Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes.

Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-kappaB- and Erk-dependent pathways. In addition, NT-stimulated NF-kappaB activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-kappaB, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-kappaB and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.

Dietary fish oil sensitizes A549 lung xenografts to doxorubicin chemotherapy.

A549 xenografts were allowed to grow in nude mice to about 5 mm in diameter, then diets were changed to modified AIN-76 diets containing 19% wt./wt. fish oil (FO) or 20% wt./wt. corn oil (CO). Ten days later dietary ferric citrate (0.3% wt./dry wt.) was added and doxoribicin (DOX) treatment (3.6 mg/kg i.v. each of the 5 days for 18 days) commenced. Treatment with DOX halted the growth of tumors in the CO fed mice. However, in those mice, which consumed FO or FO with ferric citrate, treatment with DOX caused significant tumor regression.

Smokeless tobacco extracts modulate exogenous gene expression in early passage cultured human oral epithelial cells: an in vitro system to study chemical and viral enhancer/promoter interaction.

The increased risk for cancers of the oral cavity from smokeless tobacco use may reflect the interaction of tobacco with genetic factors, such as oncogenes, and other exogenous factors, such as viruses. An in vitro system was developed based on expression of the chloramphenicol acetyltransferase (CAT) reporter gene to study interactions of chemical treatments with viral enhancer/promoters in early passage cell cultures of oral cavity-derived epithelial cells. Expression of CAT in transfected cells was significantly greater with CAT under the control of the cytomegalovirus immediate early enhancer/promoter (pCEP4/CAT) compared to the Rous sarcoma virus long terminal repeat enhancer/promoter (pRSV-cat) and the simian virus 40 (SV40) early promoter (pSV2-cat). No CAT expression was detected using corresponding control plasmids without the CAT reporter gene. Using this system, smokeless tobacco extracts prepared from either dry snuff or moist snuff delayed maximum CAT expression from Day 4 to Day 5, with sustained, significantly increased CAT expression at 12 days compared to the declining CAT expression observed in untreated control cells. Smokeless tobacco extracts can modulate intracellular gene expression. This system provides an in vitro model to test specificity of toxic agents on enhancer/promoter activity and the interaction on exogenous gene expression.

Fish oil supplementation enhanced CPT-11 (irinotecan) efficacy against MCF7 breast carcinoma xenografts and ameliorated intestinal side-effects.

The cancer chemotherapeutic efficacy of the topoisomerase I inhibitor, CPT-11 (irinotecan) is often limited by the induction of severe delayed diarrhoea. In animal studies, CPT-11 use is associated with histopathological damage to the mucosa of the small and large intestines. Results from the present study demonstrate that 60 mg CPT-11 per kg body weight (i.v. q4d x 6) halted the growth, but did not cause significant regression, of MCF7 human breast carcinoma xenografts in mice fed a diet containing 7% corn oil. However, when the diet of the MCF7-bearing mice was supplemented with 3% or 6% fish oil, the same CPT-11 treatment caused significant regression of the MCF7 xenograft. Histomorphometric analyses of intestinal mucosa of mice treated with CPT-11 and fed the diet containing 7% com oil indicated that treatment with CPT-11 induced structural changes in the intestinal mucosa which persisted at least 5 days after the last dose of CPT-11. The intestinal mucosal architecture of mice that were treated with CPT-11 and fed the diets containing fish oil was largely unchanged from the architecture of the group of mice which did not receive CPT-11. These findings indicate that fish oil supplements may be a useful adjunct to CPT-11 treatment.

Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein-Barr virus.

Numerous chemical tumor promoters induce latent Epstein Barr virus (EBV) to active replication. The effect of smokeless tobacco purified products N-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK), benzo(a)pyrene (BaP), and smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf tobacco) was tested for induction of latent EBV in Raji cells using fluorescence-activated cell sorter flow cytometry detection of the restricted component of EBV early antigen (EA-R). Concentrations of smokeless tobacco purified products or preparations were used that have carcinogenic effects in animal cell lines. There was no discernible effect for the 6-7-day duration of treatment on viability of Raji cells, or on induction of latent EBV in Raji cells. Results were comparable using paraformaldehyde- or acetone-fixed cells. There does not appear to be an in vitro effect of smokeless tobacco constituents on EBV-infected lymphocytes that may contribute to development of oral cancers.

Attenuation of the natural course of herpes simplex virus infection in human oral epithelial cell cultures by smokeless tobacco extracts suggests the possibility of a synergistic mechanism for carcinogenesis.

High prevalence of both tobacco use and latent herpes simplex virus type 1 suggests the opportunity for synergism between these agents as cocarcinogens. In this study, postprimary human oral epithelial cell cultures were infected with herpes simplex virus type 1 pretreated with 2% extracts of either loose leaf, moist, or dry snuffs. Cultures were subsequently periodically exposed to the tobacco. Parameters measured included percentage of cultures undergoing active virus production, onset and time course of cytopathic effects, and concentration of virus released into the media over time. Results showed inhibition of both herpes simplex virus-mediated cell lysis and viral replication by tobacco extracts. This is the first time that these phenomena have been demonstrated in normal human oral epithelial cells. The work described here provides evidence to support a hypothesis that herpes simplex virus type 1 and smokeless tobacco may act synergistically in oral carcinogenesis.

Application of a new preclinical drug screening system for cancer of the large bowel.

We report a prospective evaluation of three human, continuous colorectal cancer cell lines and a new semiautomated radiometric technique (Bactec system) as a primary screening procedure for cytotoxic compounds with activity against cancer of the large bowel. COLO 320DM, Ht-29, and the metastatic OM-1 colon cancer cell line that have previously been shown to yield clinically relevant information in terms of drug sensitivity patterns in humans were all tested against 11 new compounds currently being investigated in phase I or early phase II clinical trials. Our results suggest that trimetrexate, DUP-785, didemnin B, and flavone-8-acetic acid may be clinically effective for the treatment of colorectal cancer.

New screening system for selection of anticancer drugs for treatment of human colorectal cancer.

We report an evaluation of a new radiometric technique (BACTEC assay) as a potential screening system for cytotoxic compounds with activity against cancer of the large bowel. Exponentially growing cells of nine different human colorectal cancer cell lines were exposed to a variety of standard anticancer agents with or without documented clinical activity. Each drug was tested in a series of 1-h and continuous exposure studies utilizing three different concentrations. Antineoplastic effects were analyzed as a function of in vivo achievable serum concentrations, namely by a ratio of the concentration required to decrease cell growth to 10% of control to one-tenth of the peak plasma concentration in humans. Our results suggest that COLO 320DM, OM-1, and Ht-29 cells manifest responsiveness to anticancer drugs consistent with that noted in clinical studies with most agents tested. The radiometric technique provides several advantages for a screening system, including reproducibility, a good agreement with the cloning assay, speed, and low costs. The combined use of the BACTEC technique and the three colon cancer cell lines could prove useful as a screen for new anticancer compounds with activity in colorectal cancer.