Protein Kinase 억제제 첨가 후 Platelet-Activating Factor에 의하여 자극된 호중구반응의 변경

이강건(Kang-Kun Lee),고지영(Ji-Young Ko),함동석(Dong-Suk Ham),신용규(Yong-Kyoo Shin),이정수(Chung-Soo Lee) 대한약리학회 1996 대한약리학잡지 Vol.32 No.1

Roles of protein kinase C and protein tyrosine kinase in the activation of neutrophil respiratory burst, degranulation and elevation of cytosolic Ca²⁺ in platelet-activating factor (PAF)-stimulated neutrophils were investigated. Superoxide and H₂O₂ production and myeloperoxidase and acid phosphatase release in PAF-stimulated neutrophils were inhibited by protein kinase C inhibitors, staurosporine and H-7 and protein tyrosine kinase inhibitors, genistein and tyrphostin. The PAF-induced elevation of [Ca²⁺]<sub>i</sub in neutrophils was inhibited by staurosporine, genistein and methyl-2,5-dihydroxycinnamate. Staurosporine inhibited both intracellular Ca²⁺ release and Mn²⁺ influx in PAF-stimulated neutrophils. Genistein and methyl-2,5-dihydroxycinnamate inhibited Mn²⁺ influx induced by PAF, whereas their effects on intracellular Ca²⁺ release were not detected. In neutrophils preactivated by PMA, the stimulatory effect of PAF on the elevation of [Ca²⁺]_i was reduced. Protein kinase C and protein tyrosine kinase may be involved in respiratory burst, lysosomal enzyme release and Ca²⁺ mobilization in PAF-stimulated neutrophils. The elevation of [Ca²⁺]<sub>i</sub appears to be accomplished by intracullular Ca²⁺ release and Ca²⁺ influx which are differently regulated by protein kinases. Preactivation of protein kinase C appears to attenuate the stimulatory action of PAF on intracellular Ca²⁺ mobilization. Platelet-activating factor (PAF)에 의하여 자극된 호중구 respiratory burst, 탈과립과 세포질 칼슘농도의 증가에 있어 protein kinase C와 protein tyrosine kinase의 역할을 관찰하였다. PAF에 의하여 자극된 호중구에서 superoxide 및 H₂O₂의 생성과 myeloperoxidase와 acid phosphatase의 유리는 protein kinase C 억제제인 staurosporine과 H-7 그리고 protein tyrosine kinase 억제제인 genistein과 tyrphostin에 의하여 억제되었다. PAF에 의한 호중구 세포내 칼슘농도의 증가는 staurosporine, genistein과 methyl-2,5-dihydroxycinnamate에 의하여 억제 되었다. Staurosporine은 PAF에 의하여 자극된 호중구에서 세포내 칼슘유리와 망간유입을 억제 하였다. Genistein과 methyl-2,5-dihydroxycinnamate는 PAF에 의한 망간유입을 억제하였으나, 세포내 칼슘유리에 대한 이들의 효과는 관찰되지 않았다. PMA에 의하여 활성화된 호중구에서 세포내 칼슘농도의 증가에 대한 PAF의 자극효과는 감소되었다. Protein kinase C와 protein tyrosine kinase는 PAF에 의하여 자극된 호중구에서의 respiratory burst, lysosomal enzyme유리와 칼슘동원에 관여할 것으로 제시된다. 세포내 칼슘농도의 증가는 protein kinase의 영향을 다르게 받는 세포내 칼슘유리와 세포외부로 부터의 칼슘유입에 의하여 이루어질 것으로 추정된다. Protein kinase C가 활성화되어 있는 상태에서 세포내 칼슘동원에 대한 PAF의 자극작용은 감소될 것으로 시사된다.

Alteration of the Activated Responses in Platelet-Activating Factor-Stimulated Neutrophils by Protein Kinase Inhibitors

이강건,고지영,함동석,신용규,이정수,Lee, Kang-Kun,Ko, Ji-Young,Ham, Dong-Suk,Shin, Yong-Kyoo,Lee, Chung-Soo The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.1

Platelet-activating factor (PAF)에 의하여 자극된 호중구 respiratory burst, 탈과립과 세포질 칼슘농도의 증가에 있어 protein kinase C와 protein tyrosine kinase의 역할을 관찰하였다. PAF에 의하여 자극된 호중구에서 superoxide 및 $H_2O_2$의 생성과 myeloperoxidase와 acid phosphatase의 유리는 protein kinase C 억제제인 staurosporine과 H-7 그리고 protein tyrosine kinase 억제제인 genistein과 tyrphostin에 의하여 억제되었다. PAF에 의한 호중구 세포내 칼슘농도의 증가는 staurosporine, genistein과 methyl-2,5-dihydroxycinnamate에 의하여 억제 되었다. Staurosporine은 PAF에 의하여 자극된 호중구에서 세포내 칼슘유리와 망간유입을 억제 하였다. Genistein과 methyl-2,5-dihydroxycinnamate는 PAF에 의한 망간유입을 억제하였으나, 세포내 칼슘유리에 대한 이들의 효과는 관찰되지 않았다. PMA에 의하여 활성화된 호중구에서 세포내 칼슘농도의 증가에 대한 PAF의 자극효과는 감소되었다. Protein kinase C와 protein tyrosine kinase는 PAF에 의하여 자극된 호중구에서의 respiratory burst, lysosomal enzyme유리와 칼슘동원에 관여할 것으로 제시된다. 세포내 칼슘농도의 증가는 protein kinase의 영향을 다르게 받는 세포내 칼슘유리와 세포외부로 부터의 칼슘유입에 의하여 이루어질 것으로 추정된다. Protein kinase C가 활성화되어 있는 상태에서 세포내 칼슘동원에 대한 PAF의 자극작용은 감소될 것으로 시사된다. Roles of protein kinase C and protein tyrosine kinase in the activation of neutrophil respiratory burst, degranulation and elevation of cytosolic $Ca^{2+}$ in platelet-activating factor (PAF)-stimulated neutrophils were investigated. Superoxide and $H_2O_2$ production and myeloperoxidase and acid phosphatase release in PAF-stimulated neutrophils were inhibited by protein kinase C inhibitors, staurosporine and H-7 and protein tyrosine kinase inhibitors, genistein and tyrphostin. The PAF-induced elevation of $[Ca^{2+}]_i$ in neutrophils was inhibited by staurosporine, genistein and methyl-2,5-dihydroxycinnamate. Staurosporine inhibited both intracellular $Ca^{2+}$ release and $Mn^{2+}$ influx in PAF-stimulated neutrophils. Genistein and methyl-2,5-dihydroxycinnamate inhibited $Mn^{2+}$ influx induced by PAF, whereas their effects on intracellular $Ca^{2+}$ release were not detected. In neutrophils preactivated by PMA, the stimulatory effect of PAF on the elevation of $[Ca^{2+}]_i$ was reduced. Protein kinase C and protein tyrosine kinase may be involved in respiratory burst, lysosomal enzyme release and $Ca^{2+}$ mobilization in PAF-stimulated neutrophils. The elevation of $[Ca^{2+}]_i$ appears to be accomplished by intracullular $Ca^{2+}$ release and $Ca^{2+}$ influx which are differently regulated by protein kinases. Preactivation of protein kinase C appears to attenuate the stimulatory action of PAF on intracellular $Ca^{2+}$ mobilization.

토끼 뇌기저동맥에서 Protein Kinase C 길항제와 Ca<sup>2 </sub>calmodulin에 의존적인 Protein Kinase 길항제로 헤모글로빈에 의한 수축의 완화

김병남,강성돈,김종문 대한신경외과학회 1998 Journal of Korean neurosurgical society Vol.27 No.6

Regulatory Action of Protein Tyrosine Kinase in Intracellular Calcium Mobilization in C5a-stimulated Neutrophils

최원태,한은숙,이정수,Choi, Won-Tae,Han, Eun-Sook,Lee, Chung-Soo The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.3

본 연구는 C5a에 의해 자극된 호중구에서 세포내 칼슘유리와 세포외부로부터 칼슘유입에 있어 protein kinase C와 protein tyrosine kinase의 관여 여부를 조사하였다. Protein kinase C 억제제인 staurosporine과 H-7은 C5a에 의해 자극된 호중구에서 세포내 칼슘유리를 억제하였으나, 세포막을 교차한 칼슘유입이나 세포내 칼슘농도 증가에 영향을 나타내지 않았다. C5a에 의한 세포내 칼슘유리와 칼슘유입은 protein tyrosine kinase 억제제인 genistein과 methyl-2,5-dihydroxycinnamate에 의해서 억제 되었다. ADP에 의해 야기된 세포내 칼슘농도의 증가는 genistein과 methyl-2,5-dihydroxycinnamate에 의해서 억제되었으나 staurosporine과 H-7의 영향은 받지 않았다. Genistein과 methyl-2,5-dihydroxy-cinnamate는 thapsigargin을 처리한 호중구에서 칼슘유입을 감소시켰으나 이에 대한 staurosporine 과 H-7의 효과는 나타나지 않았다. 호중구를 phorbol 12-myristate 13-acetate로 전처치하였을때 세포내 칼슘증가에 미치는 C5a의 자극 효과는 감소하였다. 이상의 결과로 부터 protein tyrosine kinase는 C5a에 의해 활성화된 호중구에서 세포내 칼슘유리와 세포막을 교차한 칼슘유입의 조절에 관여할 것으로 추정된다. The present study was done to examine the involvement of protein kinase C and protein tyrosine kinase in intracellular $Ca^{2+}$ mobilization in C5a-stimulated neutrophils. Although protein kinase C inhibitors, staurosporine and H-7 inhibited intracellular $Ca^{2+}$ release in C5a-stimulated neutrophils, they did not affect $Ca^{2+}$ influx across the plasma membrane and elevation of $[Ca^{2+}]_i$ C5a-induced intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx were inhibited by protein tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate. ADP-evoked elevation of $[Ca^{2+}]_i$ was inhibited by genistein and methyl-2,5-dihydroxycinnamate but was not affectd by staurosporine and H-7. Genistein and methyl-2,5-dihydroxycinnamate reduced the store-regulated $Ca^{2+}$ influx in thapsigargin-treated neutrophils, while the effect of staurosporine and H-7 was not detected. When neutrophils were preincubated wih phorbol 12-myristate 13-acetate, the stimulatory effect of C5a on the elevation of $[Ca^{2+}]_i$ was reduced. These results suggest that protein tyrosine kinase may be involved in control of intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx across the plasma membrane in C5a-activated neutrophils.

C5a에 의해 자극된 호중구에서 세포내 칼슘동원에 대한 Protein Tyrosine Kinase의 조절작용

최원태(Won-Tae Choi),한은숙(Eun-Sook Han),이정수(Chung-Soo Lee) 대한약리학회 1996 대한약리학잡지 Vol.32 No.3

본 연구는 C5a에 의해 자극된 호중구에서 세포내 칼슘유리와 세포외부로부터 칼슘유입에 있어 protein kinase C와 protein tyrosine kinase의 관여 여부를 조사하였다. Protein kinase C 억제제인 staurosporine과 H-7은 C5a에 의해 자극된 호중구에서 세포내 칼슘유리를 억제하였으나, 세포막을 교차한 칼슘유입이나 세포내 칼슘농도 증가에 영향을 나타내지 않았다. C5a에 의한 세포내 칼슘유리와 칼슘유입은 protein tyrosine kinase 억제제인 genistein과 methyl-2,5-dihydroxycinnamate에 의해서 억제 되었다. ADP에 의해 야기된 세포내 칼슘농도의 증가는 genistein과 methyl-2,5-dihydroxycinnamate에 의해서 억제되었으나 staurosporine과 H-7의 영향은 받지 않았다. Genistein과 methyl-2,5-dihydroxy-cinnamate는 thapsigargin을 처리한 호중구에서 칼슘유입을 감소시켰으나 이에 대한 staurosporine 과 H-7의 효과는 나타나지 않았다. 호중구를 phorbol 12-myristate 13-acetate로 전처치하였을때 세포내 칼슘증가에 미치는 C5a의 자극 효과는 감소하였다. 이상의 결과로 부터 protein tyrosine kinase는 C5a에 의해 활성화된 호중구에서 세포내 칼슘유리와 세포막을 교차한 칼슘유입의 조절에 관여할 것으로 추정된다. The present study was done to examine the involvement of protein kinase C and protein tyrosine kinase in intracellular Ca²⁺ mobilization in C5a-stimulated neutrophils. Although protein kinase C inhibitors, staurosporine and H-7 inhibited intracellular Ca²⁺ release in C5a-stimulated neutrophils, they did not affect Ca²⁺ influx across the plasma membrane and elevation of [Ca²⁺]_i C5a-induced intracellular Ca²⁺ release and Ca²⁺ influx were inhibited by protein tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate. ADP-evoked elevation of [Ca²⁺]_i was inhibited by genistein and methyl-2,5-dihydroxycinnamate but was not affectd by staurosporine and H-7. Genistein and methyl-2,5-dihydroxycinnamate reduced the store-regulated Ca²⁺ influx in thapsigargin-treated neutrophils, while the effect of staurosporine and H-7 was not detected. When neutrophils were preincubated wih phorbol 12-myristate 13-acetate, the stimulatory effect of C5a on the elevation of [Ca²⁺]_i was reduced. These results suggest that protein tyrosine kinase may be involved in control of intracellular Ca²⁺ release and Ca²⁺ influx across the plasma membrane in C5a-activated neutrophils.

Bl6 Melanoma세포에서 Protein Kinase억제제들이 Cyclic AMP 경로를 통한 멜라닌 생성에 미치는 영향

차상복(Sang Bok Cha),조남영(Nam Young Cho),윤미연(Mi Yun Yoon),임혜원(Hye Won Lim),김경원(Kyoung Won Kim),박영미(Young Mi Park),이지윤(Ji Yun Lee),이진희(Jin Hee Lee),김창종(Chang Jong Kim),심상수(Sang Soo Sim) 대한약학회 2003 약학회지 Vol.47 No.1

To investigate the effect of protein klnase on melanin production via CAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in Bl6 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-Camp MSH, forskolin and 8-Br-Camp significantly increased both melanin production and tyrosinase activity in Bl6 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1μM), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, for-skolin and 8-Br-Camp with the following order of potency: MSH>forskolin>8-Br-Camp. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-Camp-stimulated Bl6 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-Camp were affected by KN-62 (calm-odulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein klnase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFrB pathway may play an important role in cyclic AMP-dependent melanin production in Bl6 melanoma cells.

Structure-based lead discovery for protein kinase C zeta inhibitor design by exploiting kinase–inhibitor complex crystal structure data and potential therapeutics for preterm labour

Qing-Chun Shao,Cui-Juan Zhang,Jie Li 대한약학회 2021 Archives of Pharmacal Research Vol.44 No.9

The protein kinase C (PKC) is a family of serine/threonine kinases with a broad range of cellular targets. Members of the PKC family participate at the diverse biologicalevents involved in cellular proliferation, differentiationand survival. The PKCisoformzeta (PKCf) is an atypicalmember that has recently been found to play an essential rolein promoting human uterine contractility and thus been raisedas a new target for treating preterm labour and other tocolyticdiseases. In this study, an integrative protocol was describedto graft hundreds of inhibitor ligands from their complexcrystal structures with cognate kinases into the active pocketof PKCf and, based on the modeled structures, to evaluate thebinding strength of these inhibitors to the non-cognate PKCfreceptor by using a consensus scoring strategy. A total of 32inhibitors with top score were compiled, and eight out of themwere tested for inhibitory potency against PKCf. Consequently,five compounds, i.e. CDK6 inhibitor fisetin, PIM1inhibitor myricetin, CDK9 inhibitor flavopiridol and PknBinhibitor mitoxantrone as well as the promiscuous kinaseinhibitor staurosporine showed high or moderate inhibitoryactivity on PKCf, with IC50 values of 58 ± 9, 1.7 ± 0.4,108 ± 17, 280 ± 47 and 0.019 ± 0.004 lM, respectively,while other three compounds, including two marketed drugsdasatinib and sunitinib as well as the Rho inhibitor fasudil,have not been detected to possess observable activity. Next,based on the modeled structure data we modified three flavonoidkinase inhibitors, i.e. fisetin, myricetin and flavopiridol,to generate a number of more potential molecularentities, two of which were found to have a moderatelyimproved activity as compared to their parent compounds.

항암제 개발: 토포아이소머라제, HSP90, mTOR 및 티로신키나제 억제제

최경철(Kyoungcheol Choi),이성호(Seongho Lee),권미지(Miji Kwon),홍세영(Seyoung Hong),박희호(Hee Ho Park),임광석(Kwang Suk Lim) 한국생물공학회 2021 KSBB Journal Vol.36 No.2

Chemical anticancer drugs that have been used for a long time for the treatment of cancer have high anticancer effects and many side effects. The side effects of anticancer drugs have also affected normal cells because they induce the death of cancer cells by inhibiting or blocking essential mechanisms for cell survival. Recently, inhibitory drugs that inhibit specific mechanisms of cancer cells are receiving a lot of attention as anticancer drugs. Inhibitory drugs have less effect on normal cells by inhibiting the activity of target proteins that are overexpressed in cancer cells. Representative anticancer inhibitors among many inhibitory drugs are mTOR inhibitors, topoisomerase inhibitors, heat shock protein 90 (HSP90) inhibitors and tyrosine kinase inhibitors. For each of these inhibitor family, new candidate inhibitor drugs are continuously being developed and clinical trials are underway. In this review, we will examine the drug mechanism of each inhibitor drug, and describe the approved drugs and drugs in clinical trials.

기초 : 톡소포자충 감염 신경교종세포의 Mitogen-Activated Protein Kinase Pathway 활성화

박병현 ( Byung Hyun Park ),이현성 ( Hyun Sung Lee ),이종수 ( Jong Soo Lee ),양승환 ( Seung Hwan Yang ),신대환 ( Dae Whan Shin ),이영하 ( Young Ha Lee ) 대한뇌종양학회 2006 대한뇌종양학회지 Vol.5 No.2

Objective:Gliomas are the most common primary brain tumors in adults. Toxoplasma gondii is an obligate intracellular parasite with a high affinity for brain cells. Mitogen-activated protein kinases(MAPKs) are known to regulate the host cell invasion, although little is known regarding MAPK signaling in Toxoplasma-infected glioma cells. Methods:U87MG and U373MG cells were infected with tachyzoites of the RH strain of T. gondii, and the kinetics of MAPK activation were determined by immunoblotting. We also determined the effects of MAPK inhibitors on glioma cell growth and T. gondii replication using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetraterazolium bromide(MTT; Sigma) method and [3H]-uracil incorporation assays. Results:There were no significant differences in the levels of unphosphorylated MAPK at different incubation times in the Toxoplasma-infected U373MG cells. T. gondii induced the activation of extracellular signal-regulated kinase(ERK) 1/2, p30MAPK, and c-Jun N-terminal kinase(JNK)1/2 within 30 minutes of infection, and showed differential kinetics of activation for each MAPK. The activation of ERK1/2, p38MAPK, and JNK1/2 in Toxoplasma-infected glioma cells was blocked by PD98059, SB202190, and SB203580, respectively. T. gondii replication was inhibited in a dose-dependent manner by the addition of MAPK inhibitors to Toxoplasma-infected glioma cells. Conclusion:Infection with T. gondii induces the activation of MAPKs in glioma cells, and this activation can be blocked by the addition of kinase-specific inhibitors. These results suggest that the MAPK pathway plays a role in the invasion of glioma cells with T. gondii.

AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin

이윤경,박송이,Young-Min Kim,Won Sup Lee,Ock Jin Park 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.3

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2-/- cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin- mediated cancer control. AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2-/- cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin- mediated cancer control.