Annexin 1 protects against apoptosis induced by serum deprivation in transformed rat retinal ganglion cells

To investigate whether Annexin 1 can protect a retinal ganglion cells line (RGC-5) from apoptosis as induced by serum deprivation. Annexin 1 location in RGC-5 cells was determined using an indirect immunofluorescent assay. Expression of Annexin 1 in RGC-5 cultures deprived of serum for 0, 2 days was semi-quantified by western blot and RT-PCR. Effects of varying concentrations of the Annexin 1 peptide fragment, Ac2-26, on the survival of the RGC-5 cells was determined, and apoptotic cells were quantified by flow cytometry. Immunoblot and RT-PCR analysis was preformed to identify caspase 3, bax and bcl-2 in RGC extracts. Annexin 1 was localized in the cytoplasm of RGC-5 cells and the expression of Annexin 1, caspase 3 and bax was upregulated in serum-deprived RGC-5 cells. Ac2-26 attenuated the apoptosis resulting from serum deprivation of RGC-5 in a concentration-dependent manner, decreased caspase 3 and bax levels and produced an increase of bcl-2 in cell lysates. Annexin 1, in specific the peptide fragment Ac2-26, may play an important role in decreasing apoptosis in serum-deprived RGC-5 cells.     

Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid.

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.     

Agmatine protects cultured retinal ganglion cells from tumor necrosis factor-alpha-induced apoptosis

AimsWe investigated the protective effects of agmatine against tumor necrosis factor (TNF)-α-induced apoptosis in transformed rat retinal ganglion cells (RGC-5 cell line).Main methodsThe RGC-5 cells were exposed to 50 ng/mL TNF-α for 48 h with or without presence of 100 μM agmatine as indicated. Cell viability was determined by lactate dehydrogenase (LDH) assay. Double staining with Hoechst 33342 and propidium iodide for morphological analysis was performed. Subsequently, using annexin V assay, the proportion of cells actively undergoing apoptosis was determined.Key findingsAfter 48 h of exposure to 50 ng/mL TNF-α, 17.00% of RGC-5 cells were lost, as evident by LDH assay. TNF-α-induced RGC-5 cell death was reduced to 8.14% with 100 μM agmatine treatment. This observed cell loss was due to apoptotic cell death, as established by annexin V assay.SignificanceOur results reveal that agmatine has neuroprotective effects against TNF-α-induced apoptosis in retinal ganglion cells in vitro.     

Apoptotic cell death kinetics in vitro depend on the cell types and the inducers used

INTRODUCTION: In vitro exposure of cells to a fluorochrome-labeled inhibitor of caspases (FLICA) labels cells after caspase activation and arrests further progress of apoptotic cell death. The labeled apoptotic cells can be quantified in relation to time of apoptosis induction with flow cytometry. Loss of membrane integrity (late apoptosis and cell death) was measured with exposure to propidium iodide (PI). From the labeling patterns with FLICA and PI the apoptotic cell death kinetics was calculated. METHODS: HL60 cells and human umbilical vein endothelial cells (HUVECs) were incubated in the presence of the fluorescent inhibitor of caspases, FAM-VAD-FMK (20 mM, FLICA) for up to 48 h. Apoptosis was induced by Camptothecin (CPT, 0.15 microM) or by a mixture of tumour necrosis factor alpha (TNF-alpha, 3 nM)-Cycloheximide (CHX, 50 microM). Samples were counterstained with PI. RESULTS: Incubation of HL60 cells with CPT induced apoptosis in 92% of cells within the first 18 h at a rate of 5% per hour while incubation with TNF-alpha/CHX resulted in apoptosis in 76% of the cells within the first 6 h at a rate of 12% per hour. Incubation of HUVECs with TNF-alpha/CHX induced apoptosis in 65% of the cells within the first 18 h at a rate of 3.7% per hour during the first 6 h of the incubation. During incubation with TNF-alpha/CHX the remaining viable HL60 cells and HUVECs entered apoptosis within 48 h at an approximate rate of 0.2 per hour. However, on the road of the cell death, HL60 cells showed a transit from the viable (FLICA-/PI-) to early (FLICA+/PI-) and further to late apoptotic phase (FLICA+/PI+), while HUVECs entered directly from the viable to the late apoptotic stage. CONCLUSION: Apoptotic turnover rate depends on the stimulus used to induce apoptosis, while the type of the cell determines the way of the transition within the apoptotic cascade.     

Rotenone-induced death of RGC-5 cells is caspase independent, involves the JNK and p38 pathways and is attenuated by specific green tea flavonoids

The aim of the present studies was to characterise cell death following inhibition of mitochondrial complex I with rotenone in a transformed cell line (RGC-5 cells) and to examine the neuroprotective properties of the flavonoids genistein, epigallocatechin gallate (EGCG), epicatechin (EC) and baicalin. Rotenone-induced cell death of RGC-5 cells results in a generation of reactive oxygen species, a breakdown of DNA, the translocation of membrane phosphatidylserine, an up-regulation of haemoxygenase-1 and is unaffected by necrostatin-1 (inhibitor of necroptosis), z-VAD-fmk (pan caspase inhibitor) or NU1025 (PARP inhibitor) but attenuated with SP600125 (JNK inhibitor). Rotenone-induced toxicity of RGC-5 cells also caused an activation of mitogen-activated kinases indicated by an up-regulation and translocation into mitochondria of p-c-Jun, pJNK and pp38. Exposure of RGC-5 cells to rotenone does not affect apoptosis inducing factor or significantly stimulate caspase-3 activity. EGCG and EC both significantly blunt rotenone toxicity of RGC-5 cells at concentrations of 50 μM while genistein and baicalin were without effect. Significantly, genistein is approximately 20 times less efficacious than EGCG (IC₅₀ 2.5 μM) and EC (IC₅₀ 1.5 μM) at inhibiting sodium nitroprusside-induced lipid peroxidation. These studies show that rotenone toxicity of RGC-5 cells is neither necroptosis nor caspase-dependent apoptosis but involves the activation of mitogen-activated kinases and is inhibited by a JNK inhibitor, EGCG and EC. Genistein attenuates lipid peroxidation less efficaciously than EC and EGCG and does not affect rotenone toxicity of RGC-5 cells.     

Modulation of tumor cell proliferation and apoptosis by polyamine depletion in cells of head and neck squamous cell carcinomas

These studies were carried out to examine the capacity of alpha-difluoromethylornithine (DFMO) to modulate cell proliferation and apoptosis in cells of squamous cell carcinomas (SCCs) of the head and neck. Exposure of cells to DFMO (5 mM for 48 h) depleted intracellular putrescine and spermidine levels (greater than 5-fold) and inhibited proliferation of the cells without manifestation of cytotoxicity as measured by a clonogenic assay. Exposure of the cells to DFMO did not influence the survival response after exposure to single-dose radiation between 0 and 10 Gy. Treatment of polyamine-depleted cells with 200 nM staurosporine amplified apoptosis 65% (1.65-fold) over that in controls, as determined by flow cytometry. The increased apoptosis after DFMO treatment was effectively inhibited by the addition of 1 mM putrescine or spermidine. Cleavage of poly(ADP-ribose) polymerase (PARP) illustrated that the staurosporine treatment induced apoptosis in the cells within 6 h. Analysis of PARP cleavage indicated that treatment with DFMO accelerated the kinetics of progression of apoptosis but did not influence the sensitivity of cells to 10 nM-1 microM staurosporine. These data suggest an involvement of endogenous polyamines in modulation of proliferation kinetics and apoptosis in human SCCs and suggest opportunities to explore new therapeutic strategies in head and neck cancer patients to be treated with radiation therapy.     

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimer’s disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48 h after 5% O₂ hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.     

Fusiogenic endogenous-retroviral syncytin-1 exerts anti-apoptotic functions in staurosporine-challenged CHO cells

Fusiogenic glycoprotein syncytin-1, expressed in human placenta, is a promising candidate for acquiring a basic knowledge of placental syncytialization. However, its cellular mode of action is unidentified. We investigated whether syncytin-1 may exert influence on apoptotic processes. Therefore, we incubated CHO cells after stable transfection with syncytin-1 (CHO-52) in the presence or absence of staurosporine (STS), a kinase inhibitor well characterized to induce apoptosis. When testing the phenotype of CHO-52 cells, we could demonstrate that the induction of apoptosis by STS was delayed over a period of up to 24 h. Furthermore, the cell death rate was decreased by approx 75% following transfection of syncytin-1 in CHO-52 compared to mock-treated cells. In detail, after 18h of incubation with 500 nM STS, 64 ± 2% of CHO-52 cells were viable compared to 16 ± 1% of CHO-mocks, after 24 h 43 ± 3% vs 5 ± 2%, respectively. CHO-52 cells exhibited a lower expression of active caspase 3 and anti-apoptotic Bcl-2 was found to be increased in CHO-52 cells at baseline and following STS treatment. Our study provides first evidence that syncytin-1 serves anti-apoptotic function under certain conditions. A lessened activation of caspase 3 and an increased expression of Bcl-2 are possible mechanisms.     

Mesenchymal stem cells attenuate hydrogen peroxide-induced oxidative stress and enhance neuroprotective effects in retinal ganglion cells

The apoptosis of retinal ganglion cells leads to visual impairment and blindness in ocular neurodegenerative diseases, especially in diabetic retinopathy (DR). Mounting evidence suggests that oxidative stress contributes to the pathogenesis of DR. In the present study, we investigated whether bone mesenchymal stem cells (BMSCs) have protective ability to relieve hydrogen peroxide (H₂O₂)-induced injury on retinal ganglion cells in vitro. An immortalized retinal ganglion cells, RGC-5 cells, were exposed to an indicated concentration of H₂O₂ for 24 h. Cell viability was analyzed by CCK-8 assay to find out a certain concentration to build H₂O₂ oxidative damage model. Morphological changes in RGC-5 cells were observed under optical microscope, and cell apoptosis was detected with Hoechst fluorescence staining. Then, BMSCs were co-cultured with RGC-5 cells in a transwell culture system for 24 h and 48 h. Flow cytometry was performed to qualify the apoptosis rate of RGC-5 cells. Conditioned medium was collected for evaluation the inflammatory cytokines by ELISA. The content of intracellular malondialdehyde (MDA) and superoxide dismutase (SOD) was assayed by thiobarbituric acid and xanthine oxidase method, respectively. qRT-PCR and ELISA were conducted for analysis of the expression changes in brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), respectively. After H₂O₂ exposure, the morphological varieties were observed as cytoplasm shrinking and paramorphia together with nuclear gathering. Meanwhile, the apoptotic cells had hyperfluorescence with Hoechst 33258 staining. Co-culture with BMSCs significantly inhibited retinal cell death. It was found that BMSCs reduced H₂O₂-induced inflammatory factors IL-1β and TNF-α, down-regulated intracellular oxidant factor MDA, up-regulated intracellular antioxidant factor SOD, and increased neurotrophins BDNF and CNTF expression. BMSCs may enhance protective effect of RGC-5 cells in H₂O₂-induced damage through improving antioxidant capacity, inhibiting pro-inflammatory cytokine secretion, and promoting neurotrophin expression.     

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H₂O₂), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H₂O₂‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright © 2014 John Wiley & Sons, Ltd.     

An epigenetic modifier results in improved in vitro blastocyst production after somatic cell nuclear transfer

The present study was designed to examine the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on development of porcine cloned embryos. Our results showed that treatment of cloned embryos derived from sow oocytes with 50 nM TSA for up to 24 h after the onset of activation could significantly improve blastocyst yield compared to the control (46.4+/-4.6% vs 17.7+/-4.9% for treated and untreated embryos, respectively; p<0.05), whereas similar cleavage rate and total cell number per blastocyst were observed. In order to assess if the improvement is cell line specific, three cell lines were tested, and for all cell lines an enhancement in blastocyst development compared to their corresponding control was observed. Our data demonstrate that TSA treatment after somatic cell nuclear transfer in the pig can significantly improve the in vitro blastocyst production.     

Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.     

The in vitro development of cloned sheep embryos treated with Scriptaid and Trichostatin (A)

Introduction and aimsAlthough, it has been success in the generation of animal clones from somatic cells in various animal species, the information related to nuclear reprogramming of cloned embryos is found to be limited. This study aims to compares the effect of both Scriptaid (SCR) and Trichostatin (A) treatments in improving cloning efficiency, and embryos developmental rate of cloned sheep embryos in vitro. Three groups were formed, i.e., one SCR group, second TSA group, with both treatment concentrations of 5 nM, 50 nM, and 500 nM, respectively, and third were control group with 0 nM. Methods: Ovaries of slaughtered sheep were collected and oocytes were recovered from antral follicles using aspiration method and in vitro maturation of oocytes were done. Then zona dissecting with micropipettes and oocyte enucleation were carried out under the micromanipulator. Later nuclear transfer, cell fusion and activation were done via cell fusion machine. Finally the embryo cultured in incubating chamber at the CO2 incubator up to 9 days. The result: In general the results showed that when the concentration increases the cleavage rate increased. The cleavage rates of the SCNT embryos treated with SCR at different concentrations are closely related to cleavage rate of embryos treated with TSA at same concentration; such as 39.47% for 500 nM TSA, 38.09% for 500 nM SCR; 18.6% for 50 nM TSA, 19.17% for 50 nM SCR, and 22.64% for 5 nM TSA, 17.18% for 5 nM SCR. As for the control group, the cleavage rate of the SCNT embryos cleavage ratewere27.47%., 30% and 30.85% respectively for bothtreatments. While there is a significant difference in TSA treatments at an eight-cell stage at the concentration (5 and 50 nM TSA) compared to the all other cleavage cell stages of (500 nM TSA and control). Also their were a differences between (50 nM of TSA) compared to the (50 nM SCR). Also there were a significant differences between the 16 cell stage at the (500 nM TSA) compared to other treatment (5 nM, 50 nM TSA and control). Regarding the SCR there were a significant difference at 8 cell stage between (5 nM SCR), compared to the other treatment (50 nM, 500 nM SCR and control). Also there were a significant difference at 16 cell stage between (50 nM, and 500 nM SCR), compared to the other treatment (5 nM SCR and control). While in the development of the embryos reach to blastocyst stage the SCR and the control group show a higher rate, in compered to TSA that did not show any development to blastocyst stage. The total SCR treatment showed (3/41 = 7.31%), and the total control showed (4/89 = 4.49%) blastula stage. It concludes that SCR improve the final development blastula stage compared to the TSA treatments that did not improved embryos reach to final developmental blastula stages may be due to spices differences or to the toxicity of TSA, especially at higher concentrations.     

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.     

Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.     

羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖(Sargassum Fusiforme Polysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western blot法测定Caspase-3酶原的变化; RT-PCR检测Caspase-3 mRNA表达。结果显示:SFPS作用lovo细胞24,36,48和72h的IC_(50)分别为375,355,178和60mg/L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征:细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5-300mg/L作用24h后,显示有凋亡细胞特有的DNA梯状条带;而500mg/L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。     

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.     

Effects of cyclopeptide C*HSDGIC* from the cyclization of PACAP (1-5) on the proliferation and UVB-induced apoptosis of the retinal ganglion cell line RGC-5

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that confers potent neurotrophic and neuroprotective effects. Cyclopeptide C*HSDGIC* (CHC), which results from the cyclization of PACAP (1-5) with disulfide, has been demonstrated to represent a potent agonist for the PACAP-specific receptor PAC1 which mediates the majority of PACAP's effects. In this study, the expression of PAC1 in a rat retinal ganglion cell line (RGC-5) was confirmed using a western blot analysis, and it was determined that CHC promoted the proliferation of RGC-5 cells using the cell counting kit-8 (CCK8) assay and flow cytometry. Furthermore, the treatment of CHC attenuated the decrease of cell viability in cells exposed to UVB irradiation. Flow cytometry and a JC-1 assay revealed that the CHC treatment protected the RGC-5 cells against UVB-induced apoptosis. In addition, similar to PACAP, the anti-apoptotic effect of CHC was related to the down-regulation of caspase-3. In summary, these results demonstrate for the first time that PAC1 is present in RGC-5 cells and that CHC, a cyclopeptide from PACAP, promotes RGC-5 cell proliferation and attenuates UVB-induced apoptosis.