digitonin and Lung-Neoplasms

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.

Topics: Cell Line, Tumor; Cell Membrane Permeability; Cell Size; Cytoskeleton; Digitonin; Humans; Lung Neoplasms; Osmotic Pressure

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

The antitumor effect of isolated lung perfusion with cisplatin was limited because the intracellular platinum concentration did not increase sufficiently. To solve this problem, digitonin, a detergent, was chosen to increase cell permeability and enhance intracellular uptake and antitumor effect. This study was designed to investigate toxicity, pharmacokinetics, and efficacy of isolated lung perfusion with the combined use of digitonin and cisplatin in Fischer 344 rats.. Systemic and local toxicities of isolated lung perfusion treatment were evaluated on the basis of body weight change, survival rate, and histologic findings. The maximal tolerated dose of digitonin was determined by assessing survival on day 21 after contralateral pneumonectomy, body weight change, and histologic findings. Pharmacokinetics were observed in a solitary lung tumor nodule model by measuring platinum concentration in tumor and normal lung tissue. The antitumor effect was evaluated by the number of tumor nodules in the left lung 21 days after isolated lung perfusion. Isolated lung perfusion was performed 7 days after 1.0 x 10(6) methylcholanthrene sarcoma cells were injected into the external jugular vein.. The maximal tolerated dose of digitonin was 20 micromol/L. Platinum concentration of tumor nodules in the digitonin-cisplatin-treated rats was 20% higher than in the cisplatin-only group (5.48 +/- 0.64 microg/g tissue versus 4.50 +/- 1.09 microg/g tissue; p = 0.067). The number of pulmonary nodules decreased significantly by digitonin use (1.3 +/- 1.5 versus 9.7 +/- 2; p < 0.0001).. Isolated lung perfusion with digitonin and cisplatin in combination was performed safely and enhanced the antitumor effect. These drugs in combination show promise for enhancing the effect of clinical isolated lung perfusion.

Topics: Animals; Cell Survival; Cisplatin; Digitonin; Dose-Response Relationship, Drug; Drug Synergism; Infusions, Intra-Arterial; Lung; Lung Neoplasms; Male; Neoplasm Transplantation; Rats; Rats, Inbred F344; Sarcoma, Experimental; Tumor Cells, Cultured

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cell Division; Cell Membrane Permeability; Digitonin; DNA, Neoplasm; Drug Screening Assays, Antitumor; Humans; Lung Neoplasms; Mice; Oligonucleotides, Antisense; Tumor Cells, Cultured

The question of whether and to what extent the in vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16-40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O2 consumption rate supported by 0.4 mM tetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7-22% excess) for ADP + glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold.

Topics: Adenosine Diphosphate; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line; Cell Membrane Permeability; Digitonin; Electron Transport Complex IV; Glutamic Acid; Humans; Kinetics; Liver Neoplasms; Lung Neoplasms; Malates; Mitochondria; Multiple Myeloma; Neuroblastoma; Osteosarcoma; Oxygen Consumption; Potassium Cyanide; Tetramethylphenylenediamine; Tumor Cells, Cultured

Topics: Adenosine; Allopurinol; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Digitonin; Glucose; Glutathione; Humans; Insulin; Lung Neoplasms; Mannitol; Mitochondria; Organ Preservation Solutions; Oxygen Consumption; Potassium Chloride; Procaine; Raffinose; Reperfusion Injury; Rotenone; Tumor Cells, Cultured; Umbilical Veins

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 subcellular forms of cytidylyltransferase (EC 2.7.7.15) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97,690 +/- 10,175. Thus, the L-Form appears to be a dimer of the Mr 45,000 catalytic subunit. The f/f degrees value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284,000 +/- 25,000. The H-Form was dissociated into L-Form by incubation of cytosol at 37 degrees C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45,000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45,000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.

Topics: Adenoma; Animals; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry, Physical; Choline-Phosphate Cytidylyltransferase; Cytosol; Digitonin; Humans; Liver; Liver Neoplasms; Lung; Lung Neoplasms; Male; Molecular Weight; Nucleotidyltransferases; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Tumor Cells, Cultured