Effects of Staurosporine and Genistein on Superoxide and HOCl Production in C5a- or PMA-activated Neutrophils

윤영철,강희정,신용규,이정수,Yun Young-Chul,Kang Hee-Jeong,Shin Yong-Kyoo,Lee Chung-Soo The Korean Society of Pharmacology 1995 대한약리학잡지 Vol.31 No.1

C5a 또는 PMA에 의하여 활성화된 호중구에서의 superoxide와 HOCl 생성에 나타내는 staurosporine, genistein과 pertussis toxin의 효과를 관찰하였다. C5a에 의한 superoxide과 $H_2O_2$의 생성은 staurosporine, genistein과 pertussis toxin에 의하여 억제되었다. PMA의 자극효과는 staurosporine에 의하여 억제되었으나 pertussis toxin에 의하여 영향을 받지 않았으며, 한편 이는 genistein에 의하여 더 촉진되었다. Staurosporine, genistein은 sodium fluoride에 의한 superoxide 생성을 억제 하였으나 pertussis toxin은 영향을 나타내지 않았다. PMA에 의한 $H_2O_2$의 생성은 staurosporine에 의하여 억제되었으나 pertussis toxin은 영향을 나타내지 않았다. Genistein은 PMA에 의한 $H_2O_2$의 생성에 자극효과를 나타내지 않았다. Staurosporine과 pertussis toxin은 C5a 또는 PMA에 의한 HOCl 생성을 억제하였으나, 이에 반하여 genistein은 자극하였다. C5a와 PMA에 의한 myeloperoxidase 유리는 genistein에 의하여 억제되었나, pertussis toxin의 효과는 나타나지 않았다. Staurosporine은 유리에 대한 PMA의 자극효과에 영향을 주지 않았다. Myeloperoxidase 활성은 genistein에 의하여 현저하게 증가되었으나 staurosporine과 pertussis toxin의 영향은 받지 않았다. 이상의 결과는 호중구의 respiratory burst가 protein kinase C와 protein tyrosine kinase에 의하여 조절된다고 제시한다. Protein kinase C의 직접적인 자극에 따른 superoxide 생성은 protein tyrosine kinase의 영향을 역으로 받을 것으로 추정된다. Genistein은 아마도 myeloperoxidase를 활성화하여 HOCl 생성을 촉진할 것으로 시사된다. Effects of staurosporine, genistein and pertussis toxin on superoxide and HOCl production in C5a- or PMA-activated neutrophils were investigated. A C5a-induced superoxide and $H_2O_2$ production was inhibited by staurosporine, genistein and pertussis toxin. The stimulatory effect of PMA was inhibited by staurosporine but was not affected by pertussis toxin, whereas it was further promoted by genistein. Staurosporine and genistein inhibited superoxide production by sodium fluoride, but pertussis toxin did not affect it. PMA-induced $H_2O_2$ production was inhibited by staurosporine but was not affected by pertussis toxin. Genistein did not show a stimulatory effect on PMA-induced $H_2O_2$ production. Staurosporine and pertussis toxin inhibited HOCl production by C5a- or PMA, whereas genistein stimulated it. C5a-or PMA-induced myeloperoxidase release was inhibited by genistein, in this response the effect of pertussis toxin was not detected. Staurosporine did not affect the stimulatory effect of PMA on the release. Myeloperoxidase activity was markedly increased by genistein but was not affected by staurosporine and pertussis toxin. These results indicate that the respiratory burst of neutrophils may be regulated by protein kinase C and protein tyrosine kinase. Superoxide production induced by the direct activation of protein kinase C might be affected by protein tyrosine kinase oppositely. Genistein probably pro-motes HOCl production by activating myeloperoxidase.

Staurosporine과 Genistein이 C5a 또는 PMA에 의하여 활성화된 호중구에서의 Superoxide와 HOCl 생성에 나타내는 영향

윤영철(Young Chul Yun),강희정(Hee Jeong Kang),신용규(Yong Kyoo Shin),이정수(Chung Soo Lee) 대한약리학회 1995 대한약리학잡지 Vol.31 No.1

C5a 또는 PMA에 의하여 활성화된 호중구에서의 superoxide와 HOCl 생성에 나타내는 staurosporine, genistein과 pertussis toxin의 효과를 관찰하였다. C5a에 의한 superoxide과 H₂O₂의 생성은 staurosporine, genistein과 pertussis toxin에 의하여 억제되었다. PMA의 자극효과는 staurosporine에 의하여 억제되었으나 pertussis toxin에 의하여 영향을 받지 않았으며, 한편 이는 genistein에 의하여 더 촉진되었다. Staurosporine, genistein은 sodium fluoride에 의한 superoxide 생성을 억제 하였으나 pertussis toxin은 영향을 나타내지 않았다. PMA에 의한 H₂O₂의 생성은 staurosporine에 의하여 억제되었으나 pertussis toxin은 영향을 나타내지 않았다. Genistein은 PMA에 의한 H₂O₂의 생성에 자극효과를 나타내지 않았다. Staurosporine과 pertussis toxin은 C5a 또는 PMA에 의한 HOCl 생성을 억제하였으나, 이에 반하여 genistein은 자극하였다. C5a와 PMA에 의한 myeloperoxidase 유리는 genistein에 의하여 억제되었나, pertussis toxin의 효과는 나타나지 않았다. Staurosporine은 유리에 대한 PMA의 자극효과에 영향을 주지 않았다. Myeloperoxidase 활성은 genistein에 의하여 현저하게 증가되었으나 staurosporine과 pertussis toxin의 영향은 받지 않았다. 이상의 결과는 호중구의 respiratory burst가 protein kinase C와 protein tyrosine kinase에 의하여 조절된다고 제시한다. Protein kinase C의 직접적인 자극에 따른 superoxide 생성은 protein tyrosine kinase의 영향을 역으로 받을 것으로 추정된다. Genistein은 아마도 myeloperoxidase를 활성화하여 HOCl 생성을 촉진할 것으로 시사된다. Effects of staurosporine, genistein and pertussis toxin on superoxide and HOCl production in C5a- or PMA-activated neutrophils were investigated. A C5a-induced superoxide and H₂O₂ production was inhibited by staurosporine, genistein and pertussis toxin. The stimulatory effect of PMA was inhibited by staurosporine but was not affected by pertussis toxin, whereas it was further promoted by genistein. Staurosporine and genistein inhibited superoxide production by sodium fluoride, but pertussis toxin did not affect it. PMA-induced H₂O₂ production was inhibited by staurosporine but was not affected by pertussis toxin. Genistein did not show a stimulatory effect on PMA-induced H₂O₂ production. Staurosporine and pertussis toxin inhibited HOCl production by C5a- or PMA, whereas genistein stimulated it. C5a-or PMA-induced myeloperoxidase release was inhibited by genistein, in this response the effect of pertussis toxin was not detected. Staurosporine did not affect the stimulatory effect of PMA on the release. Myeloperoxidase activity was markedly increased by genistein but was not affected by staurosporine and pertussis toxin. These results indicate that the respiratory burst of neutrophils may be regulated by protein kinase C and protein tyrosine kinase. Superoxide production induced by the direct activation of protein kinase C might be affected by protein tyrosine kinase oppositely. Genistein probably pro-motes HOCl production by activating myeloperoxidase.

MCF-7 세포주에서 Staurosporine 투여에 의한 세포주기 분기 변화와 p53단백 발현

남정(Jung Nam),여경아(Kyung A Yea),이해남(Hae Nam Lee),조현희(Hyun Hee Jo),류기성(Ki Sung Ryu),유영옥(Young Oak Lew),나종구(Jong Gu Rha),한구택(Ku Taek Han) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.3

Objective : We investigated the effects on the cell cycle and p53 expression by the treatment of various concentrations of staurosporine to elucidate the molecular mechanism of staurosporine induced cell cycle arrest in MCF-7 cell line. Methods : Various concentrations of staurosporine were treated in MCF-7 cells cultured with RPMI 1640 media. The harvested cells were fixed and permeabilized with 1% paraformaldehyde and absolute methanol. Then the cells were stained indirectly with anti-p53 primary antibody and FITC conjugated goat anti-mouse(GAM)-IgG secondary antibody. Sequentially DNA were stained with 0.1% RNase and PI solution. These stained cells were analyzed by the standard FACScan flow cytometer. The obtained results were analyzed further with WinList 3.0, and ModiFit LT software program. Results : MCF-7 cells were arrested mostly in G1 phase of cell cycle at 5-10 nM of staurosporine, however, the cells were arrested in G2 phase at 20-100 nM of staurosporine. The expressions of p53 protein were higher in the MCF-7 cells treated with both concentrations of 10 nM and 100 nM of staurosporine compaired with the control cells. This suggests that the p53 may be involved in the mechanism of G1 and G2M arrest of cell cycle in MCF-7 cell. Conclusion : The points of arrest in cell cycle differred depending on the concentrations of staurosporine and these cell cycle arrests at G0G1 and G2M pahse were related with p53 protein expression. It suggested that these results could be extended to study for staurosporine to be usefull as a potential anti-tumor agent.

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells

Kim, Du Sik,An, Jeong Mi,Lee, Han Gil,Seo, Su Ryeon,Kim, Seon Sook,Kim, Ju Yeon,Kang, Jeong Wan,Bae, Yun Soo,Seo, Jeong Taeg Informa Healthcare 2013 Free radical research Vol.47 No.2

<P>Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H<SUB>2</SUB>O<SUB>2</SUB> accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.</P>

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one inhibits neurite outgrowth and causes neurite retraction in PC12 cells independently of soluble guanylyl cyclase

Lee, Han Gil,Kim, So Young,Kim, Du Sik,Seo, Su Ryeon,Lee, Syng-Ill,Shin, Dong Min,De Smet, Patrick,Seo, Jeong Taeg Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of neuroscience research Vol.87 No.1

<P>The effect of the potent soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on neurite outgrowth and retraction was investigated in PC12 cells and SH-SY5Y human neuroblastoma cells. ODQ inhibited neurite outgrowth and triggered neurite retraction in the cells stimulated with nerve growth factor (NGF), staurosporine, or Y-27632. The nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had little effect on neurite outgrowth induced by Y-27632 or staurosporine. In the presence of ODQ, treatment of the cells with the cell-permeable cGMP analogue 8-bromo-cGMP failed to retrigger Y-27632- and staurosporine-induced neurite outgrowth. Furthermore, the depletion of sGC by RNA interference failed to prevent Y-27632- and staurosporine-induced neurite outgrowth. These results indicate that the NO/sGC/cGMP signaling cascade is not critically involved in ODQ-induced neurite remodeling. The MEK inhibitor PD98059 did not inhibit neurite outgrowth, and Y-27632 and staurosporine did not induce ERK phosphorylation, suggesting that the inhibitory effect of ODQ on neurite outgrowth is independent of the ERK signaling pathway. In contrast, pretreatment with dithionite or a hemin-glutathione mixture reversed the inhibitory effect of ODQ on Y-27632- and staurosporine-induced neurite outgrowth, indicating that ODQ might act on an intracellular redox-sensitive molecule. We conclude that ODQ inhibits Y-27632- and staurosporine-induced neurite outgrowth and triggers neurite retraction in an sGC-independent manner in neuronal cells and suggest that oxidation of unidentified redox-sensitive protein could be responsible for these effects. © 2008 Wiley-Liss, Inc.</P>

Staurosporine Induces ROS-Mediated Process Formation in Human Gingival Fibroblasts and Rat Cortical Astrocytes

Han Gil Lee,Du Sik Kim,Seong Ah Moon,Jeong Wan Kang,Jeong Taeg Seo 대한구강생물학회 2015 International Journal of Oral Biology Vol.40 No.1

In the present study, we investigated the effect ofstaurosporine on the formation of cellular processes in humangingival fibroblasts and rat astrocytes. Staurosporine caused arapid induction of process formation in human gingivalfibroblasts and rat astrocytes in a concentration dependentmanner. The process formation of human gingival fibroblastsand rat astrocytes was prevented by the pretreatment withN-acetylcysteine, suggesting that staurosporine-induced ROSproduction was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited thestaurosporine-induced process formation, whereas cytochalasinD, an actin filament breakdown agent, failed to suppress theformation of cellular processes. This result indicated thatpolymerization of microtubule, and not actin filament, wasresponsible for the formation of cellular processes induced bystaurosporine. In support of this hypothesis, Western blotanalysis was conducted using anti-tubulin antibody, and theresults showed that the amount of polymerized microtubule wasincreased by the treatment with staurosporine while that ofdepolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROSmediated,microtubule-dependent formation of cellularprocesses in human gingival fibroblasts and rat astrocytes.

E1B-19k의 세포내 위치와 Bax와의 Dimerization에 관한 연구

윤수한,김진영,박승우,안영환,안영민,조기홍,조경기,Yoon, Soo Han,Kim, Jin Young,Park, Seung Woo,Ahn, Young Hwan,Ahn, Young Min,Cho, Ki Hong,Cho, Kyung Gi 대한신경외과학회 2000 Journal of Korean neurosurgical society Vol.29 No.6

Purpose : The subcellular localization of E1B-19k has been known cytosol or nuclear membrane by immunohistochemical staining and could dimerize with Bax to regulate cell death also known by the in-vitro immunoprecipitation. We planed to confirm this dimerization of E1B-19k with Bax in vivo in Cos-7 cells by using green fluorescent protein. Material and Method : We cloned E1B-19k and Bax into C3-EGFP. C3-EGFP-E1B-19k, C3-EGFP-Bax, and C3-EGFP-E1B-19k and pcDNA3-Bax were transfected into Cos-7 cells. We explored location of E1B-19k and Bax, and confirmed its dimerization with Bax in transfected living healthy Cos-7 cells by following green fluorescent protein of E1B-19k on the confocal microscope. Results : E1B-19k was located diffusely in cytoplasm and in nucleus but not in mitochondria. It prevented cell death from the apoptosis by staurosporine but its location was not changed. GFP-E1B-19k is not changed its intracellular location with Bax even with staurosporine. Conclusion : These results support that E1B-19k does not localize in mitochondria nor dimerize with Bax even with staurosporine. We could anticipate E1B-19k prevent cell death via the other dimerizing partner or pathways.

Calcium overload is essential for the acceleration of staurosporine-induced cell death following neuronal differentiation in PC12 cells

Su Ryeon Seo,서정택 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.4

Differentiation of neuronal cells has been shown to accelerate stress-induced cell death, but the underlying mechanisms are not completely understood. Here, we find that early and sustained increase in cytosolic ([Ca2+]c) and mitochondrial Ca2+ levels ([Ca2+]m) is essential for the increased sensitivity to staurosporineinduced cell death following neuronal differentiation in PC12 cells. Consistently, pretreatment of differentiated PC12 cells with the intracellular Ca2+-chelator EGTA-AM diminished staurosporine-induced PARP cleavage and cell death. Furthermore, Ca2+ overload and enhanced vulnerability to staurosporine in differentiated cells were prevented by Bcl-XL overexpression. Our data reveal a new regulatory role for differentiation- dependent alteration of Ca2+ signaling in cell death in response to staurosporine.

The ubiquitin E3 ligase CHIP promotes proteasomal degradation of the serine/threonine protein kinase PINK1 during staurosporine-induced cell death

Yoo, Lang,Chung, Kwang Chul American Society for Biochemistry and Molecular Bi 2018 The Journal of biological chemistry Vol.293 No.4

<P>Mutations in the gene for the serine/threonine protein kinase PTEN-induced putative kinase 1 (PINK1) are the second most frequent cause of autosomal recessive Parkinson's disease (PD). Via its kinase activity, PINK1 regulates neuronal cell survival and mitochondrial quality control. Numerous reports have revealed that PINK1 has diverse and physiologically significant functions, and therefore its activity should be tightly regulated. However, the molecular mechanisms regulating PINK1 stability and the modulator(s) involved have not been elucidated. In this study, we demonstrate that the ubiquitin E3 ligase carboxyl terminus of Hsp70-interacting protein (CHIP) promotes PINK1 ubiquitination and decreases its steady-state levels. Moreover, PINK1 levels were strongly reduced in HEK293 and SH-SY5Y cells exposed to the apoptosis-inducer staurosporine. Of note, we found that this reduction resulted from CHIP-mediated PINK1 ubiquitination. Accordingly, siRNA-mediated CHIP knockdown reduced susceptibility to staurosporine-induced cell death. Taken together, these findings suggest that CHIP plays a role in negative regulation of PINK1 stability and may suppress PINK1's cytoprotective effect during staurosporine-induced mammalian cell death. We propose that this PINK1 regulatory pathway might contribute to Parkinson's disease pathogenesis.</P>

갑상선에서 protein kinase C에 의한 thyroxine 유리조절

김진상,Kim, Jin-shang 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.6

Previous studies suggested that the inhibition of thyroxine ($T_4$) release by ${\alpha}_1$-adrenoceptor and muscarinic receptor stimulation results in activated protein kinase C (PKC) from mouse and guinea pig thyroids. In the present study, the effect of carbachol, methoxamine, phorbol myristate acetate (PMA), and R59022 on the release of $T_4$ from the mouse, rat, and guinea pig thyroids was compared to clarify the role of PKC in the regulation of the release of $T_4$. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for $T_4$ by EIA kits. Forskolin, an adenylate cyclase activator, chlorophenylthio-cAMP sodium, a membrane permeable analog of cAMP, and isobutyl-methylxanthine, a phosphodiesterase inhibitor, like TSH (thyroid stimulating hormone), enhaced the release of $T_4$ from the mouse, rat, and guinea pig thyroids. Methoxamine, an ${\alpha}_1$-adrenoceptor agonist, inhibited the TSH-stimulated release of $T_4$ in mouse, but not rat and guinea pig thyroids. In contrast, carbachol, a muscarinic receptor agonist, inhibited the release of $T_4$ in guinea pig, but not mouse and rat thyroids. These inhibition were reversed by prazosin, an ${\alpha}_1$-adrenoceptor antagonist or atropine, a muscarinic antagonist or $M_1$- and $M_3$-muscarinic antagonists, in mouse or guinea pig thyroids. In addition, staurosporine, a PKC inhibitor, reversed methoxamine or carbachol inhibition of TSH stimulation. Furthermore, PMA, a PKC activator, and R59022, a diacylglycerol (DAG) kinase inhibitor, inhibited the TSH-stimulated release of $T_4$ in mouse, rat, and guinea pig thyroids. These inhibition were blocked by staurosporine. These findings suggest that the activation of receptor or DAG inhibits TSH-stimulated $T_4$ release through a PKC-dependent mechanism in thyroid gland.