Experimental colon carcinogenesis is facilitated by endogenous factors in the intestinal contents

The theory that endogenous factors in the intestinal contentsmay be pathogenic during large bowel carcinogenesis was testedin the dimethylhydrazine (DMH)-induced rat colon cancer model.Thirty female Wistar rats, each serving as their own control,had a surgical transection of the proximal colon with reanastomosisto the rectum, thereby excluding part of the colon from faecalcontact. All rats then received a course of DMH (40 mg/kg bodywt/wk s.c. for 10 weeks) while fed on Vivonex. This diet wasselected because it lacks known exogenous (dietary) cocarcinogens.It also produces mucosal atrophy in functioning (proximal) colon,to parallel the disuse atrophy induced in the defunctioned (distal)colon. Animals remained on the diet throughout the experimentand were killed when moribund or at 40 weeks. At necropsy, theanatomical distribution, number, size and histological typeof colon tumours were compared between functioning and defunctionedcolonic segments within the same animal. At autopsy, there weresignificantly fewer colon tumours in the defunctioned segment(P < 0.005). Furthermore, there were significantly fewercarcinomas (P <0.005) and fewer tumours >1 cm diameter(P <0.01) in this segment. The data indicate that endogenousfactors in the intestinal contents facilitate chemically-inducedcolon carcinogenesis. Luminal nutrition may be implicated.     

Helicobacter pylori infection enhances glandular stomach carcinogenesis in Mongolian gerbils treated with chemical carcinogens

Helicobacter pylori (Hp) is thought to be a stomach carcinogen from epidemiological findings. To determine the effects of infection with the bacteria on experimental carcinogenesis, a study of the glandular stomach of Mongolian gerbils (MGs) was performed. Male MGs were treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by inoculation with Hp or infected with Hp followed by N-methyl-N'-nitro-N-nitrosoguanidine administration. Animals were killed at week 50, and their excised stomachs underwent microbiological and histopathological examinations. In addition, a serological investigation was performed. The incidences of adenocarcinomas were significantly higher in animals treated with 60 or 300 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine for 10 weeks followed by Hp inoculation or Hp followed by 20 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine for 30 weeks than in the respective controls. Moreover, tumour-bearing animals had higher titres of anti-Hp antibodies than tumour-free animals. Of interest was the finding that a dose of 100 p.p.m. N-methyl-N'-nitro-N-nitrosoguanidine given to infected gerbils eradicated the Hp in about half the animals, with a concomitant reduction in the promoting effect. No tumours were found in animals infected with Hp without N-methyl-N'-nitro-N-nitrosoguanidine or non-treated gerbils. Hp infection enhances glandular stomach carcinogenesis in MGs treated with N-methyl-N'-nitro-N-nitrosoguanidine. Animals with high titres of anti-Hp antibodies are at greatest risk of developing neoplasms.     

Environmental and chemical carcinogenesis

People are continuously exposed exogenously to varying amounts of chemicals that have been shown to have carcinogenic or mutagenic properties in experimental systems. Exposure can occur exogenously when these agents are present in food, air or water, and also endogenously when they are products of metabolism or pathophysiologic states such as inflammation. It has been estimated that exposure to environmental chemical carcinogens may contribute significantly to the causation of a sizable fraction, perhaps a majority, of human cancers, when exposures are related to "life-style" factors such as diet, tobacco use, etc. This chapter summarizes several aspects of environmental chemical carcinogenesis that have been extensively studied and illustrates the power of mechanistic investigation combined with molecular epidemiologic approaches in establishing causative linkages between environmental exposures and increased cancer risks. A causative relationship between exposure to aflatoxin, a strongly carcinogenic mold-produced contaminant of dietary staples in Asia and Africa, and elevated risk for primary liver cancer has been demonstrated through the application of well-validated biomarkers in molecular epidemiology. These studies have also identified a striking synergistic interaction between aflatoxin and hepatitis B virus infection in elevating liver cancer risk. Use of tobacco products provides a clear example of cancer causation by a life-style factor involving carcinogen exposure. Tobacco carcinogens and their DNA adducts are central to cancer induction by tobacco products, and the contribution of specific tobacco carcinogens (e.g. PAH and NNK) to tobacco-induced lung cancer, can be evaluated by a weight of evidence approach. Factors considered include presence in tobacco products, carcinogenicity in laboratory animals, human uptake, metabolism and adduct formation, possible role in causing molecular changes in oncogenes or suppressor genes, and other relevant data. This approach can be applied to evaluation of other environmental carcinogens, and the evaluations would be markedly facilitated by prospective epidemiologic studies incorporating phenotypic carcinogen-specific biomarkers. Heterocyclic amines represent an important class of carcinogens in foods. They are mutagens and carcinogens at numerous organ sites in experimental animals, are produced when meats are heated above 180 degrees C for long periods. Four of these compounds can consistently be identified in well-done meat products from the North American diet, and although a causal linkage has not been established, a majority of epidemiology studies have linked consumption of well-done meat products to cancer of the colon, breast and stomach. Studies employing molecular biomarkers suggest that individuals may differ in their susceptibility to these carcinogens, and genetic polymorphisms may contribute to this variability. Heterocyclic amines, like most other chemical carcinogens, are not carcinogenic per se but must be metabolized by a family of cytochrome P450 enzymes to chemically reactive electrophiles prior to reacting with DNA to initiate a carcinogenic response. These same cytochrome P450 enzymes--as well as enzymes that act on the metabolic products of the cytochromes P450 (e.g. glucuronyl transferase, glutathione S-transferase and others)--also metabolize chemicals by inactivation pathways, and the relative amounts of activation and detoxification will determine whether a chemical is carcinogenic. Because both genetic and environmental factors influence the levels of enzymes that metabolically activate and detoxify chemicals, they can also influence carcinogenic risk. Many of the phenotypes of cancer cells can be the result of mutations, i.e., changes in the nucleotide sequence of DNA that accumulate as tumors progress. These can arise as a result of DNA damage or by the incorporation of non-complementary nucleotides during DNA synthetic processes. Based upon the disparity between the infrequency of spontaneous mutations and the large numbers of mutations reported in human tumors, it has been postulated that cancers must exhibit a mutator phenotype, which would represent an early event in cancer progression. A mutator phenotype could be generated by mutations in genes that normally function to guarantee genetic stability. These mutations presumably arise via DNA damage by environmental or endogenous agents, but it remains to be determined whether the acquisition of a mutator phenotype is a necessary event during tumor progression.     

Telomere length and telomerase activity in carcinogenesis of the stomach

Telomerase activity is generally absent in primary cell cultures and normal tissues. Telomerase is known to be induced upon immortalization or malignant transformation of human cells. In the present study, we analyzed both telomere length and telomerase activity in biopsy samples from mucosa undergoing metaplasia, adenoma and cancer of the stomach. We attempted to estimate the correlation between telomerase activity and telomere length in these tissues. Telomerase activity was estimated using the telomeric repeat amplification protocol and telomere length by Southern blot analysis. Extracts were defined as telomerase-negative when the signals were less intense than those for 10(2) KATO-III cells (positive control). We detected telomerase activity in 15%, 45% and 89% of the examined cases of intestinal metaplasia, adenoma and gastric cancer respectively. However, telomere length in the gastric mucosa became reduced as the mucosa underwent metaplasia and developed into adenoma. Gastric cancers showed a broad range of telomere length among cases. However, gastric adenomas showed the shortest telomere length. These results suggest that telomerase is expressed during the early phase (intestinal metaplasia through adenoma) of gastric carcinogenesis, although the activity at that stage is not high enough to fully restore the reduced telomeric DNA.      

Steroid receptors in dimethylhydrazine-induced colon carcinogenesis

To investigate whether gonadal hormones are involved in the tumorigenesis of dimethylhydrazine (DMH)-induced colonic neoplasms, the authors measured steroid receptors in the neoplasms. 30- or 60-day-old BD-IX rats were injected with 20 mg of 1,2-DMH per kg of body weight once a week for 20 weeks. Fifty-seven rats were sacrificed at 40 to 45 weeks after the initial injection. Androgen receptor (AR), estrogen receptor (ER), and progesterone receptor (PR) were measured in colonic neoplasms. The total number of colonic neoplasms was 274 among 57 rats, 65.8% in male rats and 34.2% in female rats. The mean number of colonic neoplasms per rat was higher in male rats, i.e., 5.6, compared with 3.5 in female rats. A slightly higher number of colonic neoplasms per rat was seen in the rats that had the initial injection at 30 days of age. The number of large colonic neoplasms with a diameter of more than 1 cm was 77 (28.1%), 74% of which were seen in male rats. Thus, a higher incidence of tumors that were also larger were seen in male rats. Histologic findings showed that 53.6% of the neoplasms were carcinomas. The highest incidence of colonic neoplasms was in the distal colon in both sexes. Most of the well-differentiated adenocarcinoma were seen in the distal colon (82.2%), whereas mucinous carcinoma and undifferentiated adenocarcinoma were prominent in the proximal colon or cecum (56.1%). In rats with a normal colon, low levels of AR and PR were determined; but ER was not found in any regions of the colon. In DMH-induced colonic cancer, the incidence as well as the concentration was higher in male rats (60.6%, 16.9 +/- 3.6 fm/mg protein), compared with female rats (40.0%, 4.6 +/- 0.8 fm/mg protein). Similar incidences and levels of ER and PR were seen in both sexes. There was no relationship between steroid receptors and histologic findings in colonic neoplasms. These results suggest that the gonadal hormones, especially androgens, appear to be involved in DMH-induced colon tumorigenesis in male rats.     

Infection, inflammation and colon carcinogenesis

While alterations of the intestinal microbiome have been described in IBD patients, with suggestions that particular species may be associated with ileal Crohn's disease, no individual bacterial species or groups have been consistently associated with either colonic Crohn's disease or ulcerative colitis. Likewise, the role that microbial biomolecular activity may play in these diseases remains unknown. Metabolomic analysis of fecal water from patients with these diseases has identified microbial population shifts suggesting that functional capacity may be more critical than microbial membership. While chronic inflammation is widely thought to be a critical factor, biomolecular pathways implicated in the development of IBD-associated colon cancer remain incompletely characterized.     

Precancerous lesion and carcinogenesis of stomach cancer

A research on pathogenesis of gastric carcinoma was carried out by observing microcarcinoma, small carcinoma, early multicarcinoma as well as the mucosa around the cancer. The results show that the site of different types of carcinoma is correlated to the type of gastric dysplasia. In this paper, various types of gastric dysplasia and multicentric histogenesis of gastric cancer are discussed.     

Selenium and cabbage and colon carcinogenesis in mice

The influence of dietary selenium and cabbage on the formation of colon tumors in female Swiss mice treated with 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8] was reported. Mice received a control diet (laboratory chow), the control diet plus selenium in the drinking water (1 mg/liter), or the control diet with added cabbage (12.8 g/100 g diet). They also received 8 weekly sc injections of DMH. The experiment was divided into two time periods: a) from 5 weeks before the first injection until 3 days after the last one (initiation period), and b) the subsequent 19.5 weeks until sacrifice of the mice (promotion period). Selenium had a strong protective effect when given during the initiation period; adenomas were reduced to a much greater extent than adenocarcinomas. The only effect of selenium supplementation in the promotion period was a small decrease in adenomas. Cabbage apparently had two opposing actions. It increased tumor incidence, particularly adenocarcinomas, if given in the initiation period, but it reduced adenoma formation considerably when given in the promotion period.     

Transplacental chemical carcinogenesis in man

This editorial was prompted by the published association of maternal diethylstilbestrol (DES) ingestion during pregnancy and subsequent development of vaginal adenocarcinoma among female offspring, and explores various factors involved in transplacental chemical carcinogenesis in humans. Known prenatal determinants of carcinogenic transmission are 1) germ cells, 2) transplantation, and 3) ionizing radiation. Other chemicals besides DES which may be implicated in transplacental carcinogenesis are cytotoxic anticancer agents, such as therapy. The hypothesis of DES-associated maternal-fetal exchange was developed as a result of physician recognition of a cluster of cases with commonality; it is hoped that further epidemiological studies, more systemitized, will lead to hypotheses regarding the epidemiology of other in utero carcinogenesis.     

Role of the RNA-binding protein HuR in colon carcinogenesis

Immunohistochemical analysis of paired tumor and normal tissue specimens revealed that the expression and cytoplasmic abundance of the RNA-binding protein HuR increased with malignancy, particularly in colon carcinomas. Interventions to modulate HuR expression in human RKO colon cancer cells altered gene expression profiles and identified beta-catenin mRNA as a novel HuR target. Subcutaneous injection of HuR-overexpressing RKO cells into nude mice produced significantly larger tumors than those arising from control populations; conversely, RKO cells expressing reduced HuR through small interference RNA- or antisense HuR-based approaches developed significantly more slowly. We propose that HuR-regulated target mRNA expression contributes to colon cancer growth. Our results suggest a pivotal function for HuR in colon carcinogenesis.     

Heterogenicity of ornithine decarboxylase during mouse colon carcinogenesis and in human colon tumors

Ornithine decarboxylase (ODC) was separated, using diethylamino-ethyl ion-exchange chromatography, into multiple peaks of activity. We investigated the isoforms of ODC during 1,2-dimethylhydrazine-induced colon carcinogenesis and in human colon tumors. ODC in both mouse and human normal-appearing colonic mucosa was consistently separated into two active peaks by diethylaminoethyl-Sepharose CL-6B column chromatography. The major peak (Peak I) contained about 75% of the mouse and 72% of the human colonic mucosal ODC activity. During and after 10 weekly injections of 1,2-dimethylhydrazine (20 mg/kg, i.p.), colonic ODC activity was significantly enhanced with induction of both peaks but with a more significant increase in Peak II. ODC activity in both 1,2-dimethylhydrazine-induced and human colon tumors was significantly higher compared with the normal colon mucosa. The chromatographic profile of tumors showed the predominance of the second peak. Furthermore, the chromatographic profile of ODC after alkaline phosphatase treatment yielded an elution of only one peak coincident with the Peak I and the disappearance of Peak II. The second peak of ODC (the phosphorylated form) may be a specific isoform associated with colon tumorigenesis and tumor growth.     

Role of Notch signaling in colon homeostasis and carcinogenesis

Colorectal cancer is a leading cause of cancer-related deaths world-wide. Despite the development of new anticancer agents, there will be an estimated 150,000 new cases and 50,000 deaths associated with this disease during the next year.((1)) This is due, in part, to the limitations of chemotherapy, resulting from drug resistance and organ system toxicities. To overcome the inherent limitations associated with standard chemotherapy techniques, the development of novel drug targets is of utmost importance in combating this disease. There is accumulating evidence that a small fraction of cancer cells, referred to as cancer stem cells, may play a critical role in the pathogenesis of this disease. In fact, the identification of cancer stem cells can be accomplished based on the expression of surface markers associated with a cancer stem-like phenotype. This stem-like phenotype includes indefinite self-replication, pluripotency, and most importantly, resistance to chemotherapeutics. Therefore, understanding the properties of cancer stem cells may ultimately lead to new therapeutic approaches. Recently, several studies have shown that Notch signaling is critical in maintaining cancer stem cell properties. This review provides a summary of colonic crypt organization and colon carcinogenesis with a focus on stem cells. Moreover, we discuss novel therapeutic strategies that are under development for targeting Notch signaling in cancer stem cells.     

Mouse models for the study of colon carcinogenesis

The study of experimental colon carcinogenesis in rodents hasa long history, dating back almost 80 years. There are manyadvantages to studying the pathogenesis of carcinogen-inducedcolon cancer in mouse models, including rapid and reproducibletumor induction and the recapitulation of the adenoma–carcinomasequence that occurs in humans. The availability of recombinantinbred mouse panels and the existence of transgenic, knock-outand knock-in genetic models further increase the value of thesestudies. In this review, we discuss the general mechanisms oftumor initiation elicited by commonly used chemical carcinogensand how genetic background influences the extent of disease.We will also describe the general features of lesions formedin response to carcinogen treatment, including the underlyingmolecular aberrations and how these changes may relate to thepathogenesis of human colorectal cancer. Abbreviations: AA, arachidonic acid; ACF, aberrant crypt foci; AOM, azoxymethane; Apc, adenomatous polyposis coli; BCAC, β-catenin-accumulated crypt; COX-2, cyclooxygenease-2; cPLA₂, cytosolic phospholipase A₂; CRC, colorectal cancer; DFMO, difluoromethylornithine; DMAB, 3,2'-dimethyl-4-aminobiphenyl; DMH, 1,2-dimethylhydrazine; DSS, dextran sodium sulfate; HCA, heterocyclic amine; IBD, inflammatory bowel disease; i.p., intraperitoneal; IQ, 2-amino-33-methylimidazo[4,5-f]quinoline; MAM, methylazoxymethanol; miRNA, microRNA; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, methylnitrosourea; ODC, ornithine decarboxylase; PGDH, 15-hydroxyprostaglandin dehydrogenase; PGE₂, prostaglandin E2; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; s.c., subcutaneous; TGF, transforming growth factor; TNF, tumor necrosis factor Received August 13, 2008; revised October 31, 2008; accepted November 20, 2008.     

DNA methylome alterations in chemical carcinogenesis

Carcinogenesis, a complex multifactorial process of the transformation of normal cells into malignant cells, is characterized by many biologically significant and interdependent alterations triggered by the mutational and/or non-mutational (i.e., epigenetic) events. One of these events, specific to all types of cancer, is alterations in DNA methylation. This review summarizes the current knowledge of the role of DNA methylation changes induced by various genotoxic chemicals (carcinogenic agents that interact with DNA) and non-genotoxic carcinogens (chemicals causing tumor by mechanisms other than directly damaging DNA) in the lung, colorectal, liver, and hematologic carcinogenesis. It also emphasizes the potential role for epigenetic changes to serve as markers for carcinogen exposure and carcinogen risk assessment.     

Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon

Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-ras(G12D) allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-ras(G12D) throughout the colon (LSL-K-ras(G12D)/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-ras(G12D) induces increased expression of procarcinogenic protein kinase C beta II (PKC beta II), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-ras(G12D) inhibits expression of procarcinogenic PKC beta II and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice.     

Thioproline prevents carcinogenesis in the remnant stomach induced by duodenal reflux

An excessive duodenal reflux induced by surgery has been widely accepted to cause gastric carcinogenesis in the remnant stomach. As one of causative factors for malignancy, N-nitroso compounds produced by enteric bacteria have been postulated. However, there is no concrete information to prove this hypothesis. This study was undertaken to elucidate the factors underlying the remnant stomach carcinogenesis, by giving thiazolidine-4-carboxylic acid (thioproline; TPRO) to the rats with duodenal reflux as a nitrite scavenger. Operated 39 animals were used, divided into 2 groups; one with a diet containing 0.5% TPRO (n=18), and the other with a diet without TPRO (n=21). Adenocarcinoma developed in 16 rats out of 21 (76.2%) of untreated rats, whereas adenocarcinoma was detected in 1 rat of the TPRO-treated rats (5.6%). TPRO thus prevented the development of gastric cancer in the remnant stomach, thereby suggesting a concern of nitroso compounds to the carcinogenesis.     

Neo-angiogenesis and the premalignant micro-circulatory augmentation of early colon carcinogenesis

Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 μm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.