• Journal of Sasang Constitutional Medicine
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• v.25 no.3
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• pp.208-217
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• 2013

Objectives Modified Yeolda-Hanso tang (MYH) is a traditional herbal formula in Korea for various diseases. MYH is containing the 10 herbs : Pueraria lobata (Willd.) Ohwi, Angelica tenuissima Nakai, Scutellaria baicalensis Georgi, Platycodon grandiflorum (Jacq), Angelicae Dahurica, Cimicifuga heracleifolia Kom, Raphanus sativa L., Polygala tenuifolia (Willd), Acorus gramineus Soland and Dimocarpus longan Lour. The 10 herbs is constituted as a ratio of the 6:4:2:1:2:2:2:4:6:6. We investigated neuroprotective effects of MYH on human neuroblastoma SH-SY5Y cells and evaluated the ability of MYH to prevent and treat for neurodegenerative diseases such as Parkinson's disease via basal autophagy enhancement. Methods Pharmacological induction of Autophagy by MYH in SH-SY5Y cells: Induction of autophagy by MYH in human neuroblastoma SH-SY5Y cells was carreid out by immunoblot analysis with several autophagy markers. SH-SY5Y cells were treated with MYH at the concentration of 400 and $800{\mu}g/ml$ for 24 hr. Specifically, the autophagosome proteins LC3 II and Atg5 levels were increased and autophagy pathway related proteins such as beclin-1, PI3 Kinase class III protein, ULK1, mTOR and AMPK were activated. Conclusions MYH can enhance the induction of autophagy through key regulator AMPK, mTOR, and Beclin-1 and it should be considered as a possible candidate of neuroprotective agents for such as Parkinson's disease.

• Molecules and Cells
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• v.38 no.2
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• pp.138-144
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• 2015

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

• Reproductive and Developmental Biology
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• v.37 no.2
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• pp.65-73
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• 2013

The objective of this study was to examine the effects of high concentrations of glucose on porcine parthenotes developing in vitro. Addition of 55 mM glucose to the culture medium of embryos at the four-cell-stage significantly inhibited blastocyst formation, resulting in fewer cells in blastocyst-stage embryos and increased levels of apoptosis and autophagy compared to control. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression of pro-apoptotic genes (Caspase 3, Bax and Bak) and autophagy genes (Atg6 and Atg8/Lc3) were increased significantly by the addition of 55 mM glucose to the culture medium compared to control. MitoTracker Green fluorescence revealed a decrease in the overall mitochondrial mass compared to control. However, the addition of 55 mM glucose had no effect on mRNA expression of the nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1. These results suggest that hyperglycemia reduced the mitochondrial content of porcine embryos developing in vitro and that this may hinder embryonic development to the blastocyst stage and embryo quality by increasing apoptosis and autophagy in these embryos.

• Animal Bioscience
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• v.37 no.2
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• pp.284-294
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• 2024

Objective: Pork is an important source of animal protein in many countries. Subtle physiochemical changes occur during pork postmortem aging. The changes of apoptosis and autophagy in pork at 6 h to 72 h after slaughter were studied to provide evidence for pork quality. Methods: In this article, morphological changes of postmortem pork was observed through Hematoxylin-eosin staining, apoptotic nuclei were observed by TdT-mediated dUTP nick end labeling assay, protein related to apoptosis and autophagy expressions were tested by western blot and LC3 level were expressed according to immunofluorescence assay. Results: In this study, we found the occurrence of apoptosis in postmortem pork, and the process was characterized by nucleus condensation and fragmentation, formation of apoptotic bodies, increase in apoptosis-related Bax/Bcl-2 levels, and activation of caspases. Autophagy reached its peak between 24 and 48 h after slaughter, accompanied by the formation of autophagosomes on the cell membrane and expression of autophagy-related proteins beclin-1, P62, LC3-I, LC3-II, and ATG5. Conclusion: Obvious apoptosis was observed at 12 h and autophagy reached its peak at 48 h. The present work provides the evidence for the occurrence of apoptosis and autophagy during postmortem aging of pork. In conclusion, the apoptosis and autophagy of muscle cells discovered in this study have important implications for pork in the meat industry.

• Biomolecules & Therapeutics
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• v.30 no.6
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• pp.616-624
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• 2022

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has been suggested as a repositioning candidate for the treatment of brain tumors. However, the efficacy of MBZ needs further study to improve the beneficial effect on the survival of those patients. In this study, we explored a novel strategy to improve MBZ efficacy using a drug combination. When glioblastoma cells were treated with MBZ, cell proliferation was dose-dependently inhibited with an IC₅₀ of less than 1 µM. MBZ treatment also inhibited glioblastoma cell migration with an IC₅₀ of less than 3 µM in the Boyden chamber migration assay. MBZ induced G2-M cell cycle arrest in U87 and U373 cells within 24 h. Then, at 72 h of treatment, it mainly caused cell death in U87 cells with an increased sub-G1 fraction, whereas polyploidy was seen in U373 cells. However, MBZ treatment did not affect ERK1/2 activation stimulated by growth factors. The marked induction of autophagy by MBZ was observed, without any increased expression of autophagy-related genes ATG5/7 and Beclin 1. Co-treatment with MBZ and the autophagy inhibitor chloroquine (CQ) markedly enhanced the anti-proliferative effects of MBZ in the cells. Triple combination treatment with temozolomide (TMZ) (another autophagy inducer) further enhanced the anti-proliferative effect of MBZ and CQ. The combination of MBZ and CQ also showed an enhanced effect in TMZ-resistant glioblastoma cells. Therefore, we suggest that the modulation of protective autophagy could be an efficient strategy for enhancing the anti-tumor efficacy of MBZ in glioblastoma cells.

• International Journal of Oral Biology
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• v.45 no.1
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• pp.1-7
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• 2020

Oral lichen planus (OLP) is a chronic inflammatory disease observed in approximately 0.5-2.2% of the population, and it is recognized as a premalignant lesion that can progress into oral squamous cell carcinoma (OSCC). The rate of malignant transformation is approximately 1.09-2.3%, and the risk factors for malignant transformation are age, female, erosive type, and tongue site location. Malignant transformation of OLP is likely related to the low frequency of apoptotic phenomena. Therefore, apoptosis-related genetic factors, like p53, BCL-2, and BAX are reviewed. Increased p53 expression and altered expression of BCL-2 and BAX were observed in OLP patients, and the malignant transformation rate in these patients was relatively higher. The involvement of microRNA (miRNA) in the malignant transformation of OLP is also reviewed. Because autophagy is involved in cell survival and death through the regulation of various cellular processes, autophagy-related genetic factors may function as factors for malignant transformation. In OLP, decreased levels of ATG9B mRNA and a higher expression of IGF1 were observed, suggesting a reduction in cell death and autophagic response. Activated IGF1-PI3K/AKT/mTor cascade may play an important role in a signaling pathway related to the malignant transformation of OLP to OSCC. Recent research has shown that miRNAs, such as miR-199 and miR-122, activate the cascade, increasing the prosurvival and proproliferative signals.

• Fisheries and Aquatic Sciences
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• v.26 no.6
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• pp.406-422
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• 2023

Sarcopenia is an age-related, progressive skeletal muscle disorder involving the loss of muscle mass and strength. Previous studies have shown that γ-aminobutyric acid (GABA) from fermented oysters aids in regulatory T cells (Tregs) cell expansion and function by enhancing autophagy, and concomitantly mediate muscle regeneration by modulating muscle inflammation and satellite cell function. The fermentation process of oysters not only increases the GABA content but also enhances the content of branched amino acids and free amino acids that aid the level of protein absorption and muscle strength, mass, and repair. In this study, the effect of GABA-enriched fermented sarco oyster extract (FSO) on reduced muscle mass and functions via Treg modulation and enhanced autophagy in aged mice was investigated. Results showed that FSO enhanced the expression of autophagy markers (autophagy-related gene 5 [ATG5] and GABA receptor-associated protein [GABARAP]), forkhead box protein 3 (FoxP3) expression, and levels of anti-inflammatory cytokines (interleukin [IL]-10 and transforming growth factor [TGF]-β) secreted by Tregs while reducing pro-inflammatory cytokine levels (IL-17A and interferon [IFN]-γ). Furthermore, FSO increased the expression of IL-33 and its receptor IL-1 receptor-like 1 (ST2); well-known signaling pathways that increase amphiregulin (Areg) secretion and expression of myogenesis markers (myogenic factor 5, myoblast determination protein 1, and myogenin). Muscle mass and function were also enhanced via FSO. Overall, the current study suggests that FSO increased autophagy, which enhanced Treg accumulation and function, decreased muscle inflammation, and increased satellite cell function for muscle regeneration and therefore could decrease the loss of muscle mass and function with aging.

• Biomolecules & Therapeutics
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• v.29 no.2
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• pp.211-219
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• 2021

Alopecia is a distressing condition caused by the dysregulation of anagen, catagen, and telogen in the hair cycle. Dermal papilla cells (DPCs) regulate the hair cycle and play important roles in hair growth and regeneration. Myristoleic acid (MA) increases Wnt reporter activity in DPCs. However, the action mechanisms of MA on the stimulation of anagen signaling in DPCs is not known. In this study, we evaluated the effects of MA on anagen-activating signaling pathways in DPCs. MA significantly increased DPC proliferation and stimulated the G2/M phase, accompanied by increasing cyclin A, Cdc2, and cyclin B1. To elucidate the mechanism by which MA promotes DPC proliferation, we evaluated the effect of MA on autophagy and intracellular pathways. MA induced autophagosome formation by decreasing the levels of the phospho-mammalian target of rapamycin (phospho-mTOR) and increasing autophagy-related 7 (Atg7) and microtubule-associated protein 1A/1B-light chain 3II (LC3II). MA also increased the phosphorylation levels of Wnt/β-catenin proteins, such as GSK3β (Ser9) and β-catenin (Ser552 and Ser675). Treatment with XAV939, an inhibitor of the Wnt/β-catenin pathway, attenuated the MA-induced increase in β-catenin nuclear translocation. Moreover, XAV939 reduced MA-induced effects on cell cycle progression, autophagy, and DPC proliferation. On the other hand, MA increased the levels of phospho (Thr202/Tyr204)-extracellular signal regulated kinases (ERK). MA-induced ERK phosphorylation led to changes in the expression levels of Cdc2, Atg7 and LC3II, as well as DPC proliferation. Our results suggest that MA promotes anagen signaling via autophagy and cell cycle progression by activating the Wnt/β-catenin and ERK pathways in DPCs.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2022.09a
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• pp.106-106
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• 2022

The cone of Red Pine (Pinus densiflora), which has been used as a drug in traditional medicine. Its ethyl acetate fraction was reported to exert antioxidant, anti-melanogenesis, and anti-inflammation activites. Apoptosis of hepatic stellate cells (LX-2) is regarding as a potential strategy for alleviation of hepatic fibrosis. We conducted to investigated whether the treatment of cone has a potential to control of some factors related in apoptosis and autophagy in cell signaling pathways. We suggest that the cone induced apoptosis through confirming the expression levels of genes (cPARP, Bcl-XL, Bax, p53, and caspase-3) in LX-2 cells. Also, the cone may regulate autophagy (LC3, p62, Beclin-1, and ATG12). Remarkably, the treatment of cone may affect to formation of autophagosomes in the immunofluorescence image in live cells. These findings suggest that the ethyl acetate fraction from the cone of Red Pine (P. densiflora) may have potential as an alternative therapeutic agent for the alleviation and prevention of liver fibrosis.

• Journal of the Korean Applied Science and Technology
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• v.37 no.1
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• pp.91-101
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• 2020

The purpose of this study was to investigate the influence of obesity on the expression of autophagy-related proteins in cardiac muscle. To this end, obesity was induced in rats through 20 weeks of high-fat diet, and the animals were then subjected to 8 weeks of treadmill exercise. Subsequently, the expression of proteins that regulate the induction of autophagy, formation of autophagosome, and fusion of autophagosome and lysosome was confirmed. Obesity was induced in the experimental animals (SD rats) through 20 weeks of high-fat diet (carbohydrate: 20%, fat: 60%, and protein: 20%), and they were subsequently subjected to 8 weeks of treadmill exercise (5 days/week, 30 min/day, 5 minutes; 8m/min, 5 minutes; 11m/min, 20 minutes; 14m/min). The experimental groups comprised the normal diet control group (ND-CON, n=10), high-fat diet comparison group (HFD-CON, n=10), and high-fat exercise group (HFD-TE, n=10). Oral glucose tolerance test was conducted before and after 8 weeks of treadmill exercise, and the area under the curve (AUC) was calculated. Through fasting insulin and fasting glucose levels, HOMA-IR, which is an index of insulin resistance, and abdominal visceral fat/body weight (AVF/BW) were calculated for comparison. Moreover, autophagy-related proteins were analyzed from cardiac tissue to investigate the effects of exercise training. Obesity was successfully induced in the HFD-CON group through long-term high-fat diet, and the HFD-CON group had higher body weight, AUC, HOMA-IR, and AVF/BW compared to the ND-CON group. The HFD-TE group, which underwent 8 weeks of treadmill exercise, showed improvements in AUC, HOMA-IR, and AVF/BW. Although the body weight tended to decrease as well, there was no statistically significant difference. mTOR and AMPK, which are involved in the induction of autophagy, both decreased in obesity but increased upon exercise. Beclin-1, BNIP3, ATG-7, p62, and LC3, which are related to the formation of autophagosomes, all increased in obesity and decreased after exercise. Cathepsin L and LAMP2, which regulate the fusion of autophagosome and lysosome, both decreased in obesity and increased upon exercise. Physical activity, including treadmill exercise, was found to induce normal autophagy and improve pathological phenomena observed in metabolic diseases. Therefore, the findings suggest the need to consider treadmill exercise as a primary means to achieve effective prevention and treatment of cardiac diseases.