flunarizine and Neuroblastoma

Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL.. We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells.. Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells.. Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood-brain barrier as did all of the positively screened drugs in this study.. Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Amlodipine; Blotting, Western; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Cell Line, Tumor; Child; Drug Evaluation, Preclinical; Flunarizine; Humans; Intracellular Space; Membrane Glycoproteins; Molecular Chaperones; Neuroblastoma; Neuronal Ceroid-Lipofuscinoses; Nicardipine; Nifedipine; Nimodipine; Potassium Chloride; RNA Interference; RNA, Small Interfering

Serum albumin protects against cell death elicited by various cytotoxic agents; however, conflicting views on the protective mechanism still remain. Hence, we have studied the ability of serum albumin to prevent apoptosis of human neuroblastoma SH-SY 5 Y cells elicited by four compounds known to release Ca(2+) from the endoplasmic reticulum, i.e. dotarizine, flunarizine, thapsigargin and cyclopiazonic acid. Spontaneous basal apoptosis, after 24 h incubation in Dulbecco's Modified Eagle Medium (DMEM) containing 10% serum, was 5%. Dotarizine (30--50 microM) enhanced basal apoptosis to 18--43%, flunarizine (30--50 microM) to 15%, thapsigargin (1--10 microM) to 21--35%, and cyclopiazonic acid (100 microM) to 10%. Serum deprivation augmented basal apoptosis to 20%. Under serum-free medium, 30 microM dotarizine or flunarizine drastically enhanced apoptosis to 63% and 68%, respectively; the increase was milder with 1 microM thapsigargin (37%) and 30 microM cyclopiazonic acid (27%). In serum-free medium, albumin (29 or 49 mg/ml) fully prevented the apoptotic effects of dotarizine, flunarizine and cyclopiazonic acid. The four compounds increased the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in fluo-4 loaded cells; such increase developed slowly to reach a plateau after several minutes, followed by a slow decline. Albumin did not modify the kinetic parameters of such increase. In the absence of serum, dotarizine, flunarizine, thapsigargin, and cyclopiazonic acid caused mitochondrial depolarization in tetramethylrhodamine ethyl ester (TMRE)-loaded cells; depolarization was inhibited by cytoprotective concentrations of albumin. These results suggest that albumin protects cells from entering into apoptosis by preventing mitochondrial depolarization. They also suggest that inhibition of mitochondrial depolarization might become a target to develop new anti-apoptotic compounds with therapeutic neuroprotective potential in stroke, Alzheimer's disease, and other neurodegenerative diseases.

Topics: Apoptosis; Benzhydryl Compounds; Calcium; Calcium Channel Blockers; Cell Line, Tumor; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Flunarizine; Humans; Membrane Potentials; Mitochondria; Neuroblastoma; Piperazines; Serum Albumin, Bovine; Thapsigargin; Time Factors

Calcium channel antagonists are drugs currently used in the treatment of neurological and cardiovascular disorders and occasionally produce parkinsonism and movement disorders as a side effect. We investigated the effects of calcium channel antagonists on the pharmacology of dopamine systems in vivo and in vitro. Flunarizine, cinnarizine, and diltiazem reduce the viability of dopamine-rich human neuroblastoma cells in vitro. These compounds plus verapamil, nifedipine, and nicardipine reduce 3H-spiperone binding to bovine striatal membranes, 3H-dopamine uptake, K(+)-induced 3H-dopamine release, and apomorphine-induced rotation, but not amphetamine-induced rotation, in 6-OH-dopamine-lesioned rats. Therefore, all calcium channel antagonists tested reduce dopamine neurotransmission in vitro and in vivo, whereas the evidence of toxicity for dopamine cells in vitro is restricted to flunarizine, cinnarizine, and diltiazem. The clinical relevance of these toxic effects may depend on several factors, including age, penetration across the blood-brain barrier, and types of calcium channels present in the different neuronal subtypes. On the other hand, the finding of dopamine-regulating properties not associated to neurotoxic effects in the dihydropyridines and verapamil provides new putative therapeutics tools for the treatment of neurologic disorders associated with dopamine hyperactivity.

Topics: Animals; Calcium Channel Blockers; Cattle; Cinnarizine; Diltiazem; Dopamine; Dose-Response Relationship, Drug; Flunarizine; Humans; In Vitro Techniques; Neuroblastoma; Rats; Tumor Cells, Cultured

Several diphenylmethylpiperazine derivatives are potential therapeutic agents for prevention of ischemic injuries in the heart and brain, because of their ability to block Ca2+ currents and their antioxidant activity. In this study, the current lead compound, U-92032 ((7-((bis-4-fluorophenyl)methyl)-1-piperazinyl)-2-(2-hydroxyethylamin o)- 4-(1-methylethyl)-2,4,6-cycloheptatrien-1-one), has been compared with flunarizine and nifedipine (well-known T- and L-type Ca2+ channel antagonists, respectively) for their effects on Ca2+ channels in a mouse neuronal cell line, N1E-115 cells, and their ability to preserve the phenomenon of long-term potentiation and to improve neurological symptoms in gerbil ischemic models. U-92032, like flunarizine, blocked transient Ba2+ currents (IBa) through T-type Ca2+ channels with no effect on nifedipine-sensitive non-inactivating currents. Transient IBa was reduced by U-92032 at a constant rate, the magnitude of which depended on the drug concentration, probably because of a time-dependent accumulation of the lipophilic drug in the membrane phase. For instance, the drug at 6 microM reduced IBa by 21% per min and abolished it in less than 5 min, about 3 times faster than flunarizine at the same concentration. Otherwise, U-92032 behaved like flunarizine, showing a use-dependent block without noticeable effects on the current-voltage relationship for transient IBa. Oral administration of U-92032 (1 and 25 mg/kg) or flunarizine (25 mg/kg), but not nifedipine (50 mg/kg), to gerbils 1 h prior to bilateral carotid artery occlusion, preserved long-term potentiation in hippocampal CA1 neurons, which were largely abolished by ischemia without the drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antioxidants; Brain Ischemia; Calcium Channel Blockers; Flunarizine; Gerbillinae; Hippocampus; Lipid Peroxidation; Long-Term Potentiation; Malondialdehyde; Mice; Neuroblastoma; Neurons; Piperazines; Rats; Tropolone; Tumor Cells, Cultured

Bradykinin-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single NG108-15 cells with indo-1-based dual-emission microfluorimetry (50% effective concentration, 16 nM). A 1-min exposure to 30 nM bradykinin completely depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store; refilling the store required extracellular Ca2+ (half time, 2 min). Refilling the IP3-sensitive store was completely blocked by 1 microM La3+ and 10 microM nitrendipine, but not 10 microM verapamil, 10 microM flunarizine, 1 microM nitrendipine, or 0.1 microM La3+. Thapsigargin irreversibly depleted the Ca2+ store and prevented its refilling (half-maximal inhibitory concentration, 3 nM). Influx of Ca2+ across the plasma membrane did not increase after depletion of the IP3-sensitive store by exposure to bradykinin, although maintained presence of the agonist produced significant Ca2+ influx. Similarly, Mn2+ and Ba2+ influx, as measured by indo-1 quenching and spectral shifts, did not increase following depletion of IP3-sensitive store. In contrast to depletion of the IP3-sensitive Ca2+ store by bradykinin, thapsigargin (10 nM) treatment produced Ca2+ and Ba2+ influx. We conclude that after Ca2+ mobilization, the IP3-sensitive Ca2+ store in NG108-15 cells is refilled with cytoplasmic Ca2+ via a thapsigargin-sensitive Ca(2+)-Mg(2+)-ATPase. Cytoplasmic Ca2+ is replenished by a persistent leak of Ca2+ across the plasma membrane. This leak is not modulated by the status of the intracellular Ca2+ store. In NG108-15 cells, agonist and thapsigargin-evoked Ca2+ entry are mediated by activation of plasmalemmal Ca2+ channels independent of the status of the IP3-sensitive intracellular Ca2+ store.

Topics: Animals; Bradykinin; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Flunarizine; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Mice; Neuroblastoma; Nitrendipine; Terpenes; Thapsigargin; Tumor Cells, Cultured; Verapamil

Tetrandrine, an alkaloid isolated from the Chinese herb, Radix stephaniae tetrandrae, has been used clinically as a hypotensive agent for a long time. Recently, several studies have demonstrated that tetrandrine behaves like a calcium entry blocker. In the present investigation, the whole cell version of the patch clamp technique was used to study the effect of tetrandrine on the type I (transient inward) calcium current in neuroblastoma cells. These results showed that tetrandrine inhibited the transient inward current, without affecting the channel kinetics. The effects of tetrandrine were dose-dependent and reversible but did not depend on the frequency of stimulation (use-dependence) or the membrane potential. These data clearly demonstrate that tetrandrine is a novel and potent antagonist of the transient inward current in neuroblastoma cells.

Topics: Alkaloids; Animals; Barium; Benzylisoquinolines; Calcium Channel Blockers; Flunarizine; Kinetics; Membrane Potentials; Mice; Neuroblastoma; Nifedipine; Tumor Cells, Cultured

The sensitivities of two types of voltage-dependent calcium channel currents in N1E-115 neuroblastoma cells to various agents were studied using the whole cell version of the patch clamp technique. Cells cultured in normal media expressed predominantly transient (T) currents whereas cells cultured in media with dimethylsulfoxide for 1 month expressed predominantly long-lasting (L) currents. Furthermore, by selecting cells with one or two short neurites it was possible to obtain cells which expressed only L channels. The dihydropyridine agonist, Bay K-8644 (5 microM), increased the amplitude of L channel currents by a factor of nearly two, whereas T channel currents were unaffected. Nifedipine (0.1 mM) significantly inhibited L channel currents, whereas T channel currents were insensitive to this treatment. Flunarizine, a diphenylpiperazine, had no effect on L channel currents but selectively inhibited T channel currents in a dose-dependent manner, with a significant effect at a concentration of 1 microM. However, flunarizine did not change the I-V relationships of T channel currents. Furthermore, the voltage dependence of T channel inactivation was shifted toward more negative potential by flunarizine. The present study provides direct evidence of the selective inhibition of T channel currents by flunarizine in N1E-115 neuroblastoma cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium Channels; Electrophysiology; Flunarizine; Mice; Microelectrodes; Neuroblastoma; Nifedipine; Tumor Cells, Cultured

An increase in susceptibility of neuroblastoma cells to calcium was inhibited when the cells were treated with hypothermia, diphenylhydantoin, chlorpromazine or pentobarbital; all of these treatments affect membrane permeability to calcium or membrane-associated cell metabolism. Diphenylhydantoin and chlorpromazine were found to have positive effects only when they were administered before the addition of calcium. This was in contrast to flunarizine, which showed a protective effect only in combination with calcium. However, leupeptin did not show any positive effect, indicating that calcium-activated neutral protease may be only slightly involved in this type of calcium-induced cell damage.

Topics: Calcium; Cell Membrane Permeability; Cell Survival; Chlorpromazine; Cold Temperature; Flunarizine; Humans; Neuroblastoma; Pentobarbital; Phenytoin; Tumor Cells, Cultured