Protection of PC12 cells from hydrogen peroxide-induced cytotoxicity by salvianolic acid B, a new compound isolated from Radix Salviae miltiorrhizae

A number of studies indicate that reactive oxygen species (ROS) are involved in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The neuroprotective effects of salvianolic acid B (SalB) from Radix Salviae miltiorrhizae (RSM) against hydrogen peroxide (H2O2)-induced rat pheochromocytoma line PC12 injury were evaluated in the present study. Vitamin E, a potent antioxidant, was employed as a positive control agent. Following exposure of cells to H2O2 (150 microM), a marked decrease in cell survival and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased levels of malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. In parallel, H2O2 caused significant elevation in intracellular Ca2+ level and caspase-3 activity, and induced apoptotic death as determined by flow cytometric assay. However, pretreatment of the cells with SalB (0.1-10 microM) prior to H2O2 exposure blocked these H2O2-induced cellular events noticeably. Moreover, SalB exhibited significantly higher potency as compared to Vitamin E. The present findings indicated that SalB exerts neuroprotective effects against H2O2 toxicity, which might be of importance and contribute to its clinical efficacy for the treatment of neurodegenerative diseases.     

Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.     

Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl(2). Exposure to CoCl(2) caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl(2) and led the reduction of DNA fragmentation and sub-G(1) fraction of cell cycle. Also, EGCG attenuated the CoCl(2)-induced disruption of mitochondrial membrane potential (DeltaPsim), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl(2)-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl(2) treatment. Taken together, the present results suggest that EGCG inhibit the CoCl(2)-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.     

Protective effect of a novel cystine C(60) derivative on hydrogen peroxide-induced apoptosis in rat pheochromocytoma PC12 cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. One of the effective ways to prevent the reactive oxygen species (ROS) mediated cellular injury is dietary or pharmaceutical augmentation of free radical scavengers. In the present study, we describe the synthesis and characterization of a novel cystine C(60) derivative (CFD). The compound was analyzed by FT-IR, (1)H NMR, (13)C NMR, LC-MS and elemental analysis. It contains five cystine moieties per C(60) molecule. This water-soluble amino-fullerene derivative was able to scavenge both superoxide and hydroxyl radical with biocompatibility. We investigated its potential protective effects on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. Cells treated with hydrogen peroxide underwent cytotoxicity and apoptotic death determined by MTT assay, flow cytometry analysis, PI/Hoechst 33342 staining and glutathione peroxidase assay. The CFD was able to reduce the accumulation of reactive oxygen species and cellular damage caused by hydrogen peroxide in PC12 cells. RF assay demonstrated that CFD could penetrate through the cell membrane and it has played its distinguished role in protecting PC12 cells against hydrogen peroxide-induced cytotoxicity. The results suggest that CFD has the potential to prevent oxidative stress-induced cell death without evident toxicity. Hence, we can hypothesize that the protective effect of CFD on hydrogen peroxide-induced apoptosis is related to its scavenger activity.     

Radiofrequency radiation at 2.856 GHz does not affect key cellular endpoints in neuron-like PC12 cells

To investigate the potential cytotoxicity of radiofrequency (RF) radiation on central nervous system, rat pheochromocytoma (PC12) cells were exposed to 2.856 GHz RF radiation at a specific absorption rate (SAR) of 4 W/kg for 8 h a day for 2 days in 35 mm Petri dishes. During exposure, the real-time variation of the culture medium temperature was monitored in the first hour. Reactive oxygen species (ROS) production, intracellular Ca²⁺ concentration, and cell apoptosis rate were assessed immediately after exposure by flow cytometry. The results showed that the medium temperature raised about 0.93 °C, but no significant changes were observed in apoptosis, ROS levels or intracellular Ca²⁺ concentration after treatment. Although several studies suggested that RF radiation does indeed cause neurological effects, this study presented inconsistent results, indicating that 2.856 GHz RF radiation exposure at a SAR of 4 W/kg does not have a dramatic impact on PC12 cells, and suggests the need for further investigation on the key cellular endpoints of other nerve cells after exposure to RF radiation.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.     

Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2+

These experiments were designed to learn the role of bradykinin induced changes in intracellular Ca²⁺ in the activation of phospholipase D activity in PC12 cells. Ionomycin at a concentration of 0.1M caused an increase in intracellular Ca²⁺ comparable to bradykinin, but had no effect on phospholipase D activity. Carbachol, ATP, and thapsigargin also increased intracellular Ca²⁺ but had no effect on phospholipase D activity. Increases in intracellular Ca²⁺ may be a necessary but not a sufficient factor in the activation of phospholipase D. To investigate this issue, the bradykinin induced increase in intracellular Ca²⁺ was blocked by preincubating the cells in Ca²⁺-free media plus EGTA or in media containing the intracellular Ca²⁺ chelator BAPTA/AM. These preincubations completely blocked the bradykinin induced increase in intracellular Ca²⁺ but only attenuated the bradykinin mediated activation of phospholipase D. Physiological increases in intracellular Ca²⁺ apparently do not mediate the effect of bradykinin on phospholipase D.     

Chloride channels involve in hydrogen peroxide-induced apoptosis of PC12 cells

Chloride channel activity is one of the critical factors responsible for cell apoptotic volume decrease (AVD). However, the roles of chloride channels in apoptosis have not been fully understood. In the current study, we assessed the role of chloride channels in hydrogen peroxide (H₂O₂)-induced apoptosis of pheochromocytoma cells (PC12). Extracellular application of H₂O₂ activated a chloride current and induced cell volume decrease in a few minutes. Incubation of cells with H₂O₂ elevated significantly the membrane permeability to the DNA dye Hoechst 33258 in 1 h and induced apoptosis of most PC12 cells tested in 24 h. The chloride channel blocker NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate) prevented appearance of H₂O₂-induced high membrane permeability and cell shrinkage, suppressed H₂O₂-activated chloride currents and protected PC12 cells from apoptosis induced by H₂O₂. The results suggest that chloride channels may contribute to H₂O₂-induced apoptosis by ways of elevation of membrane permeability and AVD in PC12 cells.     

Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells

Phospholipase D (PLD) plays an important role as an effector in the membrane lipid-mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H(2)O(2))-induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H(2)O(2) resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H(2)O(2)-induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1-butanol. Expression of PLD2, but not of PLD1, correlated with increased H(2)O(2)-induced PLD activity in a concentration- and time-dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H(2)O(2)-induced apoptosis in PC12 cells. Expression of PLD2 lipase-inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H(2)O(2)-induced cleavage and activation of caspase-3. Taken together, the results suggest that PLD2 activity is specifically up-regulated by H(2)O(2) in PC12 cells and that it plays a suppressive role in H(2)O(2)-induced apoptosis.     

Excessive reactive oxygen species induces apoptosis in fibroblasts: role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.     

Hydrogen peroxide-induced apoptosis in pc12 cells and the protective effect of puerarin

In this study, the effect of puerarin on hydrogen peroxide-induced apoptosis in PC12 cells was studied. Exposure of cells to 0.5mM H(2)O(2)may cause significant viability loss and apoptotic rate increase. When c-Myc, Bcl-2 and Bax expression and caspase-3 activity were measured, using Ac-DEVD-AMC as a substrate, the changes in these apoptosis regulatory and effector proteins suggested that the elevation of c-Myc, decrease in Bcl-2:Bax protein ratio, and caspase-3 activation all play a key role in apoptosis. When cells were treated with puerarin prior to 0.5 mM H(2)O(2)treatment, a reduction in viability loss and apoptotic rate was seen. In addition, c-Myc expression decreased and Bcl-2:Bax ratio increased. Puerarin also reduced the H(2)O(2)-induced elevation of caspase-3 activation. These results suggest that puerarin can protect neurons against oxidative stress. It can block apoptosis in its early stages via the regulation of anti- and pro-apoptotic proteins, as well as by the attenuation of caspase-3 activation in H(2)O(2)-induced PC12 cells.     

Differential Involvement of Intracellular Ca2+ in 1-Methyl-4-phenylpyridinium- or 6-Hydroxydopamine-Induced Cell Viability Loss in PC12 Cells

1-Methyl-4-phenylpyridinium (MPP⁺) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP⁺-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP⁺ or 6-OHDA evoked the elevation of intracellular Ca²⁺ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca²⁺ levels due to both toxins. The results show that the MPP⁺-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca²⁺ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca²⁺-independent manner.     

Protection of cells from oxidative stress by microsomal glutathione transferase 1

Rat liver microsomal glutathione transferase 1 (MGST1) is a membrane-bound enzyme that displays both glutathione transferase and glutathione peroxidase activities. We hypothesized that physiologically relevant levels of MGST1 is able to protect cells from oxidative damage by lowering intracellular hydroperoxide levels. Such a role of MGST1 was studied in human MCF7 cell line transfected with rat liver mgst1 (sense cell) and with antisense mgst1 (antisense cell). Cytotoxicities of two hydroperoxides (cumene hydroperoxide (CuOOH) and hydrogen peroxide) were determined in both cell types using short-term and long-term cytotoxicity assays. MGST1 significantly protected against CuOOH and against hydrogen peroxide (although less pronounced and only in short-term tests). These results demonstrate that MGST1 can protect cells from both lipophilic and hydrophilic hydroperoxides, of which only the former is a substrate. After CuOOH exposure MGST1 significantly lowered intracellular ROS as determined by FACS analysis.     

Synthesis of beta-alanine C60 derivative and its protective effect on hydrogen peroxide-induced apoptosis in rat pheochromocytoma cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially prominent in neural diseases. One of the effective ways to prevent the reactive oxygen species (ROS) mediated cellular injury is dietary or pharmaceutical augmentation of some free radical scavenger. Water-soluble amino-fullerene derivative is a novel compound that behaves as a free radical scavenger with excellent biocompatibility. In the present study, we synthesized a novel beta-alanine C(60) derivative. The product was characterized by FT-IR, (1)H NMR, (13)C NMR, LC-MS and elemental analysis. We investigated the protective effect on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death determined by MTT, flow cytometry analysis and PI/Hoechst 33342 staining. Moreover, the scavenging ability of beta-alanine C(60) derivative to reactive oxygen species both in vivo and in vitro of PC12 cells was measured. The results suggest that beta-alanine C(60) derivative has the potential to prevent oxidative stress-induced cell death without evident toxicity. Hence, on the basis of the above-mentioned studies, we can hypothesize that the protective effect of beta-alanine C(60) derivative on H(2)O(2) induced apoptosis is related to their known scavenger activity toward ROS.     

Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment

Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca(2+) concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca(2+). To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca(2+) overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1-30 microM) of quercetin to protect the cells suffering from intracellular Ca(2+) overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H(2)O(2). The cell death respectively induced by H(2)O(2) and A23187 was significantly suppressed by removal of external Ca(2+). Furthermore, quercetin greatly delayed the process of Ca(2+)-dependent cell death although it did not significantly affect the elevation of intracellular Ca(2+) concentration by H(2)O(2) and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca(2+). Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca(2+).     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Diindolylmethane at concentrations of 20–50 µM induced [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Diindolylmethane-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca²⁺]_i rise in PC3 cells by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A₂-sensitive store-operated Ca²⁺ channels. Diindolylmethane caused cell death in which apoptosis may participate.     

Antioxidant Effect of Phenelzine on MPP+-Induced Cell Viability Loss in Differentiated PC12 Cells

Phenelzine, deprenyl, and antioxidants (SOD, catalase, ascorbate, or rutin) reduced the loss of cell viability in differentiated PC12 cells treated with 250 M MPP⁺, whereas N-acetylcysteine and dithiothreitol did not inhibit cell death. Phenelzine reduced the condensation and fragmentation of nuclei caused by MPP⁺ in PC12 cells. Phenelzine and deprenyl prevented the MPP⁺-induced decrease in mitochondrial membrane potential, cytochrome c release, formation of reactive oxygen species, and depletion of GSH in PC12 cells. Phenelzine revealed a scavenging action on hydrogen peroxide and reduced the hydrogen peroxide–induced cell death in PC12 cells, whereas deprenyl did not depress the cytotoxic effect of hydrogen peroxide. Both compounds reduced the iron and EDTA-mediated degradation of 2-deoxy-d-ribose degradation. The results suggest that phenelzine attenuates the MPP⁺-induced viability loss in PC12 cells by reducing the alteration of mitochondrial membrane permeability that seems to be mediated by oxidative stress.     

Nordihydroguaiaretic acid-induced Ca2+ handling and cytotoxicity in human prostate cancer cells

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells was investigated. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. NDGA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 30 microM. The Ca2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca2+ partly decreased the NDGA-induced [Ca2+]i increase, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and intracellular Ca2+ release. NDGA-induced Ca2+ influx was independently confirmed by measuring NDGA-induced Mn2+ -coupled quench of fura-2 fluorescence. The NDGA-induced Ca2+ influx was not affected by L-type Ca2+ channel blockers. In Ca2+ -free medium, the NDGA-induced [Ca2+]i increase was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca2+]i increase. NDGA-induced intracellular Ca2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20-50 microM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca2+]i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca2+]i increase via releasing stored Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. NDGA also caused cytotoxicity at higher concentrations.