4-hydroxy-2-nonenal and Colonic-Neoplasms

Irinotecan (CPT11) is widely prescribed for treatment of various intractable cancers such as advanced and metastatic colon cancer cells, but its continuous treatment promotes the resistance development. In this study, we established CPT11-resistant variants of three human colon cancer (DLD1, RKO and LoVo) cell lines, and found that gain of the resistance elicited an up-regulation of aldo-keto reductase (AKR) 1C3 in the cells. Additionally, the sensitivity to CPT11 toxicity was decreased and increased by overexpression and knockdown, respectively, of the enzyme. Moreover, the resistant cells suppressed formation of reactive 4-hydroxy-2-nonenal by CPT11 treatment, and the suppressive effect was almost completely abolished by addition of an AKR1C3 inhibitor. These results suggest that up-regulated AKR1C3 contributes to promotion of the chemoresistance by detoxifying the reactive aldehyde. Western blot and real-time polymerase-chain reaction analyses and ATP-binding cassette (ABC) B1-functional assay revealed that, among three ABC transporters, ABCB1 was the most highly up-regulated by development of the CPT11 resistance, inferring a significant contribution of pregnane-X receptor-dependent signaling to the ABCB1 up-regulation. The combined treatment with inhibitors of AKR1C3 and ABCB1 potently sensitized the resistant cells to CPT11 and its active metabolite SN38. Taken together, our results suggest that combination of AKR1C3 and ABCB1 inhibitors is effective as adjuvant therapy to enhance CPT11 sensitivity of intractable colon cancer cells.

Topics: Aldehydes; Aldo-Keto Reductase Family 1 Member C3; ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Irinotecan; Up-Regulation

Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect.. Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)-an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis.. Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice.. These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect.

Topics: Aldehydes; Animals; Bystander Effect; Cell Line; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Enterococcus faecalis; Female; Gram-Positive Bacterial Infections; HCT116 Cells; Humans; In Situ Hybridization, Fluorescence; Intestinal Mucosa; Macrophages; Mice; Mice, Inbred NOD; Neoplasm Transplantation; Neoplastic Stem Cells

Nuclear factor erythroid 2-related factor 2 (NRF2) is the transcription factor that regulates an array of antioxidant/detoxifying genes for cellular defense. The conformational changes of Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor protein of NRF2, by various stimuli result in NRF2 liberation and accumulation in the nucleus. In the present study, we aimed to investigate the effect of KEAP1 knockdown on NRF2 target gene expression and its toxicological implication using human colon cancer cells. The stable KEAP1-knockdown HT29 cells exhibit elevated levels of NRF2 and its target gene expressions. In particular, the mRNA levels of aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, and 1B10) were substantially increased in KEAP1 silenced HT29 cells. These differential AKRs expressions appear to contribute to protection against oxidative stress. The KEAP1-knockdown cells were relatively more resistant to hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) compared to the control cells. Accordantly, we observed accumulation of 4HNE protein adducts in H2O2- or 4HNE-treated control cells, whereas KEAP1-knockdown cells did not increase adduct formation. The treatment of KEAP1-silenced cells with AKR1C inhibitor flufenamic acid increased 4HNE-induced cellular toxicity and protein adduct formation. Taken together, these results indicate that AKRs, which are NRF2-dependent highly inducible gene clusters, play a role in NRF2-mediated cytoprotection against lipid peroxide toxicity.

Topics: Aldehyde Reductase; Aldehydes; Aldo-Keto Reductases; Cell Death; Cell Survival; Colonic Neoplasms; DNA Adducts; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Oxidative Stress; Vitamin K 3

Cisplatin (cis-diamminedichloroplatinum, CDDP) is widely used for treatment of patients with solid tumors formed in various organs including the lung, prostate and cervix, but is much less sensitive in colon and breast cancers. One major factor implicated in the ineffectiveness has been suggested to be acquisition of the CDDP resistance. Here, we established the CDDP-resistant phenotypes of human colon HCT15 cells by continuously exposing them to incremental concentrations of the drug, and monitored expressions of aldo-keto reductases (AKRs) 1A1, 1B1, 1B10, 1C1, 1C2 and 1C3. Among the six AKRs, AKR1C1 and AKR1C3 are highly induced with the CDDP resistance. The resistance lowered the sensitivity toward cellular damages evoked by oxidative stress-derived aldehydes, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal that are detoxified by AKR1C1 and AKR1C3. Overexpression of AKR1C1 or AKR1C3 in the parental HCT15 cells mitigated the cytotoxicity of the aldehydes and CDDP. Knockdown of both AKR1C1 and AKR1C3 in the resistant cells or treatment of the cells with specific inhibitors of the AKRs increased the sensitivity to CDDP toxicity. Thus, the two AKRs participate in the mechanism underlying the CDDP resistance probably via detoxification of the aldehydes resulting from enhanced oxidative stress. The resistant cells also showed an enhancement in proteolytic activity of proteasome accompanied by overexpression of its catalytic subunits (PSMβ9 and PSMβ10). Pretreatment of the resistant cells with a potent proteasome inhibitor Z-Leu-Leu-Leu-al augmented the CDDP sensitization elicited by the AKR inhibitors. Additionally, the treatment of the cells with Z-Leu-Leu-Leu-al and the AKR inhibitors induced the expressions of the two AKRs and proteasome subunits. Collectively, these results suggest the involvement of up-regulated AKR1C1, AKR1C3 and proteasome in CDDP resistance of colon cancers and support a chemotherapeutic role for their inhibitors.

Topics: 20-Hydroxysteroid Dehydrogenases; 3-Hydroxysteroid Dehydrogenases; Aldehydes; Aldo-Keto Reductase Family 1 Member C3; Cell Line, Tumor; Cisplatin; Colonic Neoplasms; Drug Resistance, Neoplasm; HeLa Cells; HT29 Cells; Humans; Hydroxyprostaglandin Dehydrogenases; MCF-7 Cells; Oxidative Stress; Proteasome Endopeptidase Complex

The cancer preventive activity of vitamin E has been extensively discussed, but the activities of specific forms of tocopherols have not received sufficient attention. Herein, we compared the activities of δ-tocopherol (δ-T), γ-T, and α-T in a colon carcinogenesis model. Male F344 rats, seven weeks old, were given two weekly subcutaneous injections of azoxymethane (AOM) each at a dose of 15 mg/kg body weight. Starting 1 week before the AOM injection, the animals were maintained on a modified AIN76A diet, or the same diet containing 0.2% of δ-T, γ-T, α-T, or a γ-T-rich mixture of tocopherols (γ-TmT), until the termination of the experiment at 8 weeks after the second AOM injection. δ-T treatment showed the strongest inhibitory effect, decreasing the numbers of aberrant crypt foci by 62%. γ-T and γ-TmT were also effective, but α-T was not. Immunohistochemical analysis showed that δ-T and γ-T treatments reduced the levels of 4-hydroxynonenal and nitrotyrosine and the expression of cyclin D1 in the colon, preserved the expression of PPAR-γ, and decreased the serum levels of prostaglandin E2 and 8-isoprostane. Supplementation with 0.2% δ-T, γ-T, or α-T increased the respective levels of tocopherols and their side-chain degradation metabolites in the serum and colon tissues. Rather high concentrations of δ-T and γ-T and their metabolites were found in colon tissues. Our study provides the first evidence for the much higher cancer preventive activity of δ-T and γ-T than α-T in a chemically induced colon carcinogenesis model. It further suggests that δ-T is more effective than γ-T.

Topics: Aldehydes; alpha-Tocopherol; Animals; Anticarcinogenic Agents; Azoxymethane; Colonic Neoplasms; Cyclin D1; Dinoprost; Dinoprostone; gamma-Tocopherol; Immunohistochemistry; Male; Models, Chemical; Rats; Rats, Inbred F344; Tocopherols; Tyrosine

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.

Topics: Adipose Tissue; Aldehydes; Animals; Body Weight; Cachexia; Catalase; Colonic Neoplasms; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Gene Expression; Genes, APC; GTP Phosphohydrolases; Hindlimb; Interleukin-6; Ion Channels; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria, Muscle; Mitochondrial Proteins; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Oxidative Phosphorylation; Oxidative Stress; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Sirtuin 1; STAT3 Transcription Factor; Succinate Dehydrogenase; Superoxide Dismutase; Trans-Activators; Transcription Factors; Uncoupling Protein 3

The human aldo-keto reductase (AKR) 1B10 is suggested as a tumor marker in various solid tumors. Using colon cancer cells, we found that AKR1B10 was induced with acquisition of resistance to the anticancer drug mitomycin-c (MMC). In the resistant cells, treatment with an AKR1B10 inhibitor decreased their MMC tolerance. In the nonresistant cells, overexpression and silencing of AKR1B10 decreased and increased, respectively, susceptibility to cytotoxic effects of MMC and 4-hydroxy-2-nonenal, which was formed as a product of lipid peroxidation by MMC treatment. These results suggest a role of AKR1B10 in the development of MMC resistance, which may be mediated by its ability to detoxify cytotoxic aldehydes including 4-hydroxy-2-nonenal.

Topics: Aldehyde Reductase; Aldehydes; Aldo-Keto Reductases; Antibiotics, Antineoplastic; Caspase 3; Colonic Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Gene Knockdown Techniques; HT29 Cells; Humans; Lipid Peroxidation; Mitomycin; Reactive Oxygen Species; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Up-Regulation

Methylamine dichloramine (CH(3)NCl(2)) produced by neutrophils may promote colon tumors and colitis via architectural and oxidative changes in crypts, which are secretory granulae composed of goblet cells located in the colorectal mucosal layer. We investigated whether CH(3)NCl(2), in comparison with the other reactive oxygen species (ROS) such as H(2)O(2) and HOCl, derived from primed neutrophils in inflammatory sites in the large intestine, is a biogenic factor for the induction of colorectal disease in mice.. Male ICR-strain mice were administered each oxidant (0.5-0.7 micromol/mouse) by enema under anesthesia. The colorectal tissues were evaluated by histopathological and immunohistochemical analyses. Hemolysis and hemoglobin oxidation by the methylamine chloramines and HOCl were examined by adding them (50-400 microM) to a sheep erythrocyte suspension (1x10(8) cells/ml) and its lysate at pH 7 and 37 degrees C.. CH(3)NCl(2) oxidized erythrocyte hemoglobin more effectively than HOCl, indicating it has high cell permeability and selective oxidation ability. CH(3)NCl(2) mainly induced atrophy of crypts at 6 h after administration, while the other ROS tested did not. Furthermore, 4-hydroxy-2-nonenal (4-HNE) showed positive immunostains throughout the mucosal layer, including around the basal regions of atrophied crypts, only with CH(3)NCl(2), while positive immunostains were observed for 3-nitrotyrosine (3-NT) in the atrophied crypts and their surrounding lamina propria in the mucosal layer.. The results suggest that CH(3)NCl(2)derived from primed neutrophils may play the most important role in promoting the development of colon tumor formation and colitis by oxidative stress through its high degree of cell permeability.

Topics: Aldehydes; Animals; Chloramines; Colon; Colonic Neoplasms; Hemoglobins; Hemolysis; Hydrogen Peroxide; Hypochlorous Acid; Immunohistochemistry; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Mice; Mice, Inbred ICR; Neutrophil Activation; Oxidation-Reduction; Sheep; Tyrosine

Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H(2)O(2)), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as "Comet Assay." APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H(2)O(2), trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.

Topics: Adenomatous Polyposis Coli Protein; Aged; Aldehydes; Cell Line, Tumor; Cells, Cultured; Colon; Colonic Neoplasms; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Female; Humans; Hydrogen Peroxide; In Situ Hybridization, Fluorescence; Lipid Peroxidation; Male; Middle Aged; Oxidative Stress; ras Proteins; Tumor Suppressor Protein p53

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.

Topics: Aldehydes; Apoptosis; bcl-2-Associated X Protein; Caco-2 Cells; Cell Differentiation; Cell Proliferation; Colonic Neoplasms; Cross-Linking Reagents; Cysteine Proteinase Inhibitors; Drug Synergism; Gene Expression; Humans; Ligands; PPAR gamma; Prostaglandin D2; Rosiglitazone; Thiazolidinediones

Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0-2000 microM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 microM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 microM Fe-NTA. In contrast, 10 microM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 microM Fe-NTA increased cell growth (p < 0.05), whereas high concentrations (2000 microM) resulted in growth arrest (p < 0.05), that was accompanied by increased GSH levels (p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.

Topics: Aldehydes; Cell Proliferation; Colonic Neoplasms; DNA Damage; Ferric Compounds; Glutathione; HT29 Cells; Humans; Nitrilotriacetic Acid; Reactive Oxygen Species

The cellular production of 4-hydroxy-2-nonenal (HNE), a product of endogenous lipid peroxidation, constitutes a genotoxic risk factor for carcinogenesis. Our previous studies have shown that human HT29 colon cells developed resistance toward HNE injury after treatment with butyrate, a diet-associated gut fermentation product. This resistance was attributed to the induction of certain glutathione S-transferases (hGSTP1-1, hGSTM2-2, and hGSTA1-1) and also for the tripeptide glutathione (GSH) synthesizing enzymes. In the present study, we have investigated in HT29 cells whether hGSTA4-4, which has a high substrate specificity for HNE, was also inducible by butyrate and, thus, could contribute to the previously observed chemoresistance. In addition, we investigated if cellular depletion of GSH by L-buthionine-S,R-sulfoximine (BSO) enhances chemosensitivity to HNE injury in HT29 cells. Incubation of HT29 cells with butyrate (2-4 mM) significantly elicited a 1.8 to 3-fold upregulation of steady state hGSTA4 mRNA over 8-24 h after treatment. Moreover, 4 mM butyrate tended to increase hGSTA4-4 protein concentrations. Incubation with 100 microM BSO decreased cellular GSH levels by 77% without significant changes in cell viability. Associated with this was a 2-fold higher level of HNE-induced DNA damage as measured by the comet assay. Collectively, the results of this study and our previous work indicate that the genotoxicity of HNE is highly dependent on cellular GSH status and those GSTs that contribute toward HNE conjugation, including hGSTA4-4. Since HNE contributes to colon carcinogenesis, the favorable modulation of the GSH/GST system by butyrate may contribute to chemoprevention and reduction of the risks.

Topics: Aldehydes; Base Sequence; Butyric Acid; Cell Line, Tumor; Colonic Neoplasms; Comet Assay; DNA Primers; Glutathione; Glutathione Transferase; HT29 Cells; Humans; Mutagenicity Tests; Polymerase Chain Reaction; Substrate Specificity

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a better understanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds, which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxic effects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H(2)O(2)) in human colon cells (LT97). LT97 is a recently established cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon. The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cell microgelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity of TP53-a crucial target gene for the transition of adenoma to carcinoma-towards HNE. Expression of glutathione S-transferases (GST), which deactivates HNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cells and to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only two clones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed that both HNE and H(2)O(2) were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than in HT29clone19A and primary human colon cells. After HNE incubation, TP53 migrated more efficiently into the comet tail than the global DNA, which suggests a higher susceptibility of the TP53 gene to HNE. GST expression was significantly lower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells. In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma are common preneoplastic lesions occurring in advanced age.

Topics: Adenoma; Aldehydes; Cell Differentiation; Cell Movement; Colonic Neoplasms; Comet Assay; DNA Damage; DNA, Neoplasm; Female; Glutathione; Glutathione Transferase; Growth Inhibitors; Humans; Hydrogen Peroxide; In Situ Hybridization, Fluorescence; Karyotyping; Male; Middle Aged; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Dietary fibre sources are fermented by the gut flora to yield short-chain fatty acids (SCFA) together with degraded phytochemicals and plant nutrients. Butyrate, a major SCFA, is potentially chemoprotective by suppressing the growth of tumour cells and enhancing their differentiation. Conversely, it could lead to a positive selection pressure for transformed cells by inducing glutathione S-transferases (GST) and enhancing chemoresistance. Virtually nothing is known about how butyrate's activities are affected by other fermentation products. To investigate such interactions, a variety of dietary fibre sources was fermented with human faecal slurries in vitro, analysed for SCFA, and corresponding SCFA mixtures were prepared. HT29 colon tumour cells were treated for 72 h with individual SCFA or complex samples. The growth of cells, GST activity, and chemoresistance towards 4-hydroxynonenal were determined. Fermentation products inhibited cell growth more than the corresponding SCFA mixtures, and the SCFA mixtures were more active than butyrate, probably due to phytoprotectants and to propionate, respectively, which also inhibit cell growth. Only butyrate induced GST, whereas chemoresistance was caused by selected SCFA mixtures, but not by all corresponding fermentation samples. In summary, fermentation supernatant fractions contain compounds that: (1) enhance the anti-proliferative properties of butyrate (propionate, phytochemical fraction); (2) do not alter its capacity to induce GST; (3) prevent chemoresistance in tumour cells. It can be concluded that fermented dietary fibre sources are more potent inhibitors of tumour cell growth than butyrate alone, and also contain ingredients which counteract the undesired positive selection pressures that higher concentrations of butyrate induce in tumour cells.

Topics: Aldehydes; Butyrates; Cell Division; Colon; Colonic Neoplasms; Dietary Fiber; DNA Damage; Drug Resistance, Neoplasm; Fatty Acids, Volatile; Fermentation; Glutathione Transferase; Growth Inhibitors; HT29 Cells; Humans

Recently we reported that the supplementation of vitamin B6 to low vitamin B6 diet caused suppression in colon tumorigenesis and cell proliferation of azoxymethane-treated mice in a dose-dependent manner among 1, 7, and 14 mg pyridoxine HCl/kg diet (J. Nutr. 131: 2204-2207, 2001). To examine the mechanism of the anticolon tumor effect of vitamin B6, male ICR mice were fed the diet containing 1, 7, 14, and 35 mg pyridoxine HCl/kg diet for 22 wk and simultaneously given a weekly injection of azoxymethane for an initial 10 wk. The supplementation of vitamin B6 to a low vitamin B6 diet (1 mg pyridoxine HCl/kg) suppressed the levels of colonic 8-hydroxyguanosine and 4-hydroxynonenal and inducible nitric oxide synthase protein. The results suggest that the preventive effect of vitamin B6 against colon tumorigenesis is at least in part mediated by reducing oxidative stress and nitric oxide production.

Topics: Adjuvants, Immunologic; Aldehydes; Analysis of Variance; Animals; Azoxymethane; Carcinogens; Colonic Neoplasms; Cross-Linking Reagents; Guanosine; Male; Mice; Mice, Inbred ICR; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Vitamin B 6

This study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2-dimethylhydrazine (10 mg/kg body weight) for the initial 10 wk. Dietary supplemental sericin caused a 62% reduction in the incidence of colonic adenoma (P<0.05), but did not affect the incidence of colonic adenocarcinoma. Sericin intake significantly reduced the number of colon adenomas. Consumption of sericin significantly reduced the BrdU labeling index of colonic proliferating cells and the expression of colonic c-myc and c-fos. The levels of colonic 8-hydroxydeoxyguanosine, 4-hydroxynonenal, and inducible nitric oxide synthase protein were significantly suppressed by sericin. The results suggest that dietary sericin suppresses the development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production.

Topics: 1,2-Dimethylhydrazine; 8-Hydroxy-2'-Deoxyguanosine; Adenocarcinoma; Adenoma; Aldehydes; Animals; Apoptosis; Carcinogens; Cell Division; Colonic Neoplasms; Deoxyguanosine; Immunohistochemistry; Intestinal Mucosa; Male; Mice; Mice, Inbred ICR; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peptides, Cyclic; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Sericins

Butyrate, one of the major products of gut fermentation, is known to inhibit proliferation, induce apoptosis and differentiation, and increase phase II enzyme activities in tumor cells, whereas little information is available on protective effects in less-transformed colon cells. The aim of this study was to investigate whether the chemoprotective mechanism of glutathione S-transferase (GST) induction by butyrate could also play a role in earlier stages of colon carcinogenesis and whether chemoresistance of cells toward the endogenous genotoxic risk factor 4-hydroxy-2-nonenal (HNE) could be a consequence of butyrate treatment. As cell models, we used the human tumor cell lines HT29 and HT29 clone 19A, a differentiated subclone with properties resembling primary colon cells. We determined the expression of GSTP1 protein (enzyme-linked immunosorbent assay), the major GST in HT29, GSTP1 mRNA (Northern blotting), GST activity, intracellular glutathione, and total protein. The genotoxic impact of HNE (100-200 microM) was compared in butyrate-treated and nontreated cells using single-cell microgel electrophoresis. Our results show that GSTP1 mRNA, GSTP1 protein, GST activity, and total protein were increased (1.2- to 2.5-fold) and glutathione levels were maintained after 24-72 h of incubation with 4 mM butyrate. Moreover, a marked reduction of HNE-induced genotoxicity was caused by preincubation with butyrate. Butyrate also induced the phosphorylation of extracellular signal-regulated kinases (ERK1/2, Western blotting) after 5-30 min, which indicates a regulation of GST expression by this signal pathway. Most effects were greater in HT29 parent cells than in clone cells. In conclusion, butyrate enhances expression of GST and other proteins in both cell lines, which leads to an enhanced chemoprotection, reducing the impact of HNE genotoxicity. Thus butyrate could play a role in early and later stages of cancer prevention by reducing exposure to relevant risk factors.

Topics: Aldehydes; Anticarcinogenic Agents; Antimutagenic Agents; Butyrates; Colon; Colonic Neoplasms; DNA Damage; Enzyme Induction; Gene Expression; Glutathione; Glutathione S-Transferase pi; Glutathione Transferase; Isoenzymes; Polymorphism, Genetic; RNA, Messenger; Tumor Cells, Cultured

In our previous short-term experiment, Citrus auraptene inhibited the development of azoxymethane (AOM)-induced aberrant crypt foci, which are precursor lesions for colorectal carcinoma. In the present study, the possible inhibitory effect of dietary administration of auraptene was investigated using an animal colon carcinogenesis model with a colon carcinogen AOM. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce colon neoplasms. They also received diets containing 100 or 500 ppm auraptene for 4 weeks in groups of "initiation" feeding, starting 1 week before the first dosing of AOM. The diets containing auraptene were also given to rats for 38 weeks in groups of "postinitiation" feeding. At the termination of the study (38 weeks), dietary administration of auraptene caused dose-dependent inhibition in AOM-induced large bowel carcinogenesis. Auraptene feeding during the initiation phase reduced the incidence of colon adenocarcinoma by 49% at 100 ppm (P = 0.099) and 65% at 500 ppm (P = 0.0075). Auraptene administration during the postinitiation phase inhibited the incidence of colon adenocarcinoma by 58% at 100 ppm (P = 0.021) and 65% at 500 ppm (P = 0.0075). Also, the multiplicity of colon carcinoma was significantly reduced by initiation feeding at a dose level of 500 ppm (P < 0.01) and postinitiation feeding at a level of 100 and 500 ppm (P < 0.05 and P < 0.01, respectively). Feeding of auraptene suppressed the expression of cell proliferation biomarkers (ornithine decarboxylase activity and polyamine content) in the colonic mucosa and reduced the production of aldehydic lipid peroxidation [malondialdehyde and 4-hydroxy-2(E)-nonenal]. In addition, auraptene increased the activities of Phase II drug-metabolizing enzymes (glutathione S-transferase and quinone reductase) in the liver and colon. These findings suggest that the inhibitory effects of auraptene on AOM-induced colon tumorigenesis at the initiation level might be associated, in part, with increased activity of Phase II enzymes, and those at the postinitiation stage might be related to suppression of cell proliferation and lipid peroxidation in the colonic mucosa.

Topics: Aldehydes; Animals; Anticarcinogenic Agents; Cell Division; Citrus; Colonic Neoplasms; Coumarins; Enzyme Induction; Glutathione Transferase; Intestinal Neoplasms; Lipid Peroxidation; Male; Malondialdehyde; NAD(P)H Dehydrogenase (Quinone); Neoplasm Proteins; Ornithine Decarboxylase; Polyamines; Rats; Rats, Inbred F344