damage made in drug-treated and untreated cells and the results compared. Changes in mitochondrial membrane potential and the generation of reactive oxygen species were also documented through standard techniques. Con killed almost 50% of the cancer cells but spared normal cells significantly. Fluorescence studies revealed increased DNA nick formation and depolarized membrane potentials after drug treatment in both cell types. Caspase-3 expression levels confirmed the apoptosis-inducing potential of Con in both the NSCLC lines. Thus, overall results suggest considerable anticancer potential of Con against NSCLC in vitro, validating its use against lung cancer by practitioners of traditional medicine including homeopathy.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.28-37
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• 2016

Numerous plants have been documented to contain phenolic compounds. Thymol is one among these phenolic compounds that possess a repertoire of pharmacological activities, including anti-inflammatory, anticancer, antioxidant, antibacterial, and antimicrobial effects. Despite of the plethora of affects elicited by thymol, its activity profile on gastric cancer cells is not explored. In this study, we discovered that thymol exerts anticancer effects by suppressing cell growth, inducing apoptosis, producing intracellular reactive oxygen species, depolarizing mitochondrial membrane potential, and activating the proapoptotic mitochondrial proteins Bax, cysteine aspartases (caspases), and poly ADP ribose polymerase in human gastric AGS cells. The outcomes of this study displayed that thymol, via an intrinsic mitochondrial pathway, was responsible for inducing apoptosis in gastric AGS cells. Hence, thymol might serve as a tentative agent in the future to treat cancer.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1627-1635
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• 2013

Mitochondria often play central roles in apoptotic pathways, and disruption of the mitochondrial transmembrane potential (${\Delta}{\psi}m$) has been observed in various cells undergoing apoptosis. Human cytomegalovirus (HCMV) infection induces apoptosis in permissive cells; however, investigations of mitochondria-targeted apoptosis in HCMV-infected human foreskin fibroblast (HFF) cells have been limited. Here, we investigated the mitochondrial apoptosis pathway in HCMV-infected HFF cells. Flow cytometry analysis using JC-1 revealed that HCMV infection induces disruption of ${\Delta}{\psi}m$ in HFF cells when administered 24 h post-infection (hpi), and this disruption was maximized at 48 hpi. Moreover, cytochrome c, normally a mitochondrial inner membrane protein, was detected in cytoplasmic extracts of HCMV-infected cells, but not mock-infected cells, by western blot analysis at 24 hpi. A caspase activity assay based on fluorescence spectrophotometry using a fluorogenic substrate revealed an increase in caspase-3 activity at 48 hpi in HCMV-infected cells. Caspase-8 activity was increased at 72 hpi in HCMV-infected cells. These results imply that HCMV infection induces mitochondria-mediated apoptosis in HFF cells.

• Bulletin of the Korean Chemical Society
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• v.31 no.12
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• pp.3623-3631
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• 2010

We prepared several novel molecular transporters built on myo- and scyllo-inositol scaffolds with variations in the number of guanidine residues, linker chain lengths and patterns. Some of these transporters were found to localize in mitochondria, and the mitochondrial affinity seems to be substantially related to the scaffold stereochemistry.