• Journal of Life Science
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• v.31 no.3
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• pp.266-279
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• 2021

The present study examined the cytotoxic effects of a Smilax china L. extract (SCLE) in human cancer (A-549, MCF-7, MDA-MB-231, U87-MG, AGS, MKN-74, and SNU-601) and normal MRC-5 fibroblasts, as well as in mesenchymal stem cells derived from dental tissue (DSC). The 50% inhibitory concentration (IC50) values for SCLE were significantly (p<0.05) lower in the cancer cell lines (A-549, MCF-7, MDA-MB-231, U87-MG, AGS, MKN-74 and SNU-601) than in the MRC-5 and DSC cells. Cell growth was significantly (p<0.05) more inhibited in the cancer cell lines treated with 200 ㎍/ml SCLE than in the normal MRC-5 and DSC, and anoikis-like floating cell morphology was observed in the SCLE-treated cancer cells. The cells detached by SCLE treatment were retrieved daily and assayed for viability and telomerase activity. Cells retrieved at 4 days showed significantly decreased viability and telomerase activity (p<0.05), as well as apoptosis-like abnormal morphology, when compared to cells retrieved in the previous 3 days. The ratio of apoptosis and cells in the G1 phase was significantly (p<0.05) increased in the A-549, AGS, and MCF-7 cancer cells treated with SCLE for 4 days compared to untreated controls. However, after SCLE treatment, cell adhesion was not increased by application of an inhibitor of the associated protein kinase (ROCK) that mainly contributes to the increase in cell attachment. This suggests that the cellular detachment by SCLE is probably controlled by a Rho-independent mechanism(s). These observations indicate that SCLE readily induces anoikis in cancer cells and could serve as a potent agent for cancer chemotherapy.

• Journal of Ginseng Research
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• v.45 no.2
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• pp.295-304
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• 2021

Background: Acute promyelocytic leukemia (APL) is a hematopoietic malignancy driven by promyelocytic leukemia-retinoic acid receptor A (PML-RARA) fusion gene. The therapeutic drugs currently used to treat APL have adverse effects. 20(S)-ginsenoside Rh2 (GRh2) is an anticancer medicine with high effectiveness and low toxicity. However, the underlying anticancer mechanisms of GRh2-induced PML-RARA degradation and apoptosis in human APL cell line (NB4 cells) remain unclear. Methods: Apoptosis-related indicators and PML-RARA expression were determined to investigate the effect of GRh2 on NB4 cells. Z-VAD-FMK, LY294002, and C 87, as inhibitors of caspase, and the phosphatidylinositol 3-kinase (PI3K) and tumor necrosis factor-α (TNF-α) pathways were used to clarify the relationship between GRh2-induced apoptosis and PML-RARA degradation. Results: GRh2 dose- and time-dependently decreased NB4 cell viability. GRh2-induced apoptosis, cell cycle arrest, and caspase3, caspase8, and caspase9 activation in NB4 cells after a 12-hour treatment. GRh2-induced apoptosis in NB4 cells was accompanied by massive production of reactive oxygen species, mitochondrial damage and upregulated Bax/Bcl-2 expression. GRh2 also induced PML/PML-RARA degradation, PML nuclear bodies formation, and activation of the downstream p53 pathway in NB4 cells. Z-VAD-FMK inhibited caspase activation and significantly reversed GRh2-induced apoptosis and PML-RARA degradation. GRh2 also upregulated TNF-α expression and inhibited Akt phosphorylation. LY294002, an inhibitor of the PI3K pathway, enhanced the antitumor effects of GRh2, and C 87, an inhibitor of the TNF-α pathway, reversed NB4 cell viability, and GRh2-mediated apoptosis in a caspase-8-dependent manner. Conclusion: GRh2 induced caspase-dependent PML-RARA degradation and apoptosis in NB4 cells via the Akt/Bax/caspase9 and TNF-α/caspase8 pathways.

• Journal of the Korean Society of Laryngology, Phoniatrics and Logopedics
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• v.32 no.1
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• pp.15-23
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• 2021

Background and Objectives During speech, the vocal folds oscillate at frequencies ranging from 100-200 Hz with amplitudes of a few millimeters. Mechanical stimulation is an essential factor which affects metabolism of human vocal folds. The effect of mechanical vibration on the cellular response in the human vocal fold fibroblasts cells (hVFFs) was evaluated. Materials and Method We created a culture systemic device capable of generating vibratory stimulations at human phonation frequencies. To establish optimal cell culture condition, cellular proliferation and viability assay was examined. Quantitative real time polymerase chain reaction was used to assess extracellular matrix (ECM) related and growth factors expression on response to changes in vibratory frequency and amplitude. Western blot was used to investigate ECM and inflammation-related transcription factor activation and its related cellular signaling transduction pathway. Results The cell viability was stable with vibratory stimulation within 24 h. A statistically significant increase of ECM genes (collagen type I alpha 1 and collagen type I alpha 2) and growth factor [transforming growth factor β1 (TGF-β1) and fibroblast growth factor 1 (FGF-1)] observe under the experimental conditions. Vibratory stimulation induced transcriptional activation of NF-κB by phosphorylation of p65 subunit through cellular Mitogen-activated protein kinases activation by extracellular signal regulated kinase and p38 mitogen-activated protein kinases (MAPKs) phosphorylation on hVFFs. Conclusion This study confirmed enhancing synthesis of collagen, TGF-β1 and FGF was testified by vibratory stimulation on hVFFs. This mechanism is thought to be due to the activation of NF-κB and MAPKs. Taken together, these results demonstrate that vibratory bioreactor may be a suitable alternative to hVFFs for studying vocal folds cellular response to vibratory vocalization.

• Journal of Ginseng Research
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• v.45 no.3
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• pp.401-407
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• 2021

Background: Gintonin is an exogenous ginseng-derived G-protein-coupled lysophosphatidic acid (LPA) receptor ligand. LPA induces in vitro morphological changes and migration through neuronal LPA1 receptor. Recently, we reported that systemic administration of gintonin increases blood-brain barrier (BBB) permeability via the paracellular pathway and its binding to brain neurons. However, little is known about the influences of gintonin on in vivo neuron morphology and migration in the brain. Materials and methods: We examined the effects of gintonin on in vitro migration and morphology using primary hippocampal neural precursor cells (hNPC) and in vivo effects of gintonin on adult brain neurons using real time microscopic analysis and immunohistochemical analysis to observe the morphological and locational changes induced by gintonin treatment. Results: We found that treating hNPCs with gintonin induced morphological changes with a cell rounding following cell aggregation and return to individual neurons with time relapses. However, the in vitro effects of gintonin on hNPCs were blocked by the LPA1/3 receptor antagonist, Ki16425, and Rho kinase inhibitor, Y27632. We also examined the in vivo effects of gintonin on the morphological changes and migration of neurons in adult mouse brains using anti-NeuN and -neurofilament H antibodies. We found that acute intravenous administration of gintonin induced morphological and migrational changes in brain neurons. Gintonin induced some migrations of neurons with shortened neurofilament H in the cortex. The in vivo effects of gintonin were also blocked by Ki16425. Conclusion: The present report raises the possibility that gintonin could enter the brain and exert its influences on the migration and morphology of adult mouse brain neurons and possibly explains the therapeutic effects of neurological diseases behind the gintonin administration.

• Animal Bioscience
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• v.34 no.4
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• Animal Bioscience
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• v.34 no.3_spc
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• pp.385-392
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• 2021

Objective: The present study was conducted to investigate the effects of lycopene on growth performance, abdominal fat deposition, serum lipids levels, activities of hepatic lipid metabolism related enzymes and genes expression in broiler chickens. Methods: A total of 256 healthy one-day-old male Arbor Acres broiler chicks were randomly divided into four groups with eight replicates of eight birds each. Birds were fed basal diet supplemented with 0 (control), 100, 200, and 400 mg/kg lycopene, respectively. Results: Dietary 100 mg/kg lycopene increased the body weight at 21 day of age compared to the control group (p<0.05). Compared to the basal diet, broilers fed diet with 100 mg/kg lycopene had decreased abdominal fat weight, and broilers fed diet with 100 and 200 mg/kg lycopene had decreased abdominal fat percentage (p<0.05). Compared to control, diets with 100, 200, and 400 mg/kg lycopene reduced the levels of total triglyceride and total cholesterol in serum, and diets with 100 and 200 mg/kg lycopene reduced the level of serum low density lipoprotein cholesterol (p<0.05). The activity of fatty acid synthase (FAS) in 400 mg/kg lycopene treated broilers and the activity of acetyl-CoA carboxylase (ACC) in 100, 200, and 400 mg/kg lycopene treated broilers were lower than those fed basal diet (p<0.05). Lycopene increased the mRNA abundance of adenosine monophosphate activated protein kinase α (AMPK-α), whereas decreased the mRNA abundance of sterol regulatory element-binding protein 1, FAS, and ACC compared to the control group (p<0.05). Conclusion: Dietary lycopene supplementation can alleviate abdominal fat deposition and decrease serum lipids levels, possibly through activating the AMPK signaling pathway, thereby regulating lipid metabolism such as lipogenesis. Therefore, lycopene or lycopene-rich plant materials might be added to poultry feed to regulate lipid metabolism.

• Journal of Korean Medicine for Obesity Research
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• v.21 no.1
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• pp.10-21
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• 2021

Objectives: Benign prostatic hyperplasia (BPH) is a progressive pathological condition characterized by excessive proliferation of the prostate. In this study, we evaluated the effect of Corni Fructus water extract (CF) on the promotion of prostate cell proliferation by dihydrotestosterone (DHT). Methods: The effect of CF on the proliferation of LNCaP prostate cells was evaluated, and DHT was treated to induce an in vitro BPH model. To study the mechanism of inhibition of cell proliferation and BPH by CF, changes in the expression of key factors related to cell cycle and BPH were investigated. We further investigated the effect on the production of reactive oxygen species (ROS) and nitric oxide (NO) to evaluate the antioxidant and anti-inflammatory efficacy of CF. Results: Inhibition of LNCaP cell proliferation by CF was associated with decreased expression of cyclin D1 and cyclin A and increased expression of cyclin-dependent kinase inhibitor p21. CF also suppressed expression of BPH inducing factors such as 5α-reductase type 2 and androgen receptor (AR) as well as prostate specific antigen (PSA). Furthermore, CF significantly blocked DHT-induced LNCaP cell proliferation and effectively attenuated DHT-induced expression of BPH mediators and cyclins. In addition, CF inhibited DHT-induced oxidative and inflammatory reactions by inhibiting production of ROS and NO. Conclusion: Our results demonstrated that CF probably acted as 5α-reductase type 2 inhibitor, preventing the 5α-reductase type 2-AR signaling pathway, thereby reducing the conversion of testosterone to DHT and the expression of PSA, which is at least correlated with the antioxidant and anti-inflammatory activities of CF.

• Animal Bioscience
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• v.35 no.7
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• pp.1010-1020
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• 2022

Objective: The experiment was conducted to evaluate the effects of maternal undernutrition during late pregnancy on the expressions of genes involved in growth and development in ovine fetal perirenal brown adipose tissue (BAT). Methods: Eighteen ewes with singleton fetuses were allocated to three groups at day 90 of pregnancy: restricted group 1 (RG1, 0.33 MJ metabolisable energy [ME]/kg body weight [BW]^0.75/d, n = 6), restricted group 2 (RG2, 0.18 MJ ME/kg BW^0.75/d, n = 6), and a control group (CG, ad libitum, 0.67 MJ ME/kg BW^0.75/d, n = 6). The fetuses were removed at day 140 of pregnancy. All data were analyzed by using the analysis of variance procedure. Results: The perirenal fat weight (p = 0.0077) and perirenal fat growth rate (p = 0.0074) were reduced in RG2 compared to CG. In fetal perirenal BAT, the protein level of uncoupling protein 1 (UCP1) (p = 0.0001) was lower in RG1 and RG2 compared with CG and UCP1 mRNA expression (p = 0.0265) was decreased in RG2. The protein level of myogenic factor 5 (Myf5) was also decreased in RG2 (p = 0.0001). In addition, mRNA expressions of CyclinA (p = 0.0109), CyclinB (p = 0.0019), CyclinD (p = 0.0015), cyclin-dependent kinase 1 (CDK1) (p = 0.0001), E2F transcription factor 1 (E2F1) (p = 0.0323), E2F4 (p = 0.0101), and E2F5 (p = 0.0018) were lower in RG1 and RG2. There were decreased protein expression of peroxisome proliferator-activated receptor-γ (PPARγ) (p = 0.0043) and mRNA expression of CCAAT/enhancer-binding protein-α (C/EBPα) (p = 0.0307) in RG2 and decreased PPARγ mRNA expression (p = 0.0008) and C/EBPα protein expression (p = 0.0015) in both RG2 and RG1. Furthermore, mRNA expression of bone morphogenetic protein 4 (BMP4) (p = 0.0083) and BMP7 (p = 0.0330) decreased in RG2 and peroxisome proliferator-activated receptor co-activator-1α (PGC-1α) reduced in RG2 and RG1. Conclusion: Our observations support that repression of regulatory factors promoting differentiation and development results in the inhibition of BAT maturation in fetal perirenal fat during late pregnancy with maternal undernutrition.

• Animal Bioscience
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• v.35 no.1
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• pp.126-137
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• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

• Tuberculosis and Respiratory Diseases
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• v.85 no.2
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• pp.155-164
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• 2022

Background: The remarkable efficacy of osimertinib in non-small cell lung cancer (NSCLC) with acquired T790M mutation has been widely documented in clinical trials and real-world practice. However, some patients show primary resistance to this drug. Even patients who initially show a favorable response have inconsistent clinical outcomes later. Therefore, the aim of this study was to identify additional clinical predictive factors for osimertinib efficacy. Methods: A prospective cohort of patients with acquired T790M positive stage IV lung adenocarcinoma treated with osimertinib salvage therapy in Hallym University Medical Center were analyzed. Results: Sixty-one eligible patients were analyzed, including 38 (62%) women and 39 (64%) who never smoked. Their mean age was 63.3 years. The median follow-up after treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) was 36.0 months (interquartile range, 24.7-50.2 months). The majority (n=45, 74%) of patients were deceased. Based on univariate analysis, low baseline neutrophil-to-lymphocyte ratios (NLR), age ≥50 years, never-smoking history, stage IVA at osimertinib initiation, and prolonged response to previous TKIs (≥10 months) were associated with a significantly longer progression-free survival (PFS). Multivariate analysis showed that never-smoking status (hazard ratio [HR], 0.54; 95% confidence interval [CI], 0.30-0.98; p=0.041) and a baseline NLR less than or equal to 3.5 (HR, 0.23; 95% CI, 0.12-0.45; p<0.001) were independently associated with a prolonged PFS with osimertinib. Conclusion: Smoking history and high NLR were independent negative predictors of osimertinib PFS in patients with advanced NSCLC developing EGFR T790M resistance after the initial EGFR-TKI treatment.