Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and ß-catenin-dependent TCF transcriptional activity.

The cyclic nucleotide phosphodiesterase 10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from Apc(Min/+) mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 were evident by the ability of small-molecule inhibitors and small interfering RNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by short hairpin RNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/cGMP-dependent protein kinase signaling to suppress ß-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells.

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPß) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPß, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

Activation of ERK signaling and induction of colon cancer cell death by piperlongumine.

Piperlongumine (PPLGM) is a bioactive compound isolated from long peppers that shows selective toxicity towards a variety of cancer cell types including colon cancer. The signaling pathways that lead to cancer cell death in response to PPLGM exposure have not been previously identified. Our objective was to identify the intracellular signaling mechanisms by which PPLGM leads to enhanced colon cancer cell death. We found that PPLGM inhibited the growth of colon cancer cells in time- and concentration-dependent manners, but was not toxic toward normal colon mucosal cells at concentrations below 10 µM. Acute (0-60 min) and prolonged (24h) exposure of HT-29 cells to PPLGM resulted in phosphorylation of ERK. To investigate whether ERK signaling was involved in PPLGM-mediated cell death, we treated HT-29 cells with the MEK inhibitor U0126, prior to treating with PPLGM. We found that U0126 attenuated PPLGM-induced activation of ERK and partially protected against PPLGM-induced cell death. These results suggest that PPLGM works, at least in part, through the MEK/ERK pathway to result in colon cancer cell death. A more thorough understanding of the molecular mechanisms by which PPLGM induces colon cancer cell death will be useful in developing therapeutic strategies to treat colon cancer.

Targeting genetic alterations in protein methyltransferases for personalized cancer therapeutics.

The human protein methyltransferases (PMTs) constitute a large enzyme class composed of two families, the protein lysine methyltransferases (PKMTs) and the protein arginine methyltransferases (PRMTs). Examples have been reported of both PKMTs and PRMTs that are genetically altered in specific human cancers, and in several cases these alterations have been demonstrated to confer a unique dependence of the cancer cells on PMT enzymatic activity for the tumorigenic phenotype. Examples of such driver alterations in PMTs will be presented together with a review of current efforts towards the discovery and development of small-molecule inhibitors of these enzymes as personalized cancer therapeutics.

Selective cyclooxygenase-2 silencing mediated by engineered E. coli and RNA interference induces anti-tumour effects in human colon cancer cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required. METHODS: We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi). RESULTS: A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects. CONCLUSION: Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.

Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2).

An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and alpha-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and alpha-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and alpha-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and alpha-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-kappaB activation and higher expression of antiapoptotic NF-kappaB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.

Development of normal human colon cell cultures to identify priority unregulated disinfection by-products with a carcinogenic potential.

Research was initiated to develop an in vitro system to identify disinfection by-products with a potential to transform normal human colonocytes into malignant cells. Tribromomethane and bromochloroacetic acid, rodent colon carcinogens, dibromonitromethane and tribromonitromethane, recently identified in drinking water, and azoxymethane, a classic colon carcinogen, were tested for the ability to transform NCM460 cells. The chronic toxicity was determined for the series of trihalomethanes, haloacetic acids and halonitromethanes as well as NCM460 cell enzymatic capabilities. The order of cytotoxicity was halonitromethanes > haloacetic acids > trihalomethanes. Cytotoxicity within a series increased with the degree of bromination and decreased with the molecular weight. The genotoxicity profile was similar to that for cytotoxicity. Enzymatic analysis demonstrated that NCM460 cells possess glutathione-S transerase-1-1 and CYP450 activity similar to that measured in the large intestine. NCM460 cells were exposed to 10(-6) M of the test chemicals for three days. While NCM460 cells from all treatments had the ability to grow in soft agar to some extent, only cells exposed to azoxymethane or tribromomethane were able to grow in media lacking serum and growth factors. When sub cultured, NCM460 cells exposed to 10(-9) M azoxymethane for three weeks formed colonies with morphology distinct from untreated cells.

Role of BRAF-V600E in the serrated pathway of colorectal tumourigenesis.

There is increasing evidence for an alternative pathway of sporadic colorectal tumourigenesis that is associated with DNA microsatellite instability (MSI), due to methylation and loss of expression of the mismatch repair gene MLH1. Recent studies have highlighted a serrated pathway of colorectal cancer (CRC) in which serrated polyps with activating mutations in BRAF progress to CRCs with MSI following methylation and silencing of MLH1. The present study provides a novel mechanistic experimental model for these clinical observations. We investigated the role of BRAF activating mutation (BRAF-V600E) in colorectal tumourigenesis by studying the effects of forced expression of BRAF-V600E in the 'normal' colon epithelial NCM460 cell line and by targeting endogenous BRAF-V600E in MSI-High (MSI-H) colon cancer cell lines. The findings indicate that BRAF mutation in colon epithelial cells contributes to a gain in resistance towards apoptotic stimuli, which is likely to be an important characteristic of pre-malignant serrated lesions. BRAF-V600E also plays a role in the development and maintenance of transformed and invasive phenotypes in colon epithelial cells. Our findings also suggest that BRAF mutation potentiates promoter hypermethylation of the MLH1 gene promoter. Together, these results highlight BRAF as a potential target for therapeutic intervention in sporadic MSI-H colorectal cancers.

Consumption of an omega-3 fatty acids product, INCELL AAFA, reduced side-effects of CPT-11 (irinotecan) in mice.

INCELL AAFA, an omega-3 polyunsaturated fatty acid product containing a high concentration of long chain fatty acids, was tested for its ability to ameliorate the harmful side effects of CPT-11 chemotherapy including: leukopenia, anaemia, asthenia, weight loss and liver involvement. Four groups of mice were fed an AIN-76 diet modified to contain: 10% w/w corn oil (CO), 0% AAFA; 9% CO, 1% AAFA; 8% CO, 2% AAFA; or 7% CO, 3% AAFA. After 2 weeks on the diets, half of the mice received CPT-11 chemotherapy (60 mg kg(-1) q 4 days, i.v.) the rest of the mice received vehicle for 2 weeks. It was found that 2% AAFA in the diet of the CPT-11 treated mice: decreased apoptotic figures in the duodenal crypts; markedly suppressed the inflammatory eicosanoid, prostaglandin E(2) in the liver; prevented liver hypertrophy; improved white blood cell counts; significantly increased red blood cell counts; decreased numbers of CPT-11 induced immature red blood cell and micronuclei in red blood cells of the peripheral blood; increased eicosapentaenoic acid and docosahexaenoic acid in liver cell membranes and maintained normal grooming behaviour. Thus 2% AAFA in the diet reduced the side effects of CPT-11 treatment in mice.

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.

Seven genes that are differentially transcribed in colorectal tumor cell lines.

To identify genes which are differentially transcribed in colorectal tumor cells, we compared the two human tumor cell lines, SW480 and HCT116, with the cell line, NCM460, from normal colon epithelium as a control. Using the methods of differential display reverse transcription PCR and Northern blot hybridization, we detected the differential transcription of seven genes: cholecystokinin, reticulocalbin, Rab5 guanine nucleotide exchange factor Rabex5, caldesmon, differentiation related gene 1 (drg1), taxol resistant associated gene 3 (Trag-3) and the gene for the placental protein, diff33. The yet unidentified cDNA of the human Rabex5 gene and the 3' untranslated region of the human caldesmon gene were cloned.

Connexin-independent ganciclovir-mediated killing conferred on bystander effect-resistant cell lines by a herpes simplex virus-thymidine kinase-expressing colon cell line.

A novel gap junction-independent mechanism for ganciclovir-mediated bystander effect killing by a herpes simplex virus thymidine kinase (HSV-TK)-expressing SW620 human colon tumor cell line has been characterized. The mechanism of the HSV-TK/GCV bystander effect for many tumor cell lines has been demonstrated to be due to connexin gap junction transfer of phosphorylated ganciclovir (GCV) metabolites; however, there may be as yet uncharacterized connexin-independent mechanisms for the effect. To address this, the bystander effect was further evaluated in a panel of cell lines mixed with homologous HSV-TK-expressing cell lines, a SW620.TK cell line, or a high connexin43-expressing PA-317.TK cell line. Of the 10 cell lines tested, 4 were found to be resistant to bystander effect killing by their homologous HSV-TK-expressing cell lines and the PA-317.TK cells, but all of the cell lines were sensitive to GCV killing when mixed with the SW620.TK cells. The SW620.TK cells were then further evaluated for any indication of extracellular GCV metabolite efflux. Culture medium from SW620.TK cells labeled with [(3)H]GCV was evaluated for the presence of GCV nucleotides by ion-exchange column separation and HPLC analysis. The presence of GCV mono-, di-, and triphosphate metabolites in the medium was detected. Inclusion in the medium of inhibitors of extracellular phosphatases and ecto-ATPases increased the proportion of GCV metabolites recovered. These results indicate that phosphorylated GCV metabolites can be effluxed from SW620.TK cells and that some type of cellular uptake mechanism independent of gap junctions exists for nucleotide entry into neighboring cells.

Establishment of a human polyclonal oral epithelial cell line.

OBJECTIVE: To develop a human oral epithelial cell line to constitute a continuous source of cells readily available for human oral epithelial cell research. STUDY DESIGN: Oral epithelial cells from a 30-week gestational, stillborn male fetus were grown in serum-free medium and transfected by lipid-mediation with the shuttle vector plasmid, pZ189, containing the T-antigen coding region and replication origin from the SV40 virus. RESULTS: Resulting cultures produced foci of rapidly multiplying cells that failed to senesce, in contrast to controls. The transformed culture, designated GMSM-K, was polyclonal. The original culture possessed a normal human male karyotype, and the transformed line was largely hypotetraploid. Multiple clones, isolated from soft agar studies and low density plating, showed decreased doubling times. Electron microscopy and immunohistochemistry confirmed an epithelial phenotype. Cells did not generate tumors in nude mice. CONCLUSION: Few human epithelial cell lines are available to investigators and most are tumor-derived. The nontumor-derived GMSM-K line has value as a resource for human oral epithelial cell research.

Differentiation of islet cells in long-term culture.

Our previous studies in the hamster pancreatic cancer model have shown that exocrine pancreatic cancer arises from ductal/ductular cells, as well as from within the islets, most probably from islet precursor (stem) cells. To identify and characterize these cells, we established a long-term culture from isolated hamster islets and investigated their growth, differentiation, and expression of biomarkers. Islets maintained their original form and structure within the first 14 days in culture. However, beginning at day 7, ductular structures began to form within the islets. At day 21 in culture, acinar cells, intermediary cells, oncocytes, and cells comparable to pancreatic hepatocytes also appeared between ductular and endocrine cells. The number of duct-like cells gradually increased, whereas the number of hormone-producing cells decreased. After 35 days in culture, the exocrine cells disappeared, and undifferentiated cells formed a monolayer. These cells expressed cytokeratins, alpha1-antitrypsin, transforming growth factor-alpha, epidermal growth factor receptor, carbonic anhydrase II, vimentin, laminin, and showed binding to tomato lectin and Phaseolus vulgaris leukoagglutinin. They did not express the regulatory transcriptional factors, insulin-promoting factor 1, NKx6.1, Pax6, and NeuroD. The results thus indicate that islet cells have potential to form exocrine cells. At present, it is not clear whether these cells originate from preexisting stem cells or from transdifferentiated islet cells.

Two-drug combinations that increase apoptosis and modulate bak and bcl-X(L) expression in human colon tumor cell lines transduced with herpes simplex virus thymidine kinase.

Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-hydroxystaurosporine (UCN-01). Over a 2-4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5- to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-X(L), was observed in butyrate-, CPT-, and 7-hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-X(L). In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-X(L) protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.

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.

Effect of telomere and telomerase interactive agents on human tumor and normal cell lines.

Shortening of telomeres along with an up-regulation of telomerase is implicated in the immortality of tumor cells. Targeting either telomeres or telomerase with specific compounds has been proposed as an anticancer strategy. Because telomerase activity and telomeres are found in normal cells, telomere or telomerase targeting agents could induce side effects in normal tissues. We evaluated the effects of telomere and telomerase interactive agents in human tumor and normal cell lines to try to determine the potential side effects those agents might induce in patients. Toxicity of the G-quadruplex interactive porphyrins (TMPyP4, TMPyP2) and azidothymidine (AZT) were tested using a cell-counting technique against normal human cell lines (CRL-2115 and CRL-2120, fibroblasts; NHEK-Ad, adult keratinocytes; CCL-241, small intestinal cells; NCM 460, colonic mucosal epithelial cells) and human tumor cell lines (MDA-MB 231 and Hs 578T, breast cancer; SK-N-FI, neuroblastoma; HeLa, cervix cancer; MIA PaCa-2, pancreatic cancer; HT-29 and HCT-116, colon cancer; DU 145, prostatic cancer cell line). Telomerase activity of these cell lines was measured by a non-PCR-based conventional assay. The effects of TMPgammaP2, TMPyP4, and AZT were also evaluated against normal human bone marrow specimens, using a granulocyte-macrophage colony-forming assay (CFU-GM). AZT showed very low cytotoxic effects against normal and tumor cell lines, with the IC50 values above 200 microM. The IC50 values for TMPyP2 and TMPyP4 in normal human cell lines were in the range of 2.9-48.3 microM and 1.7-15.5 microM, respectively, whereas in tumor cell lines the IC50 values were 11.4-53 microM and 9.0-28.2 microM, respectively. Within the tissue types, keratinocytes were more sensitive to TMPyP4 than fibroblasts, and small intestinal cells were more sensitive than colonic mucosal epithelial cells. The IC50 for TMPyP2 and TMPyP4 in the normal marrow colony-forming assays were 19.3 +/- 5.1 microM and 47.9 +/-1.0 microM, respectively. In conclusion, the in vitro cytotoxicity of the telomere interactive agent TMPyP4 is comparable in human tumor and normal cell lines, which indicates that TMPyP4 could have effects on normal tissues.

Riboflavin uptake by human-derived colonic epithelial NCM460 cells.

Normal microflora of the large intestine synthesize a number of water-soluble vitamins including riboflavin (RF). Recent studies have shown that colonic epithelial cells possess an efficient carrier-mediated mechanism for absorbing some of these micronutrients. The aim of the present study was to determine whether colonic cells also possess a carrier-mediated mechanism for RF uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed colonic epithelial cells NCM460 and [(3)H]RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na(+) independent; 3) saturable as a function of concentration with an apparent K(m) of 0.14 microM and V(max) of 3.29 pmol x mg protein(-1) x 3 min(-1); 4) inhibited by the structural analogs lumiflavin and lumichrome (K(i) of 1.8 and 14.1 microM, respectively) but not by the unrelated biotin; 5) inhibited in a competitive manner by the membrane transport inhibitor amiloride (K(i) = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca(2+)/calmodulin-mediated pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellular model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that the identified transport system may be involved in the absorption of bacterially synthesized RF in the large intestine and that this source of RF may contribute toward RF homeostasis, especially that of colonocytes.

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.