• 동의생리병리학회지
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• 제22권5호
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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.

• The Korean Journal of Physiology and Pharmacology
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• 제3권5호
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• pp.521-528
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• 1999

Reactive oxygen species (ROS), generated by infiltrating neutrophils, are considered as an important regulator in the pathogenesis and deveolpment of pancreatitis. The present study aims to investigate whether neutrophils primed by $4{\beta}-phorbol\;12{\beta}-myristate\;13{\alpha}-acetate$ (PMA) affect the productions $H_2O_2$ and lipid peroxide (LPO), $NF-_{\kappa}B$ activation and cytokine production in pancreatic acinar cells, and whether these alterations were inhibited by an antioxidant, N-acetylcysteine (NAC) and superoxide dismutase (SOD). $H_2O_2$ (ferrithiocyanate method), LPO (as thiobarbiturate reactive substances), and cytokines $(IL-l{\bata},\; IL-6,\;TNF-{\alpha};\;enzyme-linked\;immunosorbent\;assay)$ and $NF-_{\kappa}B$ activation (electrophoretic mobility shift assay) were analyzed in acinar cells treated with or without PMA-primed neutrophils in the absence or presence of NAC (10 mM) or SOD (300 U/ml). As a result, the productions of H2O2, LPO and $TNF-{\alpha}$ were increased with the ratio of PMA-primed neutrophils to acinar cells while the productions of LPO, $IL-l{\beta},\;IL-6\;and\;TNF-{\alpha}$ were increased with time. PMA-primed neutrophils resulted in the activation of $NF-_{\kappa}B.$ Both NAC and SOD inhibited neutrophil-induced alterations in acinar cells. In conclusion, ROS, generated by neutrophils, activates $NF-_{\kappa}B,$ resulting in upregulation of inflammatary cytokines in acinar cells. Antioxidants might be clinically useful antiinflammatory agents by inhibiting oxidant-mediated activation of $NF-_{\kappa}B$ and decreasing cytokine production.

• Archives of Pharmacal Research
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• 제28권3호
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• pp.311-318
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• 2005

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC­PK1 cells were treated with H$_2$O$_2$ in the absence of serum to induce cell death. Subsequent to exposure to H$_2$O$_2$, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H202-treated control cells, it was observed that 0.5 $\mu$M of desipramine and 25 $\mu$M of NAC exhibited about 90 and $95\%$ of cytoprotection, respectively, against H$_2$O$_2$-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and $3\%$ respectively, when compared to $71\%$ in H$_2$O$_2$-exposed cells. Cellular glutathione level in 500 $\mu$M H202-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H$_2$O$_2$-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H$_2$O$_2$-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

• Environmental Analysis Health and Toxicology
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• 제21권3호
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• pp.275-282
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• 2006

In order to get insight into the mechanism of cadmium (Cd)-induced brain injury, we investigated the effects of Cd on the induction of COX-2 in bEnd.3 mouse brain endothelial cells. Cd induced COX-2 expression and $PGE_2$ release, which were attenuated by thiol-reducing antioxidant N-acetylcysteine (NAC) indicating oxidative components might contribute to these events. Indeed, Cd increased cellular reactive oxygen species (ROS) level and DNA binding activity of nuclear factor-kB (NF-kB), an oxidative stress sensitive transcription factor. Cd-induced $PGE_2$ production and COX-2 expression were significantly attenuated by Bay 11 7082, a specific inhibitor of NF-kB and by SB203580, a specific inhibitor of p38 mitogen activated protein kinase (MAPK). These data suggest that Cd induces COX-2 expression through activation of NF-kB and p38 MAPK, the oxidative stress-sensitive signaling molecules, in brain endothelial cells.

• Journal of Radiation Protection and Research
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• 제34권2호
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• pp.49-54
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• 2009

Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging and also contributes to their unfavorable effects in cultured cells and animal tissues. This study was conducted to investigate the effect of ionizing radiation (IR) on mtDNA deletion and the involvement of reactive oxygen species (ROS) in this process in human lung fibroblast (IMR-90) cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated with $^{137}Cs$ $\gamma$-rays and the intracellular ROS level was determined by 2',7'-dichlorofluorescein diacetate (DCFH-DA) and mtDNA common deletion (4977bp) was detected by nested PCR. Old cells at PD 55 and $H_2O_2$-treated young cells were compared as the positive control. IR increased the intracellular ROS level and mtDNA 4977 bp deletion in IMR-90 cells dose-dependently. The increases of ROS level and mtDNA deletion were also observed in old cells and $H_2O_2$-treated young cells. To confirm the increased ROS level is essential for mtDNA deletion in irradiated cells, the effects of N-acetylcysteine (NAC) on IRinduced ROS and mtDNA deletion were examined. 5 mM NAC significantly attenuated the IR-induced ROS increase and mtDNA deletion. These results suggest that IR induces the mtDNA deletion and this process is mediated by ROS in IMR-90 cells.

• Journal of Radiation Protection and Research
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• 제36권3호
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• pp.119-126
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• 2011

Mitochondrial DNA (mtDNA) deletion is a well-known marker for oxidative stress and aging, and contributes to harmful effects in cultured cells and animal tissues. mtDNA biogenesis genes (NRF-1, TFAM) are essential for the maintenance of mtDNA, as well as the transcription and replication of mitochondrial genomes. Considering that oxidative stress is known to affect mitochondrial biogenesis, we hypothesized that ionizing radiation (IR)-induced reactive oxygen species (ROS) causes mtDNA deletion by modulating the mitochondrial biogenesis, thereby leading to cellular senescence. Therefore, we examined the effects of IR on ROS levels, cellular senescence, mitochondrial biogenesis, and mtDNA deletion in IMR-90 human lung fibroblast cells. Young IMR-90 cells at population doubling (PD) 39 were irradiated at 4 or 8 Gy. Old cells at PD55, and H2O2-treated young cells at PD 39, were compared as a positive control. The IR increased the intracellular ROS level, senescence-associated ${\beta}$-galactosidase (SA-${\beta}$-gal) activity, and mtDNA common deletion (4977 bp), and it decreased the mRNA expression of NRF-1 and TFAM in IMR-90 cells. Similar results were also observed in old cells (PD 55) and $H_2O_2$-treated young cells. To confirm that a increase in ROS level is essential for mtDNA deletion and changes of mitochondrial biogenesis in irradiated cells, the effects of N-acetylcysteine (NAC) were examined. In irradiated and $H_2O_2$-treated cells, 5 mM NAC significantly attenuated the increases of ROS, mtDNA deletion, and SA-${\beta}$-gal activity, and recovered from decreased expressions of NRF-1 and TFAM mRNA. These results suggest that ROS is a key cause of IR-induced mtDNA deletion, and the suppression of the mitochondrial biogenesis gene may mediate this process.

• The Korean Journal of Physiology and Pharmacology
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• 제11권1호
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• pp.9-13
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• 2007

Antioxidant properties have been proposed as a mechanism for the putative anti-inflammatory effects of phenolic compounds. To reveal the relationship between antioxidant activity and anti-inflammatory effects of various antioxidants, we measured 1, 1-diphenyl-2-picryhydrazyl(DPPH)-reducing activity and examined the inhibitory effects on LPS-induced inflammation-related gene expression in the BV2 microglial cell line. Lipopolysaccharide(LPS)(0.2 ${\mu}g/ml$) was used with or without antioxidants to treat cells, and the regulation of iNOS and cytokine gene expression was monitored using an RNase protection assay(RPA). Although, all tested antioxidants had similar DPPH-reducing activity and inhibited nitrite production, but the curcuminoid antioxidants(ferulic acid, caffeic acid, and curcumin) inhibited LPS-induced gene expression(iNOS, $TNF-\alpha,\;IL-1{\beta}$, IL-6, and IL-1 Ra) in a concentration-dependent manner. Other tested antioxidants did not exhibit the same effects; N-acetylcysteine(NAC) only began to suppress $IL-1{\beta}$ gene expression just below the concentration at which cytotoxicity occurred. Moreover, the antioxidant potency of curcuminoids appeared to have no correlation with anti-inflammatory potency. Only curcumin could inhibit LPS-induced microglial activation at a micromolar level. These data suggest that curcumin may be a safe antioxidant possessing anti-inflammatory activity.

• The Korean Journal of Physiology and Pharmacology
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• 제24권6호
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• pp.493-502
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• 2020

Apigenin, a naturally occurring flavonoid, is known to exhibit significant anticancer activity. This study was designed to determine the effects of apigenin on two malignant mesothelioma cell lines, MSTO-211H and H2452, and to explore the underlying mechanism(s). Apigenin significantly inhibited cell viability with a concomitant increase in intracellular reactive oxygen species (ROS) and caused the loss of mitochondrial membrane potential (ΔΨm), and ATP depletion, resulting in apoptosis and necroptosis in monolayer cell culture. Apigenin upregulated DNA damage response proteins, including the DNA double strand break marker phospho (p)-histone H2A.X. and caused a transition delay at the G₂/M phase of cell cycle. Western blot analysis showed that apigenin treatment upregulated protein levels of cleaved caspase-3, cleaved PARP, p-MLKL, and p-RIP3 along with an increased Bax/Bcl-2 ratio. ATP supplementation restored cell viability and levels of DNA damage-, apoptosisand necroptosis-related proteins that apigenin caused. In addition, N-acetylcysteine reduced ROS production and improved ΔΨm loss and cell death that were caused by apigenin. In a 3D spheroid culture model, ROS-dependent necroptosis was found to be a mechanism involved in the anti-cancer activity of apigenin against malignant mesothelioma cells. Taken together, our findings suggest that apigenin can induce ROS-dependent necroptotic cell death due to ATP depletion through mitochondrial dysfunction. This study provides us a possible mechanism underlying why apigenin could be used as a therapeutic candidate for treating malignant mesothelioma.

• Biomolecules & Therapeutics
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• 제28권5호
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• pp.473-481
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• 2020

Axl receptor tyrosine kinase has been implicated in cancer progression, invasion, and metastasis in various cancer types. Axl overexpression has been observed in many cancers, and selective inhibitors of Axl, including R428, may be promising therapeutic agents for several human cancers, such as breast, lung, and pancreatic cancers. Here, we examined the cell growth inhibition mediated by R428 and auranofin individually as well as in combination in the human breast cancer cell lines MCF-7 and MDA-MB-231 to identify new advanced combination treatments for human breast cancer. Our data showed that combination therapy with R428 and auranofin markedly inhibited cancer cell proliferation. Isobologram analyses of these cells indicated a clear synergism between R428 and auranofin with a combination index value of 0.73. The combination treatment promoted apoptosis as indicated by caspase 3 activation and poly (ADP-ribose) polymerase cleavage. Cancer cell migration was also significantly inhibited by this combination treatment. Moreover, we found that combination therapy significantly increased the expression level of Bax, a mitochondrial proapoptotic factor, but decreased that of the X-linked inhibitor of apoptosis protein. Furthermore, the suppression of cell viability and induction of Bax expression by the combination treatment were recovered by treatment with N-acetylcysteine. In conclusion, our data demonstrated that combined treatment with R428 and auranofin synergistically induced apoptosis in human breast cancer cells and may thus serve as a novel and valuable approach for cancer therapy.

• 한국식품과학회지
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• 제53권4호
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• pp.463-469
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• 2021

본 연구에서는 curcumin과 관련 구조물질인 ferulic acid, DBM, THC을 사용하여 이들의 산화방지활성과 세포독성을 나타내는 활성과 구조 간의 연관성을 분석하였다. 각종 라디칼 소거활성과 지질산화 억제 반응에서 DBM은 거의 활성을 나타내지 못해, methoxy phenolic기가 중요한 기능 구조로 나타났으며 α, β-unsaturated carbonyl기도 이들의 산화방지 활성에 일부 관여하는 것으로 보인다. Curcumin 유도체들의 세포독성과 이들의 산화방지활성 간에는 연관성이 거의 없었으며, ROS의 생성에는 α, β-unsaturated carbonyl기가 중요한 역할을 담당하나 세포독성의 직접적인 원인이 아닌 것으로 판단된다. 세포독성 유발에는 β-diketone 구조가 중요한 역할을 하였으며, SOD/catalase 등에 의한 구조의 안정화가 세포독성을 더욱 강화시키는 것으로 판단된다. Curcumin과 ferulic acid의 조합처리에 의해 독성이 증가한 반면, DBM과 curcumin을 같이 처리 시 독성이 상쇄되었으며 THC과 curcumin은 서로 부가적인 세포독성을 나타냈다.