dihydropyridines and Glioma

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca

Topics: Amino Acids; Animals; Biological Transport; Brain Neoplasms; Calcium Channel Blockers; Calcium Channels, T-Type; Cell Death; Cell Line, Tumor; Dihydropyridines; Glioma; Humans; Mice; Mycotoxins; Neoplastic Stem Cells; Potassium Channels, Calcium-Activated; Proteomics; Sodium; Unfolded Protein Response

Evidence has accumulated that classic L-type Ca2+ channel blockers with a dihydropyridine structure also inhibit T-type Ca2+ channels in certain types of central and peripheral neurons and in smooth muscle cells, albeit with a lower potency. Thus beneficial therapeutic effects of dihydropyridines in cardiovascular and neurological diseases may not only be associated with L-type but also with T-type Ca2+ channel blockade. Little is known about the exact order of potency of dihydropyridine derivatives at T-type Ca2+ channels. Here we investigate the efficacy and potency of four therapeutically used compounds, i.e. nifedipine, nimodipine, nicardipine, niguldipine, in the neuroblastoma-glioma cell line NG108-15. For comparative purposes the Ca2+ channel agonist Bay K 8644 was included. Ca2+ channel currents were measured with the whole-cell voltage clamp technique. Subtype Ca2+ channel currents were separated by clamp protocol and selective blockers. T-type Ca2+ channel currents were inhibited with decreasing potency in the order niguldipine > nicardipine > nimodipine > nifedipine (IC50-values 244 nM, 2.5 microM, 9.8 microM, 39 microM), whereas L-type Ca2+ channel currents were blocked with similar potency (IC50 for nicardipine 75 nM). Bay K 8644 increased T-type Ca2+ channel current at nanomolar concentrations (i.e. 95 +/- 16% increase by 300 nM). T-type Ca2+ channel block was completely reversible with exception of the block by niguldipine. Our results indicate a variability of two orders of magnitude in potency of T-type Ca2+ channel block by the dihydropyridine derivatives investigated. It is speculated that the relation between the L- and T-type Ca2+ channel block may determine the therapeutic profile of a dihydropyridine derivative.

Topics: Animals; Brain Neoplasms; Calcium Channel Blockers; Cell Differentiation; Dihydropyridines; Glioma; Hybrid Cells; Membrane Potentials; Mice; Neuroblastoma; Rats; Tumor Cells, Cultured

The effects of nifedipine, niguldipine, nimodipine and nitrendipine on the high K+-induced intracellular Ca2+ ([Ca2+]i) transient in dibutyryl cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells were studied by using the fluorescent Ca2+ indicator fura-2. It was observed that nifedipine at the concentration of 50 microM inhibited the high K+-induced [Ca2+]i transient by about 60%; niguldipine at the concentration of 10 microM caused a reduction of about 65% in the high K+-induced calcium signal and a further increase in the concentration up to 50 microM did not result in a significant further reduction in the high K+-induced calcium signal. However, on the other hand, nimodipine and nitrendipine at 50 microM inhibited almost completely the high K+-induced [Ca2+]i transient. Consequently, it was demonstrated in the present study that nimodipine and nitrendipine inhibit both L- and N-type calcium channels and thus seem to be unique among the dihydropyridines examined in their effects on calcium channels in dibutyryl cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells, whereas nifedipine and niguldipine appear to block mainly L-type calcium channels.

Topics: Animals; Bucladesine; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Dihydropyridines; Evoked Potentials; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Nimodipine; Nitrendipine; Potassium; Rats

Multidrug resistance phenotypes in human tumours are associated with the overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with that of the non-P-glycoprotein-mediated multidrug resistance gene, MRP, which encodes a 190 kDa membrane ATP-binding protein. We have previously reported that overexpression of MRP appears to be responsible for spontaneous multidrug resistance in some human glioma cell lines (Abe et al., Int. J. Cancer, 58, 860-864, 1994). In this study, we investigated whether chemosensitising agents of P-glycoprotein-mediated multidrug resistance such as verapamil, a biscoclaurine alkaloid (cepharanthine), and a dihydropyridine analogue (NIK250) could also reverse multidrug resistance in human glioma cells. The glioma cell lines were the two MRP-expressing cell lines, T98G and IN500, an MDR1-expressing cell line, CCF-STTG1, and the MRP1 MDR1-non-expressing cell line, IN157. Verapamil and NIK250 almost completely reversed drug resistance to vincristine, etoposide and doxorubicin in T98G cells, while they also reversed drug resistance to vincristine and etoposide, but only partially to doxorubicin in IN500 cells. Cepharanthine as well as verapamil and NIK250 reversed vincristine resistance in CCF-STTG1 cells, but cepharanthine only partially reversed drug resistance in T98G and IN500 cells. The cellular accumulation of [3H]etoposide increased about 2- and 3-fold compared with control in T98G cells in the presence of verapamil and NIK250 respectively. Furthermore, the release of doxorubicin from the nuclei of T98G cells was blocked by NIK250. However, NIK250 and verapamil caused no apparent increase in vincristine accumulation in T98G cells. NIK250 or verapamil might exert inhibitory effects upon MRP function, resulting in a reversal of MRP-mediated spontaneous multidrug resistance in cultured human glioma cells.

Topics: Alkaloids; Antigens, Neoplasm; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzylisoquinolines; Dihydropyridines; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA-Binding Proteins; Doxorubicin; Drug Resistance, Multiple; Drug Synergism; Gene Expression; Glioma; Humans; Isoenzymes; Microscopy, Fluorescence; Sulfur Compounds; Tumor Cells, Cultured; Verapamil

Depolarization of differentiated neuroblastoma X glioma (NG108-15) cells with KCl (50 mM) or veratridine (50 microM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 +/- 7 to 595 +/- 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage-dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose-dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (-) stereoisomers of 202-791 showed agonist and antagonist properties, respectively. (+)-202-791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage-sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KCl treatment and also by divalent metal cations (micromolar range). High [K+]o-induced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 microM) and tetraethylammonium (10 microM). In contrast, veratridine-induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 microM). Veratridine-induced Ca2+ accumulation was slower (approximately 45 s), smaller in magnitude (approximately 30% of [K+]o-induced Ca2+ entry), and also enhanced by BAY K 8644 (approximately 50%). VSCC were identified in neuronal hybrid (NG108-15 and NCB-20) cells, but not in glial (C6BU-1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108-15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aminoquinolines; Animals; Bucladesine; Calcium; Cricetinae; Dihydropyridines; Fluorescent Dyes; Glioma; Humans; Hybrid Cells; Ion Channels; Membrane Potentials; Neuroblastoma; Potassium Chloride; Sodium; Tetrodotoxin; Veratridine

The effect of dihydropyridine agonists and antagonists on neuronal voltage sensitive calcium channels was investigated. The resting intracellular calcium concentration of synaptosomes prepared from whole brain was 110 +/- 9 nM, as assayed by the indicator quin 2. Depolarisation of the synaptosomes with K+ produced an immediate increase in [Ca2+]i. The calcium agonist Bay K 8644 and antagonist nifedipine did not affect [Ca2+]i under resting or depolarising conditions. In addition, K+ stimulated 45Ca2+ uptake into synaptosomes prepared from the hippocampus was insensitive to Bay K 8644 and PY 108-068 in normal or Na+ free conditions. In neuronally derived NG108-15 cells the enantiomers of the dihydropyridine derivative 202-791 showed opposite effects in modulating K+ stimulated 45Ca2+ uptake. (-)-R-202-791 inhibited K+ induced 45Ca2+ uptake with an IC50 of 100 nM and (+)-S-202-791 enhanced K+ stimulated uptake with an EC50 of 80 nM. These results suggest that synaptosomal voltage sensitive calcium channels either are of a different type to those found in peripheral tissues and cells of neural origin or that expression of functional effects of dihydropyridines requires different experimental conditions to those used here.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Aminoquinolines; Animals; Brain Chemistry; Calcium; Calcium Channel Blockers; Cell Line; Dihydropyridines; Electric Stimulation; Glioma; In Vitro Techniques; Ion Channels; Neuroblastoma; Neurons; Nifedipine; Potassium; Pyridines; Rats; Synaptosomes; Verapamil