8-bromocyclic-gmp and Colonic-Neoplasms

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Colonic Neoplasms; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Resistance, Neoplasm; Drug Synergism; Drugs, Chinese Herbal; Humans; Hypoxia; Male; Mice, Inbred BALB C; Nitroso Compounds; Oxadiazoles; Oxazines; Phosphodiesterase 5 Inhibitors; Plant Extracts; Reactive Oxygen Species; Serum Albumin, Human; Soluble Guanylyl Cyclase; Vardenafil Dihydrochloride

Bacterial diarrheagenic heat-stable enterotoxins induce colon cancer cell cytostasis by targeting guanylyl cyclase C (GCC) signaling. Anticancer actions of these toxins are mediated by cyclic guanosine 3',5'-monophosphate (cGMP)-dependent influx of Ca2+ through cyclic nucleotide-gated channels. However, prolonged stimulation of GCC produces resistance in tumor cells to heat-stable enterotoxin-induced cytostasis. Resistance reflects rapid (tachyphylaxis) and slow (bradyphylaxis) mechanisms of desensitization induced by cGMP. Tachyphylaxis is mediated by cGMP-dependent protein kinase, which limits the conductance of cyclic nucleotide-gated channels, reducing the influx of Ca2+ propagating the antiproliferative signal from the membrane to the nucleus. In contrast, bradyphylaxis is mediated by cGMP-dependent allosteric activation of phosphodiesterase 5, which shapes the amplitude and duration of heat-stable enterotoxin-dependent cyclic nucleotide accumulation required for cytostasis. Importantly, interruption of tachyphylaxis and bradyphylaxis restores cancer cell cytostasis induced by heat-stable enterotoxins. Thus, regimens that incorporate cytostatic bacterial enterotoxins and inhibitors of cGMP-mediated desensitization offer a previously unrecognized therapeutic paradigm for treatment and prevention of colorectal cancer.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Toxins; Calcium; Cell Line, Tumor; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Enterotoxins; Escherichia coli Proteins; Guanylate Cyclase; Humans; Phosphoric Diester Hydrolases; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.

Topics: Amino Acid Oxidoreductases; Aminoquinolines; Arginine; Barium; Biological Transport; Calcium; Carbachol; Colon; Colonic Neoplasms; Cyclic GMP; Epithelium; Guanylate Cyclase; Humans; Indoles; Intestinal Mucosa; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Second Messenger Systems; Tumor Cells, Cultured

The present study demonstrates the activation of Cl- channels in HT29 cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca2+, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast whole-cell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with half-maximal effects at 0.4 mumol/l for ATP, 60 pmol/l for neurotensin and 0.8 mumol/l for carbachol. The depolarization, which was caused by Cl- conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295 +/- 32 pS to 1180 +/- 271 pS (ATP; 10 mumol/l, n = 21) and 192 +/- 37 pS to 443 +/- 128 pS (neurotensin; 1 nmol/l, n = 8). The effects of the agonists could be mimicked by ionomycin (0.2 mumol/l), suggesting that an increase in intracellular Ca2+ was responsible for the activation of Cl- channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10 +/- 0.8 mV and the cell-attached conductance increased from 228 +/- 29 pS to 410 +/- 65 (n = 26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mumol/l) did not prevent the effects of Ca(2+)-increasing hormones and of hypotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Calcium; Carbachol; Carcinoma; Chlorides; Colonic Neoplasms; Culture Media; Cyclic GMP; Cytosol; Hypotonic Solutions; Ion Channels; Ionomycin; Membrane Potentials; Neurotensin; Nitrobenzoates; Osmolar Concentration; Tumor Cells, Cultured