• Molecules and Cells
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• v.46 no.8
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• pp.486-495
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• 2023

Lipofuscins are oxidized lipid and protein complexes that accumulate during cellular senescence and tissue aging, regarded as markers for cellular oxidative damage, tissue aging, and certain aging-associated diseases. Therefore, understanding their cellular biological properties is crucial for effective treatment development. Through traditional microscopy, lipofuscins are readily observed as fluorescent granules thought to accumulate in lysosomes. However, lipofuscin granule formation and accumulation in senescent cells are poorly understood. Thus, this study examined lipofuscin accumulation in human fibroblasts exposed to various stressors. Our results substantiate that in glucose-starved or replicative senescence cells, where elevated oxidative stress levels activate autophagy, lipofuscins predominately appear as granules that co-localize with autolysosomes due to lysosomal acidity or impairment. Meanwhile, autophagosome formation is attenuated in cells experiencing oxidative stress induced by a doxorubicin pulse and chase, and lipofuscin fluorescence granules seldom manifest in the cytoplasm. As Torin-1 treatment activates autophagy, granular lipofuscins intensify and dominate, indicating that autophagy activation triggers their accumulation. Our results suggest that high oxidative stress activates autophagy but fails in lipofuscin removal, leaving an abundance of lipofuscin-filled impaired autolysosomes, referred to as residual bodies. Therefore, future endeavors in treating lipofuscin pathology-associated diseases and dysfunctions through autophagy activation demand meticulous consideration.

• Applied Microscopy
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• v.47 no.1
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• pp.1-2
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• 2017

Professor Yoshinori Ohsumi received the 2016 Nobel Prize in Physiology or Medicine for his contribution to autophagy research, which was first studied using electron microscopy. To celebrate and commemorate this historical moment, I describe the role of electron microscopy in autophagy research and suggest a role for next-generation electron microscopy in this research field.

• Korean Journal of Microbiology
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• v.50 no.1
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• pp.27-32
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• 2014

Autophagy is one of the lysosomal degradation pathways to maintain cellular homeostasis. The damaged proteins or organelles are uptaken through extra- and intra-cellular stress, starvation and infected pathogens, subsequently, autophagosomes are fused with lysosomes to break down the molecules. Salmonella enterica serovar Typhimurium (S. Typhimurium), intracellular bacteria, cause acute gastroenteritis and food poisoning. Given that autophagy induced by S. Typhimurium plays an important role in the cells to control the infection, we identify whether the induction of autophagy with rapamycin, chemical inducer of autophagy, before infection regulates S. Typhimurium infection. After treatment of rapamycin or 3-methyladenine (3-MA), autophagy inhibitor, RAW264.7 cells were infected with S. Typhimurium. Pretretment of rapamycin decreased the growth rate of S. Typhimurium in the cells; otherwise, pretreatment of 3-MA increased the growth rate of S. Typhimurium. The expression of autophagy-related genes was significantly increased in the S. Typhimurium-infected cells pretreated with rapamycin. To examine whether induced autophagy by rapamycin control the infection with increase the production of reactive oxygen species (ROS) and nitric oxide (NO), antibacterial radical substrates were measured in infected cells followed by the treatment with either rapamycin or 3-MA. NO production increased in RAW264.7 cells; otherwise, ROS production remained unchanged during the infection. These findings suggest that inducing autophagy with rapamycin reveals antimicrobial activity as producing NO against S. Typhimurium infection in mouse macrophages.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.44 no.1
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• pp.95-101
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• 2018

The epidermis which is stratified by epithelial tissue renewal based on keratinocyte differentiation protects the organism from various environmental insults by forming a physical barrier. Autophagy is a mechanism which mediates lysosomal delivery and degradation of protein aggregates, damaged organelles and intracellular microorganisms. Recent reports have shown that autophagy has critical roles for proper terminal differentiation to stratum corneum via removing metabolic organelles and nuclei. However, whether increasing autophagy can activate epidermal differentiation is unknown. Here, we screened a library of natural single compounds and discovered that betaine specifically increased the LC3 positive cytosolic punctate vesicles and LC3-I to LC3-II conversion in HaCaT human keratinocyte cell line, indicating increased autophagy flux. mTOR pathway, which negatively regulates autophagy, was not affected by betaine treatment, suggesting betaine-induced autophagy through an mTOR-independent pathway. Betaine-induced autophagy was also observed in primary human keratinocyte and skin equivalent. Furthermore, epidermal thickness was increased in skin equivalent under betaine treatment. Overall, our finding suggests that betaine as a novel regulator of autophagy may induce epidermal turnover and improve the skin barrier abnormality of the aged epidermis.

• BMB Reports
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• v.42 no.9
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• pp.599-604
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• 2009

Selenium possesses the chemotherapeutic feature by inducing apoptosis in cancer cell with trivial side effects on normal cells. However, the mechanism in which is not clearly understood. Emerging evidence indicates the overlaps between the autophagy and the apoptosis. In this study, we have investigated the role of autophagy in selenium-induced apoptosis in NB4 cells. We find that autophagy is suppressed in NB4 cells treated by sodium selenite, as measured by electron microscope, acridine orange staining and western blot. Moreover, selenite combined with autophagy inhibitor contributes to the up-regulation of apoptosis, while the PI3K/Akt signaling pathway is down- regulated. Consistently, when the inhibitor of PI3K was applied, the autophagic level significantly decreased. In summary, sodium selenite increases NB4 cell apoptosis by autophagy inhibition through PI3K/Akt, and the inhibition of autophagy contributes to the up-regulation of apoptosis.

• Development and Reproduction
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• v.20 no.1
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• pp.1-10
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• 2016

Molecular targeting for the altered signaling pathways has been proven to be effective for the treatment of many types of human cancer, including colorectal cancer (CRC). The dual phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor BEZ235 has shown to exhibit potent antitumor activity against solid tumors. Autophagy is a cellular lysosomal catabolic process to maintain metabolic homeostasis, which has been known to be induced in response to many therapeutic agents in cancer cells. This process is negatively regulated by mTOR and often acts as prosurvival or prodeath mechanism following cancer therapeutics. The current study was designed to investigate the antiproliferation activity of BEZ235 and to evaluate the role of autophagy induced by BEZ235 using HCT15 CRC cells bearing ras oncogene mutation. We found that BEZ235 decreases cell viability, which was mostly dependent on $G_1$ arrest of cell cycle via suppression of cyclin A expression. BEZ235 affects PI3K/Akt/mTOR signaling pathway by increasing the phosphorylation of AKT at $Ser^{473}$ and RAS/RAF/MEK/ERK pathway by decreasing the phosphorylation of ERK at $Tyr^{204}$. BEZ235 also stimulated autophagy induction as evidenced by the increased expression of LC3-II and abundant acidic vesicular organelles (AVOs) in the cytoplasm. In addition, the combination of BEZ235 with autophagy inhibitor chloroquine, a known antagonist of autophagy, counteracted the antiproliferation effect of BEZ235. Thus, our study indicates that autophagy induced in response to BEZ235 treatment appears to act as cell death mechanism in HCT15 CRC cells.

• The Journal of Internal Korean Medicine
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• v.35 no.3
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• pp.317-331
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• 2014

Objectives: The root of Platycodon grandiflorum (PG) has been known to possess a range of pharmacological activities including anti-cancer, anti-inflammatory, and anti-oxidant effects. The present study was designed to investigate whether or not PG-induced cell death was connected with autophagy and apoptosis in NCI-H460 human lung cancer cells. Methods: Effects on the cell viability and apoptotic activity were quantified using MTT assays and flow cytometry analysis, respectively. Protein activation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting. ROS production and loss of mitochondria membrane potential (MMP) were checked with flow cytometry analysis. Results: Following exposure to PG, NCI-H460 cell proliferation decreased simultaneously inducing autophagic vacuoles and up-regulation of microtubule-associated protein 1 light chain 3 and beclin-1 protein expressions. Interestingly, pre-treated with autophagy inhibitors, 3-methyladenin or bafilomycin A1 further triggered reduction of cell viability. PG treatment also induced apoptosis that was related modulation of Bcl-2 family proteins, death receptors and activation of caspases. In addition, PG stimulation clearly enhanced loss of MMP and reactive oxygen species (ROS) generation. Conclusions: Our results suggest that PG elicited both autophagy and apoptosis by increasing loss of MMP and ROS production. PG induced-autophagy may play a cell protective role.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.6
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• pp.573-580
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• 2016

Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.1
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• pp.33-37
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• 2013

Autophagy is a degradation process that acts in response to environmental stressors. Recently, autophagy has been detected in normal term, preeclamptic and intrauterine growth-restricted placentas. The object of this work was to investigate the presence of autophagy in first trimester voluntary interruption of pregnancy placental villi by the expression of autophagy-related proteins, light chain 3 (LC3), and Beclin-1. In first trimester placental villi laser scanning confocal microscopy (LSCM) analysis revealed LC3 and Beclin-1 immunoreactivity prevalently located in villous cytotrophoblasts. Using LSCM, LC3, and Beclin-1 were localized to the cytoplasm of the trophoblast layer in human full-term placentas. Beclin-1 expression and LC3 activation were confirmed by western blotting. These data emphasize that autophagy activation is different among cytotrophoblasts and syncytiotrophoblasts depending on the gestational age and thus we speculate that autophagy might play a prosurvival role throughout human pregnancy.

• Molecules and Cells
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• v.40 no.9
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• pp.643-654
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• 2017

Autophagy is a degradation pathway in eukaryotic cells in which aging proteins and organelles are sequestered into double-membrane vesicles, termed autophagosomes, which fuse with vacuoles to hydrolyze cargo. The key step in autophagy is the formation of autophagosomes, which requires different kinds of vesicles, including COPII vesicles and Atg9-containing vesicles, to transport lipid double-membranes to the phagophore assembly site (PAS). In yeast, the cis-Golgi localized t-SNARE protein Sed5 plays a role in endoplasmic reticulum (ER)-Golgi and intra-Golgi vesicular transport. We report that during autophagy, sed5-1 mutant cells could not properly transport Atg8 to the PAS, resulting in multiple Atg8 dots being dispersed into the cytoplasm. Some dots were trapped in the Golgi apparatus. Sed5 regulates the anterograde trafficking of Atg9-containing vesicles to the PAS by participating in the localization of Atg23 and Atg27 to the Golgi apparatus. Furthermore, we found that overexpression of SFT1 or SFT2 (suppressor of sed5 ts) rescued the autophagy defects in sed5-1 mutant cells. Our data suggest that Sed5 plays a novel role in autophagy, by regulating the formation of Atg9-containing vesicles in the Golgi apparatus, and the genetic interaction between Sft1/2 and Sed5 is essential for autophagy.