• The Korean Journal of Physiology and Pharmacology
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• 제21권3호
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• pp.327-334
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• 2017

Epidemiologic interest in particulate matter (PM) is growing particularly because of its impact of respiratory health. It has been elucidated that PM evoked inflammatory signal in pulmonary epithelia. However, it has not been established $Ca^{2+}$ signaling mechanisms involved in acute PM-derived signaling in pulmonary fibroblasts. In the present study, we explored dust particles PM modulated intracellular $Ca^{2+}$ signaling and sought to provide a therapeutic strategy by antagonizing PM-induced intracellular $Ca^{2+}$ signaling in human lung fibroblasts MRC5 cells. We demonstrated that PM10, less than $10{\mu}m$, induced intracellular $Ca^{2+}$ signaling, which was mediated by extracellular $Ca^{2+}$. The PM10-mediated intracellular $Ca^{2+}$ signaling was attenuated by antioxidants, phospholipase blockers, polyADPR polymerase 1 inhibitor, and transient receptor potential melastatin 2 (TRPM2) inhibitors. In addition, PM-mediated increases in reactive oxygen species were attenuated by TRPM2 blockers, clotrimazole (CLZ) and N-(p-amylcinnamoyl) anthranilic acid (ACA). Our results showed that PM10 enhanced reactive oxygen species signal by measuring DCF fluorescence and the DCF signal attenuated by both TRPM2 blockers CLZ and ACA. Here, we suggest functional inhibition of TRPM2 channels as a potential therapeutic strategy for modulation of dust particle-mediated signaling and oxidative stress accompanying lung diseases.

• 약학회지
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• 제52권6호
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• pp.441-445
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• 2008

To investigate effect of caffeic acid on the intracellular reactive oxygen species production, we used DHE for intracellular superoxide anion production, DCF for intracellular ${H_2}{O_2}$ production and DHR for intracellular hydroperoxide production in Raw 264.7 cells. DPPH assay showed that antioxidant activity of caffeic acid with 39.5 ${\mu}M$ of ${IC}_{50}$ values was similar to that of ascorbic acid with 41.3 ${\mu}M$ of ${IC}_{50}$ values. Caffeic acid dose-dependently inhibited silica-induced ${H_2}{O_2}$ and hydroperoxide production but did not affect superoxide anion production in Raw 264.7 cells, which suggest that antioxidant effect of caffeic acid acts on the post-step of superoxide anion. On the other hand, caffeic acid showed a potent antioxidant effect in $lCuSO_4$-induced lipid peroxidation. Furthermore, plasma superoxide dismutase activity (3.43${\pm}$0.23 U/ml) in 10 mg/kg caffeic acid-fed mice was significantly higher than that (2.32${\pm}$0.24 U/ml) of control. From the above results, it is referred that caffeic acid appears to have potent anti-oxidant activity in both cell system and in vivo system.

• Molecules and Cells
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• 제21권1호
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• pp.121-128
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• 2006

Maitotoxin (MTX) is known as one of the most potent marine toxins involved in Ciguatera poisoning, but intracellular signaling pathways caused by MTX was not fully understood. Thus, we have investigated whether intracellular reactive oxygen species (ROS) are involved in MTX-induced cellular responses in human umbilical vein endothelial cells. MTX induced a dose-dependent increase of intracellular [$Ca^{2+}$]. MTX stimulated the production of intracellular ROS in a dose- and time-dependent manner, which was suppressed by BAPTA-AM, an intracellular $Ca^{2+}$ chelator. Ionomycin also elevated the ROS production in a dose-dependent manner. MTX elevated transamidation activity in a time-dependent manner and the activation was largely inhibited by transfection of tissue transglutaminase siRNA. The activation of tissue transglutaminase and ERK1/2 by MTX was suppressed by BAPTA-AM or ROS scavengers. In addition, MTX-induced cell death was significantly delayed by BAPTA-AM or a ROS scavenger. These results suggest that [$Ca^{2+}$]-dependent generation of intracellular ROS, at least in part, play an important role in MTX-stimulated cellular responses, such as activation of tTGase, ERK phosphorylation, and induction of cell death, in human umbilical vein endothelial cells.

• The Korean Journal of Physiology and Pharmacology
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• 제6권4호
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• pp.187-191
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• 2002

Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular $Ca^{2+}$ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular $Ca^{2+}$ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a $Ca^{2+}$ activated protein, and $Ca^{2+}$/calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of $Ca^{2+}$/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular $Ca^{2+}$ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and $Ca^{2+}$/calmodulin-dependent protein kinase II in HepG2 cells.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.540-552
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• 2019

To determine the chemopreventive potential of alyssin and iberin, the in vitro anticancer activities and molecular targets of isothiocyanates (ITCs) were measured and compared to sulforaphane in hepatocellular carcinoma cell HepG2. The SR-FTIR spectra observed a similar pattern vis-a-vis the biomolecular alteration amongst the ITCs-treated cells suggesting a similar mode of action. All of the ITCs in this study cause cancer cell death through both apoptosis and necrosis in concentration dependent manner ($20-80{\mu}M$). We found no interactions of any of the ITCs studied with DNA. Notwithstanding, all of the ITCs studied increased intracellular reactive oxygen species (ROS) and suppressed tubulin polymerization, which led to cell-cycle arrest in the S and $G_2/M$ phase. Alyssin possessed the most potent anticancer ability; possibly due to its ability to increase intracellular ROS rather than tubulin depolymerization. Nevertheless, the structural influence of alkyl chain length on anticancer capabilities of ITCs remains inconclusive. The results of this study indicate an optional, potent ITC (viz., alyssin) because of its underlying mechanisms against hepatic cancer. As a consequence, further selection and development of effective chemotherapeutic ITCs is recommended.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제44회 동계 정기학술대회 초록집
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• pp.542-542
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• 2013

저온 플라즈마를 발생시키는 대기압 마이크로-플라즈마 젯(Micro-plasma jet)을 이용하여 플라즈마와 세포와의 상호작용에 대한 연구를 진행하였다. 세포의 대사과정에서 생성되는 활성산소 종(Reactive Oxygen Species, ROS)은 세포에 산화 스트레스를 유발시킨다. 이러한 스트레스는 세포 예정사(programmed cell death)의 원인이 된다. 플라즈마 형성 기체로 헬륨, 아르곤, 질소를 사용하여 각각의 기체에 따른 세포의 형태 변화 및 세포 내 활성 산소 종의 영향을 분석하였다. 실험에 사용된 세포는 인체의 폐암 세포[Human lung cancer cell, A549]이며 플라즈마 처리 후 Intracellular ROS assay를 통하여 플라즈마에서 발생되는 활성 산소 종(Reactive Oxygen Species, ROS)이 세포 내에 들어가 활성 산소 종을 증가시키는 것을 확인하였다. 이때, 플라즈마에서 발생되는 활성 산소 종(Reactive Oxygen Species, ROS)들은 광 방출 스펙트럼(Optical Emission Spectroscopy)로 분석하였고, 기체별로 비교하여 보았다. 또한, 이 때 발생되는 플라즈마의 전류-전압 특성에 따른 optical intensity를 비교하였다.

• Molecules and Cells
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• 제46권6호
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• pp.329-336
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• 2023

Reactive oxygen species (ROS) serve as secondary messengers that regulate various developmental and signal transduction processes, with ROS primarily generated by NADPH OXIDASEs (referred to as RESPIRATORY BURST OXIDASE HOMOLOGs [RBOHs] in plants). However, the types and locations of ROS produced by RBOHs are different from those expected to mediate intracellular signaling. RBOHs produce O₂^•- rather than H₂O₂ which is relatively long-lived and able to diffuse through membranes, and this production occurs outside the cell instead of in the cytoplasm, where signaling cascades occur. A widely accepted model explaining this discrepancy proposes that RBOH-produced extracellular O₂^•- is converted to H₂O₂ by superoxide dismutase and then imported by aquaporins to reach its cytoplasmic targets. However, this model does not explain how the specificity of ROS targeting is ensured while minimizing unnecessary damage during the bulk translocation of extracellular ROS (eROS). An increasing number of studies have provided clues about eROS action mechanisms, revealing various mechanisms for eROS perception in the apoplast, crosstalk between eROS and reactive nitrogen species, and the contribution of intracellular organelles to cytoplasmic ROS bursts. In this review, we summarize these recent advances, highlight the mechanisms underlying eROS action, and provide an overview of the routes by which eROS-induced changes reach the intracellular space.

• 환경생물
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• 제21권3호
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• pp.303-307
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• 2003

Tributyltin (TBT) used world-wide in antifouling paints toy ships is a wide-spread environmental pollutant. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of toxicity in reproductive organs by TBT. In this study, we investigated that the mechanisms underlying apoptosis induced by TBT in R2C cell. Effects of TBT on intracellular $Ca^{2+}$ level and reactive oxygen species (ROS) were investigated in R2C cells by fluorescence detector. TBT significantly induced intracellular $Ca^{2+}$ level in a time-dependent manner. The rise in intracellular $Ca^{2+}$ level was followed by a time-dependent generation of reactive oxygen species (ROS) at the cytosol level. Simultaneously, TBT induced the release of cytochrome c from the mitochondrial membrane into the cytosol. Furthermore, ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular $Ca^{2+}$ chelator, indicating the important role of $Ca^{2+}$ in R2C during these early intracellular events. In addition, Z-DEVD FMB, a caspase -3 inhibitor, decreased apoptosis by TBT. Taken together, the present results indicated that the apoptotic pathway by TBT might start with an increase in intracellular $Ca^{2+}$ level, continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.

• IMMUNE NETWORK
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• 제14권5호
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• pp.241-248
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• 2014

It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-${\alpha}1$ phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-${\alpha}1$-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

• International Journal of Oral Biology
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• 제35권3호
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• pp.129-135
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• 2010

Recent studies have implicated reactive oxygen species (ROS) as determinants of the pathological pain caused by the activation of peripheral neurons. It has not been elucidated, however, how ROS activate the primary sensory neurons in the pain pathway. In this study, calcium imaging was performed to investigate the effects of NaOCl, a ROS donor, on the intracellular calcium concentration ($[Ca^{2+}]i$) in acutely dissociated dorsal root ganglion (DRG) neurons. DRG was sequentially treated with 0.2 mg/ml of both protease and thermolysin, and single neurons were then obtained by mechanical dissociation. The administration of NaOCl then caused a reversible increase in the $[Ca^{2+}]i$, which was inhibited by pretreatment with phenyl-N-tertbuthylnitrone (PBN) and isoascorbate, both ROS scavengers. The NaOCl-induced $[Ca^{2+}]i$ increase was suppressed both in a calcium free solution and after depletion of the intracellular $Ca^{2+}$ pool by thapsigargin. Additionally, this increase was predominantly blocked by pretreatment with the transient receptor potential (TRP) antagonists, ruthenium red ($50\;{\mu}M$) and capsazepine ($10\;{\mu}M$). Collectively, these results suggest that an increase in the intracellular calcium concentration is produced from both extracellular fluid and the intracellular calcium store, and that TRP might be involved in the sensation of pain induced by ROS.