Effects of staurosporine on the contraction of self-assembled constructs of goat temporomandibular joint disc cells / 华西口腔医学杂志

OBJECTIVE@#The effects of the staurosporine on contraction of self-assembled constructs and extracellular matrix syntheses of goat temporomandibular joint discs were investigated.@*METHODS@#Goat temporomandibular joint disc cells were isolated and cultured to P3, and 5.5×10⁶ cells were combined with different concentrations of staurosporine (0, 0.1, 1, 10, 100 nmol·L⁻¹) in agarose wells and cultured for one week. The samples were frozen and sectioned. Safranin-O,  Picro-sirius red and immunohistochemical staining were performed to observe the distributions of the extracellular matrix and the expression of alpha-smooth muscle actin (α-SMA). Enzyme linked immunosorbent assay (ELISA) and Blyscan kits were utilized to quan--titatively detect the contents of type Ⅰ collagen (ColⅠ) and glycosaminoglycans (GAGs).@*RESULTS@#Each group of goat temporo-mandibular joint disc cells in the agarose wells were gathered to self-assemble into a disc-shaped base for 4 hours and then to gradually contract into a round shape. The Picro-sirius red staining was strong and indicated collagen distribution. The Safranin-O staining observed GAGs throughout the entire construct. The expression of ColⅠ was strongly posi-tive in the staurosporine groups; however, the expression of α-SMA was weak. ColⅠ and GAGs contents in the stau-rosporine groups were greater than that of the control group, especially in the 10 nmol·L⁻¹ group (P<0.01).@*CONCLUSIONS@#Staurosporine has a certain effect on the shrinkage of self-assembled constructs; however, such effect is not prominent. Staurosporine contributes to the construction synthesis of extracellular matrix.

Anticancer potential of metabolic compounds from marine actinomycetes isolated from Lagos Lagoon sediment / 药物分析学报

Thirty-two actinomycetes strains were isolated from sediment samples from 12 different sites at Lagos Lagoon and identified using standard physiological and biochemical procedures as well as 16S rDNA gene sequence analysis. Secondary metabolites were extracted from the strains and their anticancer activity on the K562 (Human acute myelocytic leukemia), HeLa (cervical carcinoma), AGS (Human gastric), MCF-7 (breast adenocarcinoma) and HL-60 (Human acute promyelocytic leukemia) cell lines was determined. The metabolic extracts exhibited cytotoxicity with IC50 values ranging from 0.030 mg/mL to 4.4 mg/mL. The Streptomyces bingchenggensis ULS14 extract was cytotoxic against all the cell lines tested. The bioactivity-guided extraction and purification of the metabolic extracts from this strain yielded two purified anticancer compounds: ULDF4 and ULDF5. The structures of the extracted compounds were determined using spectroscopic analyses, including electrospray ionization mass spectrophotometer and nuclear magnetic resonance (1 Dimensional and 2 Dimensional), and were shown to be structurally similar to staurosporine and kigamicin. The IC50 of ULDF4 and ULDF5 against the HeLa cell line was 0.034 μg/mL and 0.075 μg/mL, respectively. This study is the first to reveal the anticancer potential of actinomycetes from Lagos Lagoon, which could be exploited for therapeutic purposes.

Taxonomic characterization and isolation of antitrypanosomal compound from Streptomyces sp. FACC-A032 isolated from Malaysian forest soil

Aims: The present study is aimed at taxonomic characterization and isolation of active compound MS01 from Streptomyces sp. FACC-A032 which exhibited strong antitrypanosomal activity (IC50 0.02 μg/mL). Methodology and results: Isolate FACC-A032 was characterized based on its cultural, morphological, physiological and genomic properties. Isolate FACC-A032 was tentatively identified as Streptomyces sp. Biochemical analysis of diaminopimelic acid (DAP) isomer of whole-cell hydrolysates further confirmed the isolate FACC-A032 that contained LL-DAP isomer as species belonging to the genus Streptomyces. The inoculum for submerged cultures of isolate FACCA032 was prepared from cultures on ISP2 agar. After eight days of growth at 28  2 °C and 200 rpm in fermentation medium M3, fermentation broth was extracted with butanol and the crude extracts (solvent layer) were separated and dried in vacuo. Further studies were carried out to isolate the active compound from the culture extracts of isolate FACCA032. Using bioassay-guided isolation, crude extract was partitioned based on different polarity. After which, the resulting elutes were tested for antitrypanosomal activity. The active fraction was analyzed with HPLC-DAD analysis. Based on the analysis, major peak in the active fraction was collected using HPLC preparative. Active compound MS01 was isolated and structure elucidated using NMR spectroscopy. Conclusion, significance and impact of study: Bioassay-guided isolation techniques used in this study had discovered an active antitrypanosomal compound, staurosporine, from Streptomyces sp. FACC-A032. This is the first discovery of staurosporine, a protein kinase inhibitor, from Malaysian soil actinobacteria Streptomyces sp. Therefore, the study demonstrated the potential of Malaysian soil actinobacteria as antitrypanosomal therapeutic agent.

Aspergillus fumigatus conidia inhibit early apoptosis of alveolar epithelial cells / 中华传染病杂志

Objective To observe the impact of Aspergillus fumigatus conidia on the early apoptosis of alveolar epithelial cells.Methods A549 cells was first stimulated with tumor necrosis factor-α(TNF-α)or staurosporine (STS)to induce cell apoptosis,and then co-cultured with Aspergillus fumigatus conidia of three different concentrations (5 × 10 5 ,5 × 10 6 ,5 × 10 7 )simultaneously.The changes of the apoptosis rate of A549 cells were detected by flow cytometry.Results The early apoptosis of A549 cells was increased markedly after stimulated with TNF-α ([14.94 ± 0.82 ]% vs [3.26 ± 0.74]%,χ2 =9.18,P <0.05)and STS ([18.10±1 .00]% vs [3.26±0.74]%,χ2 =12.32,P <0.05). When Aspergillus fumigatus conidia of different concentrations were added,the apoptosis rate was decreased (χ2 =31 .23,P <0.05 ).The early apoptosis of A549 cells was negatively correlated with the concentration of Aspergillus fumigatus conidia (rTNF = - 0.92,r STS = - 0.94,both P < 0.05 ). Conclusion Aspergillus fumigatus conidia inhibits the early apoptosis of A549 cells induced by TNF-αor STS and the inhibit effect increases with the concentration of Aspergillus fumigatus conidia.

ERK1/2 mediated inhibitory role of curcumin on STS-induced neurons toxic injury / 中国生化药物杂志

Objective To explore extracellular signal-regulated kinase ( ERK1/2 ) expression in the role of curcumin inhibited staurosporine (STS)-mediated neurons toxic injury through added PD098059, and to clarify ERK1/2 mediated inhibitory role of curcumin on STS-induced neurons toxic injury.Methods The neurons toxic injury model of primary cultured hippocampal neurons was established by STS.The experiment was divided into six groups:normal control group, STS model group, PD098059 +STS model group, curcumin +STS pretreatment group, curcumin+PD098059+STS treatment group and curcumin treatment group.The cell viability were determined by MTT method, lactate dehydrogenase (LDH) release rate, cell toxicity were detected, nuclear shape were observed by DAPI staining, and ERK1/2 expression were detected by Western blot method.Results The cell viability of curcumin +STS pretreatment group was significantly higher than STS model group ( P <0.001 ); the cell viability had no significant difference between PD098059 +STS model group and curcumin +PD098059 +STS treatment group;compared with curcumin +STS model group , the cell viability of curcumin +PD098059 +STS treatment group was significantly decreased ( P<0.001 ).LDH results showed that the nerve cell toxicity of curcumin +STS pretreatment group was obviously less than STS model group (P<0.001).The cell nuclear shape showed typical apoptosis morphological characteristics in STS model group, and curcumin inhibited the effect of STS mediated-neuronal apoptosis.ERK1/2 protein expression in curcumin +STS pretreatment group significantly increased compared with STS model group ( P <0.001 ) .Conclusion Curcumin inhibited STS-mediated neurons toxicity injury by up-regulating ERK1/2 expression.After PD098059 blocking the nerve cells ERK1/2 synthesis, the inhibitory action of curcumin failed to implemented, which illustrated that ERK1/2 mediated curcumin to inhibit STS-induced neuronal toxic injury.

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

In the present study, we investigated the effect of staurosporine on the formation of cellular processes in human gingival fibroblasts and rat astrocytes. Staurosporine caused a rapid induction of process formation in human gingival fibroblasts and rat astrocytes in a concentration dependent manner. The process formation of human gingival fibroblasts and rat astrocytes was prevented by the pretreatment with N-acetylcysteine, suggesting that staurosporine-induced ROS production was responsible for the process formation. Colchicine, a microtubule depolymerizing agent, inhibited the staurosporine-induced process formation, whereas cytochalasin D, an actin filament breakdown agent, failed to suppress the formation of cellular processes. This result indicated that polymerization of microtubule, and not actin filament, was responsible for the formation of cellular processes induced by staurosporine. In support of this hypothesis, Western blot analysis was conducted using anti-tubulin antibody, and the results showed that the amount of polymerized microtubule was increased by the treatment with staurosporine while that of depolymerized beta-tubulin in soluble fraction was decreased. These results indicate that staurosporine induces ROS-mediated, microtubule-dependent formation of cellular processes in human gingival fibroblasts and rat astrocytes.

Effect of heat shock protein 70 expression in Curcumin’s inhibition on STS-induced neurons injury / 中国生化药物杂志

Objective To explore the effect of heat shock protein 70(HSP70)expression in the role of Curcumin inhibited staurosporine(STS)-mediated neurons toxic injury.Methods The primary cultured hippocampal neurons was cultured in vitro and the stress damage model of STS-induced nerve cell toxicity was established.The experiment were divided into six groups according to the added drugs:normal control group,the STS model group (final concentration was 20μmol/L),Quercetin+STS model group(final concentration were 10 μmol/L and 20 μmol/L,respectively),Curcumin+STS pretreatment group(for 20μmol/L final concentration),Curcumin+Quercetin+STS treatment group(final concentration were 20μmol/L,10μmol/L and 20μmol/L,respectively)and Curcumin treatment group(final concentration was 20μmol/L).The cell viability were determined by thiazole blue (MTT)method,cell toxicity were measured by lactate dehydrogenase(LDH)release rate and HPS70 expression were detected by Western Blot. Results MTT results showed that the cell viability of Curcumin+STS pretreatment group was significantly higher than STS model group(P<0.001).Compared with Quercetin+STS model group,the cell viability of Curcumin+Quercetin+STS treatment group had little change.LDH results show that the nerve cell toxicity of Curcumin+STS pretreatment group was obviously less than that of STS model group(P<0.001).Western Blot results show that compared with STS model group,HSP70 protein expression in Curcumin+STS pretreatment group was significantly increased(P<0.001).Conclusion Curcumin can inhibit STS-mediated neurons toxicity stress damage though increasing HSP70 expression,when added Quercetin to block HSP70 expression in nerve cells,the inhibiting effect of Curcumin on STS-mediated neuron toxic stress injury is counteract.

Apoptotic-like activity of staurosporine in axenic cultures of Trypanosoma evansi / Actividad proapoptótica de la estaurosporina en cultivos axénicos de Trypanosoma evansi

Trypanosoma evansi is a blood protozoan parasite of the genus Trypanosoma which is responsible for surra (Trypanosomosis) in domestic and wild animals. This study addressed apoptotic-like features in Trypanosoma evansi in vitro. The mechanism of parasite death was investigated using staurosporine as an inducing agent. We evaluated its effects through several cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, maintenance of plasma membrane integrity, and mitochondrial trans-membrane potential. For access to these features we have used the flow cytometry and fluorescence microscopy with cultures in the stationary phase and adjusted to a density of 10(6) cells/mL. The apoptotic effect of staurosporine in T. evansi was evaluated at 20 nM final concentration. There was an increase of phosphatidylserine exposure, whereas mitochondrial potential was decreased. Moreover, no evidence of cell permeability increasing with staurosporine was observed in this study, suggesting the absence of a necrotic process. Additional studies are needed to elucidate the possible pathways associated with this form of cell death in this hemoparasite.

Trypanosoma evansi es un hemoparásito, el cual es el agente causal de la surra (tripanosomiasis) en mamíferos, perteneciente al orden Kinetoplastidae. Este estudio se oriento a caracterizar la muerte celular similar a apoptosis en cultivos in vitro de Trypanosoma evansi a través del uso del inductor esturosporina. Este efecto se evaluó a través de diversos aspectos fenotípicos de la apoptosis: el encogimiento celular, la exposición de fosfatidilserina, el mantenimiento de la integridad de la membrana plasmática y el potencial de membrana mitocondrial. Para evaluar estas características se utilizaron técnicas de citometría de flujo y microscopía de fluorescencia con cultivos en fase estacionaria ajustados a una densidad de 10(6) células/mL. El efecto apoptótico de la estaurosporina en Trypanosoma evansi fue evaluado a una concentración de 20 nM. Se evidenció un aumento de la exposición a fosfatidilserina, mientras que el potencial mitocondrial disminuyó. Por otra parte, no hay evidencias de aumento de la permeabilidad celular con estaurosporina, sugiriendo la ausencia de un proceso necrótico. Estudios adicionales son necesarios para dilucidar las posibles vías asociadas con esta forma de muerte celular en este hemoparásito.

Staurosporine induced poly (ADP-ribose) polymerase independent cell death in Dictyostelium discoideum.

In the present study D. discoideum has been used as a model organism to understand the role of poly (ADP-ribose) polymerase (PARP) in caspase independent paraptotic cell death pathways. D. discoideum lacks caspases and Bcl-2 family proteins; nevertheless it has 9 potential genes for PARP. PARP has been known to get activated in various cell death associated diseases. In this study kinetics of cell death induced by staurosporine (STS), a bacterial alkaloid, was established to unravel the role of PARP. It was found that STS induced cell death in D. discoideum did not involve PARP activation, however it involved cathepsin D. Results indicated that an alternative mechanism may be existing in D. discoideum that lacks Bcl-2 family proteins for STS induced cell death that evades Bax involvement.

Extent of extracellular signal-regulated kinases phosphorylation determines the sensitivity of hepatic stellatecells to staurosporine-induced apoptosis / 中南大学学报(医学版)

Objective:Hepatic stellate cells (HSCs) are the principal cells responsible for the development of hepatic fibrosis and cirrhosis.During the fibrotic process,HSCs undergo proliferation and transdifferentiation from a quiescent to myofibroblast-like phenotype.The fate of myofibroblastlike HSCs includes apoptosis or reversion back to a quiescent phenotype.The mechanisms involved in the apoptotic process of HSCs have yet to be determined.The purpose of the present study is to determine the effects of extracellular signal-regulated kinases (ERKs) phosphorylation on the apoptosis of HSCs induced by staurosporine.Methods:We used Western blot and flow cytometry to detect the expression level of ERK and cell apoptosis status in four rat hepatic stellate cell lines (CFSC-8B,-2G,-3H and -5H).Results:Each hepatic stellate cell line had a distinct morphology consistent with their expression level of α-SMA and that CFSC-8B cells had the highest α-SMA expression.Although all four cell types expressed similar levels of ERK1/2,phosphorylation levels were significantly higher in CFSC8B and CFSC-2G than in CFSC-3H and CFSC-5H cells.When CFSC-8B ceils (high ERK1/2 phosphorylation) and CFSC-5H cells (low ERK1/2 phosphorylation) were employed to examine staurosporine-induced apoptosis,CFSC-8B cells were significantly more sensitive.Staurosporine further increased ERK1/2 phosphorylation in both cell lines.Conclusion:ERK1/2 phosphorylation in HSCs determines the sensitivity of HSCs to staurosporine-induced apoptosis.

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.

Synergistic growth inhibition of cancer cells harboring the RET/PTC1 oncogene by staurosporine and rotenone involves enhanced cell death.

TPC-1 is a highly proliferative thyroid papillary carcinoma-derived cell line. These cells express the RET/PTC1 fusion protein, whose isoforms are characterized in this work. The bacterial alkaloid staurosporine and the plant extract rotenone are death-inducing drugs that have an inhibitory synergistic effect on the growth of TPC-1 cells. We show that this synergism is accompanied by an enhancement of the induction of cell death. Staurosporine alone induces cell cycle arrest in G1, whereas rotenone induces arrest in G2/M. We suggest that this additive pressure may drive cells to die, resulting in the synergistic interaction of the drug combination. These data emphasize the potential use of the staurosporine plus rotenone combination as an anticancer tool.

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

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 staurosporine-induced 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.

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

[Ca2+]i transients by reverse mode of cardiac Na+/Ca2+ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca2+ transients ('rundown'). Rundown of NCX1 was independent of membrane PIP2 depletion. Although the activation of protein kinase C (PKC) was observed during the Ca2+ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.

Selective Protection of Normal Proliferationg Cells Against Anti-Neoplastic Chemotherapy by Stausporine Pretreatment / 대한산부인과학회잡지

OBJECTIVE: A major limiting factor in human cancer chemotherapy is toxicity in normal cells and tissues. Our goal was to determine whether normal proliferating cells could be protected from chemotherapeutic agents by taking advantage of the differential drug sensitivity of cell cycle G1 checkpoint in normal and cancer cells. METHODS: Normal peripheral blood mononuclear cells (PBMC) and ovarian cancer cell lines (OVCAR- 3 and SKOV-3) were initially treated with 10 nM of staurosporine for 48 hours. After removal of staurosporine contained media, both PBMC and ovarian cancer cells were treated with 20 nM of paclitaxel for 24 hours. Cells were then allowed to recover in drug-free medium for 4 days. The DNA contents and cell cycle changes were detected by FACScan flow cytometer in the cells harvested whenever the medium was changed. RESULTS: After pretreatment of ovarian cancer cell lines (OVCAR-3 and SKOV-3) with 10 nM of staurosporine followed by treatment with 20 nM of paclitaxel, both OVCAR-3 and SKOV-3 cells were selectively arrested in G2M phase of cell cycle by paclitaxel and they resumed their proliferative cycle to some extents after the drugs were removed and cultured with fresh media. However. pretreatment with 10 nM of staurosporine protected normal circulating PBMC that had been induced to proliferate in vitro with phytohemagglutinin from paclitaxel. Staurosporine-induced arrest of PBMC in G0/G1 phase was reversible, and arrested cells tolerated 10 nM of paclitaxel in culture. CONCLUSION: OVCAR-3 and SKOV-3 cancer cells can be targeted specifically with paclitaxel, following staurosporine-mediated, selective and reversible G0/G1 arrest in PBMC.

The Changes of Cell Cycle Phase Fractions and Expression of p53 by the Treatment of Staurosporine in MCF-7 Cell Line / 대한산부인과학회잡지

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. CONCLUSIONS: 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.

Influence of staurosporine on catecholamine release evoked by cholinergic stimulation and membrane depolarization from the rat adrenal gland

The present study was attempted to examine the effect of staurosporine (STS) on secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from the isolated perfused rat adrenal gland and to establish its mechanism of action. The perfusion of STS (3 X 10(-7) ~3 X 10(-8) M) into an adrenal vein for 20 min produced a dose-dependent inhibition in CA secretion evoked by ACh (5.32 X 10(-3) M), high K+ (5.6 X 10(-2) M), DMPP (10(-4) M for 2 min), McN-A-343 (10(-4) M for 2 min), cyclopiazonic acid (10(-5) M for 4 min) and Bay-K-8644 (10(-5) M for 4 min). Also, in the presence of tamoxifen (2 X 10(-6) M), which is known to be a protein kinase inhibitor, CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with STS (10(-7) M) under the presence of phorbol-12,13-dibutyrate (10(-7) M), a specific activator of protein kinases (for 20 min), the inhibitory effect of STS on CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid was greatly recovered to the extent of the control release as compared to those in the presence of STS only. These results demonstrate that STS causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization, indicating strongly that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells through preventing activation of protein kinases. Furthermore, these findings also suggest that these STS-sensitive protein kinases play a modulatory role partly in regulating the rat adrenomedullary CA secretion.

The Role of Protein Kinase C in Acute Lung Injury Induced by Endotoxin / 결핵

BACKGROUND: The signal pathways and their precise roles for acute respiratory distress syndrome caused by endotoxin (ETX) has not been established. Since there has been several in vitro experiments suggesting that activation of protein kinase C (PKC) pathway may be responsible for endotoxin-induced inflammatory reaction, we performed in vivo experiments in the rats with the hypothesis that PKC-inhibition can effectively prevent endotoxin-induced acute lung injury. METHODS: We studied the role of PKC in ETX-induced ALl using PKC inhibitor (staurosporine, 5Th) in the rat. Specific pathogen free male Sprague-Dawley weighted from 165 to 270g were used for the study. Animals were divided into the normal control (NC)-, vehicle control (VC)-, ETX-, PMA (phorbolmyristateacetate)-, STP+PMA-, and STP+ETX-group. PMA (50mg/kg) or ETX (7mg/kg) was instilled through polyethylen catheter after aseptic tracheostomy with and without STP (0.2mg/kg) pretreatment. STP was injected via tail vein 30mm before intratracheal injection (IT) of PMA or ETX. Bronchoalveolar lavage (BAL) was done 3- or 6-hrs after IT of PMA or FTX respectively, to measure protein concentration, total and differential cell counts. RESULTS The results were as follows. The protein concentrations in BALF in the PMA- and ETX-group were very higher than that of VC-group (p<0.001). When animals were pretreated with STP, the %reduction of the protein concentration in BALF was 64.8 8.5 and 30.4 2.5% in the STP+PMA- and STP+ETX-group, respectively (p=0.028). There was no difference in the total cell counts between the PMA-and VC-group (p = 0.26). However the ETX-group showed markedly increased total cell counts as compared to the VC- (p=0.003) and PMA group (p=0.0027), respectively. The total cell counts in BALF were not changed after pretreatment with STP compared to the PMA- (p=0.22) and ETX-group (p=0.46). The percentage of PMN, but not alveolar macrophage, was significantly elevated in the PMA-, and ETX-group. Especially in the ETX-group, the percentage of PMN was 17 times higher than that of PMA (p<0.001). The differential cell counts was not different between the PMA and STP+PMA. On the contrary the STP+ETX-group showed decreased percentage of PMN (p = 0.016). There was no significant relationship between the protein concentration and the total or differential cell counts in each group. CONCLUSION: Pretreatment with PKC-inhibitor (staurosporine) partially but significantly inhibited ETX-in-duced ALI.

Effect of Protein Kinase C Inhibitor (Staurosporine) in Human Invasive Bladder Tumor Cells / 대한비뇨기과학회지

Protein Kinase C which is a Ca++ -activated, phospholipid - dependent enzyme phosphorylates numerous protein substrates and participates in intracellular signaling processes. Protein kinase C is associated with a wide range of biological effects including stimulus-secretion coupling, induction of cellular proliferation and differentiation, activation of nuclear transcription factors and cell surface receptors and tumor promotion. Programmed cell death, referred to apoptosis is an active, energy-dependent process in which the cell participates in its own destruction during apoptosis. There is condensation and fragmentation of nuclear chromatin, accompanied by a marked decline in total cell volume, dilation of the endoplasmic reticulum and general compacting of cellular organelles. Thereafter, there is fragmentation of both nucleus and cytoplasm to give rise to small membrane-bound vesicles known as apoptotic bodies. Protein kinase C may have the regulatory role in apoptosis. Staurosporine is a potent protein kinase C inhibitor. Staurosporine inhibited the growth of human invasive bladder tumor cells, T24 in MTT test. The survival fractions of human invasive bladder tumor cells T24 were 100.0%, 76.0%, 62.5% and 18.1% with staurosporine concentration 0nM, 10nM, 100nM and 1000nM, respectively. From the results we identified that staurosporine inhibited the growth of T24 cells markedly in a dose dependent manner(P<0.05). 12-hour exposure of T24 cells to staurosporine failed to induce DNA fragmentation at the concentrations of 0nM, 10nM and 100nM but promoted fragmentation at the concentration of 1000nM, showing typical ladder pattern on agarose gel electrophoresis. On the examination of cellular morphology, T24 cells showed the features of apoptosis such as cell shrinkage, nuclear condensation and formation of bleb and apoptotic bodies after exposure to staurosporine of 10nM, 100nM and 1000nM concentrations. These results suggest that staurosporine have remarkable cytotoxic effect against human invasive bladder tumor cells T 24 and the mechanism of cytotoxicity may be apoptosis.

Effect of adenylate cyclase inhibitor and protein kinase C inhibitor on GnRH-induced LH release and LH beta subunit biosynthesis in rat anterior pituitary cells

According to our previous studies together with others, GnRH, a hypothalamic decapeptide, has been known to be a major regulator for LH release and its subunit biosynthesis in anterior pituitary gonadotropes. But the precise mechanisms by which GnRH exerts stimulatory effects on LH release and its subunit biosynthesis have not been clearly understood. In the present study we examined the effect of GnRH on protein kinase C (PKC) activity and intracellular cAMP content in cultured anterior pituitary cells of rat to clarify whether PKC or cAMP are involved in GnRH action. Moreover, we examined the effects of staurosporine (ST), a PKC inhibitor and 2',3'-dideoxyadenosine (2',3'-DDA), an adenylate cyclase inhibitor, on LH release and steady state LH beta subunit mRNA levels in cultured anterior pituitary cells of rat. PKC activity was rapidly increased within 30 min after GnRH treatment whereas intracellular cAMP level was elevated 18 h after GnRH treatment. ST significantly inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner, showing an half maximal response at 50 nM ST. 2',3'-DDA inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner in pituitary cells. From these results, it is suggested that GnRH stimulates LH beta subunit mRNA level as well as LH release in anterior pituitary cells and this GnRH action might be mediated by PKC activation and cAMP stimulation.