digitonin and Adenocarcinoma

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.

Topics: Actins; Adenocarcinoma; Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Cytochalasin D; Cytoskeleton; Digitonin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Ionomycin; Lung Neoplasms; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Paclitaxel; Thapsigargin; Time Factors

Spicamycin (SPM), produced by Streptomyces alanosinicus, induces potent differentiation in a human leukemia cell line, HL60. One of the derivatives of SPM (SPM-D), KRN5500, has a wide range of antitumor activity against human cancer cell lines. We examined the cytotoxicity of SPM-D in small and non-small cell lung cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony assays. SPM-D was active against a wide range of lung cancer cell lines. All three cisplatin (CDDP)-resistant cell lines established in our laboratory (PC-9/CDDP, PC-14/CDDP, and H69/CDDP) showed collateral sensitivity to SPM-D with relative resistance values of 0.43, 0.34, and 0.32, respectively. Intracellular SPM-D in PC-14/CDDP was 35% higher than that for PC-14 suggesting that intracellular accumulation can explain the collateral sensitivity to SPM-D at least in PC-14/CDDP. On the other hand, in PC-9/CDDP cells, no increase of intracellular SPM-D accumulation was observed, but the conversion ratio of a metabolite (the amino nucleoside moiety of spicamycin binding with glycine, SAN-G) from SPM-D evaluated by TLC was higher as compared with that of parental PC-9 cells (45.5% versus 37%; PC-9/CDDP versus PC-9). The increased intracellular metabolism of SPM-D could explain the mechanism of collateral sensitivity in PC-9/CDDP cisplatin-resistant cell lines. To elucidate the determinant of the SPM-D-induced cytotoxicity, we established SPM-D-resistant cell lines, PC-9/SPM-D, PC-14/SPM-D, and H69/SPM-D, by exposing cells to stepwise increases in SPM-D concentration. The relative resistances of these sublines were more than 5000, 46.6, and 37.8 times those of the parental cell lines, respectively. The intracellular concentration of the active metabolite, SAN-G, was found to be decreased in the SPM-D-resistant sublines. This result indicates that the intracellular metabolism of SPM-D to SAN-G is one of the determinants of cellular sensitivity to SPM-D in these SPM-D-resistant cell lines. In conclusion, both drug accumulation and metabolism may contribute to the sensitivity/resistance to SPM-D and both may merit investigation.

Topics: Adenocarcinoma; Antibiotics, Antineoplastic; Carcinoma, Small Cell; Cell Division; Cell Membrane Permeability; Cisplatin; Digitonin; Drug Resistance; Drug Screening Assays, Antitumor; Humans; Intracellular Fluid; Lung Neoplasms; Purine Nucleosides; Tumor Cells, Cultured

The regulation of 15-pS Cl- channels by Ca(2+)-mobilizing agonists was investigated by simultaneous cell-attached patch and intracellular Ca2+ concentration ([Ca2+]i) measurements. Cells were loaded with a synthetic peptide made from the calmodulin binding domain of Ca2+/calmodulin-dependent protein kinase II. This caused inhibition of Cl- channel activity without any corresponding effect on either agonist-induced [Ca2+]i mobilization or K+ channel activation. Calmodulin therefore confers Ca2+ sensitivity to the 15-pS channel. When patches were excised from the cell, Cl- channel activity ran down. Channel rundown was not reversed by ATP or calmodulin. When recording from cell-attached patches of detergent-treated cells, similar phenomenology was observed. Therefore, other factors that are lost upon plasma membrane permeabilization are required for the functioning of Ca(2+)-dependent Cl- channels. After rundown of these channels, a large-conductance, multistate, Ca(2+)-insensitive Cl- channel was seen. The smallest subconductance state of this channel was of similar magnitude to that of the Ca(2+)-dependent Cl- channel. Furthermore, its voltage and halide sensitivities were similar to those reported for the 15-pS Cl- channel and Ca(2+)-dependent whole cell Cl- currents. Because this channel is not observed in the intact cell, this may be a remnant conductance of the Ca(2+)-sensitive 15-pS Cl- channel.

Topics: Adenocarcinoma; Calcium; Calmodulin; Cell Differentiation; Cell Membrane; Chloride Channels; Colonic Neoplasms; Digitonin; Egtazic Acid; Homeostasis; Humans; Ion Channels; Ionomycin; Kinetics; Meglumine; Membrane Potentials; Membrane Proteins; Neurotensin; Tumor Cells, Cultured