• Parasites, Hosts and Diseases
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• 제56권2호
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• pp.135-145
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• 2018

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, $CK2{\beta}$, VEGF, GCL, GST and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

• Animal Bioscience
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• 제37권7호
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• pp.1156-1167
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• 2024

Objective: MicroRNAs (miRNAs) are endogenous non-coding RNAs that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. However, the precise functional mechanism of its regulation of fat metabolism is not fully understood. Methods: We identified bta-miR-365-3p, which specifically targets the 3' untranslated region (3'UTR) of the FK506-binding protein 5 (FKBP5), and verified its mechanisms for regulating expression and involvement in adipogenesis. Results: In this study, we found that the overexpression of bta-miR-365-3p significantly decreased the lipid accumulation and triglyceride content in the adipocytes. Compared to inhibiting bta-miR-36 5-3p group, overexpression of bta-miR-365-3p can inhibit the expression of adipocyte differentiation-related genes C/EBPα and PPARγ. The dual-luciferase reporter system further validated the targeting relationship between bta-miR-365-3p and FKBP5. FKBP5 mRNA and protein expression were detected by quantitative real-time polymerase chain reaction and Western blot. Overexpression of bta-miR-365-3p significantly down-regulated FKBP5 expression, while inhibition of bta-miR-365-3p showed the opposite, indicating that bta-miR-365-3p negatively regulates FKBP5. Adenosine 5'-monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway is closely related to the regulation of cell growth and is involved in the development of bovine adipocytes. In this study, overexpression of bta-miR-365-3p significantly inhibited mRNA and protein expression of AMPK, mTOR, and SREBP1 genes, while the inhibition of bta-miR-365-3p expression was contrary to these results. Overexpression of FKBP5 significantly upregulated AMPK, mTOR, and SREBP1 gene expression, while inhibition of FKBP5 expression was contrary to the above experimental results. Conclusion: In conclusion, these results indicate that bta-miR-365-3p may be involved in the AMPK/mTOR signaling pathway in regulating Yanbian yellow cattle preadipocytes differentiation by targeting the FKBP5 gene.

• 한국발생생물학회지:발생과생식
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• 제24권2호
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• pp.113-123
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• 2020

Metformin has been widely used as an antidiabetic drug, and reported to inhibit cell proliferation in many cancers including non-small cell lung cancer (NSCLC). In NSCLC cells, metformin suppresses PI3K/AKT/mTOR signaling pathway, but effect of metformin on RAS/RAF/MEK/ERK signaling pathway is controversial; several studies showed the inhibition of ERK activity, while others demonstrated the activation of ERK in response to metformin exposure. Metformin-induced activation of ERK is therapeutically important, since metformin could enhance cell proliferation through RAS/RAF/MEK/ERK pathway and lead to impairment of its anticancer activity suppressing PI3K/AKT/mTOR pathway, requiring blockade of both signaling pathways for more efficient antitumor effect. The present study tested the combination therapy of metformin and trametinib by monitoring the alterations of regulatory effector proteins of cell signaling pathways and the effect of the combination on cell viability in NCI-H2087 NSCLC cells with NRAS and BRAF mutations. We show that metformin alone blocks PI3K/AKT/mTOR signaling pathway but induces the activation and phosphorylation of ERK. The combination therapy synergistically decreased cell viability in treatment with low doses of two drugs, while it gave antagonistic effect with high doses. These findings suggest that the efficacy of metformin and trametinib combination therapy may depend on the alteration of ERK activity induced by metformin and specific cellular context of cancer cells.

• Journal of Microbiology and Biotechnology
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• 제23권11호
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• pp.1569-1576
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• 2013

In mice, supplementation of t10,c12 conjugated linoleic acid (CLA) increases liver mass and hepatic steatosis via increasing uptake of fatty acids released from adipose tissues. However, the effects of t10,c12 CLA on hepatic lipid synthesis and the associated mechanisms are largely unknown. Thus, we tested the hypothesis that gut microbiota-producing t10,c12 CLA would induce de novo lipogenesis and triglyceride (TG) synthesis in HepG2 cells, promoting lipid accumulation. It was found that treatment with t10,c12 CLA ($100{\mu}M$) for 72 h increased neutral lipid accumulation via enhanced incorporation of acetate, palmitate, oleate, and 2-deoxyglucose into TG. Furthermore, treatment with t10,c12 CLA led to increased mRNA expression and protein levels of lipogenic genes including SREBP1, ACC1, FASN, ELOVL6, GPAT1, and DGAT1, presenting potential mechanisms by which CLA may increase lipid deposition. Most strikingly, t10,c12 CLA treatment for 3 h increased phosphorylation of mTOR, S6K, and S6. Taken together, gut microbiota-producing t10,c12 CLA activates hepatic de novo lipogenesis and TG synthesis through activation of the mTOR/SREBP1 pathway, with consequent lipid accumulation in HepG2 cells.

• 동의생리병리학회지
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• 제34권2호
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• pp.61-66
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• 2020

Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells lose their characters and acquire the properties of mesenchymal cells. EMT has been reported to exert an essential role in embryonic development. Recently, EMT has emerged as a pivotal mechanism in the metastasis of cancer and the fibrosis of chronic diseases. In particular, EMT is drawing attention as a mechanism of renal fibrosis in chronic kidney diseases such as diabetic nephropathy. In this study, we developed an EMT model by treating TGF-β1 on the podocytes, which play a key role in the renal glomerular filtration. This study explored the effects of Hwanggeum-tang (HGT) recorded in Dongeuibogam as being able to be used for the treatment of Sogal whose concept had been applied to Diabetes Mellitus (DM), on the TGF-β1-induced podocyte EMT. HGT suppressed the expression of vimentin and α-SMA, the EMT marker, in the human podocytes stimulated by TGF-β1. However, HGT increased the expression of ZO-1 and nephrin. Interestingly, HGT selectively inhibited the mTOR pathway rather than the classical Smad pathway. HGT also activated the AMPK signaling. HGT's inhibitory effect on the podocyte EMT through regulation of the mTOR pathway was achieved through the activation of AMPK, which was confirmed by comparison with cells treated with compound C (CC), an inhibitor of AMPK signaling. In conclusion, HGT can be applied to the renal fibrosis by preventing TGF-β1-induced EMT of podocytes through AMPK activation and mTOR inhibition.

• Molecules and Cells
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• 제40권11호
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• pp.837-846
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• 2017

As a component of the neurovascular unit, cerebral smooth muscle cells (CSMCs) are an important mediator in the development of cerebral vascular diseases such as stroke. Epoxyeicosatrienoic acids (EETs) are the products of arachidonic acid catalyzed by cytochrome P450 epoxygenase. EETs are shown to exert neuroprotective effects. In this article, the role of EET in the growth and apoptosis of CSMCs and the underlying mechanisms under oxygen glucose deprivation (OGD) conditions were addressed. The viability of CMSCs was decreased significantly in the OGD group, while different subtypes of EETs, especially 14,15-EET, could increase the viability of CSMCs under OGD conditions. RAPA (serine/threonine kinase Mammalian Target of Rapamycin), a specific mTOR inhibitor, could elevate the level of oxygen free radicals in CSMCs as well as the anti-apoptotic effects of 14,15-EET under OGD conditions. However, SP600125, a specific JNK (c-Jun N-terminal protein kinase) pathway inhibitor, could attenuate oxygen free radicals levels in CSMCs as well as the anti-apoptotic effects of 14,15-EET under OGD conditions. These results strongly suggest that EETs exert protective functions during the growth and apoptosis of CSMCs, via the JNK/c-Jun and mTOR signaling pathways in vitro. We are the first to disclose the beneficial roles and underlying mechanism of 14,15-EET in CSMC under OGD conditions.

• KSBB Journal
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• 제30권5호
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• pp.223-229
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• 2015

In this study, extract from Artemisia annua in L. (AAE) is known as a medicinal herb that is effective against cancer. The cell cycle is regulated by the activation of cyclin-dependent kinase (CDK)/cyclin complex. We will focus on regulation of CDK2 by cyclin E. cyclin E is associated with CDK2 to regulate progression from G1 into S phase. Akt is known to play an important role in cell proliferation and cell survival. Activation of Akt increases mTOR activity that promotes cell proliferation and cancer growth. In this study, we investigated that AAE-induced cell cycle arrest at G1/S phase in HCT116 colon cancer. Treatment of AAE shows that reduced activation of Akt decreases mTOR/Mdm2 activity and then leads to increase the activation of p53. The active p53 promotes activation of p21. p21 induces inactivation of CDK2/cyclin E complex and occurs cell cycle arrest at G1/S phase. We treated LY294002 (Akt inhibitor) and Rapamycin (mTOR inhibitor) to know the relationship between the signal transduction of proteins associated with cell cycle arrest. These results suggest that AAE induces cell cycle arrest at G1/S phase by Akt/mTOR pathway in HCT116 colon cancer cell.

• 대한화장품학회지
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• 제44권1호
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• pp.95-101
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• 2018

표피는 각질형성세포의 분화로부터 재생되어 계층화되는 상피 조직으로서 물리적 장벽을 형성함으로써 다양한 외부 오염원으로부터 개체를 보호한다. 자가포식 작용(autophagy)은 단백질 축적물, 손상된 세포 소기관, 세포내 미생물 등이 리소좀으로 운반되고 분해되도록 매개하는 기작이다. 최근 연구 결과에 의하면 자가포식 작용이 각질형성세포의 대사 기관과 핵을 제거하여 각질층으로 최종 분화하는데 중요한 역할을 하는 것이 보고 되었다. 그러나 자가포식 작용을 촉진함으로써 표피 분화를 유도할 수 있는지는 알려져 있지 않다. 본 연구에서는 천연물 유래 단일 화합물 라이브러리를 스크리닝하여 베타인(betaine)이 인간 각질형성세포주인 HaCaT 세포에서 세포질 내 LC3 punctate 소포체 및 LC3-I에서 LC3-II로의 변환을 증가시켜 자가포식 작용을 촉진함을 규명했다. 자가포식 작용의 억제 신호인 mTOR 경로는 베타인에 의해 영향을 받지 않았으므로, 베타인에 의해 유도된 자가포식 작용은 mTOR에 독립적임을 알 수 있었다. 베타인에 의해 촉진되는 자가포식 작용은 primary keratinocyte 및 skin equivalent에서도 관찰되었다. 또한, 베타인 처리된 인공피부에서 표피층 두께가 증가함을 확인하였다. 이러한 결과들로부터, 자가포식 작용의 새로운 조절소재로서 베타인이 표피의 턴오버를 촉진하여 표피의 장벽기능을 개선하고 피부노화를 방지할 수 있음을 시사한다.

• Tuberculosis and Respiratory Diseases
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• 제72권4호
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• pp.343-351
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• 2012

Background: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit this pathway are currently under development for lung cancer treatment. In the present study, we have tested whether dual inhibition of PI3K/Akt/mTOR signaling can lead to enahnced antitumor effects. We have also examined the role of autophagy during this process. Methods: We analyzed the combination effect of the mTOR inhibitor, temsirolimus, and the Akt inhibitor, GSK690693, on the survival of NCI-H460 and A549 non-small cell lung cancer cells. Cell proliferation was determined by MTT assay and apoptosis induction was evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Autophagy induction was also evaluated by acridine orange staining. Changes of apoptosis or autophagy-related proteins were evaluated by western blot analysis. Results: Combination treatment with temsirolimus and GSK690693 caused synergistically increased cell death in NCI-H460 and A549 cells. This was attributable to increased induction of apoptosis. Caspase 3 activation and poly(ADP-ribose) polymerase cleavage accompanied these findings. Autophagy also increased and inhibition of autophagy resulted in increased cell death, suggesting its cytoprotective role during this process. Conclusion: Taken together, our results suggest that the combination of temsirolimus and GSK690693 could be a novel strategy for lung cancer therapy. Inhibition of autophagy could also be a promising method of enhancing the combination effect of these drugs.

• Journal of Microbiology and Biotechnology
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• 제23권7호
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• pp.923-931
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• 2013

Rapamycin, known as an inhibitor of Target of Rapamycin (TOR), is an immunosuppressant drug used to prevent rejection in organ transplantation. Despite the close association of the TOR signaling cascade with various scopes of metabolism, it has not yet been thoroughly investigated at the metabolome level. In our current study, we applied mass spectrometric analysis for profiling primary metabolism in order to capture the responsive dynamics of the Chlamydomonas metabolome to the inhibition of TOR by rapamycin. Accordingly, we identified the impact of the rapamycin treatment at the level of metabolomic phenotypes that were clearly distinguished by multivariate statistical analysis. Pathway analysis pinpointed that inactivation of the TCA cycle was accompanied by the inhibition of cellular growth. Relative to the constant suppression of the TCA cycle, most amino acids were significantly increased in a time-dependent manner by longer exposure to rapamycin treatment, after an initial down-regulation at the early stage of exposure. Finally, we explored the isolation of the responsive metabolic factors into the rapamycin treatment and the culture duration, respectively.