• 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 Ginseng Research
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• v.44 no.4
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• pp.664-671
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• 2020

Background: Ginsenoside compound-Mc1 (Mc1) is a member of the deglycosylated ginsenosides obtained from ginseng extract. Although several ginsenosides have a cardioprotective effect, this has not been demonstrated in ginsenoside Mc1. Methods: We treated H9c2 cells with hydrogen peroxide (H₂O₂) and ginsenoside Mc1 to evaluate the antioxidant effects of Mc1. The levels of antioxidant molecules, catalase, and superoxide dismutase 2 (SOD2) were measured, and cell viability was determined using the Bcl2-associated X protein (Bax):B-cell lymphoma-extra large ratio, a cytotoxicity assay, and flow cytometry. We generated mice with high-fat diet (HFD)-induced obesity using ginsenoside Mc1 and assessed their heart tissues to evaluate the antioxidant effect and the fibrosis-reducing capability of ginsenoside Mc1. Results: Ginsenoside Mc1 significantly increased the level of phosphorylated AMP-activated protein kinase (AMPK) in the H9c2 cells. The expression levels of catalase and SOD2 increased significantly after treatment with ginsenoside Mc1, resulting in a decrease in the production of H₂O₂-mediated reactive oxygen species. Treatment with ginsenoside Mc1 also significantly reduced the H₂O₂-mediated elevation of the Bax:Bcl2 ratio and the number of DNA-damaged cells, which was significantly attenuated by treatment with an AMPK inhibitor. Consistent with the in vitro data, ginsenoside Mc1 upregulated the levels of catalase and SOD2 and decreased the Bax:B-cell lymphoma-extra large ratio and caspase-3 activity in the heart tissues of HFD-induced obese mice, resulting in reduced collagen deposition. Conclusion: Ginsenoside Mc1 decreases oxidative stress and increases cell viability in H9c2 cells and the heart tissue isolated from HFD-fed mice via an AMPK-dependent mechanism, suggesting its potential as a novel therapeutic agent for oxidative stress-related cardiac diseases.

• 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.

• Tuberculosis and Respiratory Diseases
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• v.75 no.1
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• pp.9-17
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• 2013

Background: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells. Methods: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control. Results: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-$G_1$ phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process. Conclusion: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

• Molecules and Cells
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• v.46 no.9
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• pp.545-557
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• 2023

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg²⁺-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Fe²⁺, and Fe³⁺ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg²⁺-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.11
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• pp.1589-1594
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• 2016

Piperine [(E,E)-5-(3,4-methtylenedioxyphenyl)-2,4-pentadienolypiperidide] is a principal of Piperaceae, including Piper nigrum L. and Piper longum Linn., which has been used as a spice and traditional medicine. In this study, we investigated whether or not piperine has anti-cancer effects on AGS human gastric cancer cells. The results demonstrated that piperine not only inhibited proliferation using MTT assay but also induced apoptotic bodies using DAPI assay in a dose-dependent manner in response to piperine. Expression levels of p53, Bax (pro-apoptotic), cleaved caspase-9, and cleaved-PARP increased, whereas expression levels of Bcl-2, XIAP (anti-apoptotic), and Akt decreased in a dose-dependent manner compared with the control by western blotting analysis. To identify the connection between phospo-Akt and Bcl-2 family in response to piperine, LY249002 (Akt inhibitor) was treated with piperine ($150{\mu}M$). The results were shown that expression of phospo-Akt was reduced whereas expression of Bax and cleaved-PARP increased in a dose-dependent manner. These results indicate that piperine induced apoptosis in AGS cells and may serve as a chemopreventive or therapeutic agent for human gastric cancer.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.4
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• pp.1055-1060
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• 2004

Apoptosis is the ability of cells to self-destruct by the activation of an intrinsic cellular suicide program when the cells are no longer needed or when they are seriously damaged. Morphologically, apoptosis is characterized by the appearance of membrane blebbing, cell shrinkage, chromatin condensation, DNA cleavage, and the fragmentation of the cell membrane-bound apoptotic bodies. Siegesbeckia glabrescens Makino (Siegesbeckiae Herba, SG) has been widely used as treatments for arthritis, and fever, as well as detoxification properties. The present studies were undertaken to evaluate if SG has an anti-apoptotic property. Cell viability was measured by XTT and tryphan blue stain. Morphological characteristic of human aortic smooth muscle cells(HASMC) were visualized with a phase-contrast microscope. SG significantly reduced HASMC, but not human umbilical vein endothelial cell(HUVEC), viability in a dose-dependent manner. Confluent untreated cells at 24hrs showed normal morphology, flat with a uniform polygonal shape. SG-treated cells (0.5㎎/㎖) at 24hrs showed apoptotic morphology. Cells became irregular with elongated lamellipodia, and exhibited condensed chromatin in nuclei with occasional endoucleation. There was an increase in the number of apoptotic cells rounding-up and being detached from the substrate. TUNEL staining of SG-treated cells showed dark-brown stains in nuclei and cytosol. Caspases are central components of the machinery responsible for apoptosis and are generally divided into two categories; the initiator caspases, which include caspases-2,-8,-9, and -10, and the effector caspases, which include caspases-3,-6, and -7. SG decreased anti-caspase-3 protein expression, which means activation of caspases-3 activity. It has been reported that there is a link between NO formation and apoptosis. NO production was accelerated by SG treatment in HASMC. L-NNA, NOS inhibitor, inhibited SG-induced apoptosis. These results, therefore, indicated that both caspases-3 and NO production are involved in apoptosis in smooth muscle cells. According to these results, SG may have a potential effect in the treatment of hypertensive atherosclerosis.

• Biomolecules & Therapeutics
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• v.29 no.5
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• pp.551-561
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• 2021

Thyroid cancer is the most common endocrine malignancy. Patients with well-differentiated thyroid cancers, such as papillary and follicular cancers, have a favorable prognosis. However, poorly differentiated thyroid cancers, such as medullary, squamous and anaplastic advanced thyroid cancers, are very aggressive and insensitive to radioiodine treatment. Thus, novel therapies that attenuate metastasis are urgently needed. We found that both PDGFC and PDGFRA are predominantly expressed in thyroid cancers and that the survival rate is significantly lower in patients with high PDGFRA expression. This finding indicates the important role of PDGF/PDGFR signaling in thyroid cancer development. Next, we established a SW579 squamous thyroid cancer cell line with 95.6% PDGFRA gene insertion and deletions (indels) through CRISPR/Cas9. Protein and invasion analysis showed a dramatic loss in EMT marker expression and metastatic ability. Furthermore, xenograft tumors derived from PDGFRA geneedited SW579 cells exhibited a minor decrease in tumor growth. However, distant lung metastasis was completely abolished upon PDGFRA gene editing, implying that PDGFRA could be an effective target to inhibit distant metastasis in advanced thyroid cancers. To translate this finding to the clinic, we used the most relevant multikinase inhibitor, imatinib, to inhibit PDGFRA signaling. The results showed that imatinib significantly suppressed cell growth, induced cell cycle arrest and cell death in SW579 cells. Our developed noninvasive apoptosis detection sensor (NIADS) indicated that imatinib induced cell apoptosis through caspase-3 activation. In conclusion, we believe that developing a specific and selective targeted therapy for PDGFRA would effectively suppress PDGFRA-mediated cancer aggressiveness in advanced thyroid cancers.

• Journal of Nutrition and Health
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• v.57 no.1
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• pp.43-52
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• 2024

Purpose: Cancer is the leading cause of death in Koreans, with breast cancer being the most common among women. Breast cancer readily metastasizes, and the existing treatment processes impose a significant burden on patients. This study examined whether pomegranate-derived exosome-like nanovesicles (PNVs) have anti-cancer effects by inhibiting cell infiltration and metastasis while increasing apoptosis on breast cancer MDA-MB-231 cells. Methods: Initially, exosome-like nanovesicles were isolated from pomegranate using ultracentrifugation. Subsequently, the size range of these nanovesicles was confirmed using nanoparticle tracking analysis. The ability of breast cancer MDA-MB-231 cells to internalize these natural nanovesicles was assessed with flourescence microscope. The anti-cancer effects of the PNVs were confirmed by applying various concentrations of PNVs (10, 50, 100 ㎍/mL) to MDA-MB-231 cells and systematically assessing their impact on cell viability and migration. Results: The round shape of the lipid bilayer in the PNVs was confirmed, providing crucial insights into their structural properties. We demonstrate that PNVs-associated DiD dye can be efficiently internalized by the MDA-MB-231 cells. The data showed that the PNVs inhibited cell viability, invasion rates, and migration in MDA-MB-231 cells. In addition, PNVs were absorbed into the MDA-MB-231 cells, leading to an increased expression of apoptosis proteins, such as cleaved caspase-3 and phosphorus-JNK, in a concentration-dependent manner. Furthermore, a reduction in cell infiltration and decreased expression of the transition markers MMP-2 and MMP-9 proteins were observed. Conclusion: For the first time, this study suggests that PNVs may be useful in the prevention or treatment of breast cancer by inhibiting the infiltration and metastasis of MDA-MB-231 cells and inducing apoptosis.

• Journal of Life Science
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• v.25 no.1
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• pp.53-61
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• 2015

In this study, we tried to separate the photosensitizer that induces apoptosis of leukemia cells (U937) from perilla leaves. Perilla leaves (Perilla frutescens Britt var. japonica Hara) are a popular vegetable in Korea, being rich in vitamins (A and E), GABA, and minerals. Dried perilla leaves were extracted with methanol to separate the photosensitizer by various chromatographic techniques. The structure of the isolated compound (PL9443) was identified by 1D-NMR, 2D-NMR, and FAB-mass spectroscopy. Absorbance of the UV-Vis spectrum was highest at 410 nm and was confirmed by the 330, 410, and 668 nm. PL9443 compound was determined to be pheophorbide, an ethyl ester having a molecular weight of 620. It was identified as a derivative compound of pheophorbide structure when magnesium comes away from a porphyrin ring. Observation of morphological changes in U937 cells following cell death induced by treated PL9443 compound revealed representative phenomena of apoptosis only in light irradiation conditions (apoptotic body, vesicle formation). Results from examining the cytotoxicity of PL9443 substance against U937 cells showed that inhibition rates of the cell growth were 99.9% with the concentration of 0.32 nM PL9443. Also, the caspase-3/7 activity was 99% against U937 cells with the concentration of 0.08 nM of PL9443 substance. The result of the electrophoresis was that a DNA ladder was formed by the PL9443. The PL9443 compound is a promising lead compound as a photosensitizer for photodynamic therapy of cancer.