• Journal of Ginseng Research
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• v.44 no.1
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• pp.67-78
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• 2020

Background: Gintonin (GT), a novel ginseng-derived exogenous ligand of lysophosphatidic acid (LPA) receptors, has been shown to induce cell proliferation and migration in the hippocampus, regulate calcium-dependent ion channels in the astrocytes, and reduce β-amyloid plaque in the brain. However, whether GT influences autophagy in cortical astrocytes is not yet investigated. Methods: We examined the effect of GT on autophagy in primary cortical astrocytes using immunoblot and immunocytochemistry assays. Suppression of specific proteins was performed via siRNA. LC3 puncta was determined using confocal microscopy. Results: GT strongly upregulated autophagy marker LC3 by a concentration- as well as time-dependent manner via G protein-coupled LPA receptors. GT-induced autophagy was further confirmed by the formation of LC3 puncta. Interestingly, on pretreatment with an mammalian target of rapamycin (mTOR) inhibitor, rapamycin, GT further enhanced LC3-II and LC3 puncta expression. However, GT-induced autophagy was significantly attenuated by inhibition of autophagy by 3-methyladenine and knockdown Beclin-1, Atg5, and Atg7 gene expression. Importantly, when pretreated with a lysosomotropic agent, E-64d/peps A or bafilomycin A1, GT significantly increased the levels of LC3-II along with the formation of LC3 puncta. In addition, GT treatment enhanced autophagic flux, which led to an increase in lysosome-associated membrane protein 1 and degradation of ubiquitinated p62/SQSTM1. Conclusion: GT induces autophagy via mTOR-mediated pathway and elevates autophagic flux. This study demonstrates that GT can be used as an autophagy-inducing agent in cortical astrocytes.

• 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.

• Animal Bioscience
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• v.35 no.6
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• pp.847-857
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• 2022

Objective: The effects of maternal undernutrition during midgestation on muscle fiber histology, myosin heavy chain (MyHC) expression, methylation modification of myogenic factors, and the mammalian target of rapamycin (mTOR) signaling pathway in the skeletal muscles of prenatal and postnatal goats were examined. Methods: Twenty-four pregnant goats were assigned to a control (100% of the nutrients requirement, n = 12) or a restricted group (60% of the nutrients requirement, n = 12) between 45 and 100 days of gestation. Descendants were harvested at day 100 of gestation and at day 90 after birth to collect the femoris muscle tissue. Results: Maternal undernutrition increased (p<0.05) the fiber area of the vastus muscle in the fetuses and enhanced (p<0.01) the proportions of MyHCI and MyHCIIA fibers in offspring, while the proportion of MyHCIIX fibers was decreased (p<0.01). DNA methylation at the +530 cytosine-guanine dinucleotide (CpG) site of the myogenic factor 5 (MYF5) promoter in restricted fetuses was increased (p<0.05), but the methylation of the MYF5 gene at the +274,280 CpG site and of the myogenic differentiation (MYOD) gene at the +252 CpG site in restricted kids was reduced (p<0.05). mTOR protein signals were down-regulated (p<0.05) in the restricted offspring. Conclusion: Maternal undernutrition altered the muscle fiber type in offspring, but its relationship with methylation in the promoter regions of myogenic genes needs to be elucidated.

• Journal of Ginseng Research
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• v.47 no.1
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• pp.133-143
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• 2023

Background: Past studies suggested that ginseng extracts and ginseng-derived molecules exerted significant regulatory effects on skin. However, no reports have described the effects of ginseng-derived nanoparticles (GDNPs) on skin cell proliferation and wound healing. In this study, we investigated whether GDNPs regulate the proliferation of skin cells and promote wound healing in a mouse model. Methods: GDNPs were separated and purified via differential centrifugation and sucrose/D2O gradient ultracentrifugation. GDNP uptake, cell proliferation and cell cycle progression were measured by confocal microscopy, CCK-8 assay and flow cytometry, respectively. Cell migration and angiogenic effects were assessed by the wound scratch assay and tube formation assay, respectively. ELISA was used to detect extracellular matrix secretion. The relevant signaling pathway was confirmed by western blotting. The effects of GDNPs on skin wound healing were assessed by wound observation, HE staining, and western blotting. Results: GDNPs possessed the essential features of exosomes, and they were accumulated by skin cells. Treatment with GDNPs notably enhanced the proliferation of HaCaT, BJ and HUVECs. GDNPs also enhanced the migration in HaCaT cells and HUVECs and angiogenesis in HUVECs. GDNPs increased the secretion of MMP-1, fibronectin-1, elastin-1, and COL1A1 in all three cell lines. GDNPs regulated cell proliferation through the ERK and AKT/ mTOR pathways. Furthermore, GDNPs facilitated skin wound healing and decreased inflammation in a mouse skin wound model. Conclusion: GDNPs can promote skin wound healing through the ERK and AKT/mTOR pathways. GDNPs thus represent an alternative treatment for chronic skin wounds.

• Toxicological Research
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• v.26 no.4
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• pp.261-266
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• 2010

In the workplace, the arsenic is used in the semiconductor production and the manufacturing of pigments, glass, pesticides and fungicides. Therefore, workers may be exposed to airborne arsenic during its use in manufacturing. The purpose of this study was to evaluate the potential toxicity of particulate matters (PMs) doped with arsenic (PMs-Arsenic) using a rodent model and to compare the genotoxicity in various concentrations and to examine the role of PMs-Arsenic in the induction of signaling pathway in the lung. Mice were exposed to PMs $124.4{\pm}24.5\;{\mu}g/m^3$ (low concentration), $220.2{\pm}34.5\;{\mu}g/m^3$ (middle concentration), $426.4{\pm}40.3\;{\mu}g/m^3$ (high concentration) doped with arsenic $1.4\;{\mu}g/m^3$ (Low concentration), $2.5\;{\mu}g/m^3$ (middle concentration), $5.7\;{\mu}g/m^3$ (high concentration) for 4 wks (6 h/d, 5 d/wk), respectively in the whole-body inhalation exposure chambers. To determine the level of genotoxicity, Chromosomal aberration (CA) assay in splenic lymphocytes and Supravital micronucleus (SMN) assay were performed. Then, signal pathway in the lung was analyzed. In the genotoxicity experiments, the increases of aberrant cells were concentration-dependent. Also, PMs-arsenic caused peripheral blood micronucleus frequency at high concentration. The inhalation of PMs-Arsenic increased an expression of phosphorylated Akt (p-Akt: protein kinase B) and phpsphorylated mammalian target of rapamycin (p-mTOR) at high concentration group. Taken together, inhaled PMs-Arsenic caused genotoxicity and altered Akt signaling pathway in the lung. Therefore, the inhalation of PMs-Arsenic needs for a careful risk assessment in the workplace.

• Molecules and Cells
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• v.43 no.10
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• pp.856-869
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• 2020

To elucidate the mechanism of action of HOXA11-AS in modulating the cisplatin resistance of nasopharyngeal carcinoma (NPC) cells. HOXA11-AS and miR-454-3p expression in NPC tissue and cisplatin-resistant NPC cells were measured via quantitative reverse transcriptase polymerase chain reaction. NPC parental cells (C666-1 and HNE1) and cisplatin-resistant cells (C666-1/DDP and HNE1/DDP) were transfected and divided into different groups, after which the MTT method was used to determine the inhibitory concentration 50 (IC₅₀) of cells treated with different concentrations of cisplatin. Additionally, a clone formation assay, flow cytometry and Western blotting were used to detect DDP-induced changes. Thereafter, xenograft mouse models were constructed to verify the in vitro results. Obviously elevated HOXA11-AS and reduced miR-454-3p were found in NPC tissue and cisplatin-resistant NPC cells. Compared to the control cells, cells in the si-HOXA11-AS group showed sharp decreases in cell viability and IC₅₀, and these results were reversed in the miR-454-3p inhibitor group. Furthermore, HOXA11-AS targeted miR-454-3p, which further targeted c-Met. In comparison with cells in the control group, HNE1/DDP and C666-1/DDP cells in the si-HOXA11-AS group demonstrated fewer colonies, with an increase in the apoptotic rate, while the expression levels of c-Met, p-Akt/Akt and p-mTOR/mTOR decreased. Moreover, the si-HOXA11-AS-induced enhancement in sensitivity to cisplatin was abolished by miR-454-3p inhibitor transfection. The in vivo experiment showed that DDP in combination with si-HOXA11-AS treatment could inhibit the growth of xenograft tumors. Silencing HOXA11-AS can inhibit the c-Met/AKT/mTOR pathway by specifically upregulating miR-454-3p, thus promoting cell apoptosis and enhancing the sensitivity of cisplatin-resistant NPC cells to cisplatin.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.219-226
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• 2015

The anticancer activity of a methanolic extract from lemon leaves (MLL) was assessed in MCF-7-SC human breast cancer stem cells. MLL induced apoptosis in MCF-7-SC, as evidenced by increased apoptotic body formation, sub-G1 cell population, annexin V-positive cells, Bax/Bcl-2 ratio, as well as proteolytic activation of caspase-9 and caspase-3, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Concomitantly, MLL induced the formation of acidic vesicular organelles, increased LC3-II accumulation, and reduced the activation of Akt, mTOR, and p70S6K, suggesting that MLL initiates an autophagic progression in MCF-7-SC via the Akt/mTOR pathway. Epithelial-mesenchymal transition (EMT), a critical step in the acquisition of the metastatic state, is an attractive target for therapeutic interventions directed against tumor metastasis. At low concentrations, MLL induced anti-metastatic effects on MCF-7-SC by inhibiting the EMT process. Exposure to MLL also led to an increase in the epithelial marker E-cadherin, but decreased protein levels of the mesenchymal markers Snail and Slug. Collectively, this study provides evidence that lemon leaves possess cytotoxicity and anti-metastatic properties. Therefore, MLL may prove to be beneficial as a medicinal plant for alternative novel anticancer drugs and nutraceutical products.

• Biomolecules & Therapeutics
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• v.31 no.6
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• pp.599-610
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• 2023

According to recent evidence, ferroptosis is a major cell death mechanism in the pathogenesis of kidney injury and fibrosis. Despite the renoprotective effects of classical ferroptosis inhibitors, therapeutic approaches targeting kidney ferroptosis remain limited. In this study, we assessed the renoprotective effects of melatonin and zileuton as a novel therapeutic strategy against ferroptosis-mediated kidney injury and fibrosis. First, we identified RSL3-induced ferroptosis in renal tubular epithelial HK-2 and HKC-8 cells. Lipid peroxidation and cell death induced by RSL3 were synergistically mitigated by the combination of melatonin and zileuton. Combination treatment significantly downregulated the expression of ferroptosis-associated proteins, 4-HNE and HO-1, and upregulated the expression of GPX4. The expression levels of p-AKT and p-mTOR also increased, in addition to that of NRF2 in renal tubular epithelial cells. When melatonin (20 mg/kg) and zileuton (20 mg/kg) were administered to a unilateral ureteral obstruction (UUO) mouse model, the combination significantly reduced tubular injury and fibrosis by decreasing the expression of profibrotic markers, such as α-SMA and fibronectin. More importantly, the combination ameliorated the increase in 4-HNE levels and decreased GPX4 expression in UUO mice. Overall, the combination of melatonin and zileuton was found to effectively ameliorate ferroptosis-related kidney injury by upregulating the AKT/mTOR/ NRF2 signaling pathway, suggesting a promising therapeutic strategy for protection against ferroptosis-mediated kidney injury and fibrosis.

• BMB Reports
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• v.51 no.10
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• pp.508-513
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• 2018

Fibronectin fragments found in the synovial fluid of patients with osteoarthritis (OA) induce the catabolic responses in cartilage. Nuclear high-mobility group protein Box 1 (HMGB1), a damage-associated molecular pattern, is responsible for the regulation of signaling pathways related to cell death and survival in response to various stimuli. In this study, we investigated whether changes induced by 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) in HMGB1 expression influences the pathogenesis of OA via an HMGB1-modulated autophagy signaling pathway. Human articular chondrocytes were enzymatically isolated from articular cartilage. The level of mRNA was measured by quantitative real-time PCR. The expression of proteins was examined by western blot analysis, immnunofluorescence assay, and enzyme-linked immunosorbent assay. Interaction of proteins was evaluated by immunoprecipitation. The HMGB1 level was significantly lower in human OA cartilage than in normal cartilage. Although 29-kDa FN-f significantly reduced the HMGB1 expression at the mRNA and protein levels 6 h after treatment, the cytoplasmic level of HMGB1 was increased in chondrocytes treated with 29-kDa FN-f, which significantly inhibited the interaction of HMGB1 with Beclin-1, increased the interaction of Bcl-2 with Beclin-1, and decreased the levels of Beclin-1 and phosphorylated Bcl-2. In addition, the level of microtubule-associated protein 1 light chain 3-II, an autophagy marker, was down-regulated in chondrocytes treated with 29-kDa FN-f, whereas the effect was antagonized by mTOR knockdown. Furthermore, prolonged treatment with 29-kDa FN-f significantly increased the release of HMGB1 into the culture medium. These results demonstrated that 29-kDa FN-f inhibits chondrocyte autophagy by modulating the HMGB1 signaling pathway.

• Journal of Life Science
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• v.23 no.5
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• pp.689-697
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• 2013

In the United States, about 40,000 new cases of oral cancer are diagnosed each year and nearly 7,800 patients died from it in 2012. Omega-3 polyunsaturated fatty acids have been found to have anticancer effects in a variety of cancer cell lines and animal models, but their effect in oral cancer remains unclear. This study was designed to examine the effect of docosahexaenoic acid (DHA, a kind of omega-3 fatty acid) on oral cancer cells and the molecular mechanism of its action. We found that exposure of squamous cell carcinoma-4 (SCC-4) and squamous cell carcinoma-9 (SCC-9) human oral cancer cells to DHA induced growth inhibition in a dose- and time-dependent manner. Meanwhile, in addition to the elevated levels of apoptotic markers, such as cleaved PARP, subG1 portion and TUNEL-positive nuclei, DHA led to autophagic vesicle formation and an increase in autophagic flux, indicating the involvement of both apoptosis and autophagy in the inhibitory effects of DHA on oral cancer cells. Further experiments revealed that the apoptosis and autophagy induced by DHA were linked to inhibition of mammalian target of rapamycin (mTOR) signaling by AKT inhibition and AMP-activated protein kinase (AMPK) activation in SCC-9 cells. Together, our results suggest that DHA induces apoptosis- and autophagy-associated cell death through the AMPK/AKT/mTOR signaling pathway in oral cancer cells. Thus, utilization of omega-3 fatty acids may represent a promising therapeutic approach for chemoprevention and treatment of human oral cancer.