• BMB Reports
• /
• 제51권10호
• /
• pp.526-531
• /
• 2018

Protein-Protein Interactions (PPIs) play essential roles in diverse biological processes and their misregulations are associated with a wide range of diseases. Especially, the growing attention to PPIs as a new class of therapeutic target is increasing the need for an efficient method of cell-based PPI analysis. Thus, we newly developed a robust PPI assay (SeePPI) based on the co-translocation of interacting proteins to the discrete subcellular compartment 'processing body' (p-body) inside living cells, enabling a facile analysis of PPI by the enriched fluorescent signal. The feasibility and strength of SeePPI (${\underline{S}}ignal$ ${\underline{e}}nhancement$ ${\underline{e}}xclusively$ on ${\underline{P}}-body$ for ${\underline{P}}rotein-protein$ ${\underline{I}}nteraction$) assay was firmly demonstrated with FKBP12/FRB interaction induced by rapamycin within seconds in real-time analysis of living cells, indicating its recapitulation of physiological PPI dynamics. In addition, we applied p53/MDM2 interaction and its dissociation by Nutlin-3 to SeePPI assay and further confirmed that SeePPI was quantitative and well reflected the endogenous PPI. Our SeePPI assay will provide another useful tool to achieve an efficient analysis of PPIs and their modulators in cells.

• Bulletin of the Korean Chemical Society
• /
• 제35권10호
• /
• pp.2985-2989
• /
• 2014

Autophagy is a self-digestion process in which intracellular structures are degraded in response to stress. Notably, prolonged autophagy leads to cell death. In this study, we investigated whether CX-4945, an orally available protein kinase 2 (CK2) inhibitor, induces autophagic cell death in human cervical cancer-derived HeLa cells and in human prostate cancer-derived LNCaP cells. CX-4945 treatment of both cell lines resulted in the formation of autophagosomes, in the conversion of microtubule-associated protein 1 light chain 3 (LC3), and in down-regulation of the Akt-mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (S6K) signaling cascade. Thus, pharmacologic inhibition of CK2 by CX-4945 induced autophagic cell death in human cancer cells by down-regulating Akt-mTOR-S6K. These results suggest that autophagy-inducing agents have potential as anti-cancer drugs.

• Natural Product Sciences
• /
• 제25권4호
• /
• pp.298-303
• /
• 2019

This study investigated the cytotoxic effects and mechanism of action of asiatic acid in pancreatic cancer cell lines. First, we confirmed the cell viability of MIA PaCa-2 and PANC-1 cells after asiatic acid administration for 48 and 72 h. The viability of MIA PaCa-2 and PANC-1 cells decreased in a dose-dependent manner following asiatic acid administration. To investigate the underlying mechanism, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, annexin V assay, and western blotting. Asiatic acid induced apoptosis and autophagy through activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) in MIA PaCa-2 cells. Finally, the expression of miR-17 and miR-21, known as oncogenes in pancreatic cancer, was decreased by asiatic acid. These results indicate that asiatic acid has potential as a new therapeutic agent against pancreatic cancer.

• Molecules and Cells
• /
• 제43권8호
• /
• pp.705-717
• /
• 2020

While the growth factors like insulin initiate a signaling cascade to induce conformational changes in the mechanistic target of rapamycin complex 1 (mTORC1), amino acids cause the complex to localize to the site of activation, the lysosome. The precise mechanism of how mTORC1 moves in and out of the lysosome is yet to be elucidated in detail. Here we report that microtubules and the motor protein KIF11 are required for the proper dissociation of mTORC1 from the lysosome upon amino acid scarcity. When microtubules are disrupted or KIF11 is knocked down, we observe that mTORC1 localizes to the lysosome even in the amino acid-starved situation where it should be dispersed in the cytosol, causing an elevated mTORC1 activity. Moreover, in the mechanistic perspective, we discover that mTORC1 interacts with KIF11 on the motor domain of KIF11, enabling the complex to move out of the lysosome along microtubules. Our results suggest not only a novel way of the regulation regarding amino acid availability for mTORC1, but also a new role of KIF11 and microtubules in mTOR signaling.

• Preventive Nutrition and Food Science
• /
• 제20권4호
• /
• pp.221-229
• /
• 2015

Hesperidin has been shown to possess a potential inhibitory effect on vascular formation in endothelial cells. However, the fundamental mechanism for the anti-angiogenic activity of hesperidin is not fully understood. In the present study, we evaluated whether hesperidin has anti-angiogenic effects in mouse embryonic stem cell (mES)-derived endothelial-like cells, and human umbilical vascular endothelial cells (HUVECs), and evaluated their mechanism via the AKT/mammalian target of rapamycin (mTOR) signaling pathway. The endothelial cells were treated with several doses of hesperidin (12.5, 25, 50, and $100{\mu}M$) for 24 h. Cell viability and vascular formation were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and tube formation assay, respectively. Alteration of the AKT/mTOR signaling in vascular formation was analyzed by western blot. In addition, a mouse aortic ring assay was used to determine the effect of hesperidin on vascular formation. There were no differences between the viability of mES-derived endothelial-like cells and HUVECs after hesperidin treatment. However, hesperidin significantly inhibited cell migration and tube formation of HUVECs (P<0.05) and suppressed sprouting of microvessels in the mouse aortic ring assay. Moreover, hesperidin suppressed the expression of AKT and mTOR in HUVECs. Taken together, these findings suggest that hesperidin inhibits vascular formation by blocking the AKT/mTOR signaling pathways.

• Journal of Applied Biological Chemistry
• /
• 제66권
• /
• pp.186-196
• /
• 2023

Dyglomera^® is an aqueous ethanol extract derived from the fruit and pods of Dichrostachys glomerata. A previous study has revealed that Dyglomera regulates adipogenesis and lipolysis by modulating AMP-activated protein kinase (AMPK) phosphorylation and increased expression levels of lipolysis-related proteins in white adipose tissue of high fat diet-induced mice and 3T3-L1 adipocyte cells. To further investigate mechanisms of Dyglomera, additional studies were performed using 3T3-L1 cells. Results revealed that Dyglomera downregulated adipogenesis by inhibiting the protein kinase B/mammalian target of rapamycin signaling pathway and reconfirmed that it downregulated gene expression levels of proliferator-activated receptor (PPAR)-γ, CCAAT enhancer binding protein α, sterol-regulation element-binding protein-1c. Dyglomera also reduced adipokines such as tumor necrosis factor alpha, interleukin-1β, and interleukin 6 by regulating leptin expression. Moreover, Dyglomera promoted beige-and-brown adipocyte-related phenotypes and regulated metabolism by increasing mitochondrial number and expression levels of genes such as T-box protein 1, transmembrane protein 26, PR domain 16, and cluster of differentiation 40 as well as thermogenic factors such as uncoupling protein 1, proliferator-activated receptor-gamma co-activator-1α, Sirtuin 1, and PPARα through AMPK activation. Thus, Dyglomera not only can inhibit adipogenesis, but also can promote lipolysis and thermogenesis and regulate metabolism by affecting adipokine secretion from 3T3-L1 adipocytes.

• BMB Reports
• /
• 제57권3호
• /
• pp.143-148
• /
• 2024

Pulmonary fibrosis is a serious lung disease that occurs predominantly in men. Genistein is an important natural soybean-derived phytoestrogen that affects various biological functions, such as cell migration and fibrosis. However, the antifibrotic effects of genistein on pulmonary fibrosis are largely unknown. The antifibrotic effects of genistein were evaluated using in vitro and in vivo models of lung fibrosis. Proteomic data were analyzed using nano-LC-ESI-MS/MS. Genistein significantly reduced transforming growth factor (TGF)-β1-induced expression of collagen type I and α-smooth muscle actin (SMA) in MRC-5 cells and primary fibroblasts from patients with idiopathic pulmonary fibrosis (IPF). Genistein also reduced TGF-β1-induced expression of p-Smad2/3 and p-p38 MAPK in fibroblast models. Comprehensive protein analysis confirmed that genistein exerted an anti-fibrotic effect by regulating various molecular mechanisms, such as unfolded protein response, epithelial mesenchymal transition (EMT), mammalian target of rapamycin complex 1 (mTORC1) signaling, cell death, and several metabolic pathways. Genistein was also found to decrease hydroxyproline levels in the lungs of BLM-treated mice. Genistein exerted an anti-fibrotic effect by preventing fibroblast activation, suggesting that genistein could be developed as a pharmacological agent for the prevention and treatment of pulmonary fibrosis.

• Journal of Animal Science and Technology
• /
• 제64권2호
• /
• pp.312-329
• /
• 2022

Feed cost is the main factor affecting the economic benefits of pig industry. Improving the feed efficiency (FE) can reduce the feed cost and improve the economic benefits of pig breeding enterprises. Liver is a complex metabolic organ which affects the distribution of nutrients and regulates the efficiency of energy conversion from nutrients to muscle or fat, thereby affecting feed efficiency. MicroRNAs (miRNAs) are small non-coding RNAs that can regulate feed efficiency through the modulation of gene expression at the post-transcriptional level. In this study, we analyzed miRNA profiling of liver tissues in High-FE and Low-FE pigs for the purpose of identifying key miRNAs related to feed efficiency. A total 212~221 annotated porcine miRNAs and 136~281 novel miRNAs were identified in the pig liver. Among them, 188 annotated miRNAs were co-expressed in High-FE and Low-FE pigs. The 14 miRNAs were significantly differentially expressed (DE) in the livers of high-FE pigs and low-FE pigs, of which 5 were downregulated and 9 were upregulated. Kyoto Encyclopedia of Genes and Genomes analysis of liver DE miRNAs in high-FE pigs and low-FE pigs indicated that the target genes of DE miRNAs were significantly enriched in insulin signaling pathway, Gonadotropin-releasing hormone signaling pathway, and mammalian target of rapamycin signaling pathway. To verify the reliability of sequencing results, 5 DE miRNAs were randomly selected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR results of miRNAs were confirmed to be consistent with sequencing data. DE miRNA data indicated that liver-specific miRNAs synergistically acted with mRNAs to improve feed efficiency. The liver miRNAs expression analysis revealed the metabolic pathways by which the liver miRNAs regulate pig feed efficiency.

• Molecules and Cells
• /
• 제46권11호
• /
• pp.675-687
• /
• 2023

Accumulation of pathogenic amyloid-β disrupts the tight junction of retinal pigment epithelium (RPE), one of its senescence-like structural alterations. In the clearance of amyloid-β, the autophagy-lysosome pathway plays the crucial role. In this context, mammalian target of rapamycin (mTOR) inhibits the process of autophagy and lysosomal degradation, acting as a potential therapeutic target for age-associated disorders. However, efficacy of targeting mTOR to treat age-related macular degeneration remains largely elusive. Here, we validated the therapeutic efficacy of the mTOR inhibitors, Torin and PP242, in clearing amyloid-β by inducing the autophagy-lysosome pathway in a mouse model with pathogenic amyloid-β with tight junction disruption of RPE, which is evident in dry age-related macular degeneration. High concentration of amyloid-β oligomers induced autophagy-lysosome pathway impairment accompanied by the accumulation of p62 and decreased lysosomal activity in RPE cells. However, Torin and PP242 treatment restored the lysosomal activity via activation of LAMP2 and facilitated the clearance of amyloid-β in vitro and in vivo. Furthermore, clearance of amyloid-β by Torin and PP242 ameliorated the tight junction disruption of RPE in vivo. Overall, our findings suggest mTOR inhibition as a new therapeutic strategy for the restoration of tight junctions in age-related macular degeneration.

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