• Development and Reproduction
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• v.27 no.3
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• pp.149-157
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• 2023

We investigated the involvement of autophagy with steroidogenesis in testicular Leydig cells. Human chorionic gonadotropin (hCG)-stimulated T production in Leydig cells was not remarkably altered in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Although pretreatment with 3-MA demonstrated a tendency to decrease hCG-induced T production, the differences were significant only at a higher time point of 24 h following hCG. Microtubule associated protein light chain 3 (LC3)-II was detectable in the control cells in all the experiments. The hCG-induced increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleave (P450scc) protein levels were not significantly altered by 3-MA. Leydig cells isolated from immature rat testes 12 h following hCG treatment showed relatively increased levels of LC3-II protein compared to the control group. Furthermore, LC3-II levels shown in these cells reached almost the identical to those from normal adult testes. However, LC3-II protein levels were almost comparable or even slightly lower than the controls at 48 h following hCG. Expression of StAR and P450scc was upregulated at both 12 and 48 h after hCG. We also used MA-10 cells, the mouse Leydig cell line, in this experiment. When dibutyryl cyclic-AMP was treated with MA-10 cells, P4 levels were significantly increased in the cell culture medium. However, P4 levels tended to decrease in the presence of 3-MA, but the difference was not statistically significant. This was consistent with the results of the rat Leydig cell experiments. Together, we believe that although autophagy participates in steroidogenesis and enhances steroidogenic efficacy of Leydig cells, it may not be a decisive cellular process for steroidogenesis, specifically in the mature Leydig cells.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.4
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• pp.592-598
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• 2019

Objective: Autophagy is a bulk degradation system for intracellular proteins which contributes to skeletal muscle homeostasis, according to previous studies in humans and rodents. However, there is a lack of information on the physiological role of autophagy in the skeletal muscle of meat animals. This study was planned as a pilot study to investigate changes in expression of two major autophagy-related genes, microtubule-associated protein 1 light chain $3{\beta}$ (MAP1LC3B) and autophagy related 7 (ATG7) in fattening beef cattle, and to compare them with skeletal muscle growth. Methods: Six castrated Japanese Black cattle (initial body weight: $503{\pm}20kg$) were enrolled in this study and fattened for 7 months. Three skeletal muscles, M. longissimus, M. gluteus medius, and M. semimembranosus, were collected by needle biopsy three times during the observation period, and mRNA levels of MAP1LC3B and ATG7 were determined by quantitative reverse-transcription polymerase chain reaction. The expression levels of genes associated with the ubiquitin-proteasome system, another proteolytic mechanism, were also analyzed for comparison with autophagy-related genes. In addition, ultrasonic scanning was repeatedly performed to measure M. longissimus area as an index of muscle growth. Results: Our results showed that both MAP1LC3B and ATG7 expression increased over the observation period in all three skeletal muscles. Interestingly, the increase in expression of these two genes in M. longissimus was highly correlated with ultrasonic M. longissimus area and body weight. On the other hand, the expression of genes associated with the ubiquitin-proteasome system was unchanged during the same period. Conclusion: These findings suggest that autophagy plays an important role in the growth of skeletal muscle of fattening beef cattle and imply that autophagic activity affects meat productivity.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.1-7
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• 2011

Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 112 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy.

• Biomolecules & Therapeutics
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• v.27 no.1
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• pp.34-40
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• 2019

Transglutaminase 2 (TGase 2) plays a key role in p53 regulation, depleting p53 tumor suppressor through autophagy in renal cell carcinoma. We found that microtubule-associated protein 1A/1B-light chain 3 (LC3), a hallmark of autophagy, were tightly associated with the level of TGase 2 in cancer cells. TGase 2 overexpression increased LC3 levels, and TGase 2 knockdown decreased LC3 levels in cancer cells. Transcript abundance of LC3 was inversely correlated with level of wild type p53. TGase 2 knockdown using siRNA, or TGase 2 inhibition using GK921 significantly reduced autophagy through reduction of LC3 transcription, which was followed by restoration of p53 levels in cancer cells. TGase 2 overexpression promoted the autophagy process by LC3 induction, which was correlated with p53 depletion in cancer cells. Rapamycin-resistant cancer cells also showed higher expression of LC3 compared to the rapamycin-sensitive cancer cells, which was tightly correlated with TGase 2 levels. TGase 2 knockdown or TGase 2 inhibition sensitized rapamycin-resistant cancer cells to drug treatment. In summary, TGase 2 induces drug resistance by potentiating autophagy through LC3 induction via p53 regulation in cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.3
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• pp.349-360
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• 2018

Autophagy has been studied as a therapeutic strategy for cardiovascular diseases. However, insufficient studies have been reported concerning the influence of vascular smooth muscle cells (VSMCs) through autophagy regulation. The aim of the present study was to determine the effects of VSMCs on the regulation of autophagy under in vitro conditions similar to vascular status of the equipped micro-tubule target agent-eluting stent and increased release of platelet-derived growth factor-BB (PDGF-BB). Cell viability and proliferation were measured using MTT and cell counting assays. Immunofluorescence using an $anti-{\alpha}-tubulin$ antibody was performed to determine microtubule dynamic formation. Cell apoptosis was measured by cleavage of caspase-3 using western blot analysis, and by nuclear fragmentation using a fluorescence assay. Autophagy activity was assessed by microtubule-associated protein light chain 3-II (LC-II) using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured using $H_2DCFDA$. The proliferation and viability of VSMCs were inhibited by microtubule regulation. Additionally, microtubule-regulated and PDGF-BB-stimulated VSMCs increased the cleavage of caspase-3 more than only the microtubule-regulated condition, similar to that of LC3-II, implying autophagy. Inhibitory autophagy of microtubule-regulated and PDGF-BB-stimulated VSMCs resulted in low viability. However, enhancement of autophagy maintained survival through the reduction of ROS. These results suggest that the apoptosis of conditioned VSMCs is decreased by the blocking generation of ROS via the promotion of autophagy, and proliferation is also inhibited. Thus, promoting autophagy as a therapeutic target for vascular restenosis and atherosclerosis may be a good strategy.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.48 no.2
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• pp.105-112
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• 2022

2'-fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide (HMO) present in breast milk, promoting the growth of beneficial microorganisms in the gut and aiding in the relief of allergic and inflammatory reactions. In this study, the anti-melanogenic effects of 2'-FL, and its potential for application in whitening cosmetics, were evaluated. MTT assay was performed on MNT-1 cells, human-derived melanocytes. 2'-FL was treated and replaced at 48 h intervals for 7 days, and it was confirmed that there was no cytotoxicity at 20 g/L or less, while a 40% reduction in melanin production was also observed. Western blot analysis of TYR and TYRP1, factors involved in melanogenesis, revealed that 2'-FL treatment reduced their expression levels. In addition, 2'-FL application and observation of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) revealed it was converted from LC3-I to LC3-𝚷, indicating increased autophagy. Likewise, confocal microscopy revealed an increase in LC3 puncta after 2'-FL treatment. Therefore, it is suggested that 2'-FL-mediated activation of autophagy reduces melanogenesis by inhibiting the expression levels of TYR and TYRP1 proteins. In conclusion, it has been confirmed that 2'-FL induces autophagy and suppresses melanin production, so its potential as a whitening cosmetic material is expected.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.105-109
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• 2017

Autophagy is an intracellular degradation and recycling system. Oocyte maturation is dynamic process, in which various proteins should be synthesized and degraded. In our previous study, we reported the loci of autophagosome and dynamics of autophagic activity in porcine oocytes during in vitro maturation. In this study, we verified loci of autophagosome in porcine follicular cumulus-oocyte complex by detection of microtubule-associated protein 1A/1B-light chain 3 (LC3) which is the reliable marker of autophagosome. Porcine ovary including various sizes of follicles was fixed within 1 hour after collection from slaughterhouse. After fixation, immunohistochemistry was conducted on sliced ovary tissue containing various sizes of follicles by using LC3 antibody. As a result, LC3 signal was clearly detected in both cumulus and oocytes of various sizes of follicles. We also found ring shaped signal which represent autophagosome near oocyte membrane. Most of the signals in oocytes were localized nearby cellular membrane while evenly dispersed in cumulus cells. Therefore, this result suggests that autophagy occurs in porcine COCs (cumulus-oocyte complexes) at follicular stage.

• Biomolecules & Therapeutics
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• v.22 no.5
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• pp.384-389
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• 2014

Adiponectin, an adipokine predominantly secreted from adipose tissue, exhibits diverse biological responses, including metabolism of glucose and lipid, and apoptosis in cancer cells. Recently, adiponectin has been shown to modulate autophagy as well. While emerging evidence has demonstrated that autophagy plays a role in the modulation of proliferation and apoptosis of cancer cells, the role of autophagy in apoptosis of cancer cell caused by adiponectin has not been explored. In the present study, we demonstrated that globular adiponectin (gAcrp) induces both apoptosis and autophagy in human hepatoma cell line (HepG2 cells) and breast cancer cells (MCF-7), as evidenced by increase in caspase-3 activity, Bax, microtubule-associated protein light chain 3-II (LC3 II) protein levels, and autophagosome formation. Interestingly, gene silencing of LC3B, an autophagy marker, significantly enhanced gAcrp-induced apoptosis in both HepG2 and MCF-7 cell lines, whereas induction of autophagy by rapamycin, an mTOR inhibitor, significantly prevented gAcrp-induced apoptosis in hepatoma cells HepG2. Furthermore, modulation of autophagy produced similar effects on gAcrp-induced Bax expression in HepG2 cells. These results implicate that induction of autophagy plays a regulatory role in adiponectin-induced apoptosis of cancer cells, and thus inhibition of autophagy would be a novel promising target to enhance the efficiency of cancer cell apoptosis by adiponectin.

• The Journal of Korean Obstetrics and Gynecology
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• v.28 no.2
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• pp.1-14
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• 2015

Objectives : This study was designed to investigate the effects of Taraxaci Herba (TH) on cell death in breast cancer cells. Methods : In this experiment, the effects of TH on proliferation rates, cell morphology and growth pattern, intracellular reactive oxygen species (ROS) production. In addition, the effects on nuclear condensation, fragmentation and formation of acidic vesicular organelles (AVO) in MCF-7 cells were also investigated. Finally, autophagy related with protein was observed by using western blot method. Results : TH inhibited proliferation of MCF-7 cells, TH elevated intracellular ROS levels significantly. Treatment with TH did not affect nuclear morphologies such as condensation or fragmentation. On the other hand, TH treatment effectively induced AVO. Finally, one of autophagy related with protein, Microtubule-associated proteins 1A/1B light chain 3A (MAP1LC3A, LC3) level was elevated by treatment with TH. Conclusions : These data indicate that TH is able to be used for patient with breast cancer and mechanisms are involved in autophagy through ROS generation.

• Journal of Ginseng Research
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• v.43 no.1
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• pp.86-94
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• 2019

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP-mRFP-LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC.