• Molecules and Cells
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• 제40권4호
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• pp.280-290
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• 2017

Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers ($eIF2{\alpha}$ phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in Drosophila mitigated rotenone- and MG132-induced toxicity. Taken together, these findings indicate that PTP1B inhibition may represent a novel therapeutic approach for ER stress-mediated neurodegenerative diseases.

• International Journal of Oral Biology
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• 제35권2호
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• pp.51-60
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• 2010

Few studies have evaluated the apoptosis-inducing efficacy of NaF on cancer cells in vitro but there has been no previous investigation of the apoptotic effects of NaF on human oral squamous cell carcinoma cells. In this study, we have investigated the mechanisms underlying the apoptotic response to NaF treatment in the YD9 human squamous cell carcinoma cell line. The viability of YD9 cells and their growth inhibition were assessed by MTT and clonogenic assays, respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to detect apoptosis. YD9 cells were treated with NaF, and western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, and MMP and proteasome activity assays were performed sequentially. The NaF treatment resulted in a time- and dose-dependent decrease in YD9 cell viability, a dose-dependent inhibition of cell growth, and the induction of apoptotic cell death. The apoptotic response of these cells was manifested by nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, a decreased DNA content, the release of cytochrome c into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, a significant shift of the Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF treatment resulted in the downregulation of G1 cell cyclerelated proteins, and upregulation of p53 and the Cdk inhibitor $p27^{KIP1}$. Taken collectively, our present findings demonstrate that NaF strongly inhibits YD9 cell proliferation by modulating the expression of G1 cell cycle-related proteins and inducing apoptosis via mitochondrial and caspase pathways.

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.677-682
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• 2014

Resveratrol has been examined in several model systems for potential effects against cancer. Adenosine monophosphate-activated protein kinase (AMPK) is reported to suppress proliferation in most eukaryocyte cells. Whether resveratrol via AMPK inhibits proliferation of oesophageal adenocarcinoma cells (OAC) is unknown. The aim of this study was to determine the roles of AMPK in the protective effects of resveratrol in OAC proliferation and to elucidate the underlying mechanisms. Treatment of cultured OAC derived from human subjects or cell lines with resveratrol resulted in decreased cell proliferation. Further, inhibition of AMPK by pharmacological reagent or genetical approach abolished resveratrol-suppressed OAC proliferation, reduced the level of $p27^{Kip1}$, a cyclin-dependent kinase inhibitor, and increased the levels of S-phase kinase-associated protein 2 (Skp2) of $p27^{Kip1}$-E3 ubiquitin ligase and 26S proteasome activity reduced by resveratrol. Furthermore, gene silencing of $p27^{Kip1}$ reversed resveratrol-suppressed OAC proliferation. In conclusion, these findings indicate that resveratrol inhibits Skp2-mediated ubiquitylation and 26S proteasome-dependent degradation of $p27^{Kip1}$ via AMPK activation to suppress OAC proliferation.

• Molecules and Cells
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• 제24권2호
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• pp.210-214
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• 2007

The 90-kDa heat shock protein (HSP90) normally functions as a molecular chaperone participating in folding and stabilizing newly synthesized proteins, and refolding denatured proteins. The HSP90 inhibitor geldanamycin (GA) occupies the ATP/ADP binding pocket of HSP90 so inhibits its chaperone activity and causes subsequent degradation of HSP90 client proteins by proteasomes. Here we show that GA reduces the level of endogenous c-Jun in human embryonic kidney 293 (HEK293) cells in a time and dose dependent manner, and that this decrease can be reversed by transfection of HSP90 plasmids. Transfection of HSP90 plasmids in the absence of GA increases the level of endogenous c-Jun protein, but has no obvious affect on c-Jun mRNA levels. We also showed that HSP90 prolongs the half-life of c-Jun by stabilizing the protein; the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocks the degradation of c-Jun promoted by GA. Transfection of HSP90 plasmids did not obviously alter phosphorylation of c-Jun, and a Jun-2 luciferase activity assay indicated that over-expression of HSP90 elevated the total protein activity of c-Jun in HEK293 cells. All our evidence indicates that HSP90 stabilizes c-Jun protein, and so increases the total activity of c-Jun in HEK293 cells.

• BMB Reports
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• 제48권3호
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• pp.166-171
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• 2015

We previously demonstrated that resveratrol and clofarabine elicited a marked cytotoxicity on malignant mesothelioma (MM) MSTO-211H cells but not on the corresponding normal mesothelial MeT-5A cells. Little is known of the possible molecules that could be used to predict preferential chemosensitivity on MSTO-211H cells. Resveratrol and clofarabine induced downregulation of Mcl-1 protein level in MSTO-211H cells. Treatment of cells with cycloheximide in the presence of proteasome inhibitor MG132 suggested that Mcl-1 protein levels were regulated at the post-translational step. The siRNA-based knockdown of Mcl-1 in MSTO-211H cells triggered more growth-inhibiting and apoptosis-inducing effects with the resultant cleavages of procaspase-3 and its substrate PARP, increased caspase-3/7 activity, and increased percentage of apoptotic propensities. However, the majority of the observed changes were not shown in MeT-5A cells. Collectively, these studies indicate that the preferential activation of caspase cascade in malignant cells might have important applications as a therapeutic target for MM.

• 한국자원식물학회지
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• 제30권3호
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• pp.265-271
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• 2017

In this study, we elucidated the molecular mechanism of silymarin by which silymarin may inhibits cell proliferation in human colorectal cancer cells in order to search the new potential anti-cancer target associated with the cell growth arrest. Silymarin reduced the level of c-Myc protein but not mRNA level indicating that silymarin-mediated downregulation of c-Myc may result from the proteasomal degradation. In the confirmation of silymarin-mediated c-Myc degradation, MG132 as a proteasome inhibitor attenuated c-Myc degradation by silymarin. In addition, silymarin phosphorylated the threonine-58 (Thr58) of c-Myc and the point mutation of Thr58 to alanine blocked its degradation by silymarin, which indicates that Thr58 phosphorylation may be an important modification for silymarin-mediated c-Myc degradation. We observed that the inhibition of ERK1/2, p38 and $GSK3{\beta}$ blocked the Thr58 phosphorylation and subsequent c-Myc degradation by silymarin. Finally, the point mutation of Thr58 to alanine attenuated silymarin-mediated inhibition of the cell growth. The results suggest that silymarin induces the cell growth arrest through c-Myc proteasomal degradation via ERK1/2, p38 and $GSK3{\beta}-dependent$ Thr58 phosphorylation.

• 생명과학회지
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• 제25권1호
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• pp.1-7
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• 2015

칼슘 의존적으로 활성화되는 neutral protease calpain에 의한 단백질 분해는 세포의 성장을 조절하는데 중요한 단백질들의 역할에 매우 중요한 역할을 한다. Cyclin의 분해는 세포주기의 진행을 위한 필연적인 과정이다. D-type cyclins는 외부자극이나 신호에 의하여 세포주기의 G1 초기에 합성이 된 후 cyclin-dependent kinases (cdk4 및 cdk6)와의 결합하여 세포주기 S기 진입을 촉진하는 역할을 한다. 본 연구에서는 cyclin D3 단백질이 calpain protease에 의하여 번역 후 수준에서 조절 받고 있음을 제시하였다. 본 실험의 조건에서 lovastatin과 actinomycin D가 처리된 PC-3-M 전립선 암세포에서 cyclin D3 단백질의 발현이 완전히 사라졌지만, calpain inhibitor인 LLnL의 처리에 의하여 정상 수준으로 회복되었음을 알 수 있었다. 그러나 26S proteasome의 선택적 억제제인 lactacystin, lysosome 억제제인 ammonium chloride 및 chloroquine, serine protease 억제제인 PMSF는 동일 조건에서 lovastatin과 actinomycin D 처리에 의한 cyclin D3 단백질의 발현저하를 억제하지는 못하였다. In vitro 조건에서 순수 분리된 calpain은 cyclin D3 단백질을 칼슘 농도 의존적으로 분해하였으며, cyclin D3 단백질의 반감기는 LLnL 처리에 의하여 매우 유의적으로 증가되었다. 또한 calpain 저해인자인 calpastatin의 과발현은 PC-3-M 세포에서 뿐만 아니라 NIH 3T3 섬유아세포에서도 cyclin D3 단백질의 반감기 및 안전성을 증대시켰다. 이러한 결과는 cyclin D3 단백질이 칼슘에 의해 활성화 되는 protease calpain에 의해 조절됨을 보여주는 것이다.

• 생명과학회지
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• 제30권1호
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• pp.106-112
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• 2020

아미노아실-트랜스퍼 리보핵산 합성효소-상호작용 다기능 단백질 2(AIMP2)는 여러 tRNA 합성효소들과의 결합체를 이루게 하는 기능을 하며, DNA 손상에 대한 반응으로 세포사멸 활성을 나타낼 수 있다. DNA에 손상이 발생하면 AIMP2는 MDM2 공격으로부터 p53을 보호하기 위해 MDM2에 결합한다. TGF-β 신호에서 AIMP2는 세포 핵으로 들어가 FUSE 결합 단백질(FBP)과 결합하여 c-myc을 억제한다. TNF 수용체 관련 인자 2(TRAF2)는 c-Jun N-말단 키나아제(JNK), NF-κB 및 p38 미토겐 활성화 단백질 키나아제(MAPKs)의 신호에서 실행되는 두 수용체, TNF 수용체 1과 2 사이의 중요한 중재자이다. TARF2는 TNF-α 신호에서 JNK와 NF-κB의 활성화에 필요하며, 세포사멸 신호를 막는 중재자 역할을 수행한다. 또한 TNF-α 신호에서 AIMP2는 세포사멸을 향상시킨다. 이 신호에서, AIMP2는 TRAF2를 분해하는 것으로 잘 알려진 E3 유비키틴 효소인 c-IAP1과의 결합을 향상시킨다. AIMP2, TRAF2 및 c-IAP1을 포함한 복합체의 형성은 proteasome을 매개로 하여 TRAF2의 분해를 초래한다. 이러한 연구 결과는 AIMP2가 TNF-α 신호에서 직접적인 상호작용을 통해 TRAF2를 하향 조절시켜 세포사멸을 유도할 수 있음을 시사한다.

• The Korean Journal of Physiology and Pharmacology
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• 제18권5호
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• pp.403-409
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• 2014

The Bis protein is known to be involved in a variety of cellular processes including apoptosis, migration, autophagy as well as protein quality control. Bis expression is induced in response to a number of types of stress, such as heat shock or a proteasome inhibitor via the activation of heat shock factor (HSF)1. We report herein that Bis expression is increased at the transcriptional level in HK-2 kidney tubular cells and A172 glioma cells by exposure to oxidative stress such as $H_2O_2$ treatment and oxygen-glucose deprivation, respectively. The pretreatment of HK-2 cells with N-acetyl cysteine, suppressed Bis induction. Furthermore, HSF1 silencing attenuated Bis expression that was induced by $H_2O_2$, accompanied by increase in reactive oxygen species (ROS) accumulation. Using a series of deletion constructs of the bis gene promoter, two putative heat shock elements located in the proximal region of the bis gene promoter were found to be essential for the constitutive expression is as well as the inducible expression of Bis. Taken together, our results indicate that oxidative stress induces Bis expression at the transcriptional levels via activation of HSF1, which might confer an expansion of antioxidant capacity against pro-oxidant milieu. However, the possible role of the other cis-element in the induction of Bis remains to be determined.

• Molecules and Cells
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• 제40권11호
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• pp.871-879
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• 2017

Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), $10{\mu}M$ MG132 (MG), and 10 mM caffeine + $10{\mu}M$ MG132 (CA+MG). After warming, the MPF activity of oocytes and their blastocyst formation and implantation rates in the CA, MG, and CA+MG groups were much higher than those in the untreated group. However, the cell numbers in blastocysts did not differ among groups. Analysis of the effectiveness of caffeine and MG132 for improving somatic cell nuclear transfer (SCNT) technology using cryopreserved eggs showed that supplementation did not improve the blastocyst formation rate of cloned mouse eggs. These results suggest that maintaining MPF activity after cryopreservation may have a positive effect on further embryonic development, but is unable to fully overcome cryoinjury. Thus, intrinsic factors governing the developmental potential that diminish during oocyte cryopreservation should be explored.