cyclic-gmp and Colonic-Neoplasms

Despite novel technologies, colon cancer remains undiagnosed and 25% of patients are diagnosed with metastatic colon cancer. Resistant to chemotherapeutic agents is one of the major problems associated with treating colon cancer which creates the need to develop novel agents targeting towards newer targets. A phosphodiesterase is a group of isoenzyme, which, hydrolyze cyclic nucleotides and thereby lowers intracellular levels of cAMP and cGMP leading to tumorigenic effects. Many in vitro and in vivo studies have confirmed increased PDE expression in different types of cancers including colon cancer. cAMP-specific PDE inhibitors increase intracellular cAMP that leads to activation of effector molecules-cAMP-dependent protein kinase A, exchange protein activated by cAMP and cAMP gated ion channels. These molecules regulate cellular responses and exert its anticancer role through different mechanisms including apoptosis, inhibition of angiogenesis, upregulating tumor suppressor genes and suppressing oncogenes. On the other hand, cGMP specific PDE inhibitors exhibit anticancer effects through cGMP dependent protein kinase and cGMP dependent cation channels. Elevation in cGMP works through activation of caspases, suppression of Wnt/b-catenin pathway and TCF transcription leading to inhibition of CDK and survivin. These studies point out towards the fact that PDE inhibition is associated with anti-proliferative, anti-apoptotic and anti-angiogenic pathways involved in its anticancer effects in colon cancer. Thus, inhibition of PDE enzymes can be used as a novel approach to treat colon cancer. This review will focus on cAMP and cGMP signaling pathways leading to tumorigenesis and the use of PDE inhibitors in colon cancer.

Topics: Animals; Apoptosis; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Humans; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Signal Transduction

Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

Topics: Animals; Carcinogenesis; Cell Cycle; Colonic Neoplasms; Cyclic GMP; Enterotoxins; Gastrointestinal Hormones; Genomics; Homeostasis; Hormones; Humans; Inflammation; Ligands; Mutation; Natriuretic Peptides; Paracrine Communication; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Treatment Outcome

Guanylyl cyclase C (GC-C) is predominantly expressed in intestinal epithelial cells and serves as the receptor for the gastrointestinal hormones guanylin and uroguanylin, and the heat-stable enterotoxin, the causative agent for Travellers' Diarrhea. Activation of GC-C results in an increase in intracellular levels of cGMP, which can regulate fluid and ion secretion, colon cell proliferation, and the gut immune system. This review highlights recent findings arising from studies in the GC-C knock-out mouse, along with enigmatic results obtained from the first descriptions of human disease caused by mutations in the GC-C gene. We provide some insight into these new findings and comment on areas of future study, which may enhance our knowledge of this evolutionarily conserved receptor and signaling system.

Topics: Animals; Colonic Neoplasms; Cyclic GMP; Enzyme Activation; Feeding Behavior; Homeostasis; Humans; Intestines; Mice; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide

The guanylin family of bioactive peptides consists of three endogenous peptides, including guanylin, uroguanylin and lymphoguanylin, and one exogenous peptide toxin produced by enteric bacteria. These small cysteine-rich peptides activate cell-surface receptors, which have intrinsic guanylate cyclase activity, thus modulating cellular function via the intracellular second messenger, cyclic GMP. Membrane guanylate cyclase-C is an intestinal receptor for guanylin and uroguanylin that is responsible for stimulation of Cl- and HCO3- secretion into the intestinal lumen. Guanylin and uroguanylin are produced within the intestinal mucosa to serve in a paracrine mechanism for regulation of intestinal fluid and electrolyte secretion. Enteric bacteria secrete peptide toxin mimics of uroguanylin and guanylin that activate the intestinal receptors in an uncontrolled fashion to produce secretory diarrhea. Opossum kidney guanylate cyclase is a key receptor in the kidney that may be responsible for the diuretic and natriuretic actions of uroguanylin in vivo. Uroguanylin serves in an endocrine axis linking the intestine and kidney where its natriuretic and diuretic actions contribute to the maintenance of Na+ balance following oral ingestion of NaCl. Lymphoguanylin is highly expressed in the kidney and myocardium where this unique peptide may act locally to regulate cyclic GMP levels in target cells. Lymphoguanylin is also produced in cells of the lymphoid-immune system where other physiological functions may be influenced by intracellular cyclic GMP. Observations of nature are providing insights into cellular mechanisms involving guanylin peptides in intestinal diseases such as colon cancer and diarrhea and in chronic renal diseases or cardiac disorders such as congestive heart failure where guanylin and/or uroguanylin levels in the circulation and/or urine are pathologically elevated. Guanylin peptides are clearly involved in the regulation of salt and water homeostasis, but new findings indicate that these novel peptides have diverse physiological roles in addition to those previously documented for control of intestinal and renal function.

Topics: Amino Acid Sequence; Animals; Colonic Neoplasms; Cyclic GMP; Diarrhea; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Intestinal Mucosa; Kidney Diseases; Molecular Sequence Data; Natriuretic Peptides; Peptides

Topics: Animals; Cell Proliferation; Chromatography, Liquid; Colonic Neoplasms; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Mice; Phosphodiesterase 5 Inhibitors; Sildenafil Citrate; Tandem Mass Spectrometry

There is growing interest in the potential use of phosphodiesterase (PDE) inhibitors for colorectal cancer (CRC) prevention and treatment. The present study has tested the idea that PDE inhibitors inhibit growth and viability of CRC cell lines by increasing cyclic guanosine monophosphate (cGMP) and activating cGMP-dependent protein kinase (PKG). Colon cancer cell lines and those with ectopic PKG2 expression were treated with membrane-permeable 8Br-cGMP or inhibitors of PDE5, PDE9, and PDE10a. Levels of cGMP capable of activating PKG were measured by immunoblotting for phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The effects of treatment on CRC cell proliferation and death were measured using hemocytometry with trypan blue. Treatment with 8Br-cGMP had no effect on CRC cell proliferation or death. Endogenous PKG activity was undetectable in any of the CRC cells, but expression of ectopic PKG2 conferred modest inhibition of proliferation but did not affect cell death. Extremely high concentrations of all the PDE inhibitors reduced proliferation in CRC cell lines, but none of them increased cGMP levels, and the effect was independent of PKG expression. The inability of the PDE inhibitors to increase cGMP was due to the lack of endogenous cGMP generating machinery. In conclusion, PDE inhibitors that target cGMP only reduce CRC growth at clinically unachievable concentrations, and do so independent of cGMP signaling through PKG. SIGNIFICANCE STATEMENT: A large number of in vitro studies have reported that PDE inhibitors block growth of colon cancer cells by activating cGMP signaling, and that these drugs might be useful for cancer treatment. Our results show that these drugs do not activate cGMP signaling in colon cancer cells due to a lack of endogenous guanylyl cyclase activity, and that growth inhibition is due to toxic effects of clinically unobtainable drug concentrations.

Topics: Cell Transformation, Neoplastic; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Humans; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Signal Transduction

A large body of evidence has demonstrated that cyclic-guanosine monophosphate (cGMP), signaling has anti-tumor effects that might be used for colon cancer prevention. The tumor-suppressive mechanism and the signaling components downstream of cGMP remain largely unknown. The present study has characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from human colon. PKG1 was detected in both normal and tumor tissue, where it localized exclusively to the lamina propria and stroma (respectively). In contrast, PKG2 localized specifically to the epithelium where its expression decreased markedly in tumors compared to matched normal tissue. Neither PKG isoform was detected at the RNA or protein level in established colon cancer cell lines. To test for a potential tumor-suppressor role of PKG2 in the colon epithelium, Prkg2 knockout (KO) mice were subjected to azoxymethane/dextran sulfate-sodium (AOM/DSS) treatment. PKG2 deficiency was associated with crypt hyperplasia (Ki67) and almost twice the number of polyps per mouse as wild-type (WT) siblings. In vitro culture of mouse colon epithelium as organoids confirmed that PKG2 was the only isoform expressed, and it was detected in both proliferating and differentiating epithelial compartments. Colon organoids derived from Prkg2 KO mice proliferated more rapidly and exhibited a reduced ability to differentiate compared to WT controls. Taken together our results highlight PKG2 as the central target of cGMP in the colon, where it suppresses carcinogenesis by controlling proliferation in an epithelial-cell intrinsic manner.

Topics: Animals; Azoxymethane; Carcinogenesis; Cell Proliferation; Colon; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Dextran Sulfate; Epithelium; Mice; Mice, Inbred C57BL; Mice, Knockout

This is the first work to use a newly designed Li

Topics: Animals; Aquaporin 1; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclic GMP; HT29 Cells; Humans; Ion Channel Gating; Ion Channels; Lithium; Oocytes; Signal Transduction; Xenopus laevis

Topics: Chemoprevention; Colonic Neoplasms; Cyclic GMP; Humans; Phosphodiesterase 5 Inhibitors; Receptors, Enterotoxin

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

Topics: Animals; Antioxidants; Blotting, Western; Cell Line, Tumor; Colonic Neoplasms; Cyclic GMP; Forkhead Box Protein O3; Humans; Immunohistochemistry; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Oxidative Stress; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcriptome

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3',5'-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of β-catenin to inhibit Wnt/β-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes.

Topics: Antineoplastic Agents; Apoptosis; Caco-2 Cells; Carbolines; Cell Line; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Cyclin D1; HCT116 Cells; HT29 Cells; Humans; Inhibitor of Apoptosis Proteins; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Sildenafil Citrate; Sulfones; Sulindac; Survivin; Tadalafil; Wnt Signaling Pathway

Nitric oxide (NO), an uncharged free radical is implicated in various physiological and pathological processes. The present study is an investigation on the effect of NO on proliferation, apoptosis and migration of colon cancer cells. Colon adenocarcinoma cells, WiDr, were used for the in vitro experiments. Tissues from colon adenocarcinoma, adjacent normal and inflammatory tissue and lymph node with metastasis were evaluated for iNOS, MMP-2/9 and Fra-1/Fra-2. NO increases the proliferation of cancer cells and simultaneously prevents apoptosis. Expression of MMP-2/9, RhoB and Rac-1 was enhanced by NO in a time dependent manner. Further, NO increased phosphorylation of ERK1/2 and induced nuclear translocation of Fra-1 and Fra-2. Electrophoretic mobility shift analysis and use of deletion mutant promoter constructs identified role of AP-1 in NO-mediated regulation of MMP-2/9. iNOS, MMP-2/9, Fra-1 and Fra-2 in normal and colon adenocarcinoma tissues were analyzed and it was found that increased expression of these proteins in cancer when compared to normal provides support to our in vitro findings. The study showed that the NO-cGMP-PKG promotes MMP-2/9 expression by activating ERK-1/2 and AP-1. This study reveals the insidious role of NO in imparting tumor aggressiveness.

Topics: Adenocarcinoma; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Nitric Oxide; Phosphorylation; Real-Time Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Up-Regulation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3',5',-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of β-catenin as evident by its ability to reduce β-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Blotting, Western; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Humans; Models, Molecular; Phosphodiesterase 5 Inhibitors; RNA, Small Interfering; Sulindac; TCF Transcription Factors; Transcriptional Activation

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.

Topics: Animals; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calreticulin; Colonic Neoplasms; Cyclic GMP; Cytoskeleton; Dendritic Cells; Doxorubicin; Drug Resistance, Neoplasm; HT29 Cells; Humans; Nitric Oxide; Nitric Oxide Synthase; Phagocytosis

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity, but toxicity resulting from cyclooxygenase (COX) inhibition limits their clinical use for chemoprevention. Studies suggest that the mechanism may be COX independent, although alternative targets have not been well defined. Here, we show that the NSAID sulindac sulfide (SS) inhibits cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) activity in colon tumor cell lysates at concentrations that inhibit colon tumor cell growth in vitro and in vivo. A series of chemically diverse NSAIDs also inhibited cGMP hydrolysis at concentrations that correlate with their potency to inhibit colon tumor cell growth, whereas no correlation was observed with COX-2 inhibition. Consistent with its selectivity for inhibiting cGMP hydrolysis compared with cyclic AMP hydrolysis, SS inhibited the cGMP-specific PDE5 isozyme and increased cGMP levels in colon tumor cells. Of numerous PDE isozyme-specific inhibitors evaluated, only the PDE5-selective inhibitor MY5445 inhibited colon tumor cell growth. The effects of SS and MY5445 on cell growth were associated with inhibition of β-catenin-mediated transcriptional activity to suppress the synthesis of cyclin D and survivin, which regulate tumor cell proliferation and apoptosis, respectively. SS had minimal effects on cGMP PDE activity in normal colonocytes, which displayed reduced sensitivity to SS and did not express PDE5. PDE5 was found to be overexpressed in colon tumor cell lines as well as in colon adenomas and adenocarcinomas compared with normal colonic mucosa. These results suggest that PDE5 inhibition, cGMP elevation, and inhibition of β-catenin transcriptional activity may contribute to the chemopreventive properties of certain NSAIDs.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; beta Catenin; Blotting, Western; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; HCT116 Cells; HT29 Cells; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Male; Mice; Mice, Nude; Sulindac; Transcription, Genetic; Xenograft Model Antitumor Assays

Matrix metalloproteinase-9 (MMP-9) produced by colorectal cancer cells is a critical determinant of metastatic disease progression and an attractive target for antimetastatic strategies to reduce colon cancer mortality. Cellular signaling by cyclic GMP (cGMP) regulates MMP-9 dynamics in various cell systems, and the bacterial enterotoxin receptor guanylyl cyclase C (GCC), the principle source of cGMP in colonocytes, which is overexpressed in colorectal cancers, inhibits tumor initiation and progression in the intestine. Here, we show that ligand-dependent GCC signaling through cGMP induces functional remodeling of cancer cell MMP-9 reflected by a compartmental redistribution of this gelatinase, in which intracellular retention resulted in reciprocal extracellular depletion. Functional remodeling of MMP-9 by GCC signaling reduced the ability of colon cancer cells to degrade matrix components, organize the actin cytoskeleton to form locomotory organelles and spread, and hematogenously seed distant organs. Of significance, GCC effects on cancer cell MMP-9 prevented establishment of metastatic colonies by colorectal cancer cells in the mouse peritoneum in vivo. Because endogenous hormones for GCC are uniformly deficient in intestinal tumors, reactivation of dormant GCC signaling with exogenous administration of GCC agonists may represent a specific intervention to target MMP-9 functions in colon cancer cells. The notion that GCC-mediated regulation of cancer cell MMP-9 disrupts metastasis, in turn, underscores the unexplored utility of GCC hormone replacement therapy in the chemoprevention of colorectal cancer progression.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Cyclic GMP; Epithelial Cells; Guanylate Cyclase; Humans; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neoplasm Metastasis; Peritoneal Neoplasms; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction

Although it is often assumed that the antitumor effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to inhibition of cyclooxgenase (COX) activity, specifically COX-2, there is accumulating evidence that COX-2 independent mechanisms can also play an important role. Studies with sulindac sulfone (Aptosyn) and related derivatives have revealed a novel pathway of tumor growth inhibition and apoptosis mediated by activation of the guanosine 3',5' monophosphate (cGMP)-dependent enzyme protein kinase G (PKG). The present study indicates that concentrations of the NSAIDs celecoxib, indomethacin, and meclofenamic acid that inhibit growth of SW480 human colon cancer cells inhibit subcellular cGMP-phosphodiesterase (PDE) enzymatic activity and in intact cells induce a two- to threefold increase in intracellular levels of cGMP. This is associated with phosphorylation of the protein VASP, a marker of PKG activation, activation of JNK1 and a decrease in cellular levels of cyclin D1; effects seen with other agents that cause activation of PKG in these cells. On the other hand even a high concentration of the COX-2 specific inhibitor rofecoxib (500 microM) did not inhibit growth of SW480 cells. Nor did rofecoxib inhibit cGMP-PDE activity or cause other changes related to PKG activation in these cells. Since activation of the PKG pathways by celecoxib, indomethacin, and meclofenamic acid in this cell culture system required high concentrations of these compounds, it remains to be determined whether activation of this pathway contributes to the in vivo antitumor effects of specific NSAIDs.

Topics: Apoptosis; Celecoxib; Cell Adhesion Molecules; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclin D1; Cyclooxygenase Inhibitors; Enzyme Activation; Humans; Indomethacin; Lactones; Meclofenamic Acid; Microfilament Proteins; Mitogen-Activated Protein Kinase 8; Phosphoproteins; Phosphorylation; Pyrazoles; Sulfonamides; Sulfones; Tumor Cells, Cultured

Patients with chronic inflammatory bowel disease have a high risk of colon cancer. The molecules that initiate and promote colon cancer and the cancer pathways altered remain undefined. Here, using in vitro models and a mouse model of colitis, we show that nitric oxide (NO) species induce retinoblastoma protein (pRb) hyperphosphorylation and inactivation, resulting in increased proliferation through the pRb-E2F1 pathway. NO-driven pRb hyperphosphorylation occurs through soluble guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling and is dependent on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK/ERK and phosphatidylinositol 3-kinase/AKT pathways. Our results reveal a link between NO and pRb inactivation and provide insight into molecules that can be targeted in the prevention of the inflammation-to-cancer sequence.

Topics: Animals; Colitis; Colonic Neoplasms; Cyclic GMP; E2F1 Transcription Factor; Guanylate Cyclase; HCT116 Cells; HT29 Cells; Humans; MAP Kinase Kinase Kinases; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Spermine

Exisulind (sulindac sulfone) and three highly potent derivatives, OSI-461 (CP461), OSIP486823 (CP248), and OSIP487703, inhibit growth and induce apoptosis in SW480 human colon cancer cells, with IC(50)s of 200, 2, 0.1, and 0.003 micromol/L, respectively. The latter three compounds, but not exisulind, induce marked M-phase cell cycle arrest in these cells. This effect seems to be independent of the known ability of these compounds to cause activation of protein kinase G. When tested at twice their IC(50) concentration for growth inhibition, OSI-461, OSIP486823, and OSIP487703 cause depolymerization of microtubules in interphase cells, inhibit spindle formation in mitotic cells, and induce multinucleated cells. In vitro tubulin polymerization assays indicate that all three compounds interact with tubulin directly to cause microtubule depolymerization and/or inhibit de novo tubulin polymerization. These results suggest that the dual effects of OSI-461, OSIP486823, and OSIP487703 on impairment of microtubule functions and protein kinase G activation may explain the potent antiproliferative and apoptotic effects of these compounds in cancer cells.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; Mice; Microtubules; Mitosis; Spindle Apparatus; Sulindac; Thionucleotides; Tubulin; Tumor Cells, Cultured

The nonsteroidal anti-inflammatory drug sulindac is metabolized to sulindac sulfone (exisulind), an antineoplastic agent that inhibits growth and induces apoptosis in solid tumors. In colon cancer cells, the antineoplastic effects of exisulind have been attributed, in part, to induction of cyclic guanosine 3',5'-monophosphate (cGMP) signaling through inhibition of cGMP-specific phosphodiesterases, which elevates intracellular cGMP, and novel expression of cGMP-dependent protein kinase (PKG) Ibeta, the presumed downstream effector mediating apoptosis. Here, inhibition of proliferation and induction of cell death by exisulind was dissociated from cGMP signaling in human colon cancer cells. Accumulation of intracellular cGMP produced by an exogenous cell-permeant analogue of cGMP or a potent agonist of guanylyl cyclase C yielded cytostasis without cell death. Surprisingly, the antiproliferative effects of induced cGMP accumulation were paradoxically less than additive, rather than synergistic, when combined with exisulind. Further, although exisulind induced expression of PKG Ibeta, it did not elevate intracellular cGMP and its efficacy was not altered by inhibition or activation of PKG I. Rather, PKG I induced by exisulind may mediate desensitization of cytostasis induced by cGMP. Thus, cytotoxic effects of exisulind are independent of cGMP signaling in human colon cancer cells. Moreover, combination therapies, including exisulind and agents that induce cGMP signaling, may require careful evaluation in patients with colon cancer.

Topics: Antineoplastic Agents; Caco-2 Cells; Cell Death; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Flow Cytometry; Gene Expression Regulation, Neoplastic; Guanylate Cyclase; Humans; Ligands; Models, Biological; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Signal Transduction; Sulindac; Tumor Cells, Cultured

Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, followed by irradiation with visible light, which induces cell death and apoptosis. As an important second messenger, free calcium is involved in the regulation of several cellular processes. However, the role of calcium signal in the cells after PDT is less clear. This study was to explore the role of calcium signal in apoptosis and protective mechanism of colon cancer cell line SW480 in response to 5-aminolevulinic acid (ALA)-PDT.. SW480 cells were divided into control group, light group, ALA group, and ALA-PDT group. Cell apoptosis was detected by TUNEL assay. The changes of intracellular Ca(2+) concentration were observed by confocal laser scanning microscopy (CLSM). Intracellular cAMP and cGMP concentrations were detected by radioimmunoassay. The expression of calmodulin in SW480 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of protein products and phosphorylated protein products of MEK and ERK1/2 was detected by Western blot.. Apoptosis indexes of SW480 cells at 30 min and 60 min after PDT were (25.26+/-5.04)% and (50.45+/-7.85)%, respectively. CLSM revealed that intracellular Ca(2+) concentration was 100.00+/-19.83 at 10 min after PDT, but 185.40+/-18.90 at 20 min after PDT (P<0.01). cAMP concentration of ALA-PDT group was (3.215+/-0.245) pmol/L at 30 min after irradiation, which was significantly higher than those of other groups (P<0.001). The relative contents of CaM gene of ALA-PDT group at 30, 60 and 90 min after PDT were significantly higher than those of control group, light group, and ALA group (12.60+/-1.84, 11.39+/-1.13, and 12.77+/-1.35 vs. 3.97+/-0.29, 4.28+/-0.39, and 4.51+/-0.44, P<0.001). ERK pathway of SW480 cells was activated after ALA-PDT.. Calcium signal plays an important role in ALA-PDT-induced apoptosis of SW480 cells, and can induce protective mechanism of SW480 cells by activating ERK pathway.

Topics: Aminolevulinic Acid; Apoptosis; Calcium; Calmodulin; Cell Line, Tumor; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Humans; Microscopy, Confocal; Photochemotherapy; Photosensitizing Agents; Signal Transduction

Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3':5'-cyclic monophosphate-independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.

Topics: Antibiotics, Antineoplastic; Colonic Neoplasms; Cyclic GMP; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; HT29 Cells; Humans; Isoenzymes; Multidrug Resistance-Associated Proteins; Nitrates; Nitric Oxide; Nitric Oxide Synthase; S-Nitroso-N-Acetylpenicillamine

The novel cyclic dinucleotide, 3',5'-cyclic diguanylic acid, cGpGp (c-di-GMP), is a naturally occurring small molecule that regulates important signaling mechanisms in prokaryotes. Recently, we showed that c-di-GMP has "drug-like" properties and that c-di-GMP treatment might be a useful antimicrobial approach to attenuate the virulence and pathogenesis of Staphylococcus aureus and prevent or treat infection. In the present communication, we report that c-di-GMP (50 microM) has striking properties regarding inhibition of cancer cell proliferation in vitro. c-di-GMP inhibits both basal and growth factor (acetylcholine and epidermal growth factor)-induced cell proliferation of human colon cancer (H508) cells. Toxicity studies revealed that exposure of normal rat kidney cells and human neuroblastoma cells to c-di-GMP at biologically relevant doses showed no lethal cytotoxicity. Cyclic dinucleotides, such as c-di-GMP, represent an attractive and novel "drug-platform technology" that can be used not only to develop new antimicrobial agents, but also to develop novel therapeutic agents to prevent or treat cancer.

Topics: Acetylcholine; Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Dose-Response Relationship, Drug; Epidermal Growth Factor; Growth Substances; Humans; Kidney; Models, Molecular; Neuroblastoma; Rats; Staphylococcus aureus

Guanylyl cyclase C and accumulation of cGMP induced by bacterial heat-stable enterotoxins (STs) promote colon cancer cell cytostasis, serving as a tumor suppressor in intestine. Conversely, capacitative calcium entry through store-operated calcium channels (SOCs) is a key signaling mechanism that promotes colon cancer cell proliferation. The present study revealed that proliferative signaling by capacitative calcium entry through SOCs opposes and is reciprocally coupled to cytostasis mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Elimination of capacitative calcium entry employing 2-aminoethoxydiphenylborate (2-APB), a selective inhibitor of SOCs, potentiated cytostasis induced by ST. Opposition of ST-induced cytostasis by capacitative calcium entry reflects reciprocal inhibition of guanylyl cyclase C signaling. Calcium entry through SOCs induced by the calcium-ATPase inhibitor thapsigargin or the receptor agonists UTP or carbachol inhibited guanylyl cyclase C-dependent cGMP accumulation. This effect was mimicked by the calcium ionophore ionomycin and blocked by 2-APB and intracellular 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a chelator of calcium. Moreover, regulation by capacitative calcium entry reflected ligand-dependent sensitization of guanylyl cyclase C to inhibition by that cation. Although basal catalytic activity was refractory, ST-stimulated guanylyl cyclase C was inhibited by calcium, which antagonized binding of magnesium to allosteric sites required for receptor-effector coupling. These observations demonstrate that reciprocal regulation of guanylyl cyclase C signaling by capacitative calcium entry through SOCs represents one limb of a coordinated mechanism balancing colon cancer cell proliferation and cytostasis. They suggest that combining guanylyl cyclase C agonists and SOC inhibitors offers a novel paradigm for cGMP-directed therapy and prevention for colorectal tumors.

Topics: Bacterial Toxins; Calcium; Calcium Channels; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Guanylate Cyclase; Humans; Signal Transduction

Recent studies indicate that the induction of apoptosis in human colon cancer cells by certain nonsteroidal antiinflammatory drugs involves increased expression of 15-LOX-1 and synthesis of its major product 13-S-hydroxyoctadecadienoic acid (13-S-HODE). Evidence was obtained that this occurs via a cyclooxygenase-2 (COX-2)-independent mechanism, but the actual mechanism of induction of 15-LOX-1 by these compounds is not known. There is extensive evidence that treatment of SW480 human colon cancer cells with sulindac sulfone (Exisulind, Aptosyn) or the related derivative OSI-461, both of which inhibit cyclic GMP (cGMP)-phosphodiesterases but lack COX-2 inhibitory activity, causes an increase in intracellular levels of cGMP, thus activating protein kinase G (PKG), which then activates pathways that lead to apoptosis. Therefore, in the present study, we examined the effects of various agents that cause increased cellular levels of cGMP on the expression of 15-LOX-1 in SW480 human colon cancer cells. Treatment of the cells with Exisulind, sulindac sulfide, OSI-461, the guanylyl cyclase activator YC-1, or the cell-permeable cGMP compound 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) caused an increase in cellular levels of 15-LOX-1. Exisulind, OSI-461, and 8-pCPT-cGMP also increased mRNA levels of 15-LOX-1, suggesting that the effects were at the level of transcription. The cGMP-phosphodiesterase inhibitors and YC-1 increased the production of 13-S-HODE, which is the linoleic acid metabolite of 15-LOX-1. Treatment of SW480 cells with the PKG inhibitor Rp-8-pCPT-cGMP blocked Exisulind-induced 15-LOX-1 expression. Furthermore, derivatives of SW480 cells that were engineered to stably overexpress wild-type PKG Ibeta displayed increased cellular levels of 15-LOX-1 when compared with vector control cells. Taken together, these results provide evidence that the cGMP/PKG pathway can play an important role in the induction of 15-LOX-1 expression by nonsteroidal antiinflammatory drugs and related agents.

Topics: Arachidonate 15-Lipoxygenase; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Drug Interactions; Enzyme Activation; Humans; RNA, Small Interfering; Sulindac; Thionucleotides; Transfection; Up-Regulation

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

Ras mutations are present in approximately 50% of human colorectal tumors. We have previously shown that transfection of a non-tumorigenic rat intestinal epithelial cell line, IEC18, by the K-Ras oncogene (R1 cells), resulted in malignant cell transformation. Utilizing the constantly active Ras signaling pathway to selectively target transformed but not normal cells is a plausible goal.. To selectively kill Ras transformed cells by over expressing a lethal gene using a Ras-responsive promoter.. IEC18, R1 and a number of colon cancer cell lines were transfected with luciferase (Luc) reporter gene under the control of different Ras-responsive elements. The Ras-responsive promoter Py2 contains two copies of adjacent Ets and AP I binding sites followed by a minimal promoter. Apoptotic genes (bax, caspase-8 and PKG) were cloned into the Py2 plasmids. RI cells co-transfected with expression constructs and a selected vector and then grown for 3 weeks under selection.. R1, SW480 and HCT116 with mutated c-K-Ras expressed high level of Luc activity following transfection with the Py2 element. IEC18 cell lines that do not contain this mutation expressed negligible low Luc activity. Following transfection of SW480 and R1 cells with Py2-bax, caspase-8 and PKG, there was a significant reduction in the number of colony formation.. 1. Selective over-expression of pro-apoptotic genes, inhibits the growth of Ras transformed cells, and not normal cells. 2. This gene approach therapy may become a useful, effective and safe to target Ras mutated tumor cells with sparing of the normal cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspases; Cell Line, Tumor; Colonic Neoplasms; Cyclic GMP; Gene Targeting; Genes, ras; Genes, Reporter; Genetic Therapy; Humans; Luciferases; Plasmids; Signal Transduction; Transfection; Tumor Stem Cell Assay

Mortality from colon cancer is significant with an expected 30,350 colon cancer deaths in 2005 with current treatment(s). Long-acting natriuretic peptide, vessel dilator, kaliuretic peptide, and atrial natriuretic peptide have significant anticancer effects in breast and pancreatic adenocarcinomas.. Whether these peptide hormones have anticancer effects in colon adenocarcinoma cells and whether these effects are specifically mediated by cyclic GMP has not been determined.. These peptide hormones were evaluated for anticancer effects in human colon adenocarcinoma cells and to determine whether their anticancer effects are specifically mediated by cyclic GMP.. There was a 89-97% decrease (p <0.001 for each) in colon adenocarcinoma cells within 24 h with 1 mM of these peptide hormones. There was a significant (p <0.05) decrease in human colon cancer cell number with each 10-fold increase in concentration from 1 to 1,000 microM (i.e., 1 mM) of these four peptide hormones without any proliferation in the 3 d following this decrease. These same hormones decreased DNA synthesis 65-83% (p <0.001). Cyclic GMP antibody inhibited 75- 80% of these peptides' ability to decrease colon adenocarcinoma cell number and inhibited 92-96% of their DNA synthesis effects and 97% of cyclic GMP's effects. Western blots revealed that for the first time natriuretic peptide receptors (NPR) A and C were present in colon adenocarcinoma cells.. Four peptide hormones eliminate up to 97% of colon cancer cells within 24 h with their DNA effects specifically mediated by cyclic GMP.

Topics: Adenocarcinoma; Atrial Natriuretic Factor; Cell Count; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Colonic Neoplasms; Cyclic GMP; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Natriuretic Peptides; Peptide Fragments; Protein Precursors

Epidemiological and preclinical studies demonstrate that consumption of diets high in omega-3 polyunsaturated fatty acids reduces the risk of colon cancer. Inhibition of colon carcinogenesis by omega-3 polyunsaturated fatty acids is mediated through modulation of more than one signaling pathway that alters the expression of genes involved in colon cancer growth. In our earlier studies on global gene expression with cDNA microarrays, we have shown that treatment of CaCo-2 colon cancer cells with docosahexaenoic acid (DHA) down-regulated the prostaglandin family of genes, as well as cyclooxygenase 2 expression and several cell cycle-related genes, whereas it up-regulated caspases 5, 8, 9, and 10 that are associated with apoptosis. It is known that nitric oxide activates the cyclooxygenase 2 enzyme, which plays a pivotal role in the progression of colon cancer via prostaglandin synthesis and angiogenesis. The present study was undertaken to examine the multifaceted role of DHA in the expression of inducible nitric oxide synthase (iNOS) and of related proinflammatory genes, as those have been shown to play a role in tumor progression. In addition, we aimed to identify associated target genes by DNA microarray, reverse transcription-PCR analysis, and cellular localization of iNOS expression in CaCo-2 cells. Results of this study demonstrate that treatment with DHA down-regulates iNOS in parallel with a differential expression and down-regulation of IFNs, cyclic GMP, and nuclear factor kappa B isoforms. More importantly, our findings clearly demonstrate the up-regulation of cyclin-dependent kinase inhibitors p21((Waf1/Cip1)) and p27, differentiation-associated genes such as alkaline phosphatases, and neuronal differentiation factors. These finding strongly suggest that the antitumor activity of DHA may be attributed, at least in part, to an effect on iNOS regulatory genes. In addition, our results indicate the presence of specific gene expression profiles in human colon cancer that can be used as molecular targets for chemopreventive agents.

Topics: Apoptosis; Blotting, Western; Caco-2 Cells; Cell Differentiation; Cell Division; Cell Nucleus; Colonic Neoplasms; Cyclic GMP; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cyclooxygenase 2; Docosahexaenoic Acids; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Interferons; Isoenzymes; Membrane Proteins; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oligonucleotide Array Sequence Analysis; Prostaglandin-Endoperoxide Synthases; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors

The heat-stable enterotoxin peptides (ST) produced by enterotoxigenic Escherichia coli are one of the major causes of transitory diarrhea in the developing world. Toxin binding to its receptor, guanylyl cyclase C (GC-C), results in receptor activation and the production of high intracellular levels of cGMP. GC-C is expressed in two differentially glycosylated forms in intestinal epithelial cells. Prolonged exposure of human colonic cell lines to ST peptides induces cellular refractoriness to the ST peptide, in terms of intracellular cGMP accumulation. We have investigated the mechanism of cellular desensitization in human colonic Caco2 cells, and observe that exposure of cells to ST leads to a time and dose-dependent inability of cells to respond to the peptide in terms of GC-C stimulation, both in whole cells and membranes prepared from desensitized cells. This is concomitant with a 50% reduction in ST-binding activity in desensitized cells. Desensitization was correlated with a loss of the plasma membrane-associated, hyperglycosylated 145 kDa form of GC-C, while the predominant 130 kDa form, localized both on the plasma membrane and the endoplasmic reticulum, continued to be present in ST-treated cells. Desensitized cells recovered ST-responsiveness on removal of the ST peptide, which was correlated with a reappearance of the 145 kDa form on the cell surface, following processing of the endoplasmic reticulum-associated pool of the 130 kDa form. Selective internalization of the 145 kDa form of the receptor was required for cellular desensitization, as ST-treatment of cells at 4 degrees C did not lead to refractoriness. We therefore show a novel means of regulation of cellular responsiveness to the ST peptide, whereby altering cellular levels of the differentially glycosylated forms of GC-C can lead to differential ligand-mediated activation of the receptor.

Topics: Bacterial Toxins; Blotting, Western; Caco-2 Cells; Cell Membrane; Colonic Neoplasms; Cyclic GMP; Dose-Response Relationship, Drug; Enterotoxins; Epithelial Cells; Escherichia coli Proteins; Glycosylation; Guanylate Cyclase; Hot Temperature; Humans; Molecular Weight; Precipitin Tests; Protein Binding; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Time Factors; Tumor Cells, Cultured

Nitric oxide (NO)-releasing aspirin (ASA), consisting of a traditional ASA molecule to which a NO-donating moiety is covalently bound, is a promising colon cancer chemopreventive agent. NO-ASA inhibits colon cancer cell growth more potently than ASA by inhibiting cell proliferation and enhancing cell killing. We examined in cultured human colon cancer cells the effect of NO-ASA on the beta-catenin/T-cell factor signaling pathway, nuclear factor-kappaB, and NO synthase 2 and on cyclooxygenase (COX) expression, all presumed to participate in colon carcinogenesis. Besides inhibiting cell growth, NO-ASA inhibited the beta-catenin/T-cell factor signaling pathway (IC(50), 1.1 microM), nuclear factor-kappaB DNA binding (IC(50), 7.5 microM), and NO synthase 2 expression (IC(50), 2 microM). Interestingly, NO-ASA induced COX-2 expression, although it had no effect on COX-1. COX-2 induction was accompanied by increased prostaglandin E(2) production. These effects occurred at NO-ASA concentrations below or near its IC(50) for cell growth (IC(50), 2-50 microM). The metabolism of NO-ASA by these cells is characterized by a rapid deacetylation step and the formation of a conjugate with glutathione. NO-ASA had no effect on intracellular cyclic GMP concentrations. We propose a model incorporating the pleiotropic effects of NO-ASA on cell signaling and postulate that collectively these effects may contribute to its strong chemopreventive effect.

Topics: Aspirin; beta Catenin; Cell Nucleus; Colonic Neoplasms; Cyclic GMP; Cyclooxygenase 2; Cytoskeletal Proteins; DNA, Neoplasm; HT29 Cells; Humans; Isoenzymes; Membrane Proteins; NF-kappa B; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin-Endoperoxide Synthases; Signal Transduction; Trans-Activators; Transcription Factors

Hydrophobic bile acids such as deoxycholate are known tumor promoters in the gastrointestinal tract. We have previously shown that deoxycholate induces apoptosis in colon epithelial cells and that these cells can be made resistant to deoxycholate-induced apoptosis. We now show that the nitric oxide synthase/nitric oxide/guanylate cyclase/cyclic guanosine monophosphate/cGMP-activated protein kinase (NOS/NO/GC/cGMP/PKG) signaling module contributes, in part, to the observed resistance of the cultured DOC-resistant colon epithelial cells (HCT-116R) using pharmacological inhibitors/antagonists (NS2028, Rp-8pCPT-cGMP, KT5823) of members of this signaling module. A novel finding from this study is the caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis of deoxycholate-sensitive HCT-116SA cells and the absence of guanylate cyclase alpha 1 cleavage in deoxycholate-treated HCT-116R resistant cells using Western blot analyses. This cleavage was specific to caspases as lysosomal, proteasomal, serine protease, cathepsin and calpain inhibitors failed to prevent the cleavage, whereas a general caspase inhibitor and a specific caspase-6 inhibitor did prevent guanylate cyclase alpha 1 cleavage.

Topics: Animals; Apoptosis; Caspase 6; Caspases; Colonic Neoplasms; Cyclic GMP; Deoxycholic Acid; Enzyme Inhibitors; Epithelial Cells; Guanylate Cyclase; Humans; Nitric Oxide Donors; Nitric Oxide Synthase; Reactive Oxygen Species; Tumor Cells, Cultured

These studies report on the activation and induction of cGMP-dependent protein kinase (PKG) by exisulind and analogs and test the hypothesis that PKG is involved in the induction of apoptosis in colon tumor cells. Exisulind and analogs are proapoptotic drugs developed as inhibitors of cGMP phosphodiesterase gene families 5 and 2 that have been shown to sustain increased cGMP in SW480 and HT29 cells. At concentrations that induced apoptosis, both exisulind and CP461 increased PKG activity in SW480 cell supernatants. PKG activation was dose-dependent and sustained. Activation of PKG by exisulind and analogs was also seen in the colon tumor cell lines HT29, T84, and HCT116. The guanylyl cyclase activators YC-1 and guanylin increased PKG activity secondary to increased cellular cGMP and induced apoptosis in colon tumor cells. Exisulind and CP461 had no direct effect on purified PKG activity or on basal and stimulated PKG activity from cell supernatants. An additional effect of exisulind after 8 h of drug treatment was a dose-dependent increase of PKG Ibeta protein expression. beta-Catenin, a potential new substrate for PKG, whose regulation influences apoptosis, was phosphorylated by PKG in vitro. 32P-labeled cells treated with exisulind showed increased phosphorylation of beta-catenin. These data indicate that exisulind and analogs activate and induce PKG, resulting in increased phosphorylation of beta-catenin and enhanced apoptosis to promote colon tumor cell death.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Apoptosis; beta Catenin; Blotting, Western; Cloning, Molecular; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Cytoskeletal Proteins; DNA Fragmentation; Enzyme Activators; Gastrointestinal Hormones; Humans; Mutation; Natriuretic Peptides; Peptides; Phosphoric Diester Hydrolases; Phosphorylation; Radioimmunoassay; Sulindac; Trans-Activators; Tumor Cells, Cultured

Sulindac sulfone (exisulind), although a nonsteroidal anti-inflammatory drug derivative, induces apoptosis in tumor cells by a mechanism that does not involve cyclooxygenase inhibition. SW480 colon tumor cells contain guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) isoforms of the PDE5 and PDE2 gene families that are inhibited by exisulind and new synthetic analogues. The analogues maintain rank order of potency for PDE inhibition, apoptosis induction, and growth inhibition. A novel mechanism for exisulind to induce apoptosis is studied involving sustained increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction not found with selective PDE5 or most other PDE inhibitors. Accumulated beta-catenin, shown to be a substrate for PKG, is decreased by exisulind, suggesting a mechanism to explain apoptosis induction in neoplastic cells harboring adenomatous polyposis coli gene mutations.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Antineoplastic Agents; Apoptosis; beta Catenin; Cadherins; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytoskeletal Proteins; Enzyme Activation; Humans; Isoenzymes; Kinetics; Phosphodiesterase Inhibitors; Piperidines; Protein Kinases; Quinazolines; Sulindac; Trans-Activators; Tumor Cells, Cultured

The enteric peptides, guanylin and uroguanylin, are local regulators of intestinal secretion by activation of receptor-guanylate cyclase (R-GC) signaling molecules that produce cyclic GMP (cGMP) and stimulate the cystic fibrosis transmembrane conductance regulator-dependent secretion of Cl- and HCO3-. Our experiments demonstrate that mRNA transcripts for guanylin and uroguanylin are markedly reduced in colon polyps and adenocarcinomas. In contrast, a specific uroguanylin-R-GC, R-GCC, is expressed in polyps and adenocarcinomas at levels comparable with normal colon mucosa. Activation of R-GCC by uroguanylin in vitro inhibits the proliferation of T84 colon cells and elicits profound apoptosis in human colon cancer cells, T84. Therefore, down-regulation of gene expression and loss of the peptides may interfere with renewal and/or removal of the epithelial cells resulting in the formation of polyps, which can progress to malignant cancers of the colon and rectum. Oral replacement therapy with human uroguanylin was used to evaluate its effects on the formation of intestinal polyps in the Min/+ mouse model for colorectal cancer. Uroguanylin significantly reduces the number of polyps found in the intestine of Min/+ mice by approximately 50% of control. Our findings suggest that uroguanylin and guanylin regulate the turnover of epithelial cells within the intestinal mucosa via activation of a cGMP signaling mechanism that elicits apoptosis of target enterocytes. The intestinal R-GC signaling molecules for guanylin regulatory peptides are promising targets for prevention and/or therapeutic treatment of intestinal polyps and cancers by oral administration of human uroguanylin.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli; Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Apoptosis; Caco-2 Cells; Colonic Neoplasms; Cyclic GMP; Down-Regulation; Female; Gastrointestinal Hormones; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Molecular Sequence Data; Natriuretic Peptides; Peptides; Receptors, Cell Surface; RNA, Messenger; Tumor Cells, Cultured

Sulindac sulfone (Exisulind) induces apoptosis and exhibits cancer chemopreventive activity, but in contrast to sulindac, it does not inhibit cyclooxygenases 1 or 2. We found that sulindac sulfone and two potent derivatives, CP248 and CP461, inhibited the cyclic GMP (cGMP) phosphodiesterases (PDE) 2 and 5 in human colon cells, and these compounds caused rapid and sustained activation of the c-Jun NH2-terminal kinase 1 (JNK1). Rapid activation of stress-activated protein/ERK kinase 1 (SEK1) and mitogen-activated protein kinase kinase kinase (MEKK1), which are upstream of JNK1, was also observed. Other compounds that increase cellular levels of cGMP also activated JNK1, and an inhibitor of protein kinase G (PKG), Rp-8-pCPT-cGMPS, inhibited JNK1 activation by the sulindac sulfone derivatives. Expression of a dominant-negative JNK1 protein inhibited CP248-induced cleavage of poly(ADP-ribose) polymerase, a marker of apoptosis. Thus, it appears that sulindac sulfone and related compounds induce apoptosis, at least in part, through activation of PKG, which then activates the MEKK1-SEK1-JNK1 cascade. These studies also indicate a role for cGMP and PKG in the JNK pathway.

Topics: Apoptosis; Colonic Neoplasms; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Genes, Dominant; Humans; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Biological; Protein Serine-Threonine Kinases; Signal Transduction; Sulindac; Time Factors; Tumor Cells, Cultured

Human colonic adenocarcinoma cell lines have conserved several features of the native tissue. Among these is the expression of cell surface receptors for hormones and neurotransmitters that may be involved in the regulation of proliferation and differentiation processes in these cancer cells. Here, we confirm that high-affinity binding sites for the Vasoactive Intestinal Polypeptide (VIP) and for the VIP analogue Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP), were expressed in 4 human colonic adenocarcinoma cell lines, HT29, SW403, DLD-1 and Caco-2, that spontaneously displayed variable phenotypic properties in culture. We demonstrated that after long-term treatments, VIP and PACAP significantly reduced cell proliferation in the 4 cell lines and modulated intracellular cAMP and cGMP levels. Furthermore, conspicuous differences were observed from one cell type to another concerning expression of the receptor subsets or the effects of the neuropeptides on cell growth and on cyclic nucleotides production.

Topics: Adenocarcinoma; Caco-2 Cells; Cell Division; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Growth Inhibitors; HT29 Cells; Humans; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The purification of the human peptide hormone guanylin 22-115 from blood ultrafiltrate (hemofiltrate, HF) was achieved using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) as the assay system. Screening a peptide bank generated from 5000 1 HF guanylin 22-115 was detected by its molecular mass when adequate conditions for MALDI-MS analysis were chosen. The sensitivity was even better than of the established biological assay system. In addition, the susceptibility towards solvents and salts is strongly reduced. 1.2 mg of the peptide hormone was purified from 10% of the starting material.

Topics: Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Colonic Neoplasms; Cyclic GMP; Gastrointestinal Hormones; Humans; Molecular Weight; Natriuretic Peptides; Peptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tumor Cells, Cultured; Ultrafiltration

Recently, the peptides guanylin and uroguanylin were identified as endogenous ligands of the membrane-bound guanylate cyclase C (GC-C) that is mainly expressed in the intestinal epithelium. In the present study, bioactive guanylin and uroguanylin have been prepared by solid-phase methodology using Fmoc/HBTU chemistry. The two disulfide bonds with relative 1/3 and 2/4 connectivity have been introduced selectively by air oxidation of thiol groups and iodine treatment of Cys(Acm) residues. Using this strategy, several sequential derivatives were prepared. Temperature-dependent HPLC characterization of the bioactive products revealed that guanylin-related peptides exist as a mixture of two compounds. The isoforms are interconverted within approximately 90 min, which prevents their separate characterization. This effect was not detected for uroguanylin-like peptides. Synthetic peptides were tested for their potential to activate GC-C in cultured human colon carcinoma cells (T84), known to express high levels of GC-C. The results obtained show that both disulfide bonds are necessary for GC-C activation. The presence of the amino-terminally neighboring residues of Cys104 for guanylin and Cys100 for uroguanylin has been found to be essential for GC-C stimulation. Unexpectedly, a hybrid peptide obtained from substitution of the central tripeptide AYA of guanylin by the tripeptide VNV of uroguanylin was not bioactive.

Topics: Amino Acid Sequence; Animals; Chlorides; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cyclic GMP; Enzyme Activation; Female; Gastrointestinal Hormones; Guanylate Cyclase; Humans; Ion Transport; Molecular Sequence Data; Natriuretic Peptides; Peptides; Protein Conformation; Rats; Rats, Wistar; Tumor Cells, Cultured

Escherichia coli heat-stable enterotoxin (STa) was found to bind on the surface of human colonic (COLO 205) cells. The binding of [125I]STa to cell membranes was found to be specific, reversible and saturable. Scatchard analysis of the equilibrium binding demonstrated a single class of binding sites with a Kd of 0.5 x 10(-10) M. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed the specific incorporation of [125I]STa into a single STa binding protein with a molecular mass of 95 kDa. Following incubation of COLO 205 cells with STa, a rise of intracellular cGMP was also evident.

Topics: Bacterial Toxins; Binding Sites; Carrier Proteins; Cell Membrane; Colon; Colonic Neoplasms; Cyclic GMP; Diarrhea; Enterotoxins; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Guanylate Cyclase; Humans; Kinetics; Neoplasm Proteins; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Tumor Cells, Cultured

Regulation of intestinal epithelial differentiation is critical to normal function, malignant transformation, and healing. However, the intracellular regulation of intestinal epithelial differentiation is incompletely understood. We studied the effects of intracellular cyclic AMP and cyclic GMP on brush border enzyme activity in the human Caco-2 intestinal epithelial cell using pharmacologic agonists and antagonists of cAMP and cGMP mediated pathways as probes. The stable cyclic nucleotide analogs dibutyryl cAMP and dibutyryl cGMP selectively decreased Caco-2 dipeptidyl dipeptidase specific activity while increasing alkaline phosphatase. The inhibitors of adenylate and guanylate cyclase KT5720 and KT5823 each exerted the opposite effects. Combinations of dibutyryl cAMP and dibutyryl cGMP demonstrated synergistic effects on each brush border enzyme but KT5720 and KT5823 were less than additive. Thus, cAMP and CGMP may regulate human intestinal epithelial differentiation by interacting pathways.

Topics: Adenylyl Cyclase Inhibitors; Alkaline Phosphatase; Alkaloids; Bucladesine; Caco-2 Cells; Carbazoles; Cell Line; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Dibutyryl Cyclic GMP; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Drug Synergism; Enzyme Inhibitors; Epithelium; Guanylate Cyclase; Homeostasis; Humans; Indoles; Intestines; Kinetics; Microvilli; Pyrroles

Certain pathogenic bacteria produce a family of heat stable enterotoxins (STa) which activate intestinal guanylyl cyclases, increase cGMP, and elicit life-threatening secretory diarrhea. The intracellular effector of cGMP actions has not been clarified. Recently we cloned the cDNA for a rat intestinal type II cGMP dependent protein kinase (cGK II) which is highly enriched in intestinal mucosa. Here we show that cGK II mRNA and protein are restricted to the intestinal segments from the duodenum to the proximal colon, with the highest amounts of cGK II protein in duodenum and jejunum. cGK II mRNA and protein decreased along the villus to crypt axis in the small intestine, whereas substantial amounts of both were found in the crypts of cecum. In intestinal epithelia, cGK II was specifically localized in the apical membrane, a major site of ion transport regulation. In contrast to cGK II, cGK I was localized in smooth muscle cells of the villus lamina propria. Short circuit current (ISC), a measure of Cl- secretion, was increased to a similar extent by STa and by 8-Br-cGMP, a selective activator of cGK, except in distal colon and in monolayers of T84 human colon carcinoma cells in which cGK II was not detected. In human and mouse intestine, the cyclic nucleotide-regulated Cl- conductance can be exclusively accounted for by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Viewed collectively, the data suggest that cGK II is the mediator of STa and cGMP effects on Cl- transport in intestinal-epithelia.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Biological Transport; Carcinoma; Cecum; Chlorides; Colon; Colonic Neoplasms; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Enterotoxins; Enzyme Induction; Esophagus; Humans; In Situ Hybridization; Intestinal Mucosa; Intestine, Small; Isoenzymes; Male; Membrane Proteins; Microvilli; Muscle, Smooth; Organ Specificity; Rats; Rats, Sprague-Dawley; RNA, Messenger; Stomach; Tumor Cells, Cultured

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

In in vitro studies, SR 48692, a nonpeptide neurotensin receptor antagonist, inhibited the binding of [3H] or [125I]neurotensin to membrane preparations from 10-day-old mouse brains and from HT-29 cells with Ki values of 3.9 and 8.6 nM, respectively. SR 48692 also antagonized the neurotensin-induced mobilization of intracellular calcium in HT-29 cells, in agreement with previous findings. In rat cerebellar slices SR 48692 blocked the increase in cyclic GMP levels evoked by neurotensin in a dose-dependent manner. In vivo, SR 48692 antagonized the increase in rat brain mesolimbic dopamine turnover induced by the systemically active neurotensin peptide, EI [(N-Me)Arg-Lys-Pro-Trp-tert-Leu-Leu]. No effects on dopamine turnover of either EI or SR 48692 were observed in the striatum. SR 48692 did not antagonize the EI-induced decreases in mouse body temperature and spontaneous locomotor activity (LMA) or the decreases in LMA induced by ICV-administered neurotensin. Although other explanations are possible, these findings support the hypothesis that a subtype of the NT receptor may mediate the locomotor and hypothermic actions of this peptide and that it is different from the NT receptor that is involved in dopamine turnover.

Topics: Animals; Brain Chemistry; Calcium; Carcinoma; Cells, Cultured; Colonic Neoplasms; Cyclic GMP; Dopamine; Humans; Hypothermia; Locomotion; Male; Membranes; Mice; Neurotensin; Oligopeptides; Psychotropic Drugs; Pyrazoles; Quinolines; Radioligand Assay; Rats

Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP. We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine. Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effects of cGMP and cAMP on gCl were not additive. The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation. In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function. Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate gCl in cystic fibrosis cells. Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells.

Topics: Chloride Channels; Colonic Neoplasms; Cyclic GMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Membrane Proteins; Protein Kinases; Tumor Cells, Cultured

The receptor for the Escherichia coli heat-stable enterotoxin has been characterized and partially purified from the T84 human colonic cell line. Using a novel mutant heat-stable enterotoxin peptide as a radioligand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high-affinity receptor sites was detected in T84 cells, with a Kd of 0.1 nM, similar in affinity to the receptor described in human intestinal tissue. The receptor was solubilised from T84 cell membranes and affinity cross-linking of the solubilised preparation indicated that a single species of M(r) 160,000 served as the receptor. Freshly solubilised preparations of the receptor retained heat-stable enterotoxin-activable guanylyl cyclase activity. Purification of the receptor was achieved through sequential affinity chromatography on GTP--epoxy-Sepharose and wheat-germ-agglutinin columns resulting in purification of the receptor by 3000 fold. The heat-stable enterotoxin-binding characteristics of the receptor were unchanged during the purification and silver staining of the purified receptor preparation indicated a band of M(r) 160,000, which was specifically cross-linked to the 125I-labeled mutant peptide. The purified receptor retained guanylyl cyclase activity, but the activity was not stimulated on addition of human heat-stable enterotoxin, suggesting that accessory structural factors may be involved in the activation of the guanylyl cyclase/receptor.

Topics: Bacterial Toxins; Base Sequence; Binding Sites; Chromatography, Affinity; Colonic Neoplasms; Cross-Linking Reagents; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Enterotoxins; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Guanylate Cyclase; Humans; Molecular Sequence Data; Molecular Weight; Mutagenesis; Radioligand Assay; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Solubility; Tumor Cells, Cultured

Flavonoids, compounds containing a 2-phenylbenzo(gamma)pyrane nucleus, are universally distributed among vascular plants. Even though flavonoids are ingested in a normal diet in average quantities of 1 g daily, their effects on the digestive system have only been recently investigated. This study used an in vitro model of colonic secretion, monolayers of T84 colonic adenocarcinoma cells mounted in Ussing chambers, to demonstrate that 100 mumol/L of either tangeritin or nobiletin, polymethoxylated flavonoids contained in citrus fruits, stimulated sustained electrogenic chloride secretion with a maximal short-circuit current of 3.3 microA/cm2. In contrast, naringin and hesperidin, glycosylated citrus flavonoids, stimulated minimal secretion, suggesting that carbohydrate substitutions inhibited their secretory potential. The secretion stimulated by tangeritin and nobiletin was synergistic with carbachol but not with vasoactive intestinal peptide and was inhibited by barium chloride, bumetanide, H-89, and Cl- depletion. These properties suggest that tangeritin and nobiletin stimulated Cl- secretion via the cAMP pathway; however, these flavonoids did not stimulate cAMP production to the extent seen with vasoactive intestinal peptide. These flavonoids did not autooxidize, suggesting that reactive oxygen species did not mediate this secretion. These observations suggest that dietary citrus flavonoids may modulate colonic secretion, possibly through direct interaction with intracellular secretory pathways.

Topics: Adenocarcinoma; Carbachol; Chlorides; Citrus; Colon; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Drug Synergism; Flavanones; Flavones; Flavonoids; Glycosylation; Humans; Kinetics; Tumor Cells, Cultured

Chloride channels at the apical membrane of intestinal epithelial cells are involved in the excessive fluid secretion in diarrhea and diminished secretion in cystic fibrosis (CF). Diarrhea induced by heat-stable toxin from Escherichia coli is associated with elevated guanosine 3',5'-cyclic monophosphate (cGMP) in intestinal epithelial cells, but it is unknown whether chloride secretion is regulated by cGMP directly or via cGMP-dependent protein kinase (PKG). Single-channel recordings (inside-out excised patches) from the apical membrane of T84 cells reveal a 10-pS chloride channel with a linear current-voltage relationship, which is opened when an endogenous membrane-bound PKG is activated with ATP (1 mM) and cGMP (100 microM). Soluble PKG (200 nM) isolated from bovine lung, added to the intracellular face of patches, also opens this channel. No activation occurs with Ringer solution alone or only ATP or cGMP. Addition of nonhydrolyzable forms of ATP (AMP-PNP, 1 mM) or a combination of ATP, cGMP, plus H-8 (5 microM), an inhibitor of PKG, also does not stimulate the channel. The catalytic subunit of adenosine 3',5'-cyclic mono-phosphate-dependent protein kinase (PKA, 200 nM, with 1 mM ATP) activates a channel with similar characteristics. The 10 pS channel has a PNa/PCl ratio of 0.06, an anion selectivity of Br- (1.2) greater than Cl- (1.0) greater than I- (0.8) greater than F- (0.4), and a low affinity for the chloride channel blockers, 4,4-dinitrostilbene-2,2-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Carcinoma; Chloride Channels; Chlorides; Colonic Neoplasms; Cyclic GMP; Electric Conductivity; Humans; Ion Channel Gating; Membrane Proteins; Nitrobenzoates; Protein Kinases; Stilbenes; 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

STa, the heat-stable enterotoxin of Escherichia coli, is a specific activator of membrane-bound guanylyl cyclase and stimulates secretion of Cl- in a human colonic carcinoma cell line (T84). We investigated the effect of the cholinergic agent carbachol on the secretory response to STa. T84 cell monolayers were studied under voltage-clamped conditions in modified Ussing chambers. Simultaneous addition of STa and carbachol resulted in a biphasic synergistic response characterized by a brief peak in short-circuit current (Isc) followed by a prolonged plateau phase lasting up to 90 min. A synergistic response was also seen with sequential addition of the agonists, and was altered by the order and timing of agonist addition. Pretreatment with STa enhanced the synergistic response to carbachol, while the reverse order of additions produced synergy only when STa was added during or immediately after the Isc response to carbachol. Synergy occurred only with a concentration of STa sufficient to produce an Isc response alone. However, a concentration of carbachol that caused neither an increase in Isc nor intracellular Ca2+ mobilization was sufficient to evoke a synergistic response. Addition of 8-bromoguanosine 3',5'-cyclic monophosphate also produced a synergistic Isc response with carbachol, although maximal synergism was seen with simultaneous addition. Augmentation of the intracellular Ca2+ response to carbachol by STa is not the mechanism of synergy. Although the mechanism of synergy is not understood, these studies suggest that STa-induced cGMP interacts with other second messengers to produce the synergistic response, and that multiple intracellular mediators may influence the ability of STa to cause disease.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Bacterial Toxins; Calcium; Carbachol; Carcinoma; Chlorides; Colonic Neoplasms; Cyclic GMP; Drug Stability; Drug Synergism; Escherichia coli; Histamine; Hot Temperature; Humans; Intracellular Membranes; Tumor Cells, Cultured

Topics: Adenocarcinoma; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Humans; Intestinal Obstruction

Escherichia coli heat stable enterotoxin (STa) caused Cl- secretion across T84 cell monolayers in a dose-dependent manner only when applied to the apical membrane surface and not when applied to the basolateral surface. Measurement of cAMP, cGMP, and free cytosolic Ca2+ in response to STa suggested that cGMP alone mediated the Cl- secretory response. Studies utilizing blockers of the Na+,K+-ATPase pump, a Na+,K+,Cl- cotransport system, a K+ channel, and a Cl- channel suggest that all of them participate in the Cl- secretory process induced by STa. The results suggest that the Cl- secretory response induced by STa is mediated by cGMP after the enterotoxin binds to its receptor on the apical membrane. The enterotoxin, by increasing cGMP, opens a K+ channel on the basolateral membrane as well as a Cl- channel on the apical membrane. The activation of these ion exit mechanisms, together with activations of the Na+,K+,Cl- cotransporter and the Na+,K+-ATPase pump drives Cl- exit through the Cl- channel on the apical membrane.

Topics: Bacterial Toxins; Basement Membrane; Calcium; Cell Line; Cell Membrane Permeability; Chlorides; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Enterotoxins; Escherichia coli; Escherichia coli Proteins; Humans; Ion Channels; Potassium

Levels of urine excretion of cAMP and cGMP and their ratio were studied in patients with colorectal cancer in the course of a 3-year follow-up. At 12-36 months after surgery, the cAMP/cGMP ratio was higher (mainly due to decreased cGMP level) in disease-free patients than in those with recurrence or metastases. Preoperative level of cyclic nucleotide excretion cannot serve as prognostic factor for recurrence or metastasis development whereas levels of cAMP and cGMP measured within a follow-up period may be used for monitoring the course of colorectal cancer. The cAMP/cGMP ratio tended to rise in application of drugs controlling hyperlipidemia and other signs of cancrophilia.

Topics: Adult; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Female; Follow-Up Studies; Humans; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Rectal Neoplasms; Time Factors

Association of neurotensin to its receptor in HT29 cells increases the intracellular concentration of inositol phosphates. A rapid (20-30 s), transient stimulation of inositol trisphosphate (275% of the basal level) and inositol bisphosphate (420%) is first observed, followed by a slower, stable increase in inositol monophosphate (170%). Half-maximal stimulation of the three inositol phosphates was obtained with 50-100 nM neurotensin. These results indicate that neurotensin is able to regulate intracellular Ca2+ levels in HT29 cells by using inositol trisphosphate as a second messenger.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Humans; Inositol Phosphates; Kinetics; Neurotensin; Phosphatidylinositols; Receptors, Neurotensin; Receptors, Neurotransmitter; Tetradecanoylphorbol Acetate

Tissues from tumor and normal intestine were examined in 44 patients suffering from colonic and rectal cancer. The study established elevated levels of cholesterol (chiefly, cholesterol esters) and phospholipids, as compared with normal tissue. Two groups were identified among the cancer patients on the basis of the ratio of K = (Formula: see text). Cases with a relatively low value of K revealed higher levels of body fat and hyperlipidemia. That was matched by relatively higher concentrations of cholesterol and its esters, but not phospholipids, in tumor tissue, as compared with normal one. The relationship between cyclic nucleotide metabolism disorders in neoplastic tissue and lipid metabolism in cancer patients is discussed.

Topics: Adenocarcinoma; Cholesterol; Cholesterol Esters; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Humans; Middle Aged; Phospholipids; Rectal Neoplasms

In cell cultures treated with the carbocyclic analog of adenosine (C-Ado, (+/-)-aristeromycin), the utilization of hypoxanthine and guanine has been observed to be blocked. In an attempt to define the mechanism of this inhibition, we have reexamined the metabolism of C-Ado and its effects on the metabolism of guanine and hypoxanthine. In cultures of L1210 cells, C-Ado at a concentration of 25 microM inhibited the utilization of hypoxanthine and guanine for nucleotide synthesis by more than 90% but produced little or no inhibition of the utilization of these bases in cultures of L1210/MeMPR cells which lack adenosine kinase and cannot phosphorylate C-Ado. In cultures of mammalian cells (L1210, HEp-2, and colon-26 cells), C-Ado was converted to the triphosphate (as previously observed) and also to the triphosphate of the carbocyclic analog of guanosine. The presence of coformycin in the medium at a concentration sufficient to inhibit AMP deaminase almost completely prevented the formation of carbocyclic GTP; thus, the deamination of C-Ado monophosphate is essential for the formation of phosphates of carbocyclic guanosine. Since hypoxanthine (guanine) phosphoribosyltransferase is known to be subject to end product inhibition, it was considered likely that phosphates of carbocyclic guanosine or carbocyclic inosine, present in C-Ado-treated cells, were responsible for inhibition of utilization of hypoxanthine and guanine. The 5'-phosphates of the carbocyclic analogs of inosine and guanosine were synthesized and found to be effective inhibitors of the phosphoribosyltransferase. Carbocyclic GMP was a better inhibitor than carbocyclic IMP and was also superior to GMP and IMP; the concentration of C-GMP that produced a 50% inhibition of GMP formation was approximately 1 microM. It is probable that the presence of phosphates of carbocyclic guanosine accounts for the inhibition of utilization of hypoxanthine and guanine in C-Ado-treated cells.

Topics: Adenosine; Animals; Carcinoma, Squamous Cell; Cell Line; Coformycin; Colonic Neoplasms; Cyclic GMP; Cyclic IMP; Guanine; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Inosine Nucleotides; Kinetics; Leukemia L1210; Mice; Pentosyltransferases; Ribonucleotides

Prostaglandin E2 and cyclic nucleotide levels were measured in plasma and urine of 14 patients with colonic cancer. The measurements were performed 1 day before and 8 days after the removal of the tumor by operation. There was no difference between the plasma PGE2 levels (in form of the 13, 14-dihydro-15-keto metabolite) before and after the operation, but they were significantly higher than the level of a control group. No differences before and after operation were found between plasma cyclic AMP (cAMP) levels, plasma cyclic GMP (cGMP) levels, urinary cAMP levels and urinary cGMP levels. All the cyclic nucleotide concentrations were within the normal range. No correlation could be found between the stage of the tumor spread and any of the substances analyzed. The conclusions are that plasma PGE2 and plasma and urinary cyclic nucleotides do not originate from the colonic tumor tissue, and that these substances cannot be used as tumor markers.

Topics: Adenocarcinoma; Adult; Aged; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Dinoprostone; Female; Humans; Male; Middle Aged; Prostaglandins E

This study deals with the growth effect of gastrin on two xenotransplantable human gastric carcinomas (SC-6-JCK, poorly differentiated adenocarcinoma; and St-15, mucinous adenocarcinoma) and on one colonic carcinoma (Co-3, well-differentiated adenocarcinoma). In SC-6-JCK, the treatment with s.c. injection of pentagastrin at a dose of 10 micrograms/mouse once daily for 25 days promoted the growth of the tumor transplanted in nude mice, but gastrin had no effect at all on St-15 and Co-3. In SC-6-JCK, the weight, size, and labeling index of [3H]thymidine of the tumor were significantly increased in comparison with those of the control (p less than 0.05). In SC-6-JCK, cyclic adenosine 3':5'-monophosphate (cAMP) in the tumor was increased by a single i.p. injection of pentagastrin at a dose of 20 micrograms/mouse in nude mice, but such an increase was not observed in St-15 and Co-3. Cyclic guanosine 3':5'-monophosphate in SC-6-JCK was slightly increased by gastrin treatment but was not affected in the other tumors. In SC-6-JCK, at 30 min after gastrin treatment when cAMP showed a maximum increase, the activity ratio of cAMP-dependent protein kinase in the tumor was also elevated. In vitro also, gastrin stimulated cAMP production and cAMP-dependent protein kinase activation. The data suggest that some human gastric carcinomas may have receptor for gastrin.

Topics: Adenocarcinoma; Adult; Animals; Cell Division; Cell Line; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Female; Gastrins; Humans; Kinetics; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Protein Kinases; Stomach Neoplasms; Transplantation, Heterologous

Previous studies have shown that various amine hormones are able to influence the growth rate of human colorectal carcinomas propagated as xenografts in immune-deprived mice, and it is now well known that the effects of many amine and other hormones are mediated by cyclic nucleotides, acting as second messengers within cells. In the present study the influence of various derivatives of cyclic adenosine monophosphate and cyclic guanosine monophosphate on the growth of two different lines of colorectal cancer growing in immune-deprived mice, and on the cell production rate in the colonic crypt epithelium of the rat, was assessed. Growth of each tumour line, as well as crypt-cell production, was suppressed by treatment wit N6O2' dibutyryl and N6 monobutyryl derivatives of cyclic adenosine monophosphate. Dibutyryl cyclic guanosine monophosphate, on the other hand, was found to promote the growth of Tumour HXK4 and to promote crypt cell production, but to have no significant effect on Tumour HXM2.

Topics: Adenocarcinoma; Animals; Cell Division; Colon; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Epithelium; Female; Male; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Transplantation, Heterologous

Weanling F344 rats, which were fed a diet containing 10% chrysotile (B), were studied over their life-time to determine the effects of ingested asbestos on the colon. Control groups consisted of rats fed a diet containing a 10% nonnutritive cellulose or a standard laboratory rat diet. The pathological findings in the colons of 501 rats (189 on asbestos diet, 197 on fiber control diet, and 115 on standard control diet), are reported here. Epithelial tumors of the colon (eight adenocarcinomas and one adenoma) were found in nine of the rats on study. Four of the tumors were in asbestos-fed rats, two tumors were found in the non-nutritive cellulose controls, and three tumors were found in the standard laboratory rat diet controls. The probability (based on actuarial analysis) of developing adenoma or adenocarcinomas during the 32 months of the study were 7.4% for the asbestos-fed group, 3.5% for the fiber control diet and 4.0% on the standard control diet. In addition, one malignant mesothelioma of the type induced by intraperitoneally administered asbestos was found in the asbestos-fed group. Non-neoplastic lesions of the colon were also evaluated. The cumulative risk for development of any colon-associated lesion (non-neoplastic plus neoplastic lesions) was greatest for asbestos-fed rats (17.9%), compared to 13.6% for those fed the fiber control diet and 8.2% for those fed the standard control diet. The colon tissue levels of adenosine, 3'-5'-cyclic monophosphate (cAMP) were significantly lower in the animals fed asbestos compared to the control diets. Chrysotile fibers were seen by electronmicroscopy (e.m.) in six of ten ashed colon specimens of rats fed the asbestos diet. Although the differences in numbers of tumors between the animals fed asbestos and the controls were not statistically significant at the 5% level, we felt that the combination of observations including 1) evidence of increased probability of asbestos-fed animals to develop colon lesions in general; 2) evidence of a special type of mesothelioma in rats fed asbestos; 3) evidence for a cell regulator defect (lowered cAMP levels) in colon tissues of animals fed asbestos; and 4) evidence for asbestos fiber penetration of the colonic mucosa (e.m. studies) suggest that ingested asbestos is not inert in the colon.

Topics: Adenocarcinoma; Adenoma; Animals; Asbestos; Colon; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Dietary Fiber; Female; Male; Mesothelioma; Neoplasms, Experimental; Rats; Rats, Inbred F344; Time Factors

The adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels were determined in the small and large intestinal tissue of rats that had been exposed to single and chronic administration of the colon carcinogen 1,2-dimethylhydrazine (DMH). A single subcutaneous injection of DMH resulted in a decrease in the intracellular concentration of cAMP and increase in cGMP beyond the levels which had been measured in the unexposed intestinal tissue and DMH induced intestinal adenocarcinomas. Recovery to normal concentrations of the cyclic nucleotides occurred within 30 days. Multiple exposures resulted in maintaining reduced levels of cAMP while cGMP was also found to be lowered upon the chronic administration. A possible explanation for these observations is the expansion of the crypt cell population consisting of replicating intestinal cells that occurs upon exposure to the carcinogen. These findings suggest that cyclic nucleotide alterations may represent a characteristic of the precancerous state of intestinal tissue and indicates further studies are warranted to determine whether these changes may serve as a useful marker in a screening program for colon cancer.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Dimethylhydrazines; Injections, Subcutaneous; Intestinal Neoplasms; Kinetics; Male; Precancerous Conditions; Rats

The intracellular concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) in colonic tumors induced in adult male Holtzman rats by 1,2-dimethylhydrazine (DMH) were found to be 1/2 the concentration found in normal large bowel tissue. Intracellular concentrations of guanosine 3'-5'-cyclic monophosphate (cGMP) in the neoplastic cells were twice the normal colon level. The concentrations of these two cyclic nucleotides were relatively constant throughout the normal colon. Thus, the anomalous tumor cyclic nucleotide concentrations are attributed to the specific cell population of the lesion and not to the site of development within the colon.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; Dimethylhydrazines; Female; Rats

The search for molecular changes that may be diagnostic of malignancy in the colonic epithelium is complicated by the diversity of cell types and complex cell kinetics of a tissue in which most of the cells are destined to leave within hours or days. Methods for cell separation and nuclear fractionation now permit biochemical studies of those cells that retain or regain the capacity for DNA synthesis and that are likely to include the transformed cell population. Among the changes associated with malignant transformation to be described are alterations in nuclear protein composition and metabolism, qualitative and quantitative differences in adenosine deaminase activities, activation of the guanylate/cyclic GMP system, and modification of both DNA and chromosomal proteins by alkylating carcinogens. DNA modification to produce O6-methylguanine correlates well with the incidence of tumor induction by methylazoxymethanol. Modifications of chromosomal proteins to produce methylated derivatives of lysine and arginine have been observed after the administration of 1,2-dimethylhydrazine. Such changes are likely to lead to aberrant interactions between DNA and regulatory elements in chromatin, and may not be subject to repair.

Topics: Amino Acids; Chromosomal Proteins, Non-Histone; Colon; Colonic Neoplasms; Cyclic GMP; DNA; Epithelium; Kinetics

Data from cultured cells have suggested that cyclic AMP and cyclic GMP may be important determinants of cell growth and transformation. However, few studies have examined cyclic nucleotide content and metabolism in naturally occurring tumors of man. Accordingly, in the present study we compared cAMP and cGMP levels and metabolism in carcinomas of the human colon to those of the adjacent uninvolved mucosa after therapeutic resection of these tissues. The cAMP content of the tumors, determined in samples frozen 30 min after excision, was significantly lower than that of the adjacent mucosa, when expressed on the basis of tissue wet weight, protein, or DNA content. By contrast, the cGMP content of the tumors was higher than that of the surrounding mucosa if calculated on the basis of tissue wet weight, but this difference did not persist when correction was made for the higher protein or DNA content of the tumors. Incubation of slices of mucosa or tumor with or without theophylline in vitro increased tissue cAMP and cGMP content above levels observed in frozen samples of the same tissue. However, after such incubations cAMP levels in the tumors remained clearly below that of the mucosa, while cGMP content of the two tissues did not differ. The failure of theophylline to abolish differences in cAMP content and the comparable activities of high and low Km cAMP-phosphodiesterase in homogenates of the two tissues suggested that the lower cAMP content of the tumors was a consequence of diminished cAMP synthesis rather than enhanced degradation. This possibility was supported by the reduction in basal and maximal prostaglandin E1 (PGE1)-responsive adenylate cyclase activity found in tumor homogenates relative to those of mucosa, and the lower levels of cAMP in tumor slices after incubation of the tissues with a maximal dose of PGE1 and theophylline. Since NaF-responsive adenylate cyclase activity was not significantly reduced in the tumors, the lower basal and PGE1 activities may not be related to a deficiency of the catalytic unit of the cyclase complex in this tissue. The role of reduced activity of the adenylate cyclase-cAMP system and/or reduced tissue cAMP-to-cGMP ratios in the pathogenesis of colonic carcinoma is uncertain, but these changes might favor unregulated cellular proliferation.

Topics: Adenocarcinoma; Adenylyl Cyclases; Colonic Neoplasms; Cyclic AMP; Cyclic GMP; DNA, Neoplasm; Humans; Intestinal Mucosa; Prostaglandins E; Theophylline

Topics: Breast Neoplasms; Bucladesine; Cell Migration Inhibition; Colonic Neoplasms; Culture Media; Culture Techniques; Cyclic AMP; Cyclic GMP; Drug Synergism; Humans; Leukocytes; Lung Neoplasms; Neoplasms; Proteins; Theophylline; Tuberculin Test