• Biomedical Science Letters
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• v.27 no.3
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• pp.134-141
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• 2021

c-Jun N-terminal kinases (JNKs) have a Janus face, regulating both cell apoptosis and survival. The present study focused on understanding the function of JNK in tumor development and the chemoresistance underlying JNK-mediated cancer cell survival. We identified an inhibitor of JNK1, an important regulator of cancer cell survival. Kinase assay data showed that JNK1-dependent c-Jun phosphorylation was inhibited by indirubin derivatives. In particular, indirubin-3-monoxime (I3M) directly inhibited the phosphorylation of c-Jun in vitro, with a half inhibition dose (IC₅₀) of 10 nM. I3M had a significant inhibitory effect on JNK1 activity. Furthermore, we carried out assays to determine the viability, migration, and proliferation of breast cancer cells. Our results demonstrated that cell growth, scratched wound healing, and colony forming abilities were inhibited by the JNK inhibitor SP600125 and I3M. The combination of SP600125 and I3M significantly decreased cancer cell proliferation, compared with either SP600125 or I3M alone. Our studies may provide further support for JNK1-targeting cancer therapy using the indirubin derivative I3M in breast cancer.

• BMB Reports
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• v.31 no.4
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• pp.355-363
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• 1998

MHC unrestricted, antigen nonspecific killing by $CD4^+$ T-cells against virally-infected target cells was induced following cross-linking of CD4 molecules. The cytotoxicity of antibody-activated $CD4^+$ T-cells was abolished by genistein (4',5,7-trihydroxyisoflavone), a protein tyrosine kinase (PTK) inhibitor, but not by H-7, a protein kinase C (PKC) inhibitor. Genisteintreated human or bovine peripheral blood $CD4^+$ T-cells lacked PTK activity and failed to kill virally-infected target cells even after cross-linking of CD4 molecules. The cross-linking of CD4 molecules did not induce effector cell proliferation or the transcription of TNF ${\beta}$. TNF ${\beta}$ synthesis was up-regulated by incubating antibody activated effector cells with bovine herpesvirus type 1 (BHV-1) infected D17 target cells. Anti-TNF ${\beta}$ antibody partially abrogated direct effector cell-mediated antiviral cytotoxicity. On the other hand, this antibody effectively neutralized antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on effector and target cell ratio. These findings have importance to define the mechanisms of how CD4 cytotoxic cells control viral infection.

• Korean Journal of Medicinal Crop Science
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• v.7 no.1
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• pp.37-44
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• 1999

This study was conducted to screen antitumoral activity by in vitro bioassay method using 60 Korean medicinal and food plants extracted by 80% EtOH. Antitumor activity test was applied by the PKC(protein kinase C) and antibleb formation, PLC (Phospholipase C), and colorimetric tetrazolium assay (MTT assay) methods. Chenopodjum album and black Glycine max showed high antitumoral activity by 73.5% and 81.0%, respectively, against PKC by bleb-forming assay and PKC enzyme assay on human chronic leukemia K562 cell. Black Glycine max also showed 91.2% antitumoral activity in the PLC method and the lowest $IC_{50}$ $value(4.7{\mu}g/ml)$ by MTT method against P-338 cell line. In the effect of the concentration treatment on antitumoral test, the more concentration indicated the more activity value.

• Journal of Ginseng Research
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• v.44 no.3
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• pp.461-474
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• 2020

Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown. Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues. Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.

• Nutrition Research and Practice
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• v.8 no.4
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• pp.368-376
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• 2014

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and $50{\mu}g/mL$ of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and $50{\mu}g/mL$ of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.

• The Journal of Korean Medicine
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• v.27 no.1 s.65
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• pp.23-35
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• 2006

Objectives : This study was conducted to investigate the inhibitory effects of Gaejibokryunghwan on cell proliferation in HeLa cells. Methods : Human uterine cervical carcinoma HeLa cells were cultured in the 1%, 5% and 10% concentration of Gaejibokryunghwan extract solution. All three were cultured for 24 hours, 48 hours and 72 hours each, to examine the inhibitory effects of Gaejibokryunghwan. Afterwards, we drew out the effect of Gaejibokryunghwan extract solution by making 5 analysis. First analysis was to measure the proliferation rate of cells. Second was FACS analysis. Third was to estimate the activity or caspase-3. Fourth, we used XTT assay to analyze the activation or cells. Ana lastly, a molecular biological method was used to determine activation of MAP kinase in the HeLa cells. Results : After 24, 48 and 72 hours cultivation, the proliferation of HeLa cells showed the dose-dependent decrease in all Gaejibokryunghwan extract solution groups compared to the control group. In the FACS analysis, Gaejibokryunghwan extract solution groups showed increased caspase expression compared to the control group, except for the group for 48 and 72 hours in 1 % concentrate. Caspase-3 activities were increased in all, except tile group cultured for 24 hours in 5% concentrate and the groups cultured for 48 hours in 1% and 5% concentrate. In the XTT study, 1% Gaejibokryunghwan extract solution groups showed increase compared to the control group, but other Gaejibokryunghwan extract solution containing groups showed significant decrease compared to the control after 24, 48 and 72 hours of cultivation. The expressions of MAP kinase were decreased in all Gaejibokryunghwan extract solution containing groups compared to the control group after 24, 48 and 72 hours of cultivation. Conclusions : From this study, we could suggest that Gaejibokryunghwan be available to the inhibition of proliferation of human cervical carcinoma cell line, HeLa cells in vitro.

• The Korean Journal of Pain
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• v.34 no.1
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• pp.19-26
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• 2021

Background: Prolotherapy is a proliferation therapy as an alternative medicine. A combination of dextrose solution and lidocaine is usually used in prolotherapy. The concentrations of dextrose and lidocaine used in the clinical field are very high (dextrose 10%-25%, lidocaine 0.075%-1%). Several studies show about 1% dextrose and more than 0.2% lidocaine induced cell death in various cell types. We investigated the effects of low concentrations of dextrose and lidocaine in fibroblasts and suggest the optimal range of concentrations of dextrose and lidocaine in prolotherapy. Methods: Various concentrations of dextrose and lidocaine were treated in NIH-3T3. Viability was examined with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration assay was performed for measuring the motile activity. Extracellular signal-regulated kinase (Erk) activation and protein expression of collagen I and α-smooth muscle actin (α-SMA) were determined with western blot analysis. Results: The cell viability was decreased in concentrations of more than 5% dextrose and 0.1% lidocaine. However, in the concentrations 1% dextrose (D1) and 0.01% lidocaine (L0.01), fibroblasts proliferated mildly. The ability of migration in fibroblast was increased in the D1, L0.01, and D1 + L0.01 groups sequentially. D1 and L0.01 increased Erk activation and the expression of collagen I and α-SMA and D1 + L0.01 further increased. The inhibition of Erk activation suppressed fibroblast proliferation and the synthesis of collagen I. Conclusions: D1, L0.01, and the combination of D1 and L0.01 induced fibroblast proliferation and increased collagen I synthesis via Erk activation.

• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.3
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• pp.25-40
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• 2006

Purpose : This study was undertaken to evaluate the inhibitory effects of Arisaematis rhizoma on the cell proliferation in HeLa cells. Methods : The cultured cell after treatment in the different duration in 24, 48, 72 hours with solution of 1%. 5%, 10% Arisaematis rhizoma was quantified by trypan blue exclusin method. The control group was treated with 2% FBS in the different duration in 24, 48, 72 hours. We examined DNA of activated caspase by FACS analysis, caspase-3 activity, DNA fragmentation by DNA laddering, activity of HeLa Cells by the XTT assay, activity of MAP kinase by RT-PCR analysis. Results : After 72 hours culture, the growth activities of 1%, 5%, 10% Arisaematis rhizoma-treated Hela cell were significantly reduced with control group, respectively. After 24 hours culture, the ratio of cells showing caspase activity by FACS analysis were increased in 1%, 5%, 10% Arisaematis rhizoma-treated Hela cell. It were also increased in 48 hours culture of 10% and 72 hours culture of 5%, 10% Arisaematis rhizoma-treated Hela cell. In 24, 48 and 72 hours culture, DNA fragmentations of 5%, 10% Arisaematis rhizoma-treated Hela cell were obviously observed. These results meaned that Arisaematis rhizoma induces apoptosis of HeLa cells. It was supported by increased caspase-3 activity and decreased MAP kinase activity according to time periods and concentrations of Arisaematis rhizoma solution. Conclusion : The study shows that Arisaematis rhizoma has inhibitory effect on cell proliferation and induction capacity of apoptosis of human cevical carcinoma cell line, HeLa cells, in vitro. These results suggest that Arisaematis rhizoma should be useful for treatment of human cevical carcinoma.

• KSBB Journal
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• v.28 no.2
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• pp.137-145
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• 2013

In order to develop new skin whitening agents, we prepared the $CH_2Cl_2$ layer (NGC) and BuOH layer (NGB) of 75% EtOH extract of the Nelumbinis nucifera Gaertner. We measured their tyrosinase inhibitory activity in vitro and melanin synthesis inhibitory activity in B16-F1 melanoma cells. They did not show inhibitory activity against mushroom tyrosinase but showed melanin synthesis inhibitory activity in a dose-dependent manner. In a melanin synthesis inhibition assay, NGC and NGB suppressed melanin production up to 52% and 46% at a concentration of $100{\mu}g/mL$, respectively. To elucidate the mechanism of the inhibitory effects of NGC and NGB on melanogenesis, we measured the expression of melanogenesis-related proteins by western blot assay. As a result, NGC suppressed the expression of tyrosinase, tyrosinase related protein 1 (TRP-1), tyrosinase related protein 2 (TRP-2), phosphorylated cAMP responsive element binding (p-CREB) protein, and microphthalmia associated transcription factor (MITF). And NGB inhibited the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1, TRP-2, and p-CREB expression. Moreover, NGB increased the expression of phosphorylated extracellular signal-regulated kinase (p-ERK). In addition, we examined the inhibitory effect on the glycosylation of tyrosinase. As a result, NGC and NGB inhibited the activity of ${\alpha}$-glucosidase in vitro and the glycosylation of tyrosinase in B16-F1 melanoma cells. From these results, we concluded that NGC and NGB could be used as active ingredients for skin whitening.

• BMB Reports
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• v.35 no.4
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• pp.377-383
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• 2002

Arsenic trioxide ($As_O_3$) was recently demonstrated to be an effective inducer of apoptosis in patients with relapsed acute promyelocytic leukemia (APL) as well as patients with APL in whom all-trans-retinoic acid and conventional chemotherapy failed. Chronic myelogenous leukemia cells are highly resistant to chemotherapeutic drugs. To determine if $As_O_3$ might be useful for the treatment of chronic myelogenous leukemia, we examined the ability of $As_O_3$ to induce apoptosis in K562 cells. In vitro cytotoxicity of $As_O_3$ was evaluated in K562 cells by a MTT assay: the $IC_50$ value for $As_O_3$ was determined to be $10\;{\mu}m$. When analyzed by agarose gel electorphoresis, the DNA fragments became evident after incubation of the cells with $20\;{\mu}m$ $As_O_3$ for 24 h. We also found morphological changes and chromatin condensation of the cells undergoing apoptosis. Activation of caspase-3 was observed 6 h after treatment with $20\;{\mu}m$ $As_O_3$ by a Western blot analysis. Next, we examined the MAP kinase-signaling pathway of $As_O_3$-induced apoptosis in K562 cells. $As_O_3$ at $10\;{\mu}m$ strongly induced the activation of p38, inhibited $As_O_3$ induced apoptotic cell death. These results suggest that $As_O_3$ is able to induce the apoptotic activity in K562 cells, and its apoptotic mechanism may be associated with the activation of p38.