• Asian Pacific Journal of Cancer Prevention
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• v.15 no.10
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• pp.4245-4250
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• 2014

Emerging evidence has suggested that glycolysis is enhanced in cancer-associated fibroblasts (CAF), and miR-186 is downregulated during the CAF formation. However, it is not clear whether miR-186 is involved in the regulation of glycolysis and what the role of miR-186 plays during the CAF formation. In this study, quantitative PCR analysises show miR-186 is downregulated during the CAF formation. Moreover, miR-186 targets the 3' UTR of Glut1, and its overexpression results in the degradation of Glut1 mRNA, which eventually reduces the level of Glut1 protein. On the other hand, knockdown of miR-186 increased the expression of Glut1. Both time course and dose response experiments also demonstrated that the protein and mRNA levels of Glut1 increase during CAF formation, according to Western blot and quantitative PCR analyses, respectively. Most importantly, besides the regulation on cell cycle progression, miR-186 regulates glucose uptake and lactate production which is mediated by Glut1. These observations suggest that miR-186 plays important roles in glycolysis regulation as well as cell cycle checkpoint activation.

• Applied Chemistry for Engineering
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• v.32 no.6
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• pp.647-652
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• 2021

In this study, Saussurea Lappa roots were extracted using ethanol and n-hexane, and also the effects on proliferation of human hair dermal papilla cells and fibroblast and related signaling pathway were evaluated. 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) assay was conducted for cell proliferation effect of Saussurea Lappa root extract, and extracellular signal-related kinase (ERK), serine/threonine protein kinase (Akt), wingless-related integration site (Wnt)/𝛽-catenin signaling pathway, and 5𝛼-reductase expression through western blot analysis were measured. Saussurea Lappa root extract significantly increased human hair dermal papilla cells and propagation of fibroblast, promoted phosphorylation of ERK and Akt that get involved in cell proliferation. Additionally, Saussurea Lappa root extract significantly decreased promotion of Akt phosphorylation and cell proliferation by MEK/ERK inhibitor PD98059 and PI3K/Akt inhibitor LY294002. Also, Saussurea Lappa root extract induced intranuclear 𝛽-catenin accumulation by promoting phosphorylation of 𝛽-catenin (Ser552, 675) through phosphorylation of GSK-3𝛽 (Ser9), and suppressed activation of 5𝛼-reductase type I and II. Overall, Saussurea Lappa root induces cell proliferation through vitalization of ERK and Akt route of human hair dermal papilla cells and fibroblast and apoptosis defense mechanism, and can be helpful in hair loss prevention and hair growth by vitalizing the 𝛽-catenin signaling pathway and inhibiting activation of 5𝛼-reductase, which can be used as a potential hair care products.

• Tuberculosis and Respiratory Diseases
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• v.47 no.5
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• pp.618-628
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• 1999

Background: Cell growth is a balance between cell proliferation and cell death. Insulin-like growth factor-I(IGF-I), which binds IGF-I receptor(IGF-IR), mediates cellular proliferation as a potent mitogen. IGF binding protein-3(IGFBP-3) as a circulating major IGFBP can inhibit or enhance the effects of IGF-I on cellular growth by binding IGFs. Methods: We investigated the expressions of mRNA of IGF-I and IGF-IR by northern blot and phosphorylation of IGF-IR with the treatment of IGF-I by western blot in 3T3 fibroblast cells. The cellular proliferations of 3T3 cells with the treatments of IGF-I were evaluated using $^3H$-thymidine incorporation and MTT assay. Also to observe the effect of IGFBP-3 on cellular proliferation, 3T3 cells were treated with anti-IGFBP-3 and ${\alpha}IR_3$(monoclonal antibody to IGF-IR) alone or in combination. Results: Our results demonstrated that 3T3 cells showed mRNA expressions of IGF-I and IGF-IR and the IGF-I increased phosphorylation of IGF-IR. The treatments of 3T3 cells with IGF-I increased cellular proliferation in 5 % and 1 % seruma-containing media, not in serum-free media. The addition of anti-IGFBP-3 to neutralize IGFBP-3 showed 2-fold increase of cellular proliferation, and also co-incubation of anti-IGFBP-3 and ${\alpha}IR_3$ together showed similar increase of cellular proliferation in 3T3 cells. Interestingly, when the cells were pretreated with ${\alpha}IR_3$ for 4 hr, prior to the simultaneous addition of ${\alpha}IR_3$ and anti-IGFBP-3, anti-IGFBP-3-mediated cellular proliferation was decreased to control level. All of these results suggest that free IGF-I released from IGF-I/IGFBP-3 complex would be involved in the cellular proliferation. Conclusion: IGF-I is a mitogen through the activation of IGF-IR in 3T3 cells, and IGFBP-3 could be a potent inhibitor for IGF-I action by binding IGF-I.

• Molecules and Cells
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• v.28 no.5
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• pp.489-494
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• 2009

We have previously shown that the down-regulation of protein kinase CKII activity is tightly associated with cellular senescence of human fibroblast IMR-90 cells. Here, we examined the roles of p53 and $p21^{Cip1/WAF1}$ in senescence development induced by CKII inhibition using wild-type, isogenic p53-/- and isogenic p21-/- HCT116 human colon cancer cell lines. A senescent marker appeared after staining for senescence-associated ${\beta}$-galactosidase activity in wild-type HCT116 cells treated with CKII inhibitor or $CKII{\alpha}$ siRNA, but this response was almost abolished in p53- or $p21^{Cip1/WAF1}$-null cells. Increased cellular levels of p53 and $p21^{Cip1/WAF1}$ protein occurred with the inhibition of CKII. CKII inhibition upregulated p53 and $p21^{Cip1/WAF1}$ expression at post-transcriptional level and transcription level, respectively. RB phosphorylation significantly decreased in cells treated with CKII inhibitor. Taken together, this study shows that the activation of the $p53-p21^{Cip1/WAF1}$ pathway acts as a major mediator of cellular senescence induced by CKII inhibition.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.31-37
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• 2020

We previously identified a new heparinase inhibitor fungal metabolite, named CRM646-A, which showed inhibition of heparinase and telomerase activities in an in vitro enzyme assay and antimetastatic activity in a cell-based assay. In this study, we elucidated the mechanism by which CRM646-A rapidly induced nucleus condensation, plasma membrane disruption and morphological changes by increasing intracellular Ca²⁺ levels. Furthermore, PD98059, a mitogen-activated protein kinase (MEK) inhibitor, inhibited CRM646-A-induced nucleus condensation through ERK1/2 activation in rat 3Y1 fibroblast cells. We identified CRM646-A as a Ca²⁺ ionophore-like agent with a distinctly different chemical structure from that of previously reported Ca²⁺ ionophores. These results indicate that CRM646-A has the potential to be used as a new and effective antimetastatic drug.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.268-276
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• 2017

Background: UV-B-exposed keratinocytes secrete various paracrine factors. Among these factors, basic fibroblast growth factor (bFGF) stimulates the proliferation of melanocytes. Ginsenosides, the major active compounds of ginseng, are known to have broad pharmacological effects. In this study, we examined the antiproliferative effects of ginsenosides on bFGF-induced melanocyte proliferation. Methods: We investigated the inhibitory effects of Korean Red Ginseng and ginsenosides from Panax ginseng on bFGF-induced proliferation of melan-a melanocytes. Results: When melan-a melanocytes were treated with UV-B-irradiated SP-1 keratinocytes media, cell proliferation increased. This increased proliferation of melanocytes decreased with a neutralizing anti-bFGF antibody. To elucidate the effects of ginsenosides on melanocyte proliferation induced by bFGF, we tested 15 types of ginsenoside compounds. Among them, Rh3, Rh1, F1, and CK demonstrated antiproliferative effects on bFGF-induced melanocyte proliferation after 72 h of treatment. bFGF stimulated cell proliferation via extracellular signal-regulated kinase (ERK) activation in various cell types. Western blot analysis found bFGF-induced ERK phosphorylation in melan-a. Treatment with Rh3 inhibited bFGF-induced maximum ERK phosphorylation and F1-delayed maximum ERK phosphorylation, whereas Rh1 and CK had no detectable effects. In addition, cotreatment with Rh3 and F1 significantly suppressed bFGF-induced ERK phosphorylation. Western blot analysis found that bFGF increased microphthalmia-associated transcription factor (MITF) protein levels in melan-a. Treatment with Rh3 or F1 had no detectable effects, whereas cotreatment with Rh3 and F1 inhibited bFGF-induced MITF expression levels more strongly than a single treatment. Conclusion: In summary, we found that ginsenosides Rh3 and F1 have a synergistic antiproliferative effect on bFGF-induced melan-a melanocyte proliferation via the inhibition of ERK-mediated upregulation of MITF.

• Journal of the Korea Convergence Society
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• v.9 no.12
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• pp.81-88
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• 2018

Human gingival fibroblast (HGF) is the main cell type existed in periodontium and produces a variety of inflammatory mediators by external stimuli. In this study, the anti-inflammatory activity of Porphyra yezoensis ethanol extract (PYEE) on LPS-PG lipopolysaccharide from Porphyromonas gingivalis activated HGF-1 cell. Up-regulated iNOS and COX-2 expressions by LPS-PG were significantly attenuated by PYEE treatment in a dose-dependent manner. In addition, activated nuclear factor $(NF)-{\kappa}B$ was also dose-dependently inhibited by PYEE treatment. Among upstream signaling molecules, PYEE treatment inhibited phosphorylation of c-Jun $NH_2$-terminal kinase (JNK) but did not give any effect on other molecules. On the other hand, one of phase II enzymes, NAD(P)H:quinone dehydrogenase (NQO)-1, was analyzed due to its anti-inflammatory activity, which was upregulated by PYEE treatment. Consequently, PYEE could be candidates for the prevention and treatment of periodontal diseases.

• 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 Embryo Transfer
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• v.33 no.4
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• pp.195-203
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• 2018

Interferon-tau (IFNT) is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants. Also, multiple interferon genes exist in cattle, However, molecular mechanisms of these bovine IFNT (bIFNT) genes whose expressions are limited have not been characterized. We and others have observed that expression levels of bovine subtype IFNT genes in the tissues of ruminants; thus, bIFNT1 and other new type I (bIFNTc1/c2/c3) gene co-exist during the early stages of conceptus development and non-trophoblast cells. Its genes transcription could be regulated through CDX2 and ETS2 and JUN and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. Bovine ear-derived fibroblast cells, were co-transfected with luciferase reporter constructs carrying upstream (positions -1000 to +51) regions of bIFNT1 and other new type I gene and various transcription factor expression plasmids. Compared to each - 1kb-bIFNT1/c1/c2/c3-Luc increased when this constructs were co-transfected with CDX2, ETS2, JUN and/or CREBBP. Also, Its genes was had very effect on activity by CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. However, the degree of transcriptional activation of the bIFNTc1 gene was not similar to that bIFNT1/c2/c3 gene by expression plasmid. Furthermore, Sequence analyses also revealed that the expression levels of bIFNT1/c2/c3 gene mRNAs expression were highest on day 17, 20 and 22 trophoblast and, Madin-Darby bovine kidney (MDBK), Bovine ear-derived fibroblast (EF), and endometrium (Endo) non-trophoblast cells. But, bIFNTc1 mRNA had not same expression level, bIFNTc1 lowest levels than those of IFNT1/c2/c3 gene in both trophoblast and non-trophoblast cells. These results demonstrate that bovine subtype bIFNT genes display differential, in the trophoblast and non-trophoblast cells.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.289-295
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• 2013

Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of ${\alpha}1$,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human $EF1{\alpha}$ promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human $EF1{\alpha}$ promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.