• Journal of Korean Neurosurgical Society
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• v.56 no.1
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• pp.1-4
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• 2014

Objective : The aims of our study are to evaluate the effect of curcumin on spinal cord neural progenitor cell (SC-NPC) proliferation and to clarify the mechanisms of mitogen-activated protein (MAP) kinase signaling pathways in SC-NPCs. Methods : We established cultures of SC-NPCs, extracted from the spinal cord of Sprague-Dawley rats weighing 250 g to 350 g. We measured proliferation rates of SC-NPCs after curcumin treatment at different dosage. The immuno-blotting method was used to evaluate the MAP kinase signaling protein that contains extracellular signal-regulated kinases (ERKs), p38, c-Jun $NH_2$-terminal kinases (JNKs) and ${\beta}$-actin as the control group. Results : Curcumin has a biphasic effect on SC-NPC proliferation. Lower dosage (0.1, 0.5, $1{\mu}M$) of curcumin increased SC-NPC proliferation. However, higher dosage decreased SC-NPC proliferation. Also, curcumin stimulates proliferation of SC-NPCs via the MAP kinase signaling pathway, especially involving the p-ERK and p-38 protein. The p-ERK protein and p38 protein levels varied depending on curcumin dosage (0.5 and $1{\mu}M$, p<0.05). Conclusion : Curcumin can stimulate proliferation of SC-NPCs via ERKs and the p38 signaling pathway in low concentrations.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1384-1391
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• 2006

The signal transduction system is one of the most important devices involved in maintaining life, and many protein kinases are included in the cellular signal transduction system. Finding a protein kinase inhibitor is very valuable, as it can be used to study cell biology and applied to pharmaceuticals. For the efficient and rapid screening of protein kinase inhibitors, two assay systems were combined; the nonradioactive protein kinase assay system that uses an FITC-labeled IRS-2 peptide and the cell-based paper disc assay system that uses Streptomyces griseus as the indicator strain. Among 330 kinds of herb extracts tested, the extract of Dalbergia odorifera exhibited the strongest inhibitory activity in the two assay systems and was selected for further isolation. Based on solvent extraction and many steps of chromatography, seven compounds were finally separated to homogeneity and their structures determined by $^{1}H$ and $^{13}C$ NMR spectroscopies. Four were to be flavonoids and identified as butin ($C_{15}H_{12}O_5$, Mw=272.07), 3'-hydroxymelanetin ($C_{16}H_{12}O_6$, Mw=300.06), liquiritigenin ($C_{15}H_{12}O_4$, Mw=256.07), and 2'-hydroxyformononetin ($C_{16}H_{12}O_{5}$, Mw=284.07). 3'-Hydroxymelanetin inhibited the phosphorylation of the GSK3 protein by Akt to 37% at a concentration of $10{\mu}g/ml$ and showed the strongest cytotoxicity ($ED_{50}<50{\mu}g/ml$) against the human cancer cell line HCT116. Under the same conditions, liquiritigenin also inhibited the phosphorylation of GSK3 by Akt to 26%, and its cytotoxicity against the HCT116 cell line was lower than $100{\mu}g/ml$.

• Archives of Plastic Surgery
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• v.34 no.1
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• pp.13-17
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• 2007

Purpose: To understand the pathogenesis of the disease that presents abnormally proliferated vascular endothelial cells, a model of DMH(1,2-dimethylhydrazine)-induced abnormal proliferation of HUVECs(Human Umbilical Vein Endothelial Cells) was made. We indirectly determined that Protein Kinase C(PKC) restricts the cellular proliferation and inhibits the manifestation of growth factor by using several inhibiting substances of the transmitter through our previous studies. Thereupon, we attempted to observe direct enzymatic activities of PKC and its correlation with the abnormal proliferation of vascular endothelial cells. Methods: $10^5$ HUVECs cells were applied to 6 individual well plates in three different groups; A control group cultured without treatment, a group concentrated with $0.75{\times}10^{-8}M$ DMH only, and a group treated with DMH & $5{\times}10^{-9}M$ Calphostin C, inhibitor of PKC. In analyzing the formation of intracellular PKC enzyme, protein separation was performed, and separated protein was quantitatively measured. PKC enzyme reaction was analyzed through Protein Kinase C Assay System (Promega, USA), and the results were analyzed according to Beer's law. Results: Enzymatic activity of PKC presented the highest in all reaction time of a group concentrated only with DMH, and the lowest in the control group. The group treated with DMH and the inhibitor revealed statistically lower enzymatic activity than group only with DMH in all reaction time, although higher than the control group. Conclusion: From the enzymatic aspect, most active and immediate reaction of the PKC was observed in the group concentrated with DMH only. The group treated with DMH & PKC inhibitor showed meaningful decrease. Accordingly, PKC holds a significant role in DMH-induced abnormal proliferation of vascular endothelial cells.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.10a
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• pp.17-21
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• 1995

대부분의 인슐린의 작용들은 인슐린 수용체를 통하여 이루어진다. 인슐린이 수용체에 결합하면, 수용체 고유의 tyrosine kinase 효소활성의 증가를 유발시키며, 결과적으로 세포내에 존재하는 기질 단백질, IRS-1, 의 tyrosine 잔기의 인산화를 증가시키게 된다. 이후, 여러 형태의 serine / threonine protein kinase 의 연속적인 활성화가 일어난다. 이들에 부가해서, 인슐린의 효자는 세포핵 내에까지 전달되어 유전자 발현의 조절과 같은 세포핵 고유의 활동에도 관여한다. 현재, 세포막에서 시작된 인슐린의 신호들이 세포핵까지 전달되는 정확한 기전에 대해서는 알려진 바 없지만, 최근의 연구에 의하면 MAP Kinase 와 S6 Kinase 그리고 Transcription Factor AP-1의 중요성이 제시되고 있다. 특히 유전자 조절 기전에는 핵단백질인 transcription factor의 인산화 반응이 큰 역할을 한다고 보고되고 있는바, 본 연구에서 AP-1. transcription factor 의 인산화 반응이 인슐린의 신호전달계에 미치는 역할에 대하여 고찰하였다. 요약하면, AP-1 transcription factor의 구성원인 c-Jun, c-Fos 그리고 Fos 관련 단백질들의 인산화가 인슐린에 의해 증가되며, 동시에 그들의. DNA-binding activity 와 유전자 발현의 활성이 증가됨을 밝힘으로써, AP-1 transcription factor의 인산화 반응이 인슐린의 핵 내에서의 작용기전에 중요한 역할을 함이 제시되고 있다. 또한 AP-1 의 인산화 반응에 관여하는 세포핵 protein kinase로서 Casein Kinase II 의 중요성이 밝혀졌다.

• BMB Reports
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• v.36 no.4
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• pp.421-425
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• 2003

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phosphothreonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the $K_m$ for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

• Tuberculosis and Respiratory Diseases
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• v.75 no.5
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• pp.188-198
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• 2013

Over the past decade, several kinase inhibitors have been approved based on their clinical benefit in cancer patients. Unfortunately, in many cases, patients develop resistance to these agents via secondary mutations and alternative mechanisms. To date, several major mechanisms of acquired resistance, such as secondary mutation of the epidermal growth factor receptor (EGFR) gene, amplification of the MET gene and overexpression of hepatocyte growth factor, have been reported. This review describes the recent findings on the mechanisms of primary and acquired resistance to EGFR tyrosine kinase inhibitors and acquired resistance to anaplastic lymphoma kinase inhibitors, primarily focusing on non-small cell lung carcinoma.

• The Korean Journal of Pharmacology
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• v.27 no.2
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• pp.135-144
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• 1991

Using T-lymphocytes obtained from rat peripheral blood, we found that the 44kD/pI6.8 protein was the major phosphoprotein of T-lymphocytes under basal condition, and that the 44kD/pI6.3 protein was a new phosphoprotein appeared in T-lymphocytes stimulated with ${\beta}-agonist$. The phosphorylation of the 44kD/pI6.3 protein was also induced by forskolin but inhibited by H-8 pretreatment. To clarify the character of the 44kD/pI6.3 protein, we used Con-A and kinase inhibitors, H-7 and W-7. Con-A stimulation induced phosphorylation of 44kD/pI 6.3 protein but that was inhibited by W-7 pretreatment. The phosphorytation of 44kD/pI6.3 protein was not induced by the PKC activator, PMA. Instead, the phosphorylation of 44kD/pI6.8 protein was reduced by H-7, a PKC inhibitor. From the above results,it can be concluded that the 44kD/pI6.3 protein can be a common substrate for A-kinase and CaM kinase. The two dimensional tryptic peptide mapping revealed that the 44kD/pI6.8 and 44kD/pI6.3 proteins are different.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.527-538
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• 2019

Background: Ginsenoside Rg1 was shown to exert ligand-independent activation of estrogen receptor (ER) via mitogen-activated protein kinase-mediated pathway. Our study aimed to delineate the mechanisms by which Rg1 activates the rapid ER signaling pathways. Methods: ER-positive human breast cancer MCF-7 cells and ER-negative human embryonic kidney HEK293 cells were treated with Rg1 ($10^{-12}M$, $10^{-8}M$), $17{\beta}$-estradiol ($10^{-8}M$), or vehicle. Immunoprecipitation was conducted to investigate the interactions between signaling protein and ER in MCF-7 cells. To determine the roles of these signaling proteins in the actions of Rg1, small interfering RNA or their inhibitors were applied. Results: Rg1 rapidly induced $ER{\alpha}$ translocation to plasma membrane via caveolin-1 and the formation of signaling complex involving linker protein (Shc), insulin-like growth factor-I receptor, modulator of nongenomic activity of ER (MNAR), $ER{\alpha}$, and cellular nonreceptor tyrosine kinase (c-Src) in MCF-7 cells. The induction of extracellular signal-regulated protein kinase and mitogen-activated protein kinase kinase (MEK) phosphorylation in MCF-7 cells by Rg1 was suppressed by cotreatment with small interfering RNA against these signaling proteins. The stimulatory effects of Rg1 on MEK phosphorylation in these cells were suppressed by both PP2 (Src kinase inhibitor) and AG1478 [epidermal growth factor receptor (EGFR) inhibitor]. In addition, Rg1-induced estrogenic activities, EGFR and MEK phosphorylation in MCF-7 cells were abolished by cotreatment with G15 (G protein-coupled estrogen receptor-1 antagonist). The increase in intracellular cyclic AMP accumulation, but not Ca mobilization, in MCF-7 cells by Rg1 could be abolished by G15. Conclusion: Ginsenoside Rg1 exerted estrogenic actions by rapidly inducing the formation of ER containing signalosome in MCF-7 cells. Additionally, Rg1 could activate EGFR and c-Src ER-independently and exert estrogenic effects via rapid activation of membrane-associated ER and G protein-coupled estrogen receptor.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.101-108
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• 2005

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a well-known inducer of apoptotic cell death in many tumor cells. 1RAIL is expressed in human placenta, and cytotrophoblast cells express 1RAIL receptors. However, the role of TRAIL in human placentas and cytotrophoblast cells is not. well understood. In this study a trophoblast cell line, JEG-3, was used as a model system to examine the effect of TRAIL. on key intracellular signaling pathways involved in the control of trophoblastic cell apoptosis and survival JEG-3 cells expressed receptors for 1RAIL, death receptor (DR) 4, DR5, decoy receptor (OcR) 1 and DeR2. Recombinant human TRAIL (rhTRAIL) did not have a cytotoxic effect determined by MIT assay and did not induce apoptotic cell death determined by poly-(ADP-ribose) polymerase cleavage assay. rhTRAIL induced a rapid and transient nuclear translocation of nuclear $factor-{\kappa}B(NF-{\kappa}B)$ determined by immunoblotting using nuclear protein extracts. rhTRAIL rapidly activated extracellular signal-regulated protein kinase (ERK) 1/2 as determined by immnoblotting for phospho-ERK1/2. However, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38MAPK) and Akt (protein kinase B) were not activated by rhTRAIL. The ability of 1RAIL to induce $NF-{\kappa}B$ and ERK1/2 suggests that interaction between TRAIL and its receptors may play an important role in trophoblast cell function during pregnancy.

• Journal of Radiation Industry
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• v.5 no.1
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• pp.15-20
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• 2011

Protein kinases are the most important drug targets for the treatment of numerous diseases. The involvement of protein kinase C (PKC) in many biological processes such as development, memory, cell differentiation, and proliferation has been demonstrated. PKC is recognized as an important player in carcinogenesis. Thus, a variety of PKC inhibitors have been investigated. Among them, flavonoids have been demonstrated to affect the activity of many mammalian in vitro enzyme systems. The recent investigation was performed to evaluate the inhibitory effects of hesperidin, which is a flavonoid, on the proliferation and carcinogenesis of many cancers. In this study, an efficient kinase assay based on a biochip using radio-phosphorylation was established and performed for an examination of the inhibitory effects of hesperidin on PKC activity at different concentrations of 50, 200, 500 nM. It was found that hesperidin shows inhibitory potency on PKC, and that the biochip can be used to rapidly screen kinase inhibitors resulting in the therapeutic agents.