• 생명과학회지
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• 제15권3호
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• pp.406-414
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• 2005

암세포의 유전적 불안정성은 부적절하게 활성화된 DNA수복경로와 관련되어 있다. 전이성 암은 높은 유전적 불안정성을 나타내는데, 이와 관련하여 본 연구에서는 전이성 암세포에서의 중요한 DNA수복 단백질의 하나인 DN의존성 단백질 키나아제(DNA-PK)의 발현 변화를 조사하였다. 여러 종류의 전이도가 다른 암세포들을 대상으로 한 실험에서 전이성 암세포들은 각각의 모세포에 비하여 DNA-PK 성분의 조절 소단위인 Ku70/80의 발현 및 Ku의 DNA 결합 활성이 증강되어 있었다. 또한 DNA-PK의 촉매 소단위인 DNA-PKcs의 발현 및 whole DNA-PK복합체의 kinase의 활성도 전이도가 큰 암세포에서 그 모세포보다 증강되어 있음을 알 수 있어, 전이성 암세포의 증강된 DNA수복능은 부적절한 DNA수복을 일으켜 암의 진행 및 전이를 촉진시키는 원인이 될 수 있음을 시사하였다. 한편 암세포의 표피성장인자수용체의 신호전달의 증강은 암의 침윤과 전이에 관련되어 있으며, DNA-PK의 기 기능에도 영향을 줄 수 있는 가능성이 보고 된 바 있는데, 본 연구에서는 표피성장인자수용체의 신호전달과 DNA-PK의 관련성을 명확히 밝히기 위하여 새로 개발된 EGFR tyrosine kinase inhibitor인 PKI166의 DNA-PK의 활성에 미치는 영향을 조사하였다. PKI166는 Ku70/80 및 DNA-PKcs의 발현을 억제하였고 이와 관련하여 전이성 및 항암제 다제내성 암세포에서 PKI166에 의하여 항암제에 대한 감수성을 증가시켜 항암제 내성을 나타내는 전이성 암세포 대한 치료법 연구에 DNA-PK가 분자적 표적이 될 수 있음을 밝혔다.

• BMB Reports
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• 제31권5호
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• pp.468-474
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• 1998

Membrane depolarization in PC12 cells induces calcium influx via an L-type voltage-sensitive calcium channel (L-VSCC) and increases intracellular free calcium, which leads to tyrosine phosphorylation of epidermal growth factor (EGF) receptor and the associated adaptor protein, She. This activated EGF receptor complex then can activate mitogen-activated protein (MAP) kinase, as in nerve growth factor (NGF) receptor activation. In the present study, we investigated the role of EGF receptor in the signaling pathway initiated by membrane depolarization of PC12 cells. Prolonged membrane depolarization induced phosphorylation of extracellular signal-regulated kinase (ERK) within 1 min in undifferentiated PC12 cells. Pretreatment of PC12 cells with the calcium chelator EGTA abolished depolarization-stimulated ERK phosphorylation, but NGF-induced phosphorylation of ERK was not affected. The chronic treatment of phorbol ester, which down-regulated the activity of protein kinase C (PKC), did not affect the phosphorylation of ERK upon depolarization. In the presence of an inhibitor of EGF receptor, neither depolarization nor calcium ionophore increased the level of ERK phosphorylation. These data imply that the EGF receptor is functionally necessary to activate ERK and neurite outgrowth in response to the prolonged depolarization in PC12 cells, and also that PKC is apparently not involved in this signaling pathway.

• Molecules and Cells
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• 제20권2호
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• pp.280-287
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• 2005

The insulin-mediated Ras/mitogen-activated protein (MAP) kinase cascade was examined in SK-N-BE(2) and PC12 cells, which can and cannot produce reactive oxygen species (ROS), respectively. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) was much lower in SK-N-BE(2) cells than in PC12 cells when the cells were treated with insulin. The insulin-mediated interaction of IRS-1 with Grb2 was observed in PC12 but not in SK-N-BE(2) cells. Moreover, the activity of extracellular-signal-related kinase (ERK) was much lower in SK-N-BE(2) than in PC12 cells when the cells were treated with insulin. Application of exogenous $H_2O_2$ caused increased tyrosine phosphorylation and Grb2 binding to IRS-1 in SK-N-BE(2) cells, while exposure to an $H_2O_2$ scavenger (N-acetylcysteine) or to a phophatidylinositol-3 kinase inhibitor (wortmannin), and expression of a dominant negative Rac1, decreased the activation of ERK in insulin-stimulated PC12 cells. These results indicate that the transient increase of ROS is needed to activate ERK in insulin-mediated signaling and that an inability to generate ROS is the reason for the insulin insensitivity of SK-N-BE(2) cells.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.507-513
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• 2000

ErbB3/HER3 is a cell surface receptor which belongs to the ErbB/HER subfamily of receptor protein tyrosine kinases. When expressed in NIH/3T3 cells, ErbB3 can form heterodimeric coreceptor with endogenous ErbB2. Among known intracellular effectors of the ErbB2/ErbB3 are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. In the present study, we studied relative contributions of above two distinct signaling pathways to the heregulin-induced mitogenic response via activated ErbB3. For this, clonal NIH-3T3 cell lines expressing wild-type ErbB3 and ErbB3 mutants were stimulated with $heregulin{\beta}_1$. While cyclin D1 level was markedly high and further increased by treatment of heregulin in cells expressing wild-type ErbB3, the elimination of either Shc binding or PI 3-kinase binding lowered both levels. This result was supported by the reduction of cyclin $D_1$ expression by preteatment with MAPK kinase inhibitor or PI 3-kinase inhibitor before stimulation with heregulin. In accordance with the cyclin $D_1$ expression, elimination of either Shc binding or PI 3-kinase binding reduced the heregulin-induced DNA synthesis and cell growth rate. Our results obtained by the comparison of wild-type and ErbB3 mutants indicate that the full induction of the cell cycle progression through $G_1/S$ phase by ErbB3 activation is dependent on both Shc/MAPK and PI 3-kinase signal transduction pathways.

• 한국임상수의학회지
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• 제33권4호
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• pp.187-193
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• 2016

A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to a protein in a cell. It functions as an "on" or "off" switch in many cellular functions. This study aims to show that the actions of growth factors associated with PDGFR-${\alpha}$, PDGFR-${\beta}$, VEGFR-2, c-KIT, and c-ABL, which are used in veterinary medicine, are expressed in canine intractable tumors. This study used archival cases of canine paraganglioma, gastrointestinal adenocarcinoma, hepatocellular carcinoma, and renal cell carcinoma. Tissues had been immunohistochemical analysis. The antibodies used were PDGFR-${\alpha}$, PDGFR-${\beta}$, c-kit, VEGFR-2, and c-Abl. PDGFR-${\alpha}$ was expressed only in HCC, and PDGFR-${\beta}$ was expressed in all tumors. VEGFR was also only expressed in HCC, and c-KIT has been expressed in HCC, paraganglioma, and small intestinal adenocarcinoma. c-Abl was expressed in all cancers, but was weakly expressed in paraganglioma, while more than moderately expressed in other tissues. In conclusion, this study investigated how TKIs used in human medicine can be applied to canine intractable tumors, through immunohistochemistry. The results indicate that there may be an application for TKIs in treating canine intractable tumors.

• The Korean Journal of Physiology
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• 제30권2호
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• pp.209-218
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• 1996

Acetylcholine (ACh) activates the inwardly rectifying muscarinic $K^{+}$ channel in rat atrial cells via pertussis toxin (PTX)-sensitive G-protein ($G_k$) coupled with the muscarinic receptor (mAChR). Although this $K^{+}\;(K_{ACh})$ channel function has reported to be modulated by the phosphorylation process, a kinase and phosphatase involved in these processes are still unclear. Since either PKA or PKC was not effective on this ATP-modulation, the present study examined the possible involvement of the protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in the function of the $K_{ACh}$ Channel. In the inside-out (I/O) patch preparation excised from the adult rat atrial cell, when activated by 10 ${\mu}M$ ACh in the pipette and 100 ${\mu}M$ GTP in the bath, the mean open time (${\tau}_{o}$) and the channel activity ($K_{ACh}$) was 1.13 ms (n=5) and 0.19 (n=6), respectively. Following the application of 1 mM ATP into the bath, ${\tau}_{o}$ increased by 34% (1.54 ms, n=5) and $K_{ACh}$ by 66% (0.28, n=6). Channel function elevated by ATP was lasted after washout of ATP. However, this ATP-induced increase in the $K_{ACh}$ channel function did not occur in pretreated cells with genistein ($50{\sim}100 {\mu}M$), a selective PTK inhibitor, but occurred in pretreated cells with equimolar daidzein, a negative control of the genistein. On the contrary, PTP which acts on tyrosine residue conversely reversed both ATP-induced increased ${\tau}_{o}$ by 32% (1.20 ms, n=3) and $K_{ACh}$ by 41% (0.15, n=3), respectively. Taken together, these results suggest that $K_{ACh}$ channel may, at least partly, be regulated by the tyrosyl phosphorylation, although it is unclear where this process exerts on the muscarinic signal transduction pathway comprising the mAChR-$G_{k}$-the $K_{ACh}$ channel.

• 대한한방부인과학회지
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• 제20권4호
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• pp.56-73
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• 2007

Purpose: 이 실험은 골다공증의 치료약물로 자하거의 골질재흡수 억제효과를 검토하기 위하여 설계되었다. Methods: 자하거의 골질재흡수 효과를 확인하기 위하여 생쥐의 두개골 골모세포를 이용하여 Cyclooxygenase-1(COX-1), COX-2, $TGF-{\beta}$, $L-1{\beta}$, $TNF-{\alpha}$, IL-6, prostaglandin E2등의 활성화 정도를 측정하였으며, 골조직의 미세구조적 변화를 확인하였다. Results: 자하거는 $IL-1{\beta}$, $TNF-{\alpha}$, IL-6 또는 그 세가지의 조합에 의하여 유발된 PGE2의 생성 뿐만 아니라 COX-2 mRNA 수치도 감소시켰으나 COX-1 mRNA 수치에는 영향을 주지 않았다. 이로써 자하거는 시험관내에서 그리고 생체내에서 펩티드의 인산화를 억제함으로써 골의 재흡수를 저해하였다. 그리고 자하거는 생쥐에서 $IL-1{\beta}$에 의해 유발된 고칼슘혈증을 감소시켰고, 골의 재흡수를 저해하는 경로를 통하여 골에 대한 보호효과를 보여줌으로써 조기에 난소 절제한 쥐에서 골질감소와 미세구조적 변화를 부분적으로 방지하였다. 이러한 결과는 PGE2 생성에 대한 $IL-1{\beta}$, $TNF-{\alpha}$, IL-6사이의 상승효과는 COX-2의 유전자 발현이 증가한 결과이며 이러한 tyrosine kinase가 생쥐의 두개골 골모세포에서 COX-2의 신호전달에 관계한다는 것을 보여준다. Conclusion: 자하거가 생쥐의 두개골 골모세포에서 여러 신호전달물질의 활성화를 통하여 골질재흡수를 저해하는 특성을 확인함으로써 앞으로 골다공증의 예방과 치료에 대한 추가적인 임상연구가 필요할 것으로 사료된다.

• 생명과학회지
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• 제33권11호
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• pp.859-867
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• 2023

루페올은 5환성 트리테르펜의 일종으로 많은 질병에 치료 효과가 있는 것으로 보고되었으나, 인슐린 저항성에 미치는 영향은 명확하지 않다. 본 연구에서는 3T3-L1 지방세포에서 루페올의 IRS-1 인산화 억제능을 통해 인슐린 저항성 개선효과를 조사하였다. 3T3-L1 세포를 배양하고 TNF-α를 24시간 동안 처리하여 인슐린 저항성을 유도하였다. 서로 다른 농도의 루페올(15, 30 μM) 또는 100 nM의 rosiglitazone을 처리한 세포를 배양한 후, 용해된 세포를 이용하여 western blotting을 시행하였다. 실험결과 루페올은 지방세포에서 TNF-α에 의해 유발되는 인슐린 신호전달의 음성 조절자와 염증 활성화 단백질 kinase에 대한 개선 효과를 나타냈다. 인슐린 신호전달의 음성 조절자인 PTP-1B와 JNK의 활성 및 IKK와 염증활성화 단백질키나아제의 활성을 억제하였다. 또한, 루페올은 IRS-1의 serine 인산화는 하향 조절하고 tyrosine 인산화는 상향 조절하였다. 그 후, 하향 조절된 PI3K/AKT 경로가 활성화되고, GLUT 4의 세포막 전위가 자극되어, 결과적으로 인슐린 저항성이 유도된 3T3-L1 지방세포에서에서 세포내 포도당 흡수가 증가하였다. 본 연구결과, 루페올은 3T3-L1 지방세포에서 인슐린 신호전달 및 염증 활성화 단백질 kinsase들의 음성 조절인자를 억제하여, IRS-1의 serine 인산화를 하향 조절함으로써 TNF-α 유발 인슐린 저항성을 개선할 수 있을 것으로 사료된다.

• The Journal of Korean Physical Therapy
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• 제20권3호
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• pp.53-59
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• 2008

Purpose: The non-receptor-type protein tyrosine kinase Syk (636 amino acids, 72 kDa) is ubiquitously expressed in hematopoietic stem cells and has been widely studied as a regulator and effector of B cell receptor signaling that occurs in processes such as differentiation, proliferation and apoptosis. However, the mechanism relating Syk and p38 mitogen-activated protein kinases (p38MAPK) by endothelin-1 (ET-1, 21 amino acids) stimulation in muscle cells, especially in the volume-dependent hypertensive state, remains unclear. Methods: In this study, we investigated the relationship between Syk and p38MAPK for isometric contraction and enzymatic activity by ET-1 from rat aortic smooth muscle cells and aldosterone-analogue deoxycorticosterone acetate (DOCA) hypertensive state rats (ADHR). Results: The systolic blood pressure was significantly increased in ADHR than in a control group of animals. ET-1 induced isometric contraction and phosphorylation of p38MAPK, which was increased in muscle strips from ADHR. Increased vasoconstriction and phosphorylation of p38MAPK induced by treatment with 30 nM ET-1 were inhibited by the use of 10${\mu}M$ SB203580, an inhibitor of p38MAPK from ADHR. Furthermore, ET-1 induced isometric contraction and phosphorylation of Syk and p38MAPK, which were increased in the aortic smooth muscle cells. Increased tension and phosphorylation of Syk and p38MAPK induced by ET-1 were inhibited by SB203580 from rat aortic smooth muscle cells. Conclusion: These results, suggest that the Syk activity affects ET-1-induced contraction through p38MAPK in smooth muscle cells and that the same pathway directly or indirectly is associated with volume dependent hypertension. The findings suggest the need to develop cardiovascular disease-specialized physical therapy.

• Archives of Pharmacal Research
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• 제27권7호
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• pp.683-692
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• 2004

Curcumin (diferuloylmethane) is a major naturally-occurring polyphenol of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. Curcumin has shown anti-carcinogenic activity in animal models. Curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase; and an effective inducer of heme oxygenase-1. Curcumin is also a potent inhibitor of protein kinase C(PKC), EGF(Epidermal growth factor)-receptor tyrosine kinase and LĸB kinase. Subsequently, curcumin inhibits the activation of NF(nucleor factor)KB and the expressions of oncogenes including c-jun, c-fos, c-myc, NIK, MAPKs, ERK, ELK, PI3K, Akt, CDKs and iNOS. It is proposed that curcumin may suppress tumor promotion through blocking signal transduction path-ways in the target cells. The oxidant tumor promoter TPA activates PKC by reacting with zinc thiolates present within the regulatory domain, while the oxidized form of cancer chemopreventive agent such as curcumin can inactivate PKC by oxidizing the vicinal thiols present within the catalytic domain. Recent studies indicated that proteasome-mediated degradation of cell proteins playa pivotal role in the regulation of several basic cellular processes including differentiation, proliferation, cell cycling, and apoptosis. It has been demonstrated that curcumin-induced apoptosis is mediated through the impairment of ubiquitin-proteasome pathway. Curcumin was first biotransformed to dihydrocurcumin and tetrahydrocurcumin and that these compounds subsequently were converted to monoglucuronide conjugates. These results suggest that curcumin-glucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are the major metabolites of curcumin in mice, rats and humans.