• Journal of the Korea Academia-Industrial cooperation Society
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• v.18 no.12
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• pp.521-528
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• 2017

Excessive alcohol consumption is one of the leading causes of many neurological diseases, such as dementia and Alzheimer's disease, and many efforts are under way to solve them. Red ginseng is known to enhance neuronal survival, inhibit apoptosis, and promote nerve regeneration of nerve cells. This study examined whether Rg3-enriched Korean red ginseng extract (KRG) inhibits the apoptosis of PC12 cells caused by alcohol-induced neurotoxicity and how KRG regulates several factors related to the caspase mediated pathway. In this way, the cell survival rate and apoptosis rate of PC12 cells were measured using an EZ-Cytox cell viability assay kit and flow cytometry, respectively. The expression of the apoptosis-related proteins (Bcl-2, Bid, Bax and caspase-3) were analyzed by western blotting, and the significance of the measured results was confirmed using the ANOVA method. As a result, KRG increased the expression of Bcl-2; inhibited the expression of Bid, Bax, and caspase-3; and inhibited the apoptosis of alcohol-induced PC12 cells. These results mean that the KRG-induced increase in Bcl-2 expression and down-regulation of Bid and Bax expression down-regulate caspase-3 expression, which in turn inhibits the mitochondrial apoptotic pathways. This study suggests that KRG is worth developing as a neuroprotective agent candidate.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.12
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• pp.1827-1834
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• 2014

Colon cancer is the third highest cause of death in Korea. Known dietary causes of colon cancer include a diet rich in fat and red meat as well as inadequate intake of dietary fiber, fruits, and vegetables. Therefore, recent research has focused on the anticancer effects of natural products. Opuntia humifusa is a type of prickly pear that is known to contain biologically active compounds that can be used in the treatment of diabetes mellitus, arteriosclerosis, and hyperglycemia. The aim of this study was to determine whether or not O. humifusa extract affects proliferation, cell death, and DNA fragmentation in human carcinoma HT-29 cells. O. humifusa is rich in carbohydrates, minerals (Mg, K, and Ca), and total phenolics. HT-29 cells were treated with extracts of O. humifusa at concentrations of 0, 0.25, 0.5, 1, and 2 mg/mL for 24 or 48 hours. O. humifusa extracts inhibited HT-29 cell growth in a dose-dependent manner. Hoechst 33342/PI double staining and Comet assay were performed to observe changes in nuclei of cancer cells undergoing cell death. The results of both tests showed that O. humifusa extract induced cell shrinkage, DNA fragmentation, and chromatin condensation dose-dependently in HT-29 cells. The results of this study suggest that O. humifusa extract inhibits the growth of HT-29 via induction of DNA fragmentation and chromatin condensation.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.392-402
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• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

• Journal of Ginseng Research
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• v.44 no.2
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• pp.258-266
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• 2020

Background: Oxidative stress-induced cardiomyocytes apoptosis is a key pathological process in ischemic heart disease. Glutathione reductase (GR) reduces glutathione disulfide to glutathione (GSH) to alleviate oxidative stress. Ginsenoside Rb1 (GRb1) prevents the apoptosis of cardiomyocytes; however, the role of GR in this process is unclear. Therefore, the effects of GRb1 on GR were investigated in this study. Methods: The antiapoptotic effects of GRb1 were evaluated in H9C2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, annexin V/propidium iodide staining, and Western blotting. The antioxidative effects were measured by a reactive oxygen species assay, and GSH levels and GR activity were examined in the presence and absence of the GR inhibitor 1,3-bis-(2-chloroethyl)-1-nitrosourea. Molecular docking and molecular dynamics simulations were used to investigate the binding of GRb1 to GR. The direct influence of GRb1 on GR was confirmed by recombinant human GR protein. Results: GRb1 pretreatment caused dose-dependent inhibition of tert-butyl hydroperoxide-induced cell apoptosis, at a level comparable to that of the positive control N-acetyl-L-cysteine. The binding energy between GRb1 and GR was positive (-6.426 kcal/mol), and the binding was stable. GRb1 significantl reduced reactive oxygen species production and increased GSH level and GR activity without altering GR protein expression in H9C2 cells. Moreover, GRb1 enhanced the recombinant human GR protein activity in vitro, with a half-maximal effective concentration of ≈2.317 μM. Conversely, 1,3-bis-(2-chloroethyl)-1-nitrosourea co-treatment significantly abolished the GRb1's apoptotic and antioxidative effects of GRb1 in H9C2 cells. Conclusion: GRb1 is a potential natural GR agonist that protects against oxidative stress-induced apoptosis of H9C2 cells.

• Journal of Life Science
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• v.20 no.7
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• pp.1121-1126
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• 2010

The oxidative stress induced by reactive oxygen species (ROS) may play an important role in the pathogenesis of neurodegenerative diseases. In this study, we investigated the neuroprotective effects of methanolic extracts of Prunella Spica (PSE) against $H_2O_2$-induced oxidative stress in PC12 cells. The cells exposed to $H_2O_2$-induced oxidative stress were treated with various concentrations of PSE; this treatment resulted in the induction of a dose-dependent protective effect, which was evidenced by the results of MTT reduction assay, lactate dehydrogenase (LDH) release assay, morphological assay, and colony-formation assay. Interestingly, we also observed reduction of apoptotic bodies in the Hoechst staining and flow cytometric analysis. These data show that apoptosis was significantly suppressed in the PC12 cells that were exposed to $H_2O_2$-induced oxidative stress and treated with PSE. These results suggest that Prunella Spica could be a new potential protective agent against $H_2O_2$-induced oxidative stress.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.249-255
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• 2014

Programmed cell death or apoptosis is associated with changes in $K^+$ concentration in many cell types. Recent studies have demonstrated that two-pore domain $K^+$ ($K_{2P}$) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of $K_{2P}$ channels, were found to be critical for cell death. This study was performed to identify the role of $K^+$ channels in the $H_2O_2$-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to $H_2O_2$ for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited $H_2O_2$-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (${\beta}$-mercaptoethanol) with 25 mM KCl significantly decreased the rate of $H_2O_2$-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of $K^+$ channel efflux for a short-time reduces $H_2O_2$- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of $K^+$ channels which are highly expressed in a given cell type.

• Biomolecules & Therapeutics
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• v.28 no.5
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• pp.443-455
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• 2020

The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.

• Journal of Ginseng Research
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• v.39 no.1
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• pp.46-53
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• 2015

Background: Glucocorticoids (GCs) are commonly used in many chemotherapeutic protocols and play an important role in the normal regulation of bone remodeling. However, the prolonged use of GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. In this study, effects of Korean Red Ginseng (KRG) on GC-treated murine osteoblastic MC3T3-E1 cells and a GC-induced osteoporosis mouse model were investigated. Methods: MC3T3-E1 cells were exposed to dexamethasone (Dex) with or without KRG and cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Realtime polymerase chain reaction was performed to evaluate the apoptotic gene expression; osteogenic gene expression and alkaline phosphatase (ALP) activity were also measured. Western blotting was performed to evaluate the mitogen-activated protein kinase (MAPK) proteins. A GC-induced osteoporosis animal model was used for in vivo study. Results and conclusion: The MTT assay revealed that Korean Red Ginseng (KRG) prevents loss of cell viability caused by Dex-induced apoptosis in MC3T3E1 cells. Real-time polymerase chain reaction data showed that groups treated with both Dex and KRG exhibited lower mRNA levels of caspase-3 and -9, whereas the mRNA levels of Bcl2, IAPs, and XIAP increased. Moreover, groups treated with both Dex and KRG demonstrated increased mRNA levels of ALP, RUNX2, and bone morphogenic proteins as well as increased ALP activity in MC3T3-E1 cells, compared to cells treated with Dex only. In addition, KRG increased protein kinase B (AKT) phosphorylation and decreased c-Jun N-terminal kinase (JNK) phosphorylation. Moreover, microcomputed tomography analysis of the femurs showed that GC implantation caused trabecular bone loss. However, a significant reduction of bone loss was observed in the KRG-treated group. These results suggest that the molecular mechanism of KRG in the GC-induced apoptosis may lead to the development of therapeutic strategies to prevent and/or delay osteoporosis.

• Journal of Life Science
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• v.29 no.5
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• pp.514-523
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• 2019

We investigated the gene expression patterns and the mechanisms of action of the apoptotic response by microarray analysis of human keratinocyte HaCaT cells treated with ginsenoside Rb1, a saponin of Panax ginseng C. A. Meyer. Genes related to apoptosis, the G2/M transition of the mitotic cell cycle, cell division, mitotic nuclear division, and intracellular protein transport were 2-fold up-regulated in HaCaT cells treated with the ginsenoside Rb1, whereas genes related to DNA repair, regeneration fission, and extracellular matrix organization were 2-fold down-regulated. Apoptosis signaling may be mediated by FAS and PLA2G4A, and pathway analysis indicated that STAT3 might be an upstream regulator of these genes. The activity of FAS and PLA2G4A was verified by qPCR, which showed that FAS was increased about 2-fold in HaCaT cells treated with $10{\mu}g/ml$ of ginsenoside Rb1 for 24 hr, PLA2G4A was increased about twice after 6 hours, and gene expression was increased more than 2-fold after 24 hr. Knockdown of STAT3 with siRNA decreased FAS expression and increased PLA2G4A expression but only FAS was passed from the upstream regulator STAT3. These results indicate that STAT3, which is an upstream regulator, induces apoptosis via FAS during treatment with ginsenoside Rb1.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.73-81
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• 2023

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.