• Clinical and Experimental Pediatrics
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• v.45 no.9
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• pp.1106-1113
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• 2002

Purpose : Nuclear ($factor-{\kappa}BNF-{\kappa}B$) is now recognized as playing a potential role in programmed cell death and the adaptive response to various stress. Cellular hypoxia is a primary manifestation of many cardiovascular diseases. It seems that vascular endothelial growth factor (VEGF) and insulin like growth factor-I(IGF-I) have a function as a protective molecule in the heart against several stress including hypoxia. In this study, the role of $NF-{\kappa}B$ to the cellular response and regulation of protective molecules against the acute hypoxia in the heart was studied. Methods : To cause acute hypoxic stress to the heart, Sprague Dawley rats were exposed to hypoxic chamer($N_2$ 92% and $O_2$ 8%). After the hypoxic exposure, nuclear proteins, total proteins and mRNA were isolated from heart. Translocation of the transcription factors $NF-{\kappa}B$, NF-ATc, AP-1 and NKX-2.5 were evaluated by electrophoretic mobility shift assay(EMSA). The expression of IGF-I and VEGF were studied before and after the hypoxic stress by competitive-PCR, Northern hybridization and Western hybridization. To confirm the role of the $NF-{\kappa}B$ in the heart, the rats also were pretreated with diethyl-dithiocarbamic acid(DDTC) into peritoneal cavity to block $NF-{\kappa}B$ translocation into nucleus. Results : The expression of $NF-{\kappa}B$, AP-1 and NF-ATc were increased by the hypoxic stress. Increased expression of the VEGF and IGF-I were also observed by the hypoxic stress. However, the blocking of the $NF-{\kappa}B$ translocation reduced those expressions of VEGF and IGF-I. Conclusion : These results suggest that $NF-{\kappa}B$ has a protective role against the acute hypoxia through several gene expression, especially VEGF and IGF-I in heart muscle.

• Biomolecules & Therapeutics
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• v.28 no.3
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• pp.240-249
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• 2020

Despite the therapeutic effect of mesenchymal stem cells (MSCs) in ischemic diseases, pathophysiological conditions, including hypoxia, limited nutrient availability, and oxidative stress restrict their potential. To address this issue, we investigated the effect of melatonin on the bioactivities of MSCs. Treatment of MSCs with melatonin increased the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). Melatonin treatment enhanced mitochondrial oxidative phosphorylation in MSCs in a PGC-1α-dependent manner. Melatonin-mediated PGC-1α expression enhanced the proliferative potential of MSCs through regulation of cell cycle-associated protein activity. In addition, melatonin promoted the angiogenic ability of MSCs, including migration and invasion abilities and secretion of angiogenic cytokines by increasing PGC-1α expression. In a murine hindlimb ischemia model, the survival of transplanted melatonin-treated MSCs was significantly increased in the ischemic tissues, resulting in improvement of functional recovery, such as blood perfusion, limb salvage, neovascularization, and protection against necrosis and fibrosis. These findings indicate that the therapeutic effect of melatonin-treated MSCs in ischemic diseases is mediated via regulation of PGC-1α level. This study suggests that melatonin-induced PGC-1α might serve as a novel target for MSC-based therapy of ischemic diseases, and melatonin-treated MSCs could be used as an effective cell-based therapeutic option for patients with ischemic diseases.

• BMB Reports
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• v.48 no.8
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• pp.473-478
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• 2015

The NEK6 (NIMA-related kinases 6) is reported to play po-tential roles in tumorigenesis. Although it is suggested to function in several cellular pathways, the underlying mechanism in tumorigenesis is still largely unknown. In the present study, we discovered interaction of NEK6 with Smad4, a key member of transforming growth factor beta (TGFβ) pathway. Over-expression of NEK6 in hepatocellular carcinoma (HCC) cell lines suppresses TGFβ-mediated transcription activity in a kinase activity-dependent manner. In addition, NEK6 suppresses the cell growth arrest induced by TGFβ. Mechanically, NEK6 blocks nuclear translocation of Smad4, which is essential for TGFβ function. Moreover, we identified that NEK6 could be regulated by TGFβ and hypoxia. Our study sheds new light on the roles of NEK6 in canonical TGFβ/Smad pathway and tum-origenesis. [BMB Reports 2015; 48(8): 473-478]

• Development and Reproduction
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• v.17 no.2
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• pp.99-109
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• 2013

Trophoblasts, in the placenta, play a role for placental development as well as implantation in the early pregnancy. The characteristics and functions of trophoblast are identified by their localization and potency for proliferation, differentiation, and invasion. Thus, inadequate trophoblast cell death induces trophoblast dysfunction resulting in abnormal placental development and several gynecological diseases. Recently, it was reported that increased immortalization-upregulated protein-2 (IMUP-2) by hypoxia influences trophoblast apoptosis. However, IMUP-2 function on autophagy, which is type II programmed cell death remains unclear. In this study, we analyzed IMUP-2 expression in trophoblast cells (HTR8-SVneo) and compared IMUP-2 effects on cell death including apoptosis and autophagy in trophoblast regardless of IMUP-2 expression. Increased IMUP-2 in trophoblast by IMUP-2 gene transfection induces cell death, especially, apoptosis increases more than autophagy (p<0.05). However, the decreased IMUP-2 in trophoblasts after siRNA treatment decreased apoptosis with the decreased activities of caspase 3 and 7. The expressions of LC3 and MDC as an autophagosome makers and phosphorylated mTOR, which is a negative regulator for autophagy, increased. In addition, the S phase of cell cycle increased in trophoblasts when IMUP-2 expression decreased. Taken together, the alteration of IMUP-2 can control the balance between apoptosis and autophagy of trophoblasts resulting in functional involvement in placental development and in gynecological diseases by regulating the function of trophoblasts.

• Biomolecules & Therapeutics
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• v.24 no.3
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• pp.260-267
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• 2016

Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes increased in a time-dependent manner. After re-attachment of MSC spheroids to the plastic dish, their adhesion capacity and morphology were not altered. MSC spheroids showed enhanced production of hypoxia-induced angiogenic cytokines such as vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF), and hepatocyte growth factor (HGF). In addition, spheroid culture promoted the preservation of extracellular matrix (ECM) components, such as laminin and fibronectin, in a culture time- and spheroid size-dependent manner. Furthermore, phosphorylation of AKT, a cell survival signal, was significantly higher and the expression of pro-apoptotic molecules, poly (ADP ribose) polymerase-1 (PARP-1) and cleaved caspase-3, was markedly lower in the spheroids than in MSCs in monolayers. In the murine hindlimb ischemia model, transplanted MSC spheroids showed better proliferation than MSCs in monolayer. These findings suggest that MSC spheroids promote MSC bioactivities via secretion of angiogenic cytokines, preservation of ECM components, and regulation of apoptotic signals. Therefore, MSC spheroid-based cell therapy may serve as a simple and effective strategy for regenerative medicine.

• Biomolecules & Therapeutics
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• v.26 no.5
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• pp.464-473
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• 2018

Cripto is a small glycosylphosphatidylinositol-anchored signaling protein that can detach from the anchored membrane and stimulate proliferation, migration, differentiation, vascularization, and angiogenesis. In the present study, we demonstrated that Cripto positively affected proliferation and survival of mesenchymal stem cells (MSCs) without affecting multipotency. Cripto also increased expression of phosphorylated janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), 78 kDa glucose-regulated protein (GRP78), c-Myc, and cyclin D1. Notably, treatment with an anti-GRP78 antibody blocked these effects. In addition, pretreatment with STAT3 short interfering RNA (siRNA) inhibited the increase in p-JAK2, c-Myc, cyclin D1, and BCL3 levels caused by Cripto and attenuated the pro-survival action of Cripto on MSCs. We also found that incubation with Cripto protected MSCs from apoptosis caused by hypoxia or $H_2O_2$ exposure, and the level of caspase-3 decreased by the Cripto-induced expression of B-cell lymphoma 3-encoded protein (BCL3). These effects were sensitive to down-regulation of BCL3 expression by BCL3 siRNA. Finally, we showed that Cripto enhanced expression levels of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). In summary, our results demonstrated that Cripto activated a novel biochemical cascade that potentiated MSC proliferation and survival. This cascade relied on phosphorylation of JAK2 and STAT3 and was regulated by GRP78. Our findings may facilitate clinical applications of MSCs, as these cells may benefit from positive effects of Cripto on their survival and biological properties.

• Journal of The Korean Dental Society of Anesthesiology
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• v.14 no.1
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• pp.41-47
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• 2014

Background: Autophagy is a self-eating process that is important for balancing sources of energy at critical times in development and in response stress. Autophagy also plays a protective role in removing clearing damaged intracellular organelles and aggregated proteins as well as eliminating intracellular pathogens. The purpose of the present study was to examine the protective effect of propofol against hypoxic damage using keratinocytes. Methods: Human keratinocytes (HaCaT cells) were obtained from the American Type Culture Collection. Propofol which were made by dissolving them in DMSO were kept frozen at $-4^{\circ}C$ until use. The stock was diluted to their concentration with DMEM when needed. Prior to propofol treatment cells were grown to about 80% confluence and then exposed to propofol at different concentrations (0, 25, 50, 75, $100{\mu}M$) for 2 h pretreatment. Cell viability was measured using a quantitative colorimetric assay with thiazolyl blue tetrazolium bromide (MTT assay), and fluorescence microscopy and western blot analysis were used for evaluation of autophagy processes. Results: The viability of propofol-treated HaCaT cells was increased in a dose-dependent manner. Propofol did not show any significant toxic effect on the HaCaT cells. The autophagy inhibitor, 3-methyladenine, reduced cell viability of hypoxia-injured HaCat cells. Fluorescence microscopy and western blot analysis showed propofol induce autophagy pathway signals. Conclusions: Propofol enhanced viability of hypoxia-injured HaCaT cells and we suggest propofol has cellular protective effects by autophagy signal pathway activation.

• Journal of Acupuncture Research
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• v.20 no.2
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• pp.173-183
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• 2003

이 논문(論文)은 활성 산소(ROS)의 작용(作用)을 규명하고 호도약침액(胡桃藥鍼液)이 인간의 신경교종 세포인 A172에서 화학적(化學的) 저산소증(低酸素症)으로 유발된 세포 사멸에 대해 효능이 있는지를 연구(硏究)한 것이다. 화학적(化學的) 저산소증(低酸素症)은 세포내 미토콘드리아의 전자 수송을 방해하는 antimycin A를 가진 배양세포에 의해 유발(誘發)하였다. 화학적(化學的) 저산소증(低酸素症)에 노출된 세포(細胞)는 시간과 그 양에 따라서 세포 사멸의 결과(結果)가 다르게 나타난다. 화학적 저산소증에 의해서 ROS의 생산이 증가하는데 이것은 $H_2O_2$ 소거(消去) Catalase(과산화수소를 물과 산소로 분해하는 효소)에 의해 방지(防止)된다. Catalase는 화학적 저산소증에 의해 유발(誘發)된 세포 사멸을 방지하는데 비해 DMTU는 효과적이지 않다. 지질(脂質)에 녹는 산화방지제 DPPD와 물에 녹는 산화방지제 Trolox는 세포사멸을 방지하는데 효과(效果)가 없다. 호도약침액(胡桃藥鍼液)은 그 양(量)에 의존적으로 저산소증에 의해 유발된 세포 사멸을 방지하는 효과가 있다. 즉 화학적 저산소증으로 유도된 ROS의 발생을 막고, $H_2O_2$로 유도된 세포사멸을 방지하는데 이것은 화학적 저산소증과 $H_2O_2$의해 유도된 세포사멸에 대해 호도약침액(胡桃藥鍼液)이 방지효과(防止效果)가 있다는 것을 의미한다. 이러한 결과(結果)들은 $H_2O_2$가 지질 과산화와는 무관한 메카니즘으로 저산소증(低酸素症)으로 유발(誘發)된 세포사멸을 중재하고, 따라서 호도약침액(胡桃藥鍼液)은 지질막의 과산화를 방지하기 보다는 ROS를 직접적으로 소거(消去)함으로써 방지 효과가 있다는 것을 의미한다. 더구나 화학적(化學的) 저산소증(低酸素症)은 caspase와 무관한 메카니즘으로 apoptosis를 유발(誘發)한다.

• Transactions of the Korean Society of Mechanical Engineers B
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• v.36 no.5
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• pp.533-538
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• 2012

In this study, we used an $O_2$-sensitive luminescent dye to measure the $O_2$ concentration of culture media around HeLa cells cultured in a microchannel. $[Ru(bpy)_3]^{2+}$, which dissolves easily in water and which has no phototoxic effect, was used as the $O_2$-sensitive dye. The ratiometric sensing method was applied by introducing calcein as the $O_2$-insensitive dye, in order to overcome the disadvantages of intensity-based sensing. By performing calibration with an amperometric $O_2$ sensor, we could calculate the exact concentration of $O_2$ in the culture media. We applied this technique to measure the $O_2$ concentration around the cultured cells in the microchannel. As expected, the $O_2$ concentration gradually decreased as the cells moved farther away from the channel. This method is expected to be applicable to the investigation of hypoxia, which occurs commonly in scaffolds.

• BMB Reports
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• v.34 no.4
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• pp.385-389
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• 2001

Insulin-like growth factor binding protein-1 (IGFBP-1) appears to be an important modular of the insulin growth factor (IGF) bioactivity in metabolic disease and chronic hypoxia. Treatment of desferrioxamine (Dfo), cobalt, or nickel in HepG2 cells stimulated the expression of IGFBP1 mRNA as hypoxia. However, the presence of ferric ammonium citrate (FAC) in the 1% $O_2$ decreased the upregulation of the IGFBP-1 mRNA expression. In addition, actinomycin D and cycloheximide abolished the increase in the expression of IGFBP-1 mRNA that was induced by Dfo and transition metals (cobalt and nickel). To obtain further information about the putative oxygen sensor, we postulate that putative heme proteins, responsible for the oxygen-sensing process in HepG2 cells, should be sensitive to hypoada. The mechanism of these upregulations of the IGFBP-1 mRNA expression by Dfo and transition metals was investigated by treatment with 2 mM of 4,6-dioxoheptanoic acid (DHA), an inhibitor of heme biosynthesis. The results showed that 1% $O_2$-, Dfo-, cobalt-, or nickel induced IGFBP-1 mRNA expressions in HepG2 cells were all markedly inhibited when the heme synthesis was blocked by DHA. We suggest that the IGFBP-1 mRNA expression in the HepG2 cell is regulated by 1% $O_2$, Dfo, cobalt, or nickel, implicating the involvement of the putative heme-containing oxygensensing molecule.