DNA Fragmentation Enzyme-linked Immunosorbent Assay Research Articles

Synergistic Effect of COX-2 Inhibitor on Paclitaxel-Induced Apoptosis in the Human Ovarian Cancer Cell Line OVCAR-3

PurposeCelecoxib, a highly selective cyclooxygenase-2 inhibitor, regulates apoptosis of several types of human cancer cells. The purpose of this study was to investigate whether celecoxib in combination with paclitaxel modulates apoptosis of ovarian cancer cells, and to identify the signal pathway by which celecoxib mediates apoptosis.Materials and MethodsOVCAR-3 cells were exposed to paclitaxel (20 µM) in the absence or presence of celecoxib (10 µM). Cell viability was evaluated using a Cell Counting Kit-8 assay. Apoptosis was evaluated using Annexin-V/7-aminoactinomycin D staining and a cellular DNA fragmentation enzyme-linked immunosorbent assay. Caspase-3, -9, and cleavage of poly ADP-ribose polymerase (PARP) were determined by western blotting. Expression of nuclear factor-κB (NF-κB) and vascular endothelial growth factor (VEGF) and Akt activation were assessed using reverse transcriptase-polymerase chain reaction and western blotting.ResultsCelecoxib enhanced paclitaxel-induced growth inhibition of OVCAR-3 cells. Celecoxib significantly increased paclitaxel-induced apoptosis of OVCAR-3 cells. Pretreatment with celecoxib also increased activation of caspase-9, -3 and cleaved PARP following paclitaxel-treatment. Exposure of OVCAR-3 cells to celecoxib in combination with paclitaxel resulted in downregulation of NF-κB activation and VEGF expression. Furthermore, combining celecoxib and paclitaxel inhibited phosphorylation of Akt.ConclusionOVCAR-3 cells were sensitized to paclitaxel-induced apoptosis by celecoxib through downregulation of NF-κB and Akt activation, suggesting that celecoxib may work synergistically with paclitaxel to inhibit different targets and ultimately produce anticancer effects. Combining celecoxib with paclitaxel may prove beneficial in the clinical treatment of ovarian cancer.

Intestinal barrier disruption as a cause of mortality in combined radiation and burn injury.

Nuclear disaster associated with combined radiation injury (CRI) and trauma or burns results in higher mortality than component injuries. Early death is caused by sequelae of gastrointestinal (GI) leakiness such as bacterial translocation and shock. We developed a murine model to characterize GI injury after CRI and determine the extent of barrier disruption. Animals received radiation (5.5 Gy) alone or with 15% total body surface area (TBSA) scald burn and were euthanized at 24, 48, and 72 h. Mesenteric lymph node homogenate was plated on tryptic soy agar to assess for bacterial translocation. Tight junction protein, occludin, was characterized by Western blot and immunofluorescence. Intestinal histology was evaluated, and apoptosis was quantified using histone-associated DNA fragmentation enzyme-linked immunosorbent assay and Western blot for caspase-3 and caspase-8. At 72 h, a 100-fold increase in bacterial growth after CRI was observed. Occludin colocalization was reduced by radiation exposure, with largest differences in CRI at 24 and 48 h. Histopathology exhibited increased apoptosis in radiation alone and CRI animals at 24 and 48 h (P < 0.05). Further evidence of apoptotic activity in CRI was seen at 48 h, with 3-fold increases in enzyme-linked immunosorbent assay detection relative to all groups and caspase-8 activity relative to radiation alone and sham (P < 0.05). Prolonged epithelial apoptosis and disruption of tight junctions likely contribute to gut leakiness after CRI. Subsequent bacterial translocation to mesenteric lymph node potentially leads to sepsis and death and could serve as a target for mitigating agents to improve survival from CRI.

NMDA-induced Apoptosis in Mixed Neuronal/Glial Cortical Cell Cultures

In animal models of severe ischemia, it has not been uniformly observed that anesthetics are protective. However, anesthetics have not been evaluated in the presence of a mild excitotoxic insult. We hypothesized that in the presence of a mild excitotoxic insult, 3 microm N-methyl-D-aspartate (NMDA), isoflurane may prevent apoptotic cell death. Primary mixed neuronal/glial cultures were prepared from fetal rat brains. Mature cultures were exposed to dissolved isoflurane [0 mM, 0.4 mM (1.8 minimum alveolar concentration) or 1.6 mM (7 minimum alveolar concentration)] or dizocilpine (10 microM), and NMDA (0 or 3 microM) at 37 degrees C for 30 minutes. Apoptosis was assessed using terminal-deoxy-nucleotidyl end-nick labeling oligonucleosomal DNA fragmentation enzyme-linked immunosorbent assay, and caspases-3 and -9 activation assays. NMDA (3 muM) induced apoptosis in mixed neuronal/glial cell cultures. Apoptosis induced by 3 microm NMDA was caspase-3 but not caspase-9 mediated. In the presence of a mild excitotoxic insult, this investigation showed an attenuation of apoptotic cell death by dizocilpine, but not isoflurane.

The Histidine Triad Protein Hint1 Triggers Apoptosis Independent of Its Enzymatic Activity

Hint1 is a member of the evolutionarily conserved family of histidine triad proteins that acts as a haplo-insufficient tumor suppressor inducing spontaneous tumor formation in Hint+/- and Hint-/- mouse models. However, the molecular mechanisms for the tumor-suppressing activity are poorly defined. In this respect, we have recently shown that Hint1, by interaction with Pontin and Reptin, inhibits T-cell factor/beta-catenin-mediated transcription of Wnt target genes. In this study, we have found that, after transient transfection with Hint1, SW480 and MCF-7 cells undergo apoptosis as analyzed by pro-caspase-3 and poly(ADP-ribose) polymerase cleavage, M30 CytoDEATH staining, cytochrome c release, and DNA fragmentation enzyme-linked immunosorbent assay. Hint1 is involved in the regulation of apoptotic pathways by inducing an up-regulation of p53 expression coinciding with an up-regulation of the proapoptotic factor Bax and a concomitant down-regulation of the apoptosis inhibitor Bcl-2. Bad and Puma levels remained unchanged. Further analyses revealed that Hint1 is associated with the Bax promoter and is a component of the Tip60 histone acetyltransferase complex and, in this context, appears to be involved in the regulation of Bax expression. Knockdown of Hint1 by short hairpin RNA resulted in down-regulation of p53 and Bax but had no effect on Bcl-2 expression. A mutant Hint1 (H112N) protein defective in enzymatic activity as an AMP-NH2 hydrolase was not impaired in induction of apoptosis, suggesting that the Hint1 pro-apoptotic activity is independent of the Hint1 enzymatic activity.

Agmatine Suppresses Mesangial Cell Proliferation by Modulating Polyamine Metabolism

Polyamines play an essential role in the growth and differentiation of mammalian cells. The depletion of intracellular polyamines results in the suppression of growth. Proliferation of glomerular mesangial cells (MC) is the most common pathologic change in many forms of glomerulonephritis. Agmatine is a metabolite of arginine via arginine decarboxylase (ADC), highly expressed in the kidney, and unique in its capacity to suppress intracellular polyamine levels required for proliferation. As agmatine enters mammalian cells via the polyamine transport system, its antiproliferative effects may preferentially target cells with increased proliferative kinetics. In the present study, we evaluated the antiproliferative effects of agmatine on human MC in vitro. MC proliferation was stimulated with 20% fetal bovine serum (FBS) or platelet-derived growth factor (PDGF-BB, 20 ng/ml). Cell proliferation was measured using the (4.3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) proliferation assay. Intracellular polyamine levels were assayed by high performance liquid chromatography, and cell death was assessed by cellular DNA fragmentation enzyme-linked immunosorbent assay. The MTT proliferation assay showed that agmatine significantly suppressed proliferation of human MC treated with 20% FBS or 5% FBS + PDGF as compared to human MC treated with 5% FBS. Polyamine levels were markedly lower in cells treated with agmatine, and proliferation was rescued by administration of putrescine. The fragmented DNA was hardly detected in agmatine-treated human MC. In summary, human MC stimulated to increase their proliferative kinetics are significantly more sensitive to the antiproliferative effects of agmatine than normally cultured cells. Suppressed proliferation of the agmatine-treated human MC is not due to increased cell death. These results suggest that agmatine is a promising drug candidate for the treatment of human mesangial proliferative glomerulonephritis.

Effect of gemcitabine on the expression of apoptosis-related genes in human pancreatic cancer cells

To investigate the expression of genes involved in the gemcitabine-induced cytotoxicity in human pancreatic cancer cells. A human pancreatic cancer cell line, PANC-1, was cultured. 1x10(4) PANC-1 cells were plated in 96-well microtiter plates. After being incubated for 24 h, gemcitabine was added to the medium at concentrations ranging 2.5-1000 mg/L. The AlamarBlue dye method was used for cell growth analysis. DNA fragmentation was quantitatively assayed using a DNA fragmentation enzyme-linked immunosorbent assay (ELISA) kit. PAP and TP53INP1 mRNA expression was determined using the reverse transcription-polymerase chain reaction with semi-quantitative analysis. The expression of GSK-3beta and phospho-GSK-3beta proteins was examined with Western blot analysis. The IC50 for the drug after a 48-h exposure to gemcitabine was 16 mg/L. The growth of PANC-1 cells was inhibited by gemcitabine in a concentration-dependent manner (P<0.0001) and the cell growth was also inhibited throughout the time course (P<0.0001). The DNA fragmentation rate in the gemcitabine-treated group at 48 h was 44.7%, whereas it was 25.3% in the untreated group. The PAP mRNA expression was decreased after being treated with gemcitabine, whereas the TP53INP1 mRNA was increased by the gemcitabine treatment. Western blot analysis showed that phospho- GSK-3beta (ser9) was induced by the gemcitabine treatment. Gemcitabine suppresses PANC-1 cell proliferation and induces apoptosis. Apoptosis is considered to be associated with the inhibition of PAP and GSK-3beta, and the activation of TP53INP1 and pospho-GSK-3beta (ser9).

Cannabinoids Protect Astrocytes from Ceramide-induced Apoptosis through the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway

Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central nervous system by binding to the CB(1) cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However, those studies have been performed in neurons, whereas no attention has been focused on glial cells. Here we used the pro-apoptotic lipid ceramide to induce apoptosis in astrocytes, and we studied the protective effect exerted by cannabinoids. Results show the following: (i) cannabinoids rescue primary astrocytes from C(2)-ceramide-induced apoptosis in a dose- and time-dependent manner; (ii) triggering of this anti-apoptotic signal depends on the phosphatidylinositol 3-kinase/protein kinase B pathway; (iii) ERK and its downstream target p90 ribosomal S6 kinase might be also involved in the protective effect of cannabinoids; and (iv) cannabinoids protect astrocytes from the cytotoxic effects of focal C(2)-ceramide administration in vivo. In summary, results show that cannabinoids protect astrocytes from ceramide-induced apoptosis via stimulation of the phosphatidylinositol 3-kinase/protein kinase B pathway. These findings constitute the first evidence for an "astroprotective" role of cannabinoids.

Lysophosphatidic acid protects and rescues intestinal epithelial cells from radiation- and chemotherapy-induced apoptosis

Background & Aims: We have investigated whether the phospholipid growth factor lysophosphatidic acid (LPA) could prevent intestinal epithelial cells-6 (IEC-6) from apoptosis elicited by 4 different mechanisms. The antiapoptotic effect of LPA was also tested in a mouse model of radiation-induced apoptosis. Methods: Apoptosis was elicited by serum withdrawal, exposure to camptothecin, γ-irradiation, or rat tumor necrosis factor alpha and evaluated by DNA fragmentation enzyme-linked immunosorbent assay (ELISA) and annexin V staining. Caspase-3/CPP32 activity and activation was measured by ELISA and Western blotting, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was applied to examine the expression of LPA-receptor transcripts. Mice were treated with 250 μL of 1 mmol/L LPA and exposed to whole-body γ-irradiation with a dose of 12 or 15 Gy and the number and localization of apoptotic bodies along the crypt were recorded. Results: LPA pretreatment reduced DNA fragmentation induced in all models of apoptosis. LPA rescued cells from apoptosis when applied up to 1 hour after camptothecin treatment or 2 hours after irradiation. LPA inhibited the activation of caspase-3/CPP32 and attenuated its activity. Blocking LPA1 receptors by pertussis toxin and the inhibition of epithelial growth factor receptor tyrosine kinase significantly attenuated the protective effect. In irradiated mice, oral LPA significantly reduced the number of apoptotic bodies in the crypt. Conclusions: (1) LPA prevents and rescues IEC-6 from apoptosis elicited by 4 different mechanisms. (2) This antiapoptotic activity is mediated through LPA1 and LPA2 receptors through the inhibition of caspase-3/CPP32 activation. (3) LPA protects enterocytes against radiation-induced apoptosis. This study suggests that in patients undergoing cancer therapy, dietary LPA might have therapeutically useful antiapoptotic capacity in the intestinal epithelium.GASTROENTEROLOGY 2002;123:206-216

Hematin is one of the cytotoxic factors in pancreatitis-associated ascitic fluid that causes hepatocellular injury

Background. We recently demonstrated that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s), inducing apoptosis in hepatocytes, and that PAAF induces hepatic adenosine triphosphate depletion, hepatocellular acidosis, and accumulation of hepatic intracellular sodium. Because ascitic fluid and serum from patients with hemorrhagic pancreatitis contain a lot of hematin, we aimed to test the hypothesis that hematin can induce hepatocellular injury, and then we compared its cytotoxicity with that of PAAF. Methods. In vivo effects of intraperitoneal injection of hematin into the liver of healthy rats were evaluated with in situ nick-end labeling, blood biochemical analysis, and nuclear magnetic resonance spectroscopy. In vitro cytotoxic and apoptosis-inducing activities of hematin on rat primary culture hepatocytes were investigated with a cellular proliferation assay kit and DNA fragmentation enzyme-linked immunosorbent assay, respectively. Furthermore, PAAF was fractionated with Sephacryl S-300 gel column chromatography, and cytotoxic activities of its fractions on a human hepatoma cell line (HuH-7) were compared with those of hematin. Results. Intraperitoneal injection of hematin into healthy rats caused apoptosis in the hepatocytes and elevated serum glutamate oxaloacetic transaminase and lactate dehydrogenase levels. Intraperitoneal injection of hematin also caused a significant decrease in the hepatic β-adenosine triphosphate/inorganic phosphate ratio, severe hepatic intracellular acidosis, and a significant increase of hepatic intracellular sodium (Na+) concentration, similar to the effects of PAAF. In vitro, hematin decreased hepatocyte viability and increased the DNA fragmentation of hepatocytes, similar to the effects of 10% PAAF. Albumin reversed the cytotoxic effects of hematin and PAAF on HuH-7 cells nearly completely and partially, respectively. Fractionation of PAAF and hematin by gel column chromatography revealed that the first peak of cytotoxic activity of PAAF corresponded to that of hematin and that the cytotoxic activity was reversed by albumin nearly completely. Conclusions. These results suggest that hematin is one of the cytotoxic factors in PAAF that causes hepatocellular injury and that cellular injuries caused by hematin may be involved in the development of multiple organ failure associated with severe acute pancreatitis. (Surgery 2002;131:66-74.)

Anti-apoptotic action of hepatocyte growth factor through mitogen-activated protein kinase on human aortic endothelial cells.

To investigate the molecular mechanisms of the anti-apoptotic action of hepatocyte growth factor (HGF), a novel angiogenic growth factor that may have a pivotal role in the regulation of endothelial cells, on human aortic endothelial cells. An index of cell number and death was determined using a water-soluble tetrazolium salt dye assay, DNA fragmentation enzyme-linked immunosorbent assay, and non-confocal fluorescence microscopy of nuclear staining with Hoechst 33258 and propidium iodide. Extracellular-signal-regulated protein kinase (ERK) and the p38 mitogen-activated protein kinase (p38MAPK) were analysed by Western blotting using a phospho-specific antibody. Treatment of quiescent endothelial cells with HGF resulted in significant dose-dependent increases in cell numbers and decreases in lactate dehydrogenase (LDH) release. Moreover, HGF significantly attenuated endothelial cell death induced by culture in serum-free conditions. We therefore focused on the signal transduction system, and in particular on ERK and p38MAPK. ERK was markedly phosphorylated by HGF. The contribution of ERK to cell growth was supported by the observation that addition of PD98059, a specific inhibitor of MAPK kinase, significantly attenuated the increase in endothelial cell numbers induced by HGF, in a dose-dependent manner. Similarly, PD98059 also attenuated the decrease in LDH release and DNA fragmentation by HGF under serum-free conditions. Interestingly, ERK was re-phosphorylated at 12 h after stimulation. Re-phosphorylation of ERK was the result of induction of endogenous HGF by exogenously added HGF, as addition of neutralizing anti-HGF antibody to the conditioned medium attenuated re-phosphorylation of ERK at 12 h. In contrast, although p38MAPK was also phosphorylated by HGF, SB203580, a specific inhibitor of p38MAPK, failed to change the endothelial cell growth induced by HGF. We have demonstrated that the anti-apoptotic action of HGF against endothelial cell death was mainly through phosphorylation of ERK on human endothelial cells.

Macrophage-derived transforming growth factor-β1 induces hepatocellular injury via apoptosis in rat severe acute pancreatitis

Background: The mechanism of acute pancreatitis-induced hepatocellular injury is unclear. We have observed hepatocyte apoptosis in rat acute necrotizing pancreatitis. These studies were designed to determine the mediator(s) responsible for hepatocyte apoptosis and to clarify the significance of macrophages as its source. Methods: A rat sodium deoxycholate-induced pancreatitis model was used. Immunohistochemical studies for apoptosis-inducing mediators on hepatocytes were examined in the liver and on the peritoneal macrophages. The levels of transforming growth factor-β1 (TGF-β1) were also evaluated quantitatively with an enzyme-linked immunosorbent assay. Induction of apoptosis on the hepatocytes was evaluated by in situ nick-end labeling and tissue DNA fragmentation enzyme-linked immunosorbent assay. Finally, the effects of TGF-β1 neutralization and macrophage depletion were examined. Results: In the liver and the peritoneal macrophages, strong expression of TGF-β1 was detected early in the course of pancreatitis. In sodium deoxycholate-induced pancreatitis, the levels of TGF-β1 were also elevated in the plasma (9.2 ± 0.8 ng/mL), in the pancreatitis-associated ascitic fluid (11.5 ± 0.6 ng/mL), and in the liver homogenate (2.8 ± 0.3 ng/g of liver tissue). Moreover, the amount of fragmented DNA of the liver with pancreatitis was 290% ± 20% of that with a sham operation and serum alanine aminotransferase levels elevated to 248.2 ± 67.0 IU/L. TGF-β1 neutralization partly blocked the positive labeling on the nuclei of the hepatocytes, the elevation of the amounts of fragmented DNA (205% ± 10% of sham operation), and the serum alanine aminotransferase level (144.2 ± 14.9 IU/L). On the other hand, the macrophage depletion caused a marked decrease in the TGF-β1 protein level in the plasma (4.8 ± 1.2 ng/mL) or in the pancreatitis-associated ascitic fluid (8.0 ± 1.0 ng/mL). Moreover, the macrophage depletion completely inhibited the elevation of the TGF-β1 protein level in the liver homogenate (1.5 ± 0.4 ng/g of liver tissue), and thereafter decreased the amounts of the positive labeling on the nuclei of the hepatocytes and decreased the amount of fragmented DNA (120% ± 18% of sham operation) and the serum alanine aminotransferase elevation (119.2 ± 24.2 IU/L). Conclusions: In a model of sodium deoxycholate-induced pancreatitis, macrophages are responsible for pancreatitis-induced hepatocellular injury by means of apoptosis, and macrophage-derived TGF-β1 is one of the major factors inducing the hepatocyte apoptosis. (Surgery 2000;127:641-9.)

Phosphatidylinositol 3-kinase and Akt protein kinase mediate IGF-I- and prosaptide-induced survival in schwann cells

Withdrawal of trophic factors necessary for Schwann cell survival regulates Schwann cell number during development and after nerve injury. In the present study, we identified signaling pathways involved in Schwann cell survival by prosaposin, prosaptides (peptides incorporating the neurotrophic sequence of prosaposin), and insulinlike growth factor-I (IGF-I). When postnatal Schwann cells were placed in low serum medium, cells underwent abrupt shrinkage, condensation of nuclei occurred, and smooth rounded apoptotic bodies appeared. Dose-response studies of cell death, measured by lactate dehydrogenase (LDH) release, demonstrated that both prosaptide TX14(A) and IGF-I dose dependently reduced cell death in primary Schwann cells. Histone-associated DNA fragmentation enzyme-linked immunosorbent assay, showed a 10- and 14-fold increase in apoptosis after 4 and 24 hr in low serum medium, respectively, that was reduced by prosaposin, TX14(A), or IGF-I. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, blocked the survival effects of both TX14(A) and IGF-I. In contrast, only TX14(A) anti-apoptotic activity was blocked by the MEK inhbitor, PD98059, although TX14(A) and IGF-I are potent activators of extracellular regulated kinases in Schwann cells. Phosphorylation of the PI3K signaling target, Akt, was measured; TX14(A) and IGF-I increased Akt activity by 12-fold and 22-fold, respectively, that was inhibited by LY294002. These findings indicate that prosaposin and IGF-I use the PI3K/Akt pathway to induce survival of Schwann cells.