Effect of formaldehyde and resveratrol on the viability of Vero, HepG2 and MCF-7 cells

A non-transformed (Vero) and two tumor cell lines (HepG2 and MCF-7) were treated with 10nM to 100 microM formaldehyde. Lower doses (10nM to 10 microM) enhanced the viability of the cultured cells, measured by MTT assay. Higher doses (75-100 microM) decreased viability of the cells by 50% or more. The 100 microM concentration of HCHO has been chosen for combination treatment of the three cell lines with a series of concentrations (0.2-100 microM) of resveratrol, a phytoestrogen occurring in various fruits. Resveratrol decreased the cytotoxicity of formaldehyde depending on cell line and point of time, especially in case of MCF-7 cells at 24 and 72 h, Vero cells at 24h and HepG2 cells at 48 h after treatment. Possible modes of interactions are discussed, considering the role of resveratrol in formaldehyde metabolism and also the estrogen receptor positivity of MCF-7 cells.     

Fusaproliferin production by Fusarium subglutinans and its toxicity to Artemia salina, SF-9 insect cells, and IARC/LCL 171 human B lymphocytes.

Fusarium subglutinans is an important pathogen of maize and other commodities worldwide. We examined MRC-115 and 71 other F. subglutinans strains from various geographic areas for their ability to synthesize fusaproliferin, a novel toxic sesterterpene recently isolated from F. proliferatum. Fusaproliferin production ranged from 30 to 1,500 micrograms/g of dried ground substrate, with 33 strains producing more than 500 micrograms/g. In particular, strain MRC-115 produced as much as 1,100 to 1,300 micrograms/g. In toxicity studies of two invertebrate models, fusaproliferin was toxic to Artemia salina (50% lethal dose, 53.4 microM) and to the lepidopteran cell line SF-9 (50% cytotoxic concentration, approximately 70 microM, after a 48-h exposure). Fusaproliferin was also toxic to the human nonneoplastic B-lymphocyte cell line IARC/LCL 171 (50% cytotoxic concentration, approximately 55 microM in culture in stationary phase after a 48-h exposure). Experiments performed will cells exposed at seeding suggested a possible cytostatic effect at subtoxic concentrations.     

The multifunctional DNA repair/redox enzyme Ape1/Ref-1 promotes survival of neurons after oxidative stress

Although correlative studies demonstrate a reduction in the expression of apurinic/apyrimidinic endonuclease/redox effector factor (Ape1/Ref-1 or Ape1) in neural tissues after neuronal insult, the role of Ape1 in regulating neurotoxicity remains to be elucidated. To address this issue, we examined the effects of reducing Ape1 expression in primary cultures of hippocampal and sensory neurons on several endpoints of neurotoxicity induced by H2O2. Ape1 is highly expressed in hippocampal and sensory neurons grown in culture as indicated by immunohistochemistry, immunoblotting and activity. Exposing hippocampal or sensory neuronal cultures to 25 or 50 nM small interfering RNA to Ape1 (Ape1siRNA), respectively, for 48 h, causes a reduction in immunoreactive Ape1 by approximately 65 and 54%, and an equivalent loss in endonuclease activity. The reduced expression of Ape1 is maintained for up to 5 days after the siRNA in the medium is removed, whereas exposing cultures to scrambled sequence siRNA (SCsiRNA) has no effect of Ape1 protein levels. The reduction in Ape1 significantly reduces cell viability in cultures 24 h after a 1-h exposure to 25-300 microM H2O2, compared to SCsiRNA treated controls. In cells treated with SCsiRNA, exposure to 300 microM H2O2 reduced cell viability by 40 and 30% in hippocampal and sensory neuronal cultures, respectively, whereas cultures treated with Ape1siRNA lost 93 and 80% of cells after the peroxide. Reduced Ape1 levels also increase caspase-3 activity in the cells, 2-3-fold, 60min after a 1-h exposure to 100 microM H2O2 in the cultures. Exposing neuronal cultures with reduced expression of Ape1 to 65 microM H2O2 (hippocampal) or 300 microM H2O2 (sensory) for 1h results in a 3-fold and 1.5-fold increase in the phosphorylation of histone H2A.X compared to cells exposed to SCsiRNA. Overexpressing wild-type Ape1 in hippocampal and sensory cells using adenoviral expression constructs results in significant increase in cell viability after exposure to various concentrations of H2O2. The C65A repair competent/redox incompetent Ape1 when expressed in the hippocampal and sensory cells conferred only partial protection on the cells. These data support the notion that both of functions of Ape1, redox and repair are necessary for optimal levels of neuronal cell survival.     

Prolonged exposure to palmitate impairs fatty acid oxidation despite activation of AMP-activated protein kinase in skeletal muscle cells

The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 microM of palmitate did not affect cell viability but significantly reduced FA oxidation by approximately 26.5%, approximately 43.5%, approximately 50%, and approximately 47%, respectively. Interestingly, this occurred despite significant increases in AMPK ( approximately 2.5-fold) and ACC ( approximately 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity ( approximately 38-60%). Low concentrations of palmitate (50-100 microM) caused an increase ( approximately 30%) in CPT-1 activity. However, as the concentration of palmitate increased, CPT-1 activity decreased by approximately 32% after exposure for 8 h to 800 microM of palmitate. Although FA uptake was reduced ( approximately 35%) in cells exposed to increasing palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values approximately 2.3-, approximately 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 microM palmitate, respectively. Interestingly, myotubes exposed to 400 microM of palmitate for 1 h increased basal glucose uptake and glycogen synthesis by approximately 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8 h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake ( approximately 65%) and glycogen synthesis ( approximately 30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs, such as in obesity and Type 2 Diabetes.     

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.     

Effects of fusariotoxin co-exposure on THP-1 human immune cells

Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T2), fumonisin B1 (FB1), zearalenone (ZEA), and moniliformin (MON) mycotoxins are common food and feed contaminants produced by Fusarium spp. However, while they are usually found to co-occur in a large range of commodities, only few data are available on mycotoxin co-exposure effects and cellular response mechanisms. In this study, the individual and combined toxic effects of these fusariotoxins were evaluated on the THP-1 human immune cell line as major fusariotoxins are mostly potent immunomodulators. In particular, four relevant fusariotoxin mixtures, namely DON-MON, DON-FB1, DON-ZEA, and NIV-T2, were studied using several parameters including cell viability as well as the expression of cell surface markers and the main mitogen-activated protein kinases (MAPKs). After 48 h exposure, a reduction of cell viability in a dose-dependent manner was observed for T2, the most cytotoxic mycotoxin, followed by NIV, DON, MON, FB1, and ZEA. Regarding mycotoxin mixtures, they mainly showed antagonism on cell viability reduction. Interestingly, at concentrations inhibiting 50% of cell viability, most viable cells exhibited surface marker loss and thus became potentially non-functional. In addition, during the first 18 h of exposure, the effects of mycotoxin mixtures on early cell apoptosis and necrosis were found to be different from those induced by the toxins alone. At the molecular level, after 1 h exposure of individual and combined mycotoxins, the three main MAPK signaling pathways (p38, SAPK/JNK, and ERK1/2) were activated, highlighting a fast reaction of the exposed cells even at low cytotoxicity levels.     

Cytotoxicity induced by nivalenol, deoxynivalenol, and fumonisin B1 in the SF-9 insect cell line

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9)

The toxicity of sixteen fungal metabolites produced by some entomopathogenic fungi or biological control fungi agents was evaluated on lepidopteran Spodoptera frugiperda (SF-9) cell line by Trypan blue dye exclusion and MTT-colorimetric assay, after 48 h of incubation. No statistical difference was found between IC50values (50% Inhibiting Concentration) and CC50 values (50% Cytotoxicity Concentration) obtained by MTT test and Trypan blue dye exclusion for each fungal metabolite. By MTT assay, the cytotoxicity ranking was fusarenon X (IC50 0.3 microM) = diacetoxyscirpenol (IC50 0.5 microM) = beauvericin (IC50 2.5 microM) = nivalenol (IC50 5.3 microM) = enniatin (IC50 6.6 microM) > or = gliotoxin (IC50 7.5 microM) > zearalenone (IC50 17.5 microM) > deoxynivalenol (IC50 47.6 microM). By Trypan blue dye exclusion the cytotoxicity ranking was fusarenon X (CC50 0.4 microM) = diacetoxyscirpenol (CC50 1.1 microM) beauvericin = (CC50 3.0 microM)=gliotoxin (CC50 4.0 microM) = enniatin (CC50 6.7 microM) > or = nivalenol (CC50 9.5 microM) > zearalenone (CC50 18.3 microM) > deoxynivalenol (CC50 45.0 microM). The comparison with other bioassays showed that the SF-9 insect cell line could represent a further tool to screen for the toxic effects of fungal metabolites especially for beauvericin, gliotoxin, and zearalenone.     

Lethal activity of beauvericin,a Beauveria bassiana mycotoxin,against the two‐spotted spider mites,Tetranychus urticae Koch

The entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin is known to produce a broad range of secondary metabolites. Beauvericin, a cyclic hexadepsipeptide, is the best known mycotoxin produced by B. bassiana; however, reports discussing the insecticidal activity of beauvericin per se are limited. In this study, we assessed the lethal activity of beauvericin against Tetranychus urticae Koch (Acarina: Tetranychidae). In addition, screening for suitable application of the mycotoxin against T. urticae on greenhouse strawberries is discussed. Beauvericin was able to control successfully T. urticae where concentrations of 10, 100 and 1,000 µg/g recorded mortalities of 84%, 100% and 100%, respectively, against motile stages. Furthermore, beauvericin inhibited egg hatching up to 83.3%, 69.3% and 53.3%, respectively, using the same concentrations under laboratory conditions. Under greenhouse conditions, the efficacy recorded was 52.6%, 85.7%, 72.4% and 72.4% at 1, 3, 7 and 10 days post‐inoculation, respectively. Beauvericin was efficacious under greenhouse conditions since the application increased strawberry yields while showing no phytotoxicity and ecotoxicological risk. Resistance to beauvericin was not detected initially at the unselected strain of T. urticae. Yet, the laboratory selection of populations of T. urticae exposed to beauvericin resulted in relatively resistant T. urticae strain that displayed no cross‐resistance to cyflumetofen and bifenazate. The acaricidal activity of beauvericin documented in this study would increase the efficacy of integrated pest management strategies.     

Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.     

OSBPa,a predicted oxysterol binding protein of Dictyostelium,is required for regulated entry into culmination

In our previous papers [e.g. Sato et al., J. Biol. Chem. 273 (1998) 21455-21462], we have shown that prodigiosins can uncouple various H(+)-ATPases through their H(+)/Cl(-) symport activity. BE-18591 is an enamine of 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde (tambjamine group antibiotics) which resembles the prodigiosins. We found that BE-18591 was a new group of antibiotics that uncouples various H(+)-ATPases: it inhibited proton pump activities with IC(50)s of about 1-2 nM (about 20 pmol/mg protein) for submitochondrial particles as well as gastric vesicles and of 230 nM (about 230 pmol/mg protein) for lysosomes, but it had little effect on their ATP hydrolyses (up to 10 microM), a property of H(+)/Cl(-) symport activity. At low concentrations (<1 microM), BE-18591 inhibited immunoproliferation, the IC(50) of lipopolysaccharide-stimulated mouse splenocytes was 38 nM, that of Concanavalin A-stimulated cells was 230 nM. Gastritis of rabbits was also inhibited. At higher concentrations (>1 microM), BE-18591 induced neurite outgrowth (15% induction in 48 h at 4 microM), inhibited bone resorption (approximately 35% in 48 h at 10 microM) and caused cell death (approximately 30% in 48 h at 4 microM) but with little apoptosis.     

Acute toxicity of cadmium and copper in hepatopancreas cells from the Roman snail (Helix pomatia)

The toxic effects of cadmium (Cd) and copper (Cu) on cellular metabolism and cell morphology were investigated in isolated hepatopancreas cells from the Roman snail (Helix pomatia). Cell viability was unaffected during 1 h of incubation with 100 microM Cd, but was significantly reduced from 93% in controls to 87% and 85% with 100 microM Cu and 500 microM Cd, respectively. The adverse effect of 500 microM Cd on cell viability was not observed in cells isolated from Cd pretreated snails. Oxygen consumption remained constant in the presence of 100 microM Cu but was inhibited by 38% after 1 h of exposure to 500 microM Cd. Hepatopancreas cells showed enhanced formation of reactive oxygen species when exposed to 100 microM Cu, but not in the presence of Cd. Morphologically, an increase in cell volume of Cd-exposed cells was noted, while cell membrane bleb formation was induced by both metals. The latter may have been induced by metal effects on the actin filamentous network of the cells which showed distinct actin-staining within the blebs at the cell surface. Overall, our data indicate that both Cd and Cu are acutely toxic for hepatopancreas cells form the Roman snail with Cu being more toxic than Cd.     

A 1,4-dihydropyridine derivative reduces DNA damage and stimulates DNA repair in human cells in vitro

Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiation-induced mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at 14.9+/-1.2 and 10.3+/-0.8mM, respectively, but did not show a cytotoxic effect at concentrations <100 microM. Alkaline single-cell gel electrophoresis (comet) assays showed that AV-153 reduced the number of DNA strand breaks in untreated cells and also in cells exposed to 2 Gy of gamma-radiation, 100 microM ethylmethane sulfonate (EMS), or 100 microM H2O2. DNA damage was reduced by up to 87% at AV-153 concentrations between 1 and 10nM, and a positive dose-effect relationship was seen between 0.01 and 1 nM. Comparison of the kinetics of DNA strand-break rejoining in the presence and absence of AV-153 revealed a considerable influence on the rate of repair. In view of the resemblance of this compound's structure to that of dihydronicotinamide, a substrate for poly(ADP-rybose)polymerase, the modulation of DNA repair by AV-153 could involve an influence on poly(ADP)ribosylation.     

Cell density influences the effect of celecoxib in two carcinoma cell lines.

It has been reported that when ovarian carcinoma cell lines are exposed to various concentrations of celecoxib, a COX-2 inhibitor, cell growth is decreased in a dose dependant manner. To examine further the effect of celecoxib, different cell densities of two carcinoma cell lines were exposed to various concentrations of celecoxib. LNCAP prostate and CAOV3 ovarian carcinoma cells were obtained from the American Type Culture Collection and maintained in Rosewell Park Memorial Institute 1640 and Dulbeceo's modified Eagle's medium, respectively. Each cell line was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotic-antimycotic solution, and placed in a humidified atmosphere containing 5% CO2 at 37 degrees C. After each cell line reached a confluency of 70-80%, 1,000, 2,000, 3,000, 5,000, 7,000 and 10,000 cells/well were seeded in 96 well plates in 100 microl medium/per well for 24 h. Each cell line was exposed to the same concentrations of celecoxib (10-100 microM) at each cell density for 72 h. Cell growth was assessed using a tetrazolium conversion assay. A significant decrease compared to controls was observed in cell growth at each cell density of LNCAP and CAOV3 cells plated with >or=30 microM and >or=50 microM celecoxib, respectively. When the cell growth curves were compared for each cell density at the same concentration of celecoxib, a significant decrease in cell growth was observed when LNCAP cells were plated at 10,000 cells/well and exposed to 10-100 microM celecoxib. At a cell density >or= 5,000 LNCAP cells/well, the inhibitory effect of celecoxib was less. Similarly, a significant decrease in cell growth was observed in CAOV3 cells plated at 1,000 cells/well compared to other cell numbers plated at the same drug concentrations. At a cell density of > 5,000 CAOV3 cells/well, the inhibitory effect of celecoxib was significantly less compared to other cell densities at the same concentration. We observed a more sensitive decrease in cell growth in both carcinoma lines studied at a cell density of 1,000 cells/well with exposure to 10-100 microM celecoxib. Both carcinoma cell lines were less sensitive at a cell density of 5,000 cells/well. Our results suggest that the inhibitory effect of celecoxib may be affected by cell density. Therefore, careful attention must be paid to determining the appropriate cell density for cytotoxicity studies.     

alpha-Bisabolol, a nontoxic natural compound, strongly induces apoptosis in glioma cells

In this study, alpha-bisabolol, a sesquiterpene alcohol present in natural essential oil, was found to have a strong time- and dose-dependent cytotoxic effect on human and rat glioma cells. After 24 h of treatment with 2.5-3.5 microM alpha-bisabolol, the viability of these cells was reduced by 50% with respect to untreated cells. Furthermore, the viability of normal rat glial cells was not affected by treatment with alpha-bisabolol at the same concentrations as above. Glioma cells treated with high concentration of alpha-bisabolol (10 microM) resulted in a 100% cell death. Judging from hypo-G1 accumulation, poly(ADP-ribose) polymerase cleavage, and DNA ladder formation, the cytotoxicity triggered by alpha-bisabolol resulted from apoptosis induction. Moreover, the dissipation of mitochondrial-inner transmembrane potential and the release of cytochrome c from mitochondria indicated that, in these glioma cells, apoptosis occurred through an intrinsic pathway. As pointed out by the experimental results, alpha-bisabolol may be considered a novel compound able to inhibit glioma cell growth and survival.     

pRb suppresses camptothecin-induced apoptosis in human osteosarcoma Saos-2 cells by inhibiting c-Jun N-terminal kinase.

This paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.     

Cytotoxicity of commonly used nitroxide radical spin probes

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 microM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30-50 microM) none of the lipid soluble spin probes tested was cytotoxic. At 100 microM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.     

Effect of 1-methyl-4-phenylpyridinium ion (MPP+) on catecholamine levels and activity of related enzymes in clonal rat pheochromocytoma PC12h cells

1-Methyl-4-phenylpyridinium ion (MPP+), a metabolite of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to reduce dopamine (DA) level and the activity of enzymes related to its metabolism in clonal rat pheochromocytoma PC12h cells. After 6 days' culture in the presence of 1 mM and 100 microM MPP+, DA content in PC12h cells was reduced markedly, but with MPP+ at concentrations lower than 10 microM, DA levels in the cells did not change. The amounts of 3,4-dihydrophenylacetic acid (DOPAC), a metabolite of DA were reduced markedly in culture medium and in PC12h cells cultured with MPP+ at concentrations higher than 1 microM. MPP+ was found to reduce the enzyme activity of tyrosine hydroxylase (TH), monoamine oxidase (MAO) and aromatic L-aminoacid decarboxylase (AADC). In the presence of MPP+ at concentrations higher than 10 microM, reduction of TH activity in the cells was more pronounced than reduction of cell protein or of the activity of a non-specific enzyme, beta-galactosidase. With 1 mM and 100 microM MPP+, MAO activity was reduced to about 30% of that in control cells. Reduction was observed with MPP+ at concentrations higher than 1 microM. AADC was the most sensitive to MPP+ and its activity was reduced markedly in the cells cultured with 100 nM MPP+. These results indicate that MPP+ inhibits not only the biosynthesis of catecholamines, but also the enzyme participating in their catabolism in cells, and may thus perturb catecholamine levels in the brain.