• BMB Reports
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• 제40권6호
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• pp.888-894
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• 2007

Src homology (SH) domains of phospholipase C-$\gamma1$ (PLC-$\gamma1$) impair NGF-mediated PC12 cells differentiation. However, whether the enzymatic activity is also implicated in this process remains elusive. Here, we report that the enzymatic activity of phospholipase C-$\gamma1$ (PLC-$\gamma1$) is at least partially involved to the blockage of neuronal differentiation via an abrogation of MAPK activation, as well as sustained Akt activation. By contrast, Overexpression of WT-PLC-$\gamma1$ exhibited sustained NGF-induced MAPK activation, and triggered transient Akt activation resulting in profound inhibition of neurite outgrowth. However, lipase-inactive mutant (LIM) PLC-$\gamma1$ cells fail to suppress neurite outgrowth, although it contains intact SH domains, specifically enhancing the expression of cyclin D1 and p21 proteins, which regulate the function of retinoblastoma Rb protein. These observations show that the lipase inactive mutant of PLC-$\gamma1$ does not alter NGF-induced neuronal differentiation via enzymatic inability and the modulation of cell cycle regulatory proteins independent on SH3 domain.

• BMB Reports
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• 제33권2호
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• pp.97-102
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• 2000

Phosphoinositide-specific phospholipase C-${\gamma}1$ (PLC-${\gamma}1$) is a pivotal mediator in the signal transduction cascades induced by many growth factors. Using a yeast two-hybrid system, heat-shock protein 90 (Hsp90) was identified as a PLC-${\gamma}1$-binding protein. A co-immunoprecipitation experiment, using anti-PLC-${\gamma}1$ antibody, demonstrated an in vivo interaction between Hsp90 and PLC-${\gamma}1$ in the NIH-3T3 cells. The interaction in NIH-3T3 was unaffected by the PDGF treatment, inducing phosphorylation and activation of PLC-${\gamma}1$. Direct interaction between Hsp90 and PLC-${\gamma}1$ was confirmed by in vitro binding experiments using purified Hsp90 and PLC-${\gamma}1$. Furthermore, Hsp90 increased the $PIP_2$-hydrolyzing activity of PLC-${\gamma}1$ up to 2-fold at $0.1{\mu}M$ in vitro. Taken together, we show for the first time, the interaction of PLC-${\gamma}1$ with Hsp90, both in vivo and in vitro. We suggest that Hsp90 may play a role in PLC-${\gamma}1$-mediated signal transduction.

• Journal of Veterinary Science
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• 제21권5호
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• pp.72.1-72.10
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• 2020

Background: Fenbendazole, a dewormer drug, is used widely in the clinical treatment of parasite infections in animals. Recent studies have shown that fenbendazole has substantial effects on tumor growth, immune responses, and inflammatory responses, suggesting that fenbendazole is a pluripotent drug. Nevertheless, the antiviral effects have not been reported. Fenbendazole can disrupt microtubules, which are essential for multiple viruses infections, suggesting that fenbendazole might have antiviral effects. Objectives: This study examined whether fenbendazole could inhibit bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures. Methods: The effects of fenbendazole on viral production, transcription of the immediate early (IE) genes, viron-associated protein expression, and the cellular signaling PLC-γ1/Akt pathway were assessed using distinct methods. Results: Fenbendazole could inhibit BoHV-1 productive infections significantly in MDBK cells in a dose-dependent manner. A time-of-addition assay indicated that fenbendazole affected both the early and late stages in the virus replication cycles. The transcription of IE genes, including BoHV-1 infected cell protein 0 (bICP0), bICP4, and bICP22, as well as the synthesis of viron-associated proteins, were disrupted differentially by the fenbendazole treatment. The treatment did not affect the cellular signaling pathway of PLC-γ1/Akt, a known cascade playing important roles in virus infection. Conclusions: Overall, fenbendazole has antiviral effects on BoHV-1 replication.

• 동의생리병리학회지
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• 제18권2호
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• pp.553-560
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• 2004

In the present study, we investigated the anti-proliferative effects of aqueous extract of Wikyung-tang(WKT) on the growth of human lung carcinoma cell line A549. WKT treatment declined the cell viability and proliferation of A549 cells in a concentration-dependent manner. The anti-proliferative effects by WKT treatment in A549 cells was associated with morphological changes such as membrane shrinking and cell rounding up. WKT treatment induced an inhibition and/or degradation of apoptotic target proteins such poly(ADP-ribose) polymerase (PARP) and phospholipase C-γ1 (PLC-γ1). WKT treatment did not affect the levels of other Bcl-2 family gene products, such as Bcl-2, Bax and Bad. Western blot analysis and RT-PCT data revealed that the levels of tumor suppressor p53 and cyclin-dependent kinase inhibitor p21 were induced by WKT treatment in A549 cells. Additionally, WKT treatment induced the down-regulation of telomerase reverse transcriptase mRNA (hTERT) expression of A549 cells, however, the levels of other telomere-regulatory gene products were not affected. Taken together, these findings suggest that WKT-induced inhibition of human lung cancer cell proliferation is associated with the induction of apoptotic cell death via regulation of several major growth regulatory gene products and WKT may have therapeutic potential in human lung cancer.

• BMB Reports
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• 제37권6호
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• pp.720-725
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• 2004

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-${\gamma}1$ has two putative PH domains, an $NH_2$-terminal (PH1) and a split PH domain ($nPH_2$ and $cPH_2$). We previously reported that the split PH domain of PLC-${\gamma}1$ binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)$P_2$) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)$P_2$, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-${\gamma}1$ $nPH_2$ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-${\gamma}1$ nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-${\gamma}1$ molecules showed reduced PI(4,5)$P_2$ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both $PH_1$ and $nPH_2$ domains are responsible for membrane-targeted translocation of PLC-${\gamma}1$ upon serum stimulation. Together, our data reveal that the amino acid residues $Pro^{500}$ and $His^{503}$ are critical for binding of PLC-${\gamma}1$ to one of its substrates, PI(4,5)$P_2$ in the membrane.

• 생명과학회지
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• 제16권4호
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• pp.612-617
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• 2006

포스포리파제 C-감마1$(phospholipase\;C-{\gamma}\;1;\;PLC-{\gamma}\;1)$은 활성화될 경우 세포 내의 이차전령인 inositol 1,4,5-trisphosphate$(IP_3)$와 diacylglycerol(DG)을 생성하는 중요한 세포 신호전달 분자이다. 튜불린은 미세소관과 방추사의 주요 구성 단백질로서 알파형과 베타형의 두 가지 동위형이 있는데 이들은 모든 진핵세포에 존재하면서 이형 이합체를 형성한다. 이 중 베타형 튜불린은 사람의 경우 6종의 또 다른 동위형이 존재하는 것으로 밝혀졌는데 이들은 각 조직에서 그 발현양상이 서로 다르게 나타난다. 이전의 연구에서 우리들은 $PLC-{\gamma}\;1$과 4종의 베타튜불린 동위형 즉, ${\beta}1$, ${\beta}2$, ${\beta}3$ 및 ${\beta}6$이 세포 내에서 서로 결합할 수 있으며 또한 외부의 자극이 전달될 경우 이들 4종의 동위형이 $PLC-{\gamma}\;1$을 활성화시켜 준다는 사실을 보고한 바 있다. 이번 실험에서는 이전의 연구에서 조사하지 못하였던 베타 튜불린의 나머지 두 가지 동위형 즉, ${\beta}4$및 ${\beta}5$가 $PLC-{\gamma}\;1$에 결합하여 $PLC-{\gamma}\;1$의 활성을 증가시켜줌으로서 세포 내에서의 신호전달계를 조절하고 있음을 확인하였다. 이 결과는 이전의 연구결과와 연관 지워볼 때, 6종의 모든 베타형 튜불린은 세포 외부의 자극이 있을 경우 $PLC-{\gamma}\;1$을 활성화시켜줌을 시사한다.

• 동의생리병리학회지
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• 제18권4호
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• pp.1147-1152
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• 2004

To investigate the anti-cancer effects of aqueous extract of Cheonkumwikyung-tang (CKWKT) on the growth of human lung carcinoma cell line A549, we performed various biochemical experiments such as the effects of CKWKT on the cell proliferation and viability, the morphological changes, the effects on expression of apoptosis and cell growth-regulatory gene products. Results obtained are as follow; CKWKT treatment declined the cell viability and proliferation of A549 cells in a concentration-dependent manner. The anti-proliferative effect by CKWKT treatment in A549 cells was associated with morphological changes such as membrane shrinking and cell rounding up. CKWKT treatment induced apoptotic cell death of A549 cells in a concentration-dependent manner, which was associated with inhibition and/or degradation of apoptotic target proteins such poly(ADP-ribose) polymerase, β-catenin and phospholipase C-γ1. Western blot analysis revealed that the levels cyclin-dependent kinase inhibitor p21 expression were induced by CKWKT treatment in A549 cells. Taken together, these findings suggest that CKWKT-induced inhibition of human lung cancer cell proliferation is associated with the induction of apoptotic cell death via regulation of several major growth regulatory gene products and CKWKT may have therapeutic potential in human lung cancer.

• 생약학회지
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• 제50권4호
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• pp.277-284
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• 2019

Rumex crispus is known to have anticancer, antioxidant, antibacterial, and bone loss inhibitory activities. Mast cells are critical immune cells that induce a type 1 IgE-mediated allergic reaction. However, there are no reports of inhibitory effects of Rumex crispus on mast cells and allergic reactions. In this study, we performed some experiments to investigate whether Rumex crispus ethanol extract(RCE) has any inhibitory effect on antigen-induced type I allergic response in vitro and in vivo. RCE inhibited degranulation of IgE-mediated mast cells(IC₅₀, ~57 ㎍/ml) and cytokine production such as TNF-α and IL-4 in a dose-dependent manner. In vivo, RCE significantly inhibited passive cutaneous anaphylaxis(PCA)(ED₅₀, ~198 mg/kg) in mice. Furthermore, RCE inhibited degranulation of MCs in ear tissue of mice with PCA. Mechanism studies showed that RCE inhibited the activation of Syk and Syk-dependent pathway such as LAT, PLC-γ, Akt, and MAP Kinase. Our results demonstrate for the first time that RCE inhibits type I hypersensitive response by suppressing the activity of Syk in mast cells, thereby reducing degranulation and cytokine production. Taken together, RCE could be used as a novel therapeutic material to suppress allergic diseases.

• 대한의생명과학회지
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• 제25권4호
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• pp.302-312
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• 2019

The aim of this work was to investigate the effect of black tea extract (BTE) on collagen -induced platelet aggregation. In this study, BTE (10~500 ㎍/mL) was shown to inhibit platelet aggregation via thromboxane A₂ (TXA₂) down-regulation by blocking cyclooxygenase-1 (COX-1) activity. Also, BTE decreased intracellular Ca²⁺ mobilization ([Ca²⁺]_i). Additionally, BTE enhanced the levels of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are aggregation-inhibiting molecules. BTE inhibited the phosphorylation of phospholipase C (PLC) γ2 and syk activated by collagen. BTE regulated platelet aggregation via cAMP-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) Ser¹⁵⁷. The anti-platelet effects of BTE in high fat diet (HFD)-induced obese rats were evaluated. After eight weeks of BTE treatment (300 and 600 mg/kg), the platelet aggregation rate in the treated groups was significantly less than that in the HFD-fed control group. Also, BTE exhibited a hepatoprotective effect and did not exert hepatotoxicity. Therefore, these data suggest that BTE has anti-platelet effects on collagen-stimulated platelet aggregation and may have therapeutic potential for the prevention of platelet-mediated thrombotic diseases.

• 생명과학회지
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• 제25권11호
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• pp.1255-1264
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• 2015

천궁(C. officinale)은 예로부터 민간처방 약재로 사용되었으며, 항염증, 항산화, 항암 및 신생혈관억제 등의 효능을 가지는 것으로 알려져 있다. 하지만 혈구암세포에서 apoptosis 유발과 관련된 분자생물학적 기전에 대해서는 명확히 밝혀져 있지 않다. 본 연구에서는 인체 혈구암세포인 U937 세포에서 천궁의 열수, 에탄올 및 메탄올 추출물(WECO, EECO 및 MECO)이 유발하는 항암효과 및 항암기전을 조사하였다. 먼저 WECO, EECO 및 MECO가 유발하는 증식억제 정도를 조사한 결과 EECO가 가장 뛰어난 효능을 가진다는 것을 알 수 있었으며, 이러한 현상이 apoptosis 유발에 의한 것임을 annexin-V 염색, apoptotic body 형성, DNA 단편화 및 MMP 소실 등을 통하여 확인하였다. EECO 처리에 의한 apoptosis 유발에는 DR4의 발현 증가와 함께 cIAP-1, Bcl-2 및 total Bid의 발현감소가 관여하였으며, caspases-3, -8 및 -9의 활성화와 함께 caspases-3의 기질 단백질인 PARP, β-catenin 및 PLC γ1의 단편화도 관찰되었다. 또한 EECO는 AMPK signaling pathway를 활성화시키는 것으로 나타났으며, AMPK 억제제인 compound C를 이용하여 AMPK의 활성을 억제하였을 경우 EECO에 의하여 유발되었던 apoptosis가 현저하게 감소되는 것으로 나타났다. 이상의 결과를 살펴볼 때 인체 혈구암세포인 U937 세포에서 EECO에 의하여 유발되는 apoptosis는 AMPK가 중요한 조절자로서 작용하는 것으로 생각된다.