Cell Death Enzyme-linked Immunosorbent Assay Research Articles

RTMS Regulates the Balance Between Proliferation and Apoptosis of Spinal Cord Derived Neural Stem/Progenitor Cells.

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive technique that uses electromagnetic fields to stimulate the brain. rTMS can restore an impaired central nervous system and promote proliferation of neural stem/progenitor cells (NSPCs), but optimal stimulus parameters and mechanisms underlying these effects remain elusive. The purpose of this study is to investigate the effect of different rTMS stimulus parameters on proliferation and apoptosis of spinal cord-derived NSPCs, the expression of brain-derived neurotrophic factor (BDNF) after rTMS, and the potentially underlying pathways. NSPCs were isolated from mice spinal cord and stimulated by different frequencies (1/10/20 Hz), intensities (0.87/1.24/1.58 T), and number of pulses (400/800/1,500/3,000) once a day for five consecutive days. NSPC proliferation was analyzed by measuring the neurosphere diameter and Brdu staining, apoptosis was detected by cell death enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and NSPC viability was assessed by cell counting kit-8 assay. We found that specific parameters of frequency (1/10/20 Hz), intensity (1.24/1.58 T), and number of pulses (800/1,500/3,000) promote proliferation and apoptosis (p < 0.05 for all), but 20 Hz, 1.58 T, and 1,500 pulses achieved the optimal response for the NSPC viability. In addition, rTMS significantly promoted the expression of BDNF at the mRNA and protein level, while also increasing Akt phosphorylation (Thr308 and Ser473; p < 0.05). Overall, we identified the most appropriate rTMS parameters for further studies on NSPCs in vitro and in vivo. Furthermore, the effect of magnetic stimulation on NSPC proliferation might be correlated to BDNF/Akt signaling pathway.

Safety of anti-VEGF treatments in a diabetic rat model and retinal cell culture.

PurposeTo analyze the safety of different concentrations of anti-VEGF on retinal cells.MethodsNon-diabetic and streptozotocin (STZ)-induced diabetic rats received intravitreal rat anti-VEGF injections that had final vitreous concentrations of 0, 0.0625, 0.125 (clinical dose), and 0.25 mg/mL. Rats were also injected with the clinical dose of ranibizumab. TUNEL assay was performed on sectioned eyes to evaluate apoptotic cells. In vitro, rat retinal cell cultures were exposed to 0, 0.0625, 0.125 (clinical dose), and 0.25 mg/mL of ranibizumab for 48 and 72 hrs. Cellular metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, necrosis by lactate dehydrogenase (LDH), and apoptosis by cell death enzyme-linked immunosorbent assay (ELISA).ResultsDiabetic rats had a significant increase (p<0.03) in apoptotic cell death at half the clinical dose, at the clinical dose, and at double the clinical dose. In vitro, MTT showed a significant decrease (p<0.04) in cellular metabolic activity at the clinical dose and double the clinical dose compared to control at 48 and 72 hrs. LDH showed a significant increase (p<0.04) in necrosis at the clinical dose and double the clinical dose compared to control at 48 and 72 hrs. ELISA showed a significant increase (p<0.04) in apoptosis at half the clinical dose, at the clinical dose, and double the clinical dose, compared to control at 48 and 72 hrs.ConclusionsAnti-VEGF treatment may be potentially detrimental to the retina by decreasing cellular metabolic activity and increasing cytotoxicity of retinal cells. The results provide a cautionary note to monitor both the retina and optic nerve status in patients undergoing frequent injections.

Scrophularia Atropatana Extract Reverses TP53 Gene Promoter Hypermethylation and Decreases Survivin Antiapoptotic Gene Expression in Breast Cancer Cells

Background:In many cases of breast cancer, the aberrant methylation of TP53 gene leads to uncontrolled cell proliferation and apoptosis inhibition. Moreover, expression of oncogenes which are under the control of P53 protein could be altered. Survivin as a conspicuous example of this category plays important roles in tumorigenesis, drug resistance and apoptosis inhibition. The present study was done to reveal the effects of Scrophularia atropatana extract on epigenetic situation of TP53 gene promoter and the expression levels of anti-apoptotic gene, survivin and its potential for production of cancer epi-drugs.Methods:Cytotoxic effect of dichloromethane extracts of Scrophularia plant on MCF-7 cell line was assessed in our previous study. Cell death ELISA (enzyme-linked immunosorbent assay) and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) tests were used to investigate the occurrence of apoptosis in the treated cells. Methylation Specific PCR (MSP) was employed to assess the changes in methylation status of the TP53 gene promoter. Furthermore, quantitative real time PCR was utilized to evaluate the resulting changes in TP53 and survivin genes expression.Results:Cell death ELISA and TUNEL assays confirmed the occurrence of apoptosis. MSP test revealed a significant change in the methylation status of TP53 promoter. QRT-PCR showed an increased TP53 gene expression in the treated cells while a significant decrease in survivin mRNA was evident.Conclusions:According to the outcomes, dichloromethane extract of S. atropatana returned the TP53 gene promoter hypermethylation to normal state. This plant could be a promising source for production of epi-drugs due to its apoptotic effects and reversal of TP53 epigenetic alterations.

Pharmaceutically inhibiting polo-like kinase 1 exerts a broad anti-tumour activity in retinoblastoma cell lines.

Retinoblastoma is the most common malignant cancer of the eye in children. Although metastatic retinoblastoma is rare, cure rates for this advanced disease remain below 50%. High-level polo-like kinase 1 expression in retinoblastomas has previously been shown to be correlated with adverse outcome parameters. Polo-like kinase 1 is a serine/threonine kinase involved in cell cycle regulation at the G2/M transition. Polo-like kinase 1 inhibition has been demonstrated to have anti-tumour effects in preclinical models of several paediatric tumours. Here, we assessed its efficacy against retinoblastoma cell lines. Expression of polo-like kinase 1 was determined in a panel of retinoblastoma cell lines by polymerase chain reaction and western blot analysis. We analysed viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT assay), proliferation (5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay), cell cycle progression (propidium iodid staining) and apoptosis (cell death enzyme-linked immunosorbent assay) in three retinoblastoma cell lines after treatment with two adenosine triphosphate-competitive polo-like kinase 1 inhibitors, BI6727 or GSK461364. Activation of polo-like kinase 1 downstream signalling components including TP53 were assessed. Treatment of retinoblastoma cells with either BI6727 or GSK461364 reduced cell viability and proliferative capacity and induced both cell cycle arrest and apoptosis. Polo-like kinase 1 inhibition also induced the p53 signalling pathway. Analysis of key players in cell cycle control revealed that low nanomolar concentrations of either polo-like kinase 1 inhibitor upregulated cyclin B1 and increased activated cyclin-dependent kinase 1 (phosphorylated at Y15) in retinoblastoma cell lines. These preclinical data indicate that polo-like kinase 1 inhibitors could be useful as components in rationally designed chemotherapy protocols to treat patients with metastasized retinoblastoma in early phase clinical trials.

Echinophora platyloba DC (Apiaceae) crude extract induces apoptosis in human prostate adenocarcinoma cells (PC 3).

Prostate cancer is the second leading malignancy worldwide and the second prominent cause of cancer-related deaths among men. Therefore, there is a serious necessity for finding advanced alternative therapeutic measures against this lethal malignancy. In this article, we report the cytotoxicity and the mechanism of cell death of the methanolic extract prepared from Echinophora platyloba DC plant against human prostate adenocarcinoma PC 3 cell line and Human Umbilical Vein Endothelial Cells HUVEC cell line. Cytotoxicity and viability of the methanolic extract were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and dye exclusion assay. Cell death enzyme-linked immunosorbent assay (ELISA) was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and DNA fragmentation gel electrophoresis. E. platyloba could decrease cell viability in malignant cells in a dose- and time-dependent manner. The IC50 values against PC 3 were determined as 236.136 ± 12.4, 143.400 ± 7.2, and 69.383 ± 1.29 μg/ml after 24, 36, and 48 h, respectively, but there was no significant activity in HUVEC normal cell (IC50 > 800 μg/ml). Morphological characterizations and DNA laddering assay showed that the methanolic extract treated cells displayed marked apoptotic characteristics such as nuclear fragmentation, appearance of apoptotic bodies, and DNA laddering fragment. Increase in an early apoptotic population was observed in a dose-dependent manner. PC 3 cell death elicited by the extract was found to be apoptotic in nature based a clear indication of TUNEL assay and gel electrophoresis DNA fragmentation, which is a hallmark of apoptosis. In summary, the E. platyloba extract attenuated the human prostate adenocarcinoma cell proliferation in vitro possibly by inducing apoptosis. E. platyloba is likely to be valuable for the treatment of human prostate adenocarcinoma.

Bitter melon juice activates cellular energy sensor AMP-activated protein kinase causing apoptotic death of human pancreatic carcinoma cells.

Prognosis of pancreatic cancer is extremely poor, suggesting critical needs for additional drugs to improve disease outcome. In this study, we examined efficacy and associated mechanism of a novel agent bitter melon juice (BMJ) against pancreatic carcinoma cells both in culture and nude mice. BMJ anticancer efficacy was analyzed in human pancreatic carcinoma BxPC-3, MiaPaCa-2, AsPC-1 and Capan-2 cells by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, cell death enzyme-linked immunosorbent assay and annexin/propidium iodide assays. BMJ effect on apoptosis regulators was assessed by immunoblotting. In vivo BMJ efficacy was evaluated against MiaPaCa-2 tumors in nude mice, and xenograft was analyzed for biomarkers by immunohistochemistry (IHC). Results showed that BMJ (2-5% v/v) decreases cell viability in all four pancreatic carcinoma cell lines by inducing strong apoptotic death. At molecular level, BMJ caused caspases activation, altered expression of Bcl-2 family members and cytochrome-c release into the cytosol. Additionally, BMJ decreased survivin and X-linked inhibitor of apoptosis protein but increased p21, CHOP and phosphorylated mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2 and p38) levels. Importantly, BMJ activated adenosine monophosphate-activated protein kinase (AMPK), a biomarker for cellular energy status, and an AMPK inhibitor (Compound C) reversed BMJ-induced caspase-3 activation suggesting activated AMPK involvement in BMJ-induced apoptosis. In vivo, oral administration of lyophilized BMJ (5mg in 100 µl water/day/mouse) for 6 weeks inhibited MiaPaCa-2 tumor xenograft growth by 60% (P < 0.01) without noticeable toxicity in nude mice. IHC analyses of MiaPaCa-2 xenografts showed that BMJ also inhibits proliferation, induces apoptosis and activates AMPK in vivo. Overall, BMJ exerts strong anticancer efficacy against human pancreatic carcinoma cells, both in vitro and in vivo, suggesting its clinical usefulness.

Effects of Oxidative Stress on Intestinal Type I Insulin-Like Growth Factor Receptor Expression

Oxidative stress activates multiple signaling transduction pathways, including the phosphatidylinositol 3-kinase (PI3-K), in an injured intestine as occurs in necrotizing enterocolitis (NEC). We have previously shown that hydrogen peroxide (H2O2)-induced PI3-K activation is significantly enhanced with exogenous insulin-like growth factor (IGF)-1 in intestinal epithelial cells. However, the effects of oxidative stress on IGF receptor type I (IGF-IR) activation and expression in the neonatal intestine during NEC are unknown. Intestinal sections from neonates undergoing bowel resections (control = 3, NEC = 20) were analyzed for IGF-IR expression. NEC was induced in newborn mouse pups using hypoxia and hyperosmolar feeds, and distal small bowel segments were analyzed for IGF-IR expression (control = 3, NEC = 7). H2O2 was used to induce oxidative stress in rat (RIE-1) and fetal human (FHs74 Int) intestinal epithelial cells. Phosphorylation of IGF-IR, Akt, a downstream effector of PI3-K, and IGF-IR levels were determined by Western blotting. Flow cytometry, immunofluorescence, immunohistochemistry, IGF-IR tyrosine phosphorylation array, cell death enzyme-linked immunosorbent assay, and Western blotting were used to determine the IGF-IR expression. An increased IGF-IR expression was noted in intestinal sections from NEC as well as murine model of NEC. H2O2 treatment rapidly activated IGF-IR and increased the expression in RIE-1 and FHs74 Int cells. Inhibition of IGF-IR resulted in significant RIE-1 cell apoptosis during oxidative stress. IGF-IR tyrosine phosphorylation array showed the recruitment of several key SH2 domain-containing proteins and oncogenes to the IGF-IR tyrosine kinase domain in H2O2-treated RIE-1 cells. IGF-IR-mediated activation of intracellular signaling may play a critical role during oxidative stress-induced apoptosis in NEC.

Apigenin Attenuates 2-Deoxy-D-ribose-Induced Oxidative Cell Damage in HIT-T15 Pancreatic .BETA.-Cells

Glucose toxicity contributes to progressive β-cell failure and the development of overt diabetes. Oxidative stress is an important aspect of glucose toxicity in pancreatic β-cells. We investigated whether the flavonoid apigenin protects pancreatic β-cells from 2-deoxy-D-ribose (dRib)-induced oxidative cell damage. HIT-T15 pancreatic β-cells were cultured with or without apigenin in the presence of dRib. Time- and dose-dependent cell viability was monitored using a cell counting kit (CCK-8), while the induction of apoptosis was analyzed using a cell death enzyme-linked immunosorbent assay (ELISA) kit. Mitochondrial membrane potential (ΔΨ(m)) was determined using the JC-1 kit. Intracellular oxidative stress was measured by fluorometric analysis of DCFH oxidation using 2',7'-dichlorofluorescin diacetate (DCFH-DA) as the probe. In addition, the DNA binding activity of the oxidative stress-related transcriptional factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) were analyzed. dRib reduced cell survival and ΔΨ(m), while it markedly increased intracellular levels of reactive oxygen species (ROS), apoptosis, and the activity of the oxidative stress-related transcription factors NF-κB and AP-1. However, pretreatment of cells with apigenin attenuated all the dRib-induced effects. The anti-oxidants, N-acetyl-L-cysteine (NAC) and alpha lipoic acid (ALA), also prevented both dRib-induced oxidative damage and activation of NF-κB and AP-1. Taken together, these results suggest that apigenin attenuates dRib-induced cell damage in pancreatic β-cells via oxidative stress-related signaling.

CHM-1, a New Vascular Targeting Agent, Induces Apoptosis of Human Umbilical Vein Endothelial Cells via p53-mediated Death Receptor 5 Up-regulation

CHM-1 (2'-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone) has been identified as a potent antitumor agent in human hepatocellular carcinoma; however, its role in tumor angiogenesis is unclear. This study investigated the effects of CHM-1 and the mechanisms by which it exerts its antiangiogenic and vascular disrupting properties. Using a xenograft model antitumor assay, we found that CHM-1 significantly inhibits tumor growth and microvessel formation. Flow cytometry, immunofluorescence microscopy, and cell death enzyme-linked immunosorbent assay kit revealed that CHM-1 inhibits growth of human umbilical vein endothelial cells (HUVEC) by induction of apoptotic cell death in a concentration-dependent manner. CHM-1 also suppresses HUVEC migration and capillary-like tube formation. We were able to correlate CHM-1-induced apoptosis in HUVEC with the cleavage of procaspase-3, -7, and -8, as well as with the cleavage of poly(ADP-ribose) polymerase by Western blotting assay. Such sensitization was achieved through up-regulation of death receptor 5 (DR5) but not DR4 or Fas. CHM-1 was also capable of increasing the expression level of p53, and most importantly, the induction of DR5 by CHM-1 was abolished by p53 small interfering RNA. Taken together, the results of this study indicate that CHM-1 exhibits vascular targeting activity associated with the induction of DR5-mediated endothelial cell apoptosis through p53 up-regulation, which suggests its potential as an antivascular and antitumor therapeutic agent.

Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

Phosphorylation of Ephrin-B1 Regulates Dissemination of Gastric Scirrhous Carcinoma

Interaction of the Eph family of receptor protein tyrosine kinase and its ligand ephrin family induces bidirectional signaling via cell-cell contacts. High expression of B-type ephrin is frequently found in various cancer cells, and their expression levels are associated with high invasion of tumors and poor prognosis. However, whether ephrin-B1 actually promotes invasion of cancer cells in vivo has not been shown. We investigated the involvement of ephrin-B1 in regulating the invasiveness of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Reduction of ephrin-B1 expression by short inter-fering RNA or overexpression of phosphorylation-defective mutant suppressed migration and invasion of scirrhous gastric cancer cells in vitro without affecting tumor cell proliferation and apoptosis. Blocking of tyrosine phosphorylation of ephrin-B1 attenuates not only dissemination of cancer cells injected intraperitoneally but also local invasion and dissemination of orthotopically implanted cancer cells in the gastric wall of nude mice. Furthermore, blocking of ephrin-B1 phosphorylation attenuated the activation of Rac1 GTPase in these invasive gastric cancer cells. Our results suggest that tyrosine phosphorylation of ephrin-B1 promotes invasion of cancer cells in vivo and is a potential therapeutic target in some types of gastrointestinal cancers.

Leptin acts as a mitogenic and antiapoptotic factor for colonic cancer cells

Obesity is associated with increased levels of leptin. The mitogenic actions of leptin have been identified in various cell types. Because obesity may be a risk factor for colonic cancer, the proliferative and antiapoptotic effects of leptin on colonic cancer cells and the role of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) signalling were investigated. Three human colonic cancer cell lines (T(84), HT29/Cl.19A and Caco-2) were treated with leptin. Cell proliferation was measured using the XTT colorimetric assay and apoptosis by a cell death enzyme-linked immunosorbent assay. Inhibitors of MAPK and PI3-K were used to evaluate the role of these signalling pathways. Phosphorylation of the downstream components extracellular signal-regulated kinase (ERK) 1/2 and Akt was detected by western blotting. Leptin increased cell number in all cell lines in a dose-dependent manner and reduced the number of apoptotic cells in a cell line-dependent manner. Leptin also caused ERK1/2 and Akt phosphorylation. Pretreatment with inhibitors of MAPK and PI3-K inhibited these responses, attenuated the mitogenic action of leptin and abolished its antiapoptotic effects. Chronic increases in leptin concentration may enhance the growth of colonic cancers via MAPK and PI3-K pathways. These effects of leptin could provide a link between obesity and colonic cancer, and may represent a target for anticancer drug development.

L-Carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture

1-Methyl-4-phenylpyridinium ion (MPP +), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson’s disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. l-Carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of l-carnitine against the toxicity of MPP + in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 μM MPP + alone or co-incubated with l-carnitine. MPP + produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)–positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP + with l-carnitine significantly reduced MPP +-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP + decreased the ratio of BCL-X L to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. l-Carnitine blocked these effects of MPP + suggesting its potential therapeutic utility in degenerative disorders such as Parkinson’s disease, Alzheimer’s disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.

Role of tumor necrosis factor-α in myocardial dysfunction and apoptosis during hindlimb ischemia and reperfusion

Peripheral vascular surgery involving limb ischemia/reperfusion is associated with tumor necrosis factor-alpha production and an increased risk of cardiac complications. The objective of this study was to investigate the role of tumor necrosis factor-alpha in myocardial apoptosis and dysfunction following hindlimb ischemia/reperfusion. Randomized perspective animal study. Research laboratory. Adults male tumor necrosis factor-alpha(-/-) and littermate wild-type mice. Bilateral hindlimb ischemia/reperfusion was induced in wild-type and tumor necrosis factor-alpha(-/-) mice using tourniquet occlusion. After 2 hrs of hindlimb ischemia, the tourniquets were released, allowing reperfusion for 0.5-24 hrs. In wild-type mice, hindlimb ischemia/reperfusion resulted in myocardial depression early during the reperfusion period (p < .05). These effects were temporally correlated with enhanced levels of myocardial and plasma tumor necrosis factor-alpha. All variables were restored to baseline levels by 24 hrs of reperfusion. Myocardial apoptosis, assessed by cell death enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining, and caspase-3 activity, was also significantly higher at 6 hrs of reperfusion (p < .05) but returned to baseline levels by 24 hrs. Interestingly, cardiac dysfunction and myocardial apoptosis were abolished in tumor necrosis factor-alpha mice subjected to the same degree of hindlimb ischemia/reperfusion as the wild-type mice. Treatment of etanercept restored cardiac function in wild-type mice. Tumor necrosis factor-alpha contributes significantly to myocardial dysfunction and apoptosis in hindlimb ischemia/reperfusion. Although a causal link between myocardial apoptosis and cardiac dysfunction is not established, our study does suggest that tumor necrosis factor-alpha may be a potential therapeutic target for cardiac injury in clinical situations involving prolonged remote ischemia/reperfusion.

Antitumor activity of a novel recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand1

To investigate the antitumor activity and safety of a novel recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmh TRAIL). Antitumor activity of rmh TRAIL was evaluated by using several tumor cell lines by MTT assay in vitro, and by using a mouse xenograft model in vivo. rmh TRAIL-induced apoptosis in tumor cells was detected by cell death enzyme-linked immunosorbent assay (ELISA), TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry. The safety of rmh TRAIL was also evaluated in several normal human cell lines. At the concentration of 0.32-1 000 ng/mL, rmh TRAIL remarkably inhibited the proliferation of 5 tumor cell lines from lung, colon, and breast cancer compared with wild type (wt TRAIL) in vitro, whereas at the concentration of 1 ng/mL-10 microg/mL, rmh TRAIL showed no or mild cytotoxicity in the normal cell lines. rmh TRAIL (3, 15 mg/kg, ip, once daily for 10 d) exerted a significant inhibition on the growth of xenograft tumor NCI-H460 in nude mice compared with the saline group (P<0.01), and was more potent than wt TRAIL, a positive control. The apoptosis of NCI-H460 cells was markedly induced in a concentration-dependent and time-dependent manner after rmh TRAIL treatment. The percentage of apoptotic cells induced by rmh TRAIL in NCI-H460 cells was significantly higher than that by wt TRAIL. rmh TRAIL provided potent antitumor activity in vivo and in vitro, whereas most normal human cells were resistant to rmh TRAIL. The results suggested that rmh TRAIL might be a useful anticancer agent in future.

Functional and phenotypic differences between glioblastoma multiforme—derived and normal human brain endothelial cells

Glioblastomas multiforme (GBMs) are hypervascular tumors characterized by endothelial cell (EC) proliferation. There is increasing evidence that ECs that infiltrate systemic tumors are different from normal blood vessel cells; whether this difference is seen in the central nervous system between GBM and normal brain tissue is not known. The goal of this investigation was to characterize and compare the functional and phenotypic properties of GBM-associated ECs and normal brain ECs. Human ECs were isolated from fresh tissue specimens, purified using flow cytometry, and characterized by immunostaining. Proliferation was measured by determining bromodeoxyuridine incorporation and Ki-67 staining, and by performing the monotetrazolium assay. The migration rate of the cells was determined using the modified Boyden chamber technique. Apoptosis was evaluated by performing the TUNEL assay, cell death enzyme-linked immunosorbent assay (ELISA), and annexin V staining. Growth factor production was analyzed using the ELISA technique. The brain tumor ECs differed from normal brain ECs morphologically and by their expression and distribution of specific markers (that is, vascular endothelial cadherin [VE-cadherin] and CD31). Functional differences between the two cell populations were also evident. The brain tumor ECs proliferated more slowly and underwent less apoptosis than normal brain ECs; however, the tumor ECs migrated faster than the normal ECs. The normal ECs were sensitive to growth factors such as vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), whereas the tumor ECs were not. In addition, the brain tumor ECs constitutively produced higher levels of ET-1 and VEGF, compared with the normal ECs. The data demonstrated that ECs derived from normal brain and from GBMs have significant phenotypic and functional distinctions. Further characterization of brain tumor ECs is essential for efficient antiangiogenic treatment of gliomas.

Phorbol Ester-induced Apoptosis of C4-2 Cells Requires Both a Unique and a Redundant Protein Kinase C Signaling Pathway

Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.

The role of the N-methyl- d-aspartate receptor in ketamine-induced apoptosis in rat forebrain culture

Recent data suggest that anesthetic drugs may cause widespread and dose-dependent apoptotic neurodegeneration during development. The window of vulnerability to this neurotoxic effect, particularly with N-methyl- d-aspartate (NMDA) antagonists such as ketamine, is restricted to the period of synaptogenesis. The purposes of this study are to determine whether treatment of forebrain cultures with ketamine results in a dose-related increase in neurotoxicity and whether upregulation of NMDA receptor subunit NR1 promotes ketamine-induced apoptosis. Forebrain cultures were treated for 12 h with 0.1, 1, 10 and 20 μM ketamine or co-incubated with NR1 antisense oligonucleotide (2 μM). After washout of the ketamine, cultures were kept in serum-containing medium (in presence of glutamate) for 24 h. Application of ketamine (10 and 20 μM) resulted in a substantial increase in DNA fragmentation as measured by cell death enzyme-linked immunosorbent assay, increased number of terminal dUTP nick-end labeling positive cells, and a reduction in mitochondrial metabolism of the dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. No significant effect was seen in the release of lactate dehydrogenase, indicating that cell death presumably occurred via an apoptotic mechanism. Co-incubation of ketamine with NR1 antisense significantly reduced ketamine-induced apoptosis. Western analysis showed that neurotoxic concentrations of ketamine increased Bax and NR1 protein levels. NR1 antisense prevented this increase caused by ketamine, suggesting that ketamine-induced cell death is associated with a compensatory upregulation of the NMDA receptor. These data suggest that NR1 antisense offers neuroprotection from apoptosis in vitro, and that upregulation of the NR1 following ketamine administration is, at least, partially responsible for the observed apoptosis.

Enamel matrix derivative inhibits TNF-α–induced apoptosis in osteoblastic MC3T3-E1 cells

The purpose of this study was to examine the effect of enamel matrix derivative (EMD) on TNF-alpha-induced apoptosis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured at an initial density of 5000/cm 2 in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and allowed to adhere for 24 hours. Medium was then changed into DMEM with 0.5% FBS. After 16 hours, cells were treated with EMD (100 microg/mL) alone, tumor necrosis factor alpha (TNF-alpha) (20 ng/mL) alone, transforming growth factor beta 1 (TGF-beta1) (10 ng/mL) alone, TNF-alpha plus TGF-beta1, or TNF-alpha plus EMD. Cells cultured with DMEM and 0.5% FBS served as control. Following 24-hour incubation, apoptosis was assessed by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay, and quantified by cell death enzyme-linked immunosorbent assay (ELISA). Both TUNEL assay and cell death ELISA show that TNF-alpha induces apoptosis in MC3T3-E1 cells. TNF-alpha increases cell death by approximately 2-fold, which is attenuated by both EMD and TGF-beta1. Like TGF-beta1, EMD protects osteoblasts from inflammation-induced apoptosis.

Mechanisms of sodium nitroprusside-induced death in human chondrocytes.

The nitric oxide (NO) donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in human chondrocytes. This study compares SNP-induced chondrocyte death and SNP-activated signaling mechanisms with apoptosis induced by CD95 activation. Sodium nitroprusside increased cell death dose-dependently. Compared to CD95 stimulation, SNP induced only low levels of internucleosomal DNA fragmentation as measured by cell-death enzyme-linked immunosorbent assay (ELISA). However, SNP caused substantial nuclear DNA cleavage, as evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL). Caspase-3 processing in response to SNP was not detected. The pancaspase inhibitor Z-VAD.FMK partially abrogated the TUNEL signal but did not block cell death or internucleosomal DNA fragmentation. The caspase-3-specific inhibitor Ac-DEVD-CHO did not inhibit the SNP-induced TUNEL signal or internucleosomal DNA fragmentation. DNA degradation was not blocked by the p38 inhibitor SB 202190 but by the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine. The results of this study support the hypothesis that the phenotype and mechanisms of SNP-induced chondrocyte death are distinct from apoptosis induction via CD95.