Epidermal Growth Factor Reduces the Development of Necrotizing Enterocolitis in a Neonatal Rat Model

American Journal of Physiology. Gastrointestinal and Liver Physiology. Jan, 2002  |  Pubmed ID: 11751169

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-alpha mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC.

Role of Mitochondrial Complexes I and II, Reactive Oxygen Species and Arachidonic Acid Metabolism in Deoxycholate-induced Apoptosis

Cancer Letters. Mar, 2002  |  Pubmed ID: 11825660

Bile acids are promoters of colon cancer; however, the mechanism(s) of action of this tumor promoter are largely unknown. Bile acids induce apoptosis in colon epithelial cells and it is probable that the modulation of apoptosis contributes, in part, to colon carcinogenesis. We tested the hypothesis that damage to mitochondria is an upstream event in sodium deoxycholate (NaDOC)-induced apoptosis and that a pro-oxidant state of the cell favors survival. NaDOC-induced damage to mitochondria was assessed by a decrease in mitochondrial membrane potential using flow cytometry and an increase in megamitochondria formation using transmission electron microscopy. We found that inhibition of mitochondrial complexes I and II with rotenone and thenoyltrifluoroacetone, respectively, dramatically protected HT-29 cells against NaDOC-induced apoptosis. Antioxidants (e.g. lazaroids U-74389G and U-8389G), however, sensitized cells to NaDOC-induced apoptosis, in spite of a reduction in reactive oxygen/nitrogen species. Lazaroid pre-treatment caused a marked decrease in NaDOC-induced activation of the anti-apoptotic transcription factor, NF-kappaB, which may provide the basis for the sensitization to apoptosis caused by these antioxidants. Inhibitors of arachidonic acid metabolism (e.g. esculetin, sulindac sulfide, NS-398) also sensitized HT-29 cells to NaDOC-induced apoptosis. These results indicate that the life/death decision is the result of a shift in the balance between specific anti-apoptotic and pro-apoptotic factors, respectively, that may have significance to colon carcinogenesis.

Detoxification of the Polyamine Analogue N1-ethyl-N11-[(cycloheptyl)methy]-4,8-diazaundecane (CHENSpm) by Polyamine Oxidase

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. May, 2002  |  Pubmed ID: 12006544

Analogues of the naturally occurring polyamines, alkylated on both terminal amines, are being developed as anticancer drugs. Because bisalkylated derivatives of putrescine (1,4-diaminobutane) are potent inhibitors of the flavin adenine dinucleotide-dependent polyamine oxidase (PAO), we asked whether PAO could detoxify synthetic bisalkylated polyamines with chain lengths longer than putrescine.

Up-regulation of IL-18 and IL-12 in the Ileum of Neonatal Rats with Necrotizing Enterocolitis

Pediatric Research. Jun, 2002  |  Pubmed ID: 12032269

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Because the proinflammatory cytokines IL-18, IL-12, and interferon (IFN)-gamma have been implicated in other diseases of the small intestine, we hypothesized that these cytokines would play an important role in NEC pathogenesis. NEC was induced in newborn rats via enteral feeding with rat milk substitute and asphyxia and cold stress (RMS). Dam-fed, asphyxia- and cold-stressed littermates were used as controls (DF). After 96 h, the distal ileum was removed from all animals and processed to determine expression and localization of IL-18, IL-12, and IFN-gamma using real-time reverse transcriptase PCR and immunohistology. IL-18 and IL-12 mRNA from the RMS group were increased (p < or = 0.05) compared with DF controls, and there was a correlation between increasing IL-18 and IL-12 mRNA levels and progression of tissue damage (r = 0.629 and 0.588, respectively; p < or = 0.05). Immunohistology revealed IL-18 in the cytoplasm of villi and crypt enterocytes and IL-12-positive monocytes/macrophages were increased with disease progression (r = 0.503, p < or = 0.05). No differences in the number of IFN-gamma-positive cells were observed between groups. These data demonstrate up-regulation of IL-18 and IL-12 in experimental NEC and a correlation between production of these proinflammatory cytokines and progression of tissue damage.

DNA Repair/pro-apoptotic Dual-role Proteins in Five Major DNA Repair Pathways: Fail-safe Protection Against Carcinogenesis

Mutation Research. Jun, 2002  |  Pubmed ID: 12052432

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage. Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33(ING1b)); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.

Patchy Field Defects of Apoptosis Resistance and Dedifferentiation in Flat Mucosa of Colon Resections from Colon Cancer Patients

Annals of Surgical Oncology. Jun, 2002  |  Pubmed ID: 12052764

Abnormal areas in normal-appearing flat colonic mucosa (field defects) may predispose individuals to colon cancer. Markers of field defects would indicate cancer risk.

Long-term Effects of Ovarian Follicular Depletion in Rats by 4-vinylcyclohexene Diepoxide

Reproductive Toxicology (Elmsford, N.Y.). Nov-Dec, 2002  |  Pubmed ID: 12401505

4-Vinylcyclohexene diepoxide (VCD) destroys preantral ovarian follicles in rats. Female 28-day Fisher 344 (F344) rats were dosed (30 days) with VCD (80 mg/kg per day, i.p.) or vehicle, and animals were evaluated for reproductive function at subsequent time points for up to 360 days. At each time point animals were killed, and ovaries and plasma collected. VCD reduced (P<0.05) the number of preantral follicles by day 30 relative to control. There were no ultrastructural differences in morphology between VCD-treated and control ovaries. Circulating FSH levels in VCD-treated animals were greater (days 120, 240, and 360, P<0.05) than in controls. Cyclicity was disrupted in the VCD-treated group by day 360. These results show that VCD-induced follicular destruction in rats is associated with a sequence of events (loss of preantral follicles, increased plasma FSH, and cyclic disruption) preceding premature ovarian senescence that is similar to events that occur during the onset of menopause in women.

Imexon Activates an Intrinsic Apoptosis Pathway in RPMI8226 Myeloma Cells

Anti-cancer Drugs. Nov, 2002  |  Pubmed ID: 12439337

Imexon is a new antitumor agent with high activity in multiple myeloma. This drug induces apoptosis, oxidative stress and mitochondrial alterations. However, it was unknown whether imexon activates an intrinsic apoptotic pathway that is associated with activation of caspase-9 or an extrinsic pathway that is induced by receptor-mediated signals such as Fas ligand characterized by caspase-8 activation. In addition, we wanted to investigate the effect of imexon on Bcl-2 family proteins. In RPMI8226 myeloma cells, imexon activated caspase-9 and -3 in a time- and concentration-dependent manner. In contrast, cleavage of procaspase-8 was observed late and only after exposure to very high concentrations of imexon. Confocal microscopy confirmed that caspase-3 is also activated after treatment with imexon. High imexon concentrations activated caspase-3 and -9 at 12 h, while caspase-8 activation occurred only at 48 h. Imexon cytotoxicity was unchanged in three RPMI8226 cell lines with different levels (low, medium and high) of FAS expression. Similarly, the levels of Bcl-2, Bax and Bcl-xL were unchanged in imexon-treated cells. However, Bcl-xL was translocated to the mitochondria. These data suggest that imexon-induced oxidation activates the intrinsic or mitochondrial pathway of apoptosis, involving cytochrome release and activation of caspase-9 and -3.

Molecular and Cellular Characterization of Imexon-resistant RPMI8226/I Myeloma Cells

Molecular Cancer Therapeutics. Jan, 2002  |  Pubmed ID: 12467213

Imexon is an aziridine-containing iminopyrrolidone with selective growth-inhibitory potency for multiple myeloma. Our previous research indicates that imexon induces mitochondrial alterations, oxidative stress, and apoptosis. This drug represents an interesting model drug with a nonmyelosuppressive profile to study the basic mechanisms leading to antitumor activity and resistance. The major purpose of this study was to characterize an imexon-resistant RPMI8226/I cell line that was developed from RPMI8226 cells by continuous exposure to imexon. No significant differences were observed in the sensitivity to several cytotoxic drugs, including mitoxantrone, mitomycin C, melphalan, methotrexate, cytarabine, cisplatin, vincristine, and paclitaxel, in the imexon-resistant cells. However, RPMI8226/I cells were cross-resistant to arsenic trioxide, doxorubicin, fluorouracil, etoposide, irinotecan, and especially IFN-alpha. The data from DNA microarray and Western blot analyses indicated that the levels of antiapoptotic proteins Bcl-2 and thioredoxin-2, which reside mainly in the mitochondria, are increased in RPMI8226/I cells. In addition, increased levels of lung resistance protein were detected in imexon-resistant cells. Expression of P-glycoprotein was not detected in RPMI8226/I cells. No loss of mitochondrial membrane potential or increase in the levels of reactive oxygen species was observed in RPMI8226/I cells after exposure to imexon; however, the levels of glutathione are increased in the RPMI8226/I cells. Transmission electron microscopy revealed significant changes in the mitochondrial morphology of RPMI8226/I cells, whereas no ultrastructural changes were observed in other cellular compartments. Imexon-resistant RPMI8226/I myeloma cells appear to have a unique mechanism of resistance that is associated with morphological alterations of mitochondria, increased protection against oxidative stress, elevated levels of glutathione, and enhanced expression of antiapoptotic mitochondrial proteins.

Development and Molecular Characterization of HCT-116 Cell Lines Resistant to the Tumor Promoter and Multiple Stress-inducer, Deoxycholate

Carcinogenesis. Dec, 2002  |  Pubmed ID: 12507930

Evidence from live cell bioassays shows that the flat mucosa from patients with colon cancer exhibits resistance to bile salt-induced apoptosis. Three independent cell lines derived from the colonic epithelial cell line HCT-116 were selected for resistance to bile salt-induced apoptosis. These cell lines were developed as tissue culture models of apoptosis resistance. Selection was carried out for resistance to apoptosis induced by sodium deoxycholate (NaDOC), the bile salt found in highest concentrations in human fecal water. Cultures of HCT-116 cells were serially passaged in the presence of increasing concentrations of NaDOC. The resulting apoptosis resistant cells were able to grow at concentrations of NaDOC (0.5 mM) that cause apoptosis in a few hours in unselected HCT-116 cells. These cells were then analyzed for changes in gene expression. Observations from cDNA microarray, 2-D gel electrophoresis/MALDI-mass spectroscopy, and confocal microscopy of immunofluorescently stained preparations indicated underexpression or overexpression of numerous genes at either the protein or mRNA level. Genes that may play a role in apoptosis and early stage carcinogenesis have been identified as upregulated in these cell lines, including Grp78, Bcl-2, NF-kappaB(p50), NF-kappaB(p65), thioredoxin peroxidase (peroxiredoxin) 2, peroxiredoxin 4, maspin, guanylate cyclase activating protein-1, PKCzeta, EGFR, Ras family members, PKA, PI(4,5)K, TRAF2 and BIRC1 (IAP protein). Under-expressed mRNAs included BNIP3, caspase-6, caspase-3 and serine protease 11. NF-kappaB was constitutively activated in all three resistant cell lines, and was responsible, in part, for the observed apoptosis resistance, determined using antisense oligonucleotide strategies. Molecular and cellular analyses of these resistant cell lines has suggested potential mechanisms by which apoptosis resistance may develop in the colonic epithelium in response to high concentrations of hydrophobic bile acids that are associated with a Western-style diet. These analyses provide the rationale for the development of hypothesis-driven intermediate biomarkers to assess colon cancer risk on an individual basis.

Nicotine Increases Oxidative Stress, Activates NF-kappaB and GRP78, Induces Apoptosis and Sensitizes Cells to Genotoxic/xenobiotic Stresses by a Multiple Stress Inducer, Deoxycholate: Relevance to Colon Carcinogenesis

Chemico-biological Interactions. Mar, 2003  |  Pubmed ID: 12606154

Epidemiologic studies indicate that environmental (smoking) and dietary factors (high fat) contribute to carcinogenesis in many organ systems. The aim of our study was to test the hypothesis that nicotine, a component of cigarette smoke, and sodium deoxycholate (NaDOC), a cytotoxic bile salt that increases in concentration in the gastrointestinal tract after a high fat meal, induce similar cellular stresses and that nicotine may enhance some of the NaDOC-induced stresses. We found that nicotine, at 0.8 microM, the very low sub-micromolar level occurring in the tissues of smokers: (1). increases oxidative stress; (2). activates NF-kappaB, a redox-sensitive transcription factor; (3). activates the 78 kD glucose regulated protein promoter, an indication of endoplasmic reticulum stress; (4). induces apoptosis; (5). enhances the ability of NaDOC to activate the 153 kD growth arrest and DNA damage promoter, an indication of increased genotoxic stress; and (6). enhances the ability of NaDOC to activate the xenobiotic response element. Our findings have applicability to G.I. cancer, in general, since smoking is a risk factor in the development of esophageal, pancreatic, gastric and colon cancer, and these cancers are also promoted by bile acids.

Perils of Immunohistochemistry: Variability in Staining Specificity of Commercially Available COX-2 Antibodies on Human Colon Tissue

Digestive Diseases and Sciences. Jan, 2003  |  Pubmed ID: 12645811

We assessed the immunohistochemical (IHC) staining patterns of three commercially available COX-2 antibodies in human tissues. The location of positive stain in sequential serial 4-mu sections of formalin fixed, paraffin embedded tissue differed considerably. Staining patterns ranged from diffuse cytoplasmic to occasional perinuclear staining. Thus, marked variability in staining results from use of different antibodies, making it important to consider the antibody used when comparing reports of COX-2 staining from different investigators.

Deoxycholate, an Endogenous Tumor Promoter and DNA Damaging Agent, Modulates BRCA-1 Expression in Apoptosis-sensitive Epithelial Cells: Loss of BRCA-1 Expression in Colonic Adenocarcinomas

Nutrition and Cancer. 2003  |  Pubmed ID: 12925308

Deoxycholate, a bile salt present at high levels in the colonic lumen of individuals on a high-fat diet, is a promoter of colon cancer. Deoxycholate also causes DNA damage. BRCA-1 functions in repair of DNA and in induction of apoptosis. We show that, when cultured cells of colonic origin are exposed to deoxycholate at different concentrations, BRCA-1 expression is induced at a low noncytotoxic concentration (10 microM) but is strongly inhibited at higher cytotoxic concentrations ( > or =100 microM). Indication of phosphorylation of BRCA-1 by deoxycholate (100 microM) at a lower dose was seen by Western blot analysis, whereas, at a higher dose, deoxycholate (200 and 300 microM) caused a complete loss of BRCA-1 expression. We show that BRCA-1 is substantially lower in colon adenocarcinomas from five patients compared with associated non-neoplastic colon tissue from the same patients, suggesting that the loss of BRCA-1 expression contributes to the malignant phenotype. In the non-neoplastic colon tissue, BRCA-1 was localized to the nongoblet cells. Our results imply that reduced expression of BRCA-1 may be associated with carcinoma of the colon.

A Proteomic Study of Resistance to Deoxycholate-induced Apoptosis

Carcinogenesis. May, 2004  |  Pubmed ID: 14729586

The development of apoptosis resistance appears to be an important factor in colon carcinogenesis. To gain an understanding of the molecular pathways altered during the development of apoptosis resistance, we selected three cell lines for resistance to induction of apoptosis by deoxycholate, an important etiologic agent in colon cancer. We then evaluated gene expression levels for 825 proteins in these resistant lines, compared with a parallel control line not subject to selection. Eighty-two proteins were identified as either over-expressed or under-expressed in at least two of the resistant lines, compared with the control. Thirty-five of the 82 proteins (43%) proved to have a known role in apoptosis. Of these 35 proteins, 21 were over-expressed and 14 were under-expressed. Of those that were over-expressed 18 of 21 (86%) are anti-apoptotic in some circumstances, of those that were under-expressed 11 of 14 (79%) are pro-apoptotic in some circumstances. This finding suggests that apoptosis resistance during selection among cultured cells, and possibly in the colon during progression to cancer, may arise by constitutive over-expression of multiple anti-apoptotic proteins and under-expression of multiple pro-apoptotic proteins. The major functional groups in which altered expression levels were found are post-translational modification (19 proteins), cell structure (cytoskeleton, microtubule, actin, etc.) (17 proteins), regulatory processes (11 proteins) and DNA repair and cell cycle checkpoint mechanisms (10 proteins). Our findings, overall, bear on mechanisms by which apoptosis resistance arises during progression to colon cancer and suggest potential targets for cancer treatment. In addition, assays of normal-appearing mucosa of colon cancer patients, for over- or under-expression of genes found to be altered in our resistant cell lines, may allow identification of early biomarkers of colon cancer risk.

Alpha-Tocopheryl Succinate Sensitizes Established Tumors to Vaccination with Nonmatured Dendritic Cells

Cancer Immunology, Immunotherapy : CII. Jul, 2004  |  Pubmed ID: 14991239

Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors.

Increased Expression and Secretion of Interleukin-6 in Patients with Barrett's Esophagus

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2004  |  Pubmed ID: 15041721

Barrett's esophagus (BE) is a common premalignant lesion of the distal part of the esophagus that arises as a consequence of chronic duodenogastroesophageal reflux. Interleukin (IL)-6 is a pleiotropic cytokine that regulates immune defense mechanisms and hematopoiesis. In addition, IL-6 may also be involved in malignant transformation and tumor progression. IL-6 has been shown to inhibit apoptosis. The major aim of this study was to evaluate expression of IL-6 in BE at the protein and mRNA levels. In addition, we tested whether proteins that are associated with IL-6 signaling, phosphorylated signal transducer and activator of transcription 3 and two antiapoptotic proteins, Bcl-x(L) and Mcl-1, are also expressed in the same tissues.

Epidermal Growth Factor Reduces Intestinal Apoptosis in an Experimental Model of Necrotizing Enterocolitis

American Journal of Physiology. Gastrointestinal and Liver Physiology. Apr, 2005  |  Pubmed ID: 15528252

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC+EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury.

Apoptosis Resistance in Barrett's Esophagus: Ex Vivo Bioassay of Live Stressed Tissues

The American Journal of Gastroenterology. Feb, 2005  |  Pubmed ID: 15667503

Barrett's esophagus (BE) is a premalignant lesion of the distal esophagus in which squamous epithelial cells are replaced by metaplastic intestinal-like columnar epithelium that contains goblet cells. The factors that contribute to the progression from normal squamous mucosa to BE, Barrett's dysplasia, and adenocarcinoma are not well understood at the molecular level. Since reflux of bile acids is associated with BE development, we speculate that cells with an apoptosis-resistant phenotype are selected after long-term repeated exposure to pulses of bile acids. This will result in the survival of cells with unrepaired DNA damage, and a consequent increase in genomic instability leading to cancer progression. The major goal of this study is to compare sensitivity to apoptosis induced by the bile acid, deoxycholate (DOC), a known inducer of apoptosis, in normal esophageal squamous epithelium, normal colon epithelium, and BE.

Assessment of Apoptosis by Immunohistochemical Markers Compared to Cellular Morphology in Ex Vivo-stressed Colonic Mucosa

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society. Feb, 2005  |  Pubmed ID: 15684335

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.

Crypt-restricted Loss and Decreased Protein Expression of Cytochrome C Oxidase Subunit I As Potential Hypothesis-driven Biomarkers of Colon Cancer Risk

Cancer Epidemiology, Biomarkers & Prevention : a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. Sep, 2005  |  Pubmed ID: 16172211

There is an increasing demand for the development of intermediate biomarkers to assess colon cancer risk. We previously determined that a live cell bioassay, which assesses apoptosis resistance in the nonneoplastic colonic mucosa, detects approximately 50% of patients with colon cancer. A hypothesis-driven biomarker that reflects apoptosis resistance in routine formalin-fixed, paraffin-embedded tissue would be easier to use. Cytochrome c oxidase is a critical enzyme that controls mitochondrial respiration and is central to apoptosis. We did an immunohistochemical study of cytochrome c oxidase subunit I expression in 46 colonic mucosal samples from 16 patients who had undergone a colonic resection. These included five patients without evidence of colonic neoplasia (three normal and two diverticulitis), three patients with tubulovillous adenomas, and eight patients with colonic adenocarcinomas. Analysis of aberrancies in expression of cytochrome c oxidase subunit I showed that, compared with nonneoplasia, the patients with neoplasia had a higher mean incidence of crypts having decreased expression (1.7 versus 22.8, P = 0.03) and a higher mean incidence having crypt-restricted loss (0.6 versus 3.2, P = 0.06). The percentage with segmented loss was low and was similar in the two groups. Combining these results, the mean % normal (i.e., with none of the three types of abnormality) was 96.7 in nonneoplasia versus only 73.2 in patients with neoplasia (P = 0.02). It should be noted that a defect in cytochrome c oxidase subunit I immunostaining was not detected in all biopsy samples from each patient for whom some abnormality was found, indicating a "patchiness" in the cytochrome c oxidase subunit I field defect. As a result of this "patchiness," the increased variability in the incidence of crypt-restricted loss of cytochrome c oxidase subunit I expression was a statistically significant feature of the neoplasia group. Crypt-restricted loss of cytochrome c oxidase subunit I has not been previously reported in colonic mucosa and is presumably the result of a crypt-restricted stem cell mutation. Decreased cytochrome c oxidase subunit I expression also significantly correlated with apoptosis resistance, a factor known to contribute to carcinogenesis. The results suggest, however, that aberrant cytochrome c oxidase subunit I expression may be a better biomarker than loss of apoptosis competence for increased colon cancer risk.

Docetaxel Induces Cell Death Through Mitotic Catastrophe in Human Breast Cancer Cells

Molecular Cancer Therapeutics. Oct, 2005  |  Pubmed ID: 16227398

Apoptosis has long been considered to be the prevailing mechanism of cell death in response to chemotherapy. Currently, a more heterogeneous model of tumor response to therapy is acknowledged wherein multiple modes of death combine to generate the overall tumor response. The resulting mechanisms of cell death are likely determined by the mechanism of action of the drug, the dosing regimen used, and the genetic background of the cells within the tumor. This study describes a nonapoptotic response to docetaxel therapy in human breast cancer cells of increasing cancer progression (MCF-10A, MCF-7, and MDA-mb-231). Docetaxel is a microtubule-stabilizing taxane that is being used in the clinic for the treatment of breast and prostate cancers and small cell carcinoma of the lung. The genetic backgrounds of these cells were characterized for the status of key pathways and gene products involved in drug response and cell death. Cellular responses to docetaxel were assessed by characterizing cell viability, cell cycle checkpoint arrest, and mechanisms of cell death. Mechanisms of cell death were determined by Annexin V binding and scoring of cytology-stained cells by morphology and transmission electron microscopy. The primary mechanism of death was determined to be mitotic catastrophe by scoring of micronucleated cells and cells undergoing aberrant mitosis. Other, nonapoptotic modes of death were also determined. No significant changes in levels of apoptosis were observed in response to docetaxel.

The Role of NO Synthases in Arginine-dependent Small Intestinal and Colonic Carcinogenesis

Molecular Carcinogenesis. Feb, 2006  |  Pubmed ID: 16329147

Arginine is catabolized by NOS2 and other nitric oxide synthases to form nitric oxide. We evaluated the roles of dietary arginine and Nos2 in Apc-dependent intestinal tumorigenesis in Min mice with and without a functional Nos2 gene. NOS2 protein was expressed only in intestinal tissues of Apc(Min/+) Nos2+/+ mice. NOS3 expression was higher in intestinal tissues of mice lacking Nos2, mainly in the small intestine. When diet was supplemented with arginine (0.2% and 2% in drinking water), lack of Nos2 results in decreased tumorigenesis in both small intestine and colon. In Nos2 knockout mice, supplemental arginine (up to 2%) caused a decrease in small intestinal tumor number and size. The arginine-dependent decrease was associated with an increase in nitrotyrosine formation and apoptosis in the region of intestinal stem cells. Mice expressing Nos2 did not show these changes. These mice did, however, show an arginine-dependent increase in colon tumor number and incidence, while no effect on apoptosis was seen. These changes were associated with increased nitrotyrosine formation in epithelial cells. Mice lacking Nos2 did not show changes in tumorigenesis or nitrotyrosine formation, while demonstrating an arginine-dependent increase in apoptosis. These data suggest that Nos2 and dietary arginine have significant effects on intestinal and colonic tumorigenesis in Min mice. In both tissues, loss of Nos2 is associated with decreased tumorigenesis when mice are supplemented with dietary arginine. In the small intestine, Nos2 prevents the arginine-induced decrease in tumor number and size, which is associated with NOS3 expression and increased apoptosis. In the colon, Nos2 is required for the arginine-induced increase in tumor number and incidence.

Identification of S-nitrosylated Proteins After Chronic Exposure of Colon Epithelial Cells to Deoxycholate

Proteomics. Mar, 2006  |  Pubmed ID: 16404723

Apoptosis resistance, a condition favoring genomic instability, is associated with higher risk of colorectal cancer. Deoxycholate (DOC) is a hydrophobic bile salt found in high concentrations in colon cancer patients, and induces apoptosis in cultured colonic cells and ex vivo in colonic biopsies. We showed previously that the chronic exposure of colon cancer cells to increasing concentrations of DOC leads to apoptosis resistance, and the suggested mechanism involves oxidative/nitrosative stress. Nitric oxide (NO) is a key signaling molecule that regulates cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, and several proteins are regulated through this PTM, including the caspases, the main effectors of apoptosis. Here, we performed a proteomics study in the DOC-induced apoptosis-resistant colon cell line, HCT-116RC. Its profile of S-nitrosylated proteins was compared to a control cell line not exposed to DOC. Eighteen differentially S-nitrosylated proteins were identified in the HCT-116RC cell line, 14 of these are novel targets of S-nitrosylation not previously reported. These proteins include cytoskeletal and signaling proteins, metabolic enzymes, chaperones, and redox- and differentiation-related proteins. These results broaden our knowledge of potential signal transduction pathways that may lead to the development of new biomarkers and therapy targets.

Role of Polyamines in Arginine-dependent Colon Carcinogenesis in Apc(Min) (/+) Mice

Molecular Carcinogenesis. Oct, 2006  |  Pubmed ID: 16705737

We evaluated the role of polyamines in arginine-dependent intestinal tumorigenesis in Apc(Min) (/+) mice. Arginine is a substrate for ornithine synthesis and thus can influence polyamine production. Supplementing the diet with arginine increased intestinal and colonic polyamine levels and colonic carcinogenesis. Inhibiting polyamine synthesis with D,L-alpha-diflouromethylornithine (DFMO) decreased small intestinal and colonic polyamine pools. In mice provided basal diet, but not when supplemented with arginine, DFMO decreased small intestinal tumor number and burden, and increased intestinal apoptosis. In mice provided supplemental arginine in the diet, DFMO induced late apoptosis and decreased tumorigenesis in the colon. DFMO slightly reduced tumor incidence, number, and size while significantly decreasing tumor burden and grade. These changes in colon tumorigenesis did not occur in mice not provided supplemental arginine. Our study indicates that polyamines play unique roles in intestinal and colonic carcinogenesis in Apc(Min) (/+) mice. Inhibition of polyamine synthesis suppresses the arginine-dependent risk of colon tumorigenesis, resulting in apoptosis induction and decreased tumorigenesis, in this murine model.

Reduced Pms2 Expression in Non-neoplastic Flat Mucosa from Patients with Colon Cancer Correlates with Reduced Apoptosis Competence

Applied Immunohistochemistry & Molecular Morphology : AIMM / Official Publication of the Society for Applied Immunohistochemistry. Jun, 2006  |  Pubmed ID: 16785784

Pms2 protein is a component of the DNA mismatch repair complex responsible both for post-replication correction of DNA nucleotide mispairs and for early steps in apoptosis. Germline mutations in DNA mismatch repair genes give rise to hereditary non-polyposis colon cancer, which accounts for about 4% of colon cancers. However, little is known about the expression of mismatch repair proteins in relation to sporadic colon cancer, which accounts for the great majority of colon cancers. Multiple samples were taken from the non-neoplastic flat mucosa of colon resections from patients with no colonic neoplasia, a tubulovillous adenoma, or an adenocarcinoma. Expression of Pms2 was assessed using semiquantitative immunohistochemistry. Apoptosis was assessed in polychrome-stained epoxy sections using morphologic criteria. Samples from patients without colonic neoplasia had moderate to strong staining for Pms2 in cell nuclei at the base of crypts, while samples from 2 of the 3 colons with a tubulovillous adenoma, and from 6 of the 10 colons with adenocarcinomas, showed reduced Pms2 expression. Samples from patients with an adenocarcinoma that had reduced Pms2 expression also exhibited reduced apoptosis capability in nearby tissue samples, evidenced when this paired tissue was stressed ex vivo with bile acid. Reduced Pms2 expression in the colonic mucosa may be an early step in progression to colon cancer. This reduction may cause decreased mismatch repair, increased genetic instability, and/or reduced apoptotic capability. Immunohistochemical determination of reduced Pms2 expression, upon further testing, may prove to be a promising early biomarker of risk of progression to malignancy.

Pathogenesis of Diarrhea in the Adult: Diagnostic Challenges and Life-threatening Conditions

European Journal of Gastroenterology & Hepatology. Oct, 2006  |  Pubmed ID: 16957509

The mechanisms that regulate ionic balance, fluid absorption and secretion in the gastrointestinal tract are complex. Disturbance of this homeostatic state by bile acids, bacterial enterotoxins and neoplasm-derived secretagogues, for example, can lead to diarrhea. Determining the causes of chronic diarrhea in individual patients may, therefore, represent a diagnostic challenge. The gastrointestinal tract has finely tuned mechanisms to maintain ionic balance, fluid absorption and secretion. To achieve this homeostasis, various ionic transport proteins/complexes are targeted to the apical, basal or basolateral membranes of epithelial cells. Na, K and Cl ion transport proteins are regulated by endogenous activators (e.g. cAMP, PGE2, Ca), dietary-related factors (e.g. bile acids) and through post-translational modifications (e.g. phosphorylation). In certain pathophysiologic states, this homeostatic ionic/fluid exchange becomes dysfunctional as a result of failure of compensatory pro-absorptive/anti-secretory mechanisms. The excessive secretion of Na and Cl ions followed by the release of a large amount of H2O into the colonic lumen results in diarrhea, which may be acute or chronic, and can be life-threatening if not ameliorated. Diverse infectious organisms (e.g. bacteria, protozoa, viruses) utilize different mechanisms to cause intestinal disease and diarrhea. These pathophysiologic mechanisms include alterations in ion transport, disruption of tight junctions and elicitation of inflammatory responses. Studies of patients infected with certain pathogens, those with ulcerative colitis and those harboring extra-colonic or colonic neoplasms have elucidated some of these pathophysiologic mechanisms of diarrhea. Determining the etiology of chronic diarrhea in specific cases may be a diagnostic challenge for both gastroenterologists and primary care physicians.

Abnormal Expression of Biomarkers in Incompletely Ablated Barrett's Esophagus

Annals of Surgery. Dec, 2006  |  Pubmed ID: 17122630

The aim of this study was to evaluate expression of cancer risk-associated biomarkers in columnar epithelium at squamocolumnar junctions produced by an ablation procedure and proton pump inhibitors in incompletely ablated Barrett's esophagus (BE) patients that were nondysplastic prior to ablation.

Bile Acids in Combination with Low PH Induce Oxidative Stress and Oxidative DNA Damage: Relevance to the Pathogenesis of Barrett's Oesophagus

Gut. Jun, 2007  |  Pubmed ID: 17145738

Barrett's oesophagus is a premalignant condition associated with an increased risk for the development of oesophageal adenocarcinoma (ADCA). Previous studies indicated that oxidative damage contributes to the development of ADCA.

Deoxycholate-induced Colitis is Markedly Attenuated in Nos2 Knockout Mice in Association with Modulation of Gene Expression Profiles

Digestive Diseases and Sciences. Mar, 2007  |  Pubmed ID: 17253130

Nos2 knockout mice were compared to wild-type mice for susceptibility to colitis in response to a diet supplemented with deoxycholate, a bile acid increased in the colon of individuals on a high-fat diet. Wild-type mice fed a fat-related diet, supplemented with 0.2% DOC, develop colonic inflammation associated with increases in nitrosative stress, proliferation, oxidative DNA/RNA damage, and angiogenesis, as well as altered expression of numerous genes. However, Nos2 knockout mice fed a diet supplemented with deoxycholate were resistant to these alterations. In particular, 35 genes were identified whose expression was significantly altered at the mRNA level in deoxycholate-fed Nos2(+/+) mice but not in deoxycholate-fed Nos2(-/-) mice. Some of these alterations in NOS2-dependent gene expression correspond to those reported in human inflammatory bowel disease. Overall, our results indicate that NOS2 expression is necessary for the development of deoxycholate-induced colitis in mice, a unique dietary-related model of colitis.

MRI-measured Water Mobility Increases in Response to Chemotherapy Via Multiple Cell-death Mechanisms

NMR in Biomedicine. Oct, 2007  |  Pubmed ID: 17265424

Numerous pre-clinical and clinical reports have demonstrated that the MRI-measured apparent diffusion coefficient of water (ADC) increases early in the response to a wide variety of anti-cancer therapies. It has been proposed that this increase in ADC generally results from an increase in the tumor extracellular volume fraction leading to a greater degree of unrestricted water motion. Furthermore, an increase in extracellular volume has been ascribed to the cell shrinkage that occurs early in the process of programmed cell death. However, other modes of death can be initiated soon after beginning therapy. These other modes of death include mitotic catastrophe and necrosis, and may also involve changes in the fraction of water with unrestricted motion. This work examines whether MRI-measured ADC is altered in response to therapies that induce cell death via non-apoptotic mechanisms and correlates ADC changes with cell death modalities regionally within the tumor. Apoptotic responses were limited to the tumor periphery in apoptosis-proficient tumors. Apoptosis was not observed in deficient tumors. Mitotic catastrophe was observed after treatment at the periphery and deeper into the tumor. Necrosis was the predominant response in the center of the tumor. ADC changes were moderate in the periphery and larger in the center. The results indicate that early and significant changes in ADC can occur in concert with mitotic catastrophe and lytic necrosis in the absence of apoptosis. Hence, changes in ADC may be a generalized measure of cytotoxic response to chemotherapy.

Activation of the Interleukin-6/STAT3 Antiapoptotic Pathway in Esophageal Cells by Bile Acids and Low PH: Relevance to Barrett's Esophagus

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Sep, 2007  |  Pubmed ID: 17875759

The molecular factors contributing to the development of Barrett's esophagus (BE) are unclear. Our previous studies showed that BE tissues secrete interleukin-6 (IL-6) and express proteins associated with IL-6 signaling, including IL-6 receptor, activated signal transducer and activators of transcription 3 (STAT3), and antiapoptotic proteins Bcl-x(L) and Mcl-1. Here, we test the hypothesis that bile acids and gastric acids, two components of refluxate associated with gastresophageal reflux disease, activate the IL-6/STAT3 pathway.

A Novel Dietary-related Model of Esophagitis and Barrett's Esophagus, a Premalignant Lesion

Nutrition and Cancer. 2007  |  Pubmed ID: 18001217

Barrett's esophagus (BE) is a premalignant lesion in which columnar epithelium (containing goblet cells) replaces esophageal squamous cells. Previous evidence suggested that hydrophobic bile acids and zinc deficiency each play a role in BE development. We fed wild-type C57BL/6 mice a zinc-deficient diet containing the hydrophobic bile acid, deoxycholic acid for various times up to 152 days. All mice fed this diet developed esophagitis by 69 days on the diet and 63% of the mice on this diet for 88 to 152 days also developed a BE-like lesion. Esophageal tissues showed thickened mucosa, increased proliferation, and increased expression of markers associated with oxidative and nitrosative stress. The newly formed BE-like lesions expressed Mucin-2, a marker of columnar differentiation. They also showed translocation of the p65 subunit of nuclear factor-kappaB and beta -catenin to the nucleus and typical histological changes associated with BE lesions. This mouse model of esophagitis and BE is expected to contribute to a deeper understanding of BE pathogenesis and to strategies for prevention of BE progression to cancer.

Field Defects in Progression to Gastrointestinal Tract Cancers

Cancer Letters. Feb, 2008  |  Pubmed ID: 18164807

A field of defective tissue may represent a pre-malignant stage in progression to many cancers. However, field defects are often overlooked in studies of cancer progression through assuming tissue at some distance from the cancer is normal. We indicate, however, the generality of field defects in gastrointestinal cancers, including cancers of the oropharynx, esophagus, stomach, bile duct, pancreas, small intestine and colon/rectum. Common features of these field defects are reduced apoptosis competence, aberrant proliferation and genomic instability. These features are often associated with high bile acid exposure and may explain the association of dietary-related factors with cancer progression.

Hydrophobic Bile Acids, Genomic Instability, Darwinian Selection, and Colon Carcinogenesis

Clinical and Experimental Gastroenterology. 2008  |  Pubmed ID: 21677822

Sporadic colon cancer is caused predominantly by dietary factors. We have selected bile acids as a focus of this review since high levels of hydrophobic bile acids accompany a Western-style diet, and play a key role in colon carcinogenesis. We describe how bile acid-induced stresses cause cell death in susceptible cells, contribute to genomic instability in surviving cells, impose Darwinian selection on survivors and enhance initiation and progression to colon cancer. The most likely major mechanisms by which hydrophobic bile acids induce stresses on cells (DNA damage, endoplasmic reticulum stress, mitochondrial damage) are described. Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. The multiple mechanisms by which hydrophobic bile acids contribute to genomic instability are discussed, and include oxidative DNA damage, p53 and other mutations, micronuclei formation and aneuploidy. Since bile acids and oxidative stress decrease DNA repair proteins, an increase in DNA damage and increased genomic instability through this mechanism is also described. This review provides a mechanistic explanation for the important link between a Western-style diet and associated increased levels of colon cancer.

Expression of Bile Acid Transporting Proteins in Barrett's Esophagus and Esophageal Adenocarcinoma

The American Journal of Gastroenterology. Feb, 2009  |  Pubmed ID: 19174784

Barrett's esophagus (BE) is a metaplastic lesion characterized by replacement of the normal squamous epithelium by columnar intestinal epithelium containing goblet cells. It is speculated that this process is an adaptation to protect cells from components of refluxate, such as gastric acid and bile acids. In contrast to the normal squamous epithelium, enterocytes of the distal ileum are adapted to transport bile acids from the intestinal lumen. Several bile acid transporters are utilized for effective removal of bile acids, including the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP), and the multidrug-resistant protein 3 (MRP3). We hypothesized that one of the possible functions of newly arising metaplastic epithelium, in the esophagus, is to transport bile acids. Our major goal was to evaluate the expression of bile acid transporters in normal squamous epithelium, BE with different grades of dysplasia, and esophageal adenocarcinoma (EAC).

Bile Acids As Endogenous Etiologic Agents in Gastrointestinal Cancer

World Journal of Gastroenterology : WJG. Jul, 2009  |  Pubmed ID: 19610133

Bile acids are implicated as etiologic agents in cancer of the gastrointestinal (GI) tract, including cancer of the esophagus, stomach, small intestine, liver, biliary tract, pancreas and colon/rectum. Deleterious effects of bile acid exposure, likely related to carcinogenesis, include: induction of reactive oxygen and reactive nitrogen species; induction of DNA damage; stimulation of mutation; induction of apoptosis in the short term, and selection for apoptosis resistance in the long term. These deleterious effects have, so far, been reported most consistently in relation to esophageal and colorectal cancer, but also to some extent in relation to cancer of other organs. In addition, evidence is reviewed for an association of increased bile acid exposure with cancer risk in human populations, in specific human genetic conditions, and in animal experiments. A model for the role of bile acids in GI carcinogenesis is presented from a Darwinian perspective that offers an explanation for how the observed effects of bile acids on cells contribute to cancer development.

Deoxycholate, an Endogenous Cytotoxin/genotoxin, Induces the Autophagic Stress-survival Pathway: Implications for Colon Carcinogenesis

Journal of Toxicology. 2009  |  Pubmed ID: 20130808

We report that deoxycholate (DOC), a hydrophobic bile acid associated with a high-fat diet, activates the autophagic pathway in non-cancer colon epithelial cells (NCM-460), and that this activation contributes to cell survival. The DOC-induced increase in autophagy was documented by an increase in autophagic vacuoles (detected using transmission electron microscopy, increased levels of LC3-I and LC3-II (western blotting), an increase in acidic vesicles (fluorescence spectroscopy of monodansycadaverine and lysotracker red probes), and increased expression of the autophagic protein, beclin-1 (immunohistochemistry/western blotting). The DOC-induced increase in beclin-1 expression was ROS-dependent. Rapamycin (activator of autophagy) pre-treatment of NCM-460 cells significantly (P < .05) decreased, and 3-MA (inhibitor of autophagy) significantly (P < .05) increased the cell loss caused by DOC treatment, alone. Rapamycin pre-treatment of the apoptosis-resistant colon cancer cell line, HCT-116RC (developed in our laboratory), resulted in a significant decrease in DOC-induced cell death. Bafilomycin A(1) and hydroxychloroquine (inhibitors of the autophagic process) increased the DOC-induced percentage of apoptotic cells in HCT-116RC cells. It was concluded that the activation of autophagy by DOC has important implications for colon carcinogenesis and for the treatment of colon cancer in conjunction with commonly used chemotherapeutic agents.

Exposure of Mouse Colon to Dietary Bile Acid Supplement Induces Sessile Adenomas

Inflammatory Bowel Diseases. May, 2010  |  Pubmed ID: 20135625

Hydrophobic Bile Acid-induced Micronuclei Formation, Mitotic Perturbations, and Decreases in Spindle Checkpoint Proteins: Relevance to Genomic Instability in Colon Carcinogenesis

Nutrition and Cancer. 2010  |  Pubmed ID: 20661832

We show, for the first time, that hydrophobic bile acids cause aberrations of the mitotic machinery of colon cells that can give rise to aneuploidy, the chromosomal perturbations common in colon tumors. First, we show that DOC induces a statistically significant fourfold increase in the number of micronuclei in NCM-460 cells (a noncancerous colon cell line) and a threefold increase in the number of micronuclei in binucleated HT-29 colon cancer cells using the cytokinesis block micronucleus assay. Second, we observed mitotic aberrations after DOC treatment, including improper alignment of chromosomes at the metaphase plate, lagging chromosomes during anaphase, anaphase/telophase chromatin bridges, multipolar divisions, and formation of polynucleated cells. It was determined that there was a statistically significant threefold increase in the number of aberrant metaphases after short-term and long-term exposure of HT-29 and HCT-116 cells, respectively. Third, we showed with Western blots and immunohistochemistry that a likely basis for these mitosis-related perturbations included decreased expression of the spindle checkpoint proteins, Mad2, BubR1, and securin. Fourth, results of DOC treatment on nocodazole-challenged cells further indicated deficiencies in activation of the spindle assembly checkpoint. This study provides mechanisms by which hydrophobic bile acids can induce genomic instability in colon epithelial cells.

Carcinogenicity of Deoxycholate, a Secondary Bile Acid

Archives of Toxicology. Aug, 2011  |  Pubmed ID: 21267546

High dietary fat causes increased bile acid secretion into the gastrointestinal tract and is associated with colon cancer. Since the bile acid deoxycholic acid (DOC) is suggested to be important in colon cancer etiology, this study investigated whether DOC, at a high physiologic level, could be a colon carcinogen. Addition of 0.2% DOC for 8-10 months to the diet of 18 wild-type mice induced colonic tumors in 17 mice, including 10 with cancers. Addition of the antioxidant chlorogenic acid at 0.007% to the DOC-supplemented diet significantly reduced tumor formation. These results indicate that a high fat diet in humans, associated with increased risk of colon cancer, may have its carcinogenic potential mediated through the action of bile acids, and that some dietary anti-oxidants may ameliorate this carcinogenicity.

Molecular and Cellular Pathways Associated with Chromosome 1p Deletions During Colon Carcinogenesis

Clinical and Experimental Gastroenterology. 2011  |  Pubmed ID: 21753893

Chromosomal instability is a major pathway of sporadic colon carcinogenesis. Chromosome arm 1p appears to be one of the "hot spots" in the non-neoplastic mucosa that, when deleted, is associated with the initiation of carcinogenesis. Chromosome arm 1p contains genes associated with DNA repair, spindle checkpoint function, apoptosis, multiple microRNAs, the Wnt signaling pathway, tumor suppression, antioxidant activities, and defense against environmental toxins. Loss of 1p is dangerous since it would likely contribute to genomic instability leading to tumorigenesis. The 1p deletion-associated colon carcinogenesis pathways are reviewed at the molecular and cellular levels. Sporadic colon cancer is strongly linked to a high-fat/low-vegetable/low-micronutrient, Western-style diet. We also consider how selected dietary-related compounds (eg, excess hydrophobic bile acids, and low levels of folic acid, niacin, plant-derived antioxidants, and other modulatory compounds) might affect processes leading to chromosomal deletions, and to the molecular and cellular pathways specifically altered by chromosome 1p loss.

Maspin is a Deoxycholate-inducible, Anti-apoptotic Stress-response Protein Differentially Expressed During Colon Carcinogenesis

Clinical and Experimental Gastroenterology. 2011  |  Pubmed ID: 22162927

Increased maspin expression in the colon is related to colon cancer risk and patient survival. Maspin is induced by the hydrophobic bile acid, deoxycholate (DOC), which is an endogenous carcinogen and inducer of oxidative stress and DNA damage in the colon. Persistent exposure of colon epithelial cells, in vitro, to high physiologic levels of DOC results in increased constitutive levels of maspin protein expression associated with the development of apoptosis resistance. When an apoptosis-resistant colon epithelial cell line (HCT-116RC) developed in the authors' laboratory was treated with a maspin-specific siRNA probe, there was a statistically significant increase in apoptosis compared to treatment with an siRNA control probe. These results indicate, for the first time, that maspin is an anti-apoptotic protein in the colon. Immunohistochemical evaluation of maspin expression in human colonic epithelial cells during sporadic colon carcinogenesis (131 human tissues evaluated) indicated a statistically significant increase in maspin protein expression beginning at the polyp stage of carcinogenesis. There was no statistically significant difference in maspin expression between hyperplastic/adenomatous polyps and colonic adenocarcinomas. The absence of "field defects" in the non-neoplastic colonic mucosa of patients with colonic neoplasia indicates that maspin may drive the growth of tumors, in part, through its anti-apoptotic function.