Inhibitory Effect of PKC412 Against Human Acute Leukemia Cell Line HL-60 Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the effects and mechanisms of PKC412 inhibitor on proliferation and apoptosis of HL-60 cell line.@*METHODS@#CCK-8 assay was used to detect the effect of PKC412 on the proliferation of HL-60 cells at different concentrations; Wright-Giemsa staining was used to estimated the effect of PKC412 on the apoptosis of HL-60 cells; the mRNA expression of BCL-2 and P53 genes was detected by qRT-PCR, the expression of BCL-2 and P53 proteins was detected by Western blot. HL-60 cells were injected into mouse caudal vein to construct acute myeloid leukemia model, PKC412 was administered to tail vein for 31.25 nmol/kg, normal saline was injected into the same site of the mice as control group, and the inhibitory effect of PKC412 on HL-60 cells in mice was observed. ELISA assay was used to detect the effect of PKC412 on the inflammatory factors of TNF-α and TGF-β in tumor mice.@*RESULTS@#PKC412 could inhibit the proliferation of HL-60 cell, which was in a dose dependent manner(r=0.9973) (IC50 was 0.31 μmol/L), and induce apoptosis of HL-60 cells. After HL-60 cell was treated by PKC412 for 48 h the expression of BCL-2 gene was down regulated(0.417±0.044 vs 0.933±0.033, t=9.347, P0.05) as compared with control group. And the expression of BCL-2 protein was decreased, while the expression of P53 protein was increased. PKC412 could inhibited the growth of HL-60 tumor cells in vivo, the survival rate of mice after administration was 50% and the weight was increased as compared with that in control group(18.02±0.403 g vs 16.44±0.562 g, t=2.272, P=0.0356). The secretion of TNF-α and TGF-β cytokine in serum and spleen cells in PKC412 group was significantly lower than that in control group (P<0.05).@*CONCLUSION@#PKC412 can induce apoptosis of HL-60 cells by inhibiting the expression level of BCL-2 gene, PKC412 administration in vivo can inhibit the growth of the tumors.

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.

Effect of Anti-vascular Endothelial Growth Factor Antibody on the Survival of Cultured Retinal Ganglion Cells

PURPOSE: To investigate the effects of anti-vascular endothelial growth factor (VEGF) antibody on the survival of retinal ganglion cell (RGC)-5 cells differentiated with staurosporine under oxidative stress. METHODS: We used real-time polymerase chain reaction and Western blot to confirm the expression of VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 in RGC-5 cells differentiated with staurosporine for 6 hours. The differentiated RGC-5 cells were treated with 800 µM hydrogen peroxide (H₂O₂) for 24 hours to induce oxidative stress. Then, the survival rate of RGC-5 was confirmed by lactate dehydrogenase assay at each concentration (0, 0.01, 0.1, and 1 mg) using bevacizumab as the anti-VEGF antibody. The expression of VEGF, VEGFR-1, and VEGFR-2 was confirmed using real-time polymerase chain reaction. RESULTS: VEGF, VEGFR-1, and VEGFR-2 were all expressed in differentiated RGC-5 cells. When RGC-5 cells were simultaneously treated with bevacizumab and 800 µM H₂O₂, survival of RGC-5 decreased with bevacizumab concentration. VEGF expression in RGC-5 cells increased with increasing concentration of bevacizumab. Similar patterns were observed for VEGFR-1 and VEGFR-2, but the degree of increase was smaller than that for VEGF. CONCLUSIONS: When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these in vitro study results, caution should be exercised with bevacizumab treatment.

TRIM29 Overexpression Promotes Proliferation and Survival of Bladder Cancer Cells through NF-κB Signaling / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: TRIM29 overexpression has been reported in several human malignancies and showed correlation with cancer cell malignancy. The aim of the current study is to examine its clinical significance and biological roles in human bladder cancer tissues and cell lines. MATERIALS AND METHODS: A total of 102 cases of bladder cancer tissues were examined for TRIM29 expression by immunohistochemistry. siRNA and plasmid transfection were performed in 5637 and BIU-87 cell lines. Cell Counting Kit-8, flow cytometry, western blot, and real-time polymerase chain reaction were performed to examine its biological roles and mechanism in bladder cancer cells. RESULTS: We found that TRIM29 overexpression showed correlation with invading depth (p=0.0087). Knockdown of TRIM29 expression in bladder cancer cell line 5637 inhibited cell growth rate and cell cycle transition while its overexpression in BIU-87 cells accelerated cell proliferation and cell cycle progression. TRIM29 overexpression also inhibited cell apoptosis induced by cisplatin. In addition, we demonstrated that TRIM29 depletion decreased while its overexpression led to upregulated expression of cyclin D1, cyclin E, and Bcl-2. We also showed that TRIM29 knockdown inhibited protein kinase C (PKC) and nuclear factor κB (NF-κB) signaling while its overexpression stimulated the PKC and NF-κB pathways. BAY 11-7082 (NF-κB inhibitor) partly attenuated the effect of TRIM29 on expression of cyclin and Bcl-2. Treatment with PKC inhibitor staurosporine resulted in ameliorated TRIM29 induced activation of NF-κB. CONCLUSION: The current study demonstrated that TRIM29 upregulates cyclin and Bcl family proteins level to facilitate malignant cell growth and inhibit drug-induced apoptosis in bladder cancer, possibly through PKC–NF-κB signaling pathways.

Cell-in-Cell Death Is Not Restricted by Caspase-3 Deficiency in MCF-7 Cells / 한국유방암학회지

PURPOSE: Cell-in-cell structures are created by one living cell entering another homotypic or heterotypic living cell, which usually leads to the death of the internalized cell, specifically through caspase-dependent cell death (emperitosis) or lysosome-dependent cell death (entosis). Although entosis has attracted great attention, its occurrence is controversial, because one cell line used in its study (MCF-7) is deficient in caspase-3. METHODS: We investigated this issue using MCF-7 and A431 cell lines, which often display cell-in-cell invasion, and have different levels of caspase-3 expression. Cell-in-cell death morphology, microstructures, and signaling pathways were compared in the two cell lines. RESULTS: Our results confirmed that MCF-7 cells are caspase-3 deficient with a partial deletion in the CASP-3 gene. These cells underwent cell death that lacked typical apoptotic properties after staurosporine treatment, whereas caspase-3-sufficient A431 cells displayed typical apoptosis. The presence of caspase-3 was related neither to the lysosome-dependent nor to the caspase-dependent cell-in-cell death pathway. However, the existence of caspase-3 was associated with a switch from lysosome-dependent cell-in-cell death to the apoptotic cell-in-cell death pathway during entosis. Moreover, cellular hypoxia, mitochondrial swelling, release of cytochrome C, and autophagy were observed in internalized cells during entosis. CONCLUSION: The occurrence of caspase-independent entosis is not a cell-specific process. In addition, entosis actually represents a cellular self-repair system, functioning through autophagy, to degrade damaged mitochondria resulting from cellular hypoxia in cell-in-cell structures. However, sustained autophagy-associated signal activation, without reduction in cellular hypoxia, eventually leads to lysosome-dependent intracellular cell death.

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.

Research Progress on Treating Acute Myeloid Leukemia by Midostaurin / 中国实验血液学杂志

FLT3 gene mutations occurred in approximately 30% of acute myeloid leukemia (AML) patients, which is closely associated with the occurrence, development and poor prognosis of AML. The therapy targeting at FLT3 mutations might be a promising treatment for AML. Midostaurin can inhibit the activities of III receptor tyrosine kinase encoded by FLT3 gene, induce cell cycle arrest and has a apoptotic effect on primitive AML cells of FLT3 -mutant, FLT3 wild-type and the expression of FLT3 mutated receptor. In view of this, the association between FLT3 mutations and AML, and research advances and clinical applications of midostaurin on the treatment of AML especially for FLT3 mutated AML, are reviewed.

Expression of TonEBP by Hypertonic and Hyperosmolar Stress in RGC-5 Cells

PURPOSE: In order to determine whether the Tonicity responsive enhancer binding protein (TonEBP) is expressed by hypertonic and hyperosmolar stress, TonEBP expression was investigated in the retinal ganglion cell (RGC) line, RGC-5 cells. METHODS: After RGC-5 cells were cultured by Staurosporine, TonEBP expression was measured with Western immunoblotting analysis and real-time reverse transcription-polymerase chain reaction in 50 mM NaCl, 100 mM mannitol, 50 mM glucose, or 100 mM glucose at 3, 6, 12, and 24 hours after exposure to each environment. RESULTS: In this study, the protein expression of TonEBP was determined to be statistically significantly checked in 50 mM NaCl after 3, and 6 hours, in 100 mM mannitol after 6 hours, and in 100 mM glucose after 3, and 6 hours. TonEBP messenger Ribonucleic acid (mRNA) expression was determined to be statistically significantly checked in 50 mM NaCl after 3 hours, in 100 mM mannitol after 3, and 24 hours, and in 50 mM glucose after 3, and 24 hours. CONCLUSIONS: These results suggested that TonEBP was expressed by hypertonic and hyperosmolar stress at the protein and mRNA levels. Further studies are nedded to determine the role of TonEBP and the mechanism of expression and regulation of TonEBP.

Calbindin-D28K Prevents Staurosporin-induced Bax Cleavage and Membrane Permeabilization

Calbindin-D28K has been implicated in the regulation of neuronal cell death. Previously, we demonstrated that calbindin-D28K prevents staurosporine (STS)-induced caspase activation and subsequent apoptosis in a neuronal cell line. However, the role of calbindin-D28K in STS-induced activation of calpain and necrotic cell death was not identified. Staurosporine induced the elevation of intracellular calcium after 1 hr of treatment. Overexpression of calbindin-D28K and presence of a calcium chelator, BAPTA, prevented the increase of calcium in STS-treated cells. Cleavage of Bax by calpain was prevented by the overexpressed calbindin-D28K. Permeabilization of the plasma membrane, a factor in necrosis, as well as apoptotic change of the nucleolus induced by STS, was prevented by calbindin-D28K. Thus, our study suggests that calbindin-D28K may exert its protective functions by preventing calpain activation in necrotic cell death, in addition to its effect on the caspase-apoptosis pathway.

Tumor Necrosis Factor-Associated Protein 1 (TRAP1) is Released from the Mitochondria Following 6-hydroxydopamine Treatment

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Most cases are sporadic and its etiology is incompletely understood. However, increasing evidence suggests that oxidative stress and mitochondrial dysfunction may be involved in the pathogenesis of Parkinson's disease. The aim of this study was to investigate changes in mitochondrial protein profiles during dopaminergic neuronal cell death using two-dimensional gel electrophoresis in conjunction with mass spectrometry. Several protein spots were found to be significantly altered following treatment of MN9D dopaminergic neuronal cells with 6-hydroxydopamine (6-OHDA). Among several identified candidates, TNF receptor-associated protein 1 (TRAP1), a mitochondrial molecular chaperone, was released from the mitochondria into the cytosol in MN9D cells as well as primary cultures of dopaminergic neurons following 6-OHDA treatment. This event was drug-specific in that such apoptotic inducers as staurosporine and etoposide did not cause translocation of TRAP1 into the cytosol. To our knowledge, the present study is the first to demonstrate the drug-induced subcellular translocation of TRAP1 during neurodegeneration. Further studies delineating cellular mechanism associated with this phenomenon and its functional consequence may provide better understanding of dopaminergic neurodegeneration that underlies PD pathogenesis.

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.

Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15 percent increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52 percent compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

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.

Effects of staurosporine on the proliferation and apoptosis of prostate cancer PC-3 cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of staurosporine (ST) on the proliferation and apoptosis of prostate cancer PC-3 cells.</p><p><b>METHODS</b>Prostate cancer PC-3 cells were treated in vitro with ST at 10(-8) mol/L. The expressions of cyclin A and cyclin D1 proteins in the cells were detected by Western blot, the effect of ST on the proliferation of the cells determined by MTT assay and plate colony formation, the apoptosis of the cells examined by flow cytometry, and their morphological changes observed under the light microscope.</p><p><b>RESULTS</b>ST treatment markedly decreased the expressions of cyclin A and cyclin D1 in the PC-3 cells, and significantly inhibited the growth of the PC-3 cells (19.35%) at 48 h. (F = 31.06, P < 0.01). The colony formation rate of the PC-3 cells was (37.10 +/- 3.43) % in the ST group, significantly lower than (64.80 +/- 4.34) % in the control (chi2 = 14.59, P < 0.05) and (62.80 +/- 4.36) % in the DMSO group (chi2 = 12.50, P < 0.05), while the apoptosis rate of the cells was remarkably higher in the ST group ([19.6 +/- 2.20] %) than in the control ([5.33 +/- 1.40] %) and the DMSO group ([5.50 +/- 0.96] %) (F = 104.36, P < 0.01). Under the light microscope, the ST-treated cells were round with indistinct margins as compared with those of the other two groups.</p><p><b>CONCLUSION</b>ST could significantly inhibit the proliferation and induce the apoptosis of PC-3 cells.</p>

Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7 cells

Neurotrophins protect neurons against excitotoxicity; however the signaling mechanisms for this protection remain to be fully elucidated. Here we report that activation of the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway is critical for protection of hippocampal cells from staurosporine (STS) induced apoptosis, characterized by nuclear condensation and activation of the caspase cascade. Both nerve growth factor (NGF) and brain-derived growth factor (BDNF) prevent STS-induced apoptotic morphology and caspase-3 activity by upregulating phosphorylation of the tropomyosin receptor kinase (Trk) receptor. Inhibition of Trk receptor by K252a altered the neuroprotective effect of both NGF and BDNF whereas inhibition of the p75 neurotrophin receptor (p75NTR) had no effect. Impairment of the PI3K/Akt pathway or overexpression of dominant negative (DN)-Akt abolished the protective effect of both neurotrophins, while active Akt prevented cell death. Moreover, knockdown of Akt by si-RNA was able to block the survival effect of both NGF and BDNF. Thus, the survival action of NGF and BDNF against STS-induced neurotoxicity was mediated by the activation of PI3K/Akt signaling through the Trk receptor.

Estaurosporinas de Eudistoma vannamei: química e bioatividade / Staurosporines of Eudistoma vannamei: chemistry and bioactivity

Eudistoma vannamei (Millar, 1977) é uma ascídia endêmica do litoral do Nordeste brasileiro, largamente encontrado nas praias rochosas do estado do Ceará. Previamente, o extrato bruto apresentou um interessante perfil em termos de bioatividade. O fracionamento bioguiado identificou uma mistura 1:1 altamente citotóxica, contendo dois derivados inéditos de estaurosporina, 2-hidroxi-7-oxoestaurosporina (I) e 3-hidroxi-7-oxoestaurosporina (II). IC50 para I/II e estaurosporina (STP) foram obtidas após 72h de incubação com diversas linguagens de células tumorais, utilizando-se o ensaio do MTT, e em linfócitos humanos normais, através do ensaio de AlamarBlue. I/II superou a citotoxicidade de STP em 7 vezes, em média, para as células tumorais, ao passo que mostrou-se tão ativa quanto frente as células normais. Uma análise cinética sobre a progressão de ciclo celular, ativação de resposta a dano e vias de reparo de DNA, e indução de apoptose de células HL-60 (leucemia) foi conduzida com 40 ou 80ng/mL I/II e acessada por citometria de fluxo e western blotting...

Pro-protein convertase-2/carboxypeptidase-E mediated neuropeptide processing of RGC-5 cell after in vitro ischemia / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5 (RGC-5) cells, pro-protein convertase-2 (PC2), carboxypeptidase-E (CPE) and preproneuropeptide Y (preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed.</p><p><b>METHODS</b>The RGC-5 cell was differentiated in 0.1 mumol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation (OGD). The acute or chronic OGD-induced cell death rates were obtained by using PI or TUNEL staining. The protein expression levels were determined by using the Western blot method and PC2 activity analysis.</p><p><b>RESULTS</b>The ischemia caused substantial cell death in an OGD dose-dependent manner. In the cells, proPC2 and preproNPY protein levels gradually increased whereas proCPE gradually decreased. After OGD, PC2 activity was decreased. In the conditioned medium, proPC2 and PC2 proteins gradually decreased whereas proCPE, CPE, and preproNPY proteins gradually increased.</p><p><b>CONCLUSION</b>These results demonstrated that OGD inhibited the neuropeptide pro-protein processing system by reducing PC2 activity and the maturation of proPC2. The aggregation of the pro-proteins and the increase of the active CPE excision adversely exacerbated the cell injury. The pro-protein processing system might play a critical role in the ischemic stress of RGC-5 cells.</p>

Both R(-)- and S(+)-Citalopram Attenuate Neuronal Apoptosis in Murine Cortical Cell Culture / 대한정신약물학회지

OBJECTIVE: Several lines of evidence have shown that neuronal injury might represent a possible pathway to mood disorders. The present study was performed to examine the possibility that the neuroprotective effects of R(-)- and S(+)-citalopram depend on types of neuronal cell death. METHODS: Free radical neurotoxicity was induced in mixed cortical cell cultures by continuous exposure to 30 micrometer Fe++ for 24 hours, which produced hydroxyl radicals via a Fenton reaction. Cultures measuring excitotoxicity were exposed for 24 hours to 50 micrometer NMDA. Neuronal apoptosis was induced by 100 nM staurosporine exposure for 24 hours. Neuronal death was analyzed 24 hours later by measuring the efflux of lactate dehydrogenase (LDH) into the bathing medium or by counting viable neurons after staining with trypan blue. RESULTS: Co-treatment with R(-)-or S(+)-citalopram prevented staurosporine-induced apoptosis of cultured cortical cells, as well as activation of caspase-3. In contrast to the differences between enantiomers found in serotonin uptake inhibition and in the in vivo behavioral depression model, both enantiomers in this study showed similar protective effects on staurosporine-induced apoptosis. Mixed cortical cell cultures exhibited marked swelling of the neuronal cell bodies accompanied by widespread neuronal death during the 24 hours following exposure to 30 micrometer Fe++ or 50 micrometer NMDA that was not sensitive to either the R(-)-or S(+)-enantiomers of citalopram. CONCLUSION: These features suggest that the neuroprotective effect of citalopram depend on type of neuronal injury.

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.

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress in vitro

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. Alter differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 microM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the Terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury.