FAT, BILE ACIDS, AND BACTERIA

Several lines of evidence suggest that diets containing large percentages of fat predispose to CRC, especially in the descending and sigmoid colon. Colon cancer rates are high in populations whose total fat intake is high and are lower in those who consume less fat. On average, fat (saturated plus unsaturated) constitutes 40% to 45% of total calorie intake in Western countries with high rates of CRC, whereas in low-risk populations, fat accounts for only 10% to 15% of dietary calories. Case-control and cohort studies also have suggested that the incidence and mortality rates from colon cancer, and in some cases rectal cancer, are positively correlated with dietary fat, but these findings are less convincing than data from descriptive epidemiologic studies.

Early trials also often failed to take into account total energy intake. A prospective study assessed the relationship of meat, fat, and fiber intake among 88,751 women aged 34 to 59 years. After adjustment for total energy intake, the intake of animal fat was significantly correlated with the risk of colon cancer. The intake ratio of red meat to chicken and fish was strongly associated with increased incidence of colon cancer, perhaps owing to differences in their fat composition. Similar findings have been reported that correlate the intake of saturated fat and the ratios of red meat to chicken and fish intake with both the incidence and recurrence of colorectal adenomas in women. Cohort studies and a combined analysis of 13 case-control studies that adjusted for total energy intake, however, failed to provide clear-cut evidence for the association between dietary fat and CRC observed in earlier studies. Studies that specifically examine the association between intake of saturated or animal fat and CRC suggest a stronger association than with total fat.

Animal studies lend additional support for the role of dietary fat in the development of colon cancer. These studies usually involve the injection of carcinogens such as 1,2-dimethylhydrazine (DMH) or azoxymethane into rodents fed various diets. Animals fed a variety of polyunsaturated and saturated fats develop greater numbers of carcinogen-induced colonic adenocarcinomas than do those on low-fat diets. The amount and source of dietary fat might affect tumor development in such studies; fatty acids derived from polyunsaturated fish oils (ω-3 fatty acids) and monosaturated olive oil might not promote tumors to the extent that other polyunsaturated fats do.

It has been proposed that dietary fat enhances cholesterol and bile acid synthesis by the liver, thereby increasing the amounts of these sterols in the colon. Colonic bacteria convert these compounds to secondary bile acids, cholesterol metabolites, and other potentially toxic metabolic compounds. Population studies demonstrate increased excretion of sterol metabolites and fecal bile acids in groups that consume a high-fat, low-fiber Western diet compared with other groups, and high fecal bile acid levels are found in some patients with CRC. Dietary fat also has been shown to increase the excretion of secondary bile acids in carcinogen-treated rats; secondary bile acids do not act as primary carcinogens but as potent promoters of colon carcinogenesis in such animal models. Little is known about how lipid and sterol metabolites promote tumors, but both bile acids and free fatty acids have been shown to damage the colonic mucosa and increase the proliferative activity of its epithelium. Dietary consumption of high amounts of corn oil and beef fat increase colonic ornithine decarboxylase levels, which are associated with rapidly proliferating mucosa.

Activation of protein kinase C (PKC) by bile acids in colonic mucosa also might represent an important intracellular event by which bile acids provoke a proliferative response. Mucin alterations are a common feature of colonic neoplasia, and alterations in MUC2 mucin have been associated with tumor progression in the colon. Bile acids induce mucin expression in human colon carcinoma cells by increasing MUC2 transcription through a process involving mitogen-activated protein (MAP) kinase-independent, PKC-dependent activation of the transcription factor AP-1.⁴ Bile acids can, in addition, induce release of arachidonate and conversion of arachidonic acid to prostaglandins in the mucosa, which can enhance cell proliferation.⁵ Preclinical and clinical evidence indicate that nonsteroidal anti-inflammatory drugs (NSAIDs), which reduce prostaglandin synthesis, reduce the incidence of large bowel cancer (discussed later); inhibition of the inducible enzyme cyclooxygenase (COX)-2 may be particularly important in this regard.⁶

Certain fatty acids could promote carcinogenesis by altering membrane fluidity after being incorporated into cell membranes. Bacterial enzymes such as 7-α-dehydroxylase (which converts cholic to deoxycholic acid), β-glucuronidase, nitroreductase, and azoreductase may be induced by a high-fat diet and also could convert compounds ingested in the diet to active carcinogens (see later).

An inverse relationship has been reported between physical activity and risk for CRC in men; obesity is associated with elevated risk of CRC. Serum cholesterol and β-lipoprotein levels have been positively correlated with the development of colorectal adenomas and CRC, but this association has not been demonstrated consistently, and serum cholesterol levels can decline before the development of colon cancer. Adiponectin is a hormone secreted by adipose tissue, serum levels of which are inversely correlated with obesity and hyperinsulinemia. Variants of adiponectin and adiponectin receptor genes have been correlated with differences in CRC risk.⁷

FIBER

Epidemiologic, case-control, and animal studies suggest that dietary fiber protects against the development of colon cancer. Dietary fiber is plant material that resists digestion and that is composed of a heterogeneous mix of carbohydrates (e.g., cellulose, hemicellulose, pectin) and noncarbohydrates (e.g., lignin). Although the protective role of fiber is not completely clear, epidemiologic studies correlate high-fiber intake with a lower incidence of colorectal neoplasia.^8,9 The majority of observational-epidemiologic and case-control studies support the protective effect of fiber-rich diets; however, these data do not define the relationship between fiber-rich food and the importance of nonfiber vegetable components, nutrients, and micronutrients in fruits and vegetables. The effect of fiber components on different portions of the large bowel also can vary. This might explain, in part, the inability to demonstrate a protective effect of fiber in several randomized, controlled trials that have examined the ability of fiber supplementation to prevent adenoma recurrence.^10,11

Two large prospective observational studies, including one from the Prostate, Lung, Colon and Ovarian Cancer Trial (PLCO) found that increased fiber intake is significantly associated with reduced risk of colorectal neoplasia.⁸ Another analysis of 13 prospective cohort studies included in the Pooling Project of Prospective Studies of Diet and Cancer demonstrated that dietary fiber intake was inversely associated with risk of CRC in age-adjusted analyses, but that after accounting for other dietary factors, high intake of dietary fiber was not associated with a reduced risk of CRC.¹²

Investigators postulate that fibers such as cereal bran exert their protective role by increasing stool bulk, thereby diluting carcinogens and promoters of carcinogenesis, enhancing their elimination, and minimizing their duration of mucosal contact by decreasing intestinal transit time. Increased fiber intake, in the form of whole wheat and rye bread, also reduces the concentration of fecal secondary bile acids and fecal mutagens in healthy subjects. Animal studies also have demonstrated a decreased incidence of colonic tumors in DMH-treated rats fed diets high in fiber and fiber components, such as wheat bran, cellulose, and hemicellulose. Cellulose and hemicellulose decrease the levels of bacterial metabolic enzymes, such as β-glucuronidase, in experimental animals and can diminish the activation of carcinogens or cocarcinogens. Furthermore, some fiber components can bind to toxic or carcinogenic substances, perhaps decreasing their contact with the colonic mucosa. Fiber components also are fermented by fecal flora to short-chain fatty acids, thereby decreasing colonic pH and potentially inhibiting carcinogenesis.

CARCINOGENS AND FECAL MUTAGENS, VITAMINS, AND MICRONUTRIENTS

The possibility that specific genotoxic carcinogens might play a role in the genesis of CRC was raised when it was noted that the stools of certain persons exhibited mutagenic activity for bacteria in vitro. Mutagenic activity often is present in the feces of populations at high risk for large bowel cancer and is low or absent in low-risk populations. A specific group of highly unsaturated reactive compounds synthesized by colonic bacteria, fecapentaenes, might play a role in large bowel carcinogenesis. It also has been recognized that “charbroiled” meat and fish, and to a lesser extent fried foods, contain powerful mutagenic compounds. The structures of these compounds resemble a heterocyclic amine known to cause colon cancer in rodents. Metabolites similar to those of fried meat and fish are being sought as mutagens in human stools. A possible association between rectal cancer and beer- and ale-drinking also has been noted. A two-fold-to-three-fold increase in CRC also has been observed in automotive pattern and model makers, but the specific carcinogenic agent in this industrial environment has not yet been identified.

The exact nature of genotoxic carcinogens that might act in the human colon remains speculative, but the identification of such compounds could provide a basis for intervention and primary prevention of CRC. Limited data suggest that foods rich in carotene (vitamin A) and vitamin C could act as antioxidants in the chemoprevention of colon cancer, but prospective trials have failed to demonstrate such an effect. Other areas that merit further exploration in the prevention of CRC include the role of yellow-green cruciferous vegetables and the role of micronutrients including selenium salts, vitamin E, and folic acid. A good deal of attention has been given to a possible role for dietary calcium in preventing colon cancer (discussed next).

CALCIUM AND VITAMIN D

Epidemiologic, clinical, and laboratory evidence suggest that calcium intake might protect against carcinogenesis in the colon. Calcium has numerous biological effects that might reduce colon carcinogenesis, including actions on the cell cycle, cyclic adenosine monophosphate (cAMP), calmodulin, tyrosine kinases, ornithine decarboxylase, and e-cadherin. The calcium-sensing receptor expressed in the intestine senses extracellular calcium with effects on differentiation and proliferation. The potential chemopreventive activity of calcium was suggested originally by epidemiologic studies reporting an inverse relationship between CRC and intake of vitamin D and calcium. Dietary calcium supplementation in the form of low-fat dairy foods can affect a variety of intermediate biomarkers thought to be associated with tumor progression in the colon, and supplemental calcium plus vitamin D alters preneoplastic features of colorectal adenomas.¹³ Although the relationship between calcium intake and a lower incidence of colonic adenomas and carcinomas has not been uniformly demonstrated, observational studies overall suggest a protective effect. A pooling project of 10 cohort studies strongly suggested that calcium intake is inversely related to the risk of CRC.¹⁴

Further support for the beneficial effect of calcium in preventing large bowel cancer comes from numerous animal studies. Abnormal proliferation occurs in neoplastic and preneoplastic lesions in the colon. The increase in colonocyte proliferation stimulated by intrarectal instillation of deoxycholate and free fatty acids or by dietary supplementation with cholic acid may be ameliorated by oral calcium supplementation in laboratory animals. Studies in rodents fed high-fat diets also demonstrate a reduction in the number of carcinogen-induced tumors in animals receiving supplemental calcium in their diet. This reduction may be true especially for tumors containing K-ras mutations, a finding also suggested by a study in humans. Ornithine decarboxylase, an enzyme involved in polyamine biosynthesis and elevated in preneoplastic states, is reduced in rat colonic mucosa incubated with calcium in vitro, and supplemental calcium suppresses elevated levels of this enzyme in the mucosa of elderly patients with adenomatous polyps. It has been suggested that dietary calcium binds to ionized fatty acids and bile acids in the intestine, converting them to insoluble calcium compounds incapable of stimulating epithelial proliferation. Calcium increases fecal excretion of both phosphate and bile acids and modifies relative amounts of bile acids in bile. In addition, calcium in milk products is capable of precipitating luminal cytotoxic surfactants, thereby inhibiting their effects on colonic mucosa. These potential beneficial effects of calcium have not been observed uniformly, however, and studies of the effects of calcium on the rectal mucosa have not always demonstrated a reduction in the proliferation rates. In other studies, calcium supplementation normalized the distribution of proliferating cells in the colonic crypt without affecting the rate of proliferation in the colorectal mucosa.

Vitamin D₃ metabolites and analogs have been shown to play an important role in the regulation of a number of important cellular processes, including proliferation, differentiation, and apoptosis, in addition to their established role in mineral homeostasis. These steroid compounds have rapid effects that do not involve gene transcription or protein synthesis, as well as genomic effects involving the vitamin D receptor and other transcription factors.^15,16 Vitamin D can modulate more than 200 genes involved in cell cycle regulation, growth factor signaling, protection against oxidative stress, bile acid and xenobiotic metabolism, cell adhesion, DNA repair, angiogenesis, inflammation, and immune function. Effects of vitamin D and its metabolites have been demonstrated in normal and malignant colonocytes, and several potential mechanisms have been suggested by which these compounds might prevent carcinogenesis in the colon. Dietary supplementation with calcium and vitamin D in rodents fed colon tumor-inducing Western diets significantly reduced tumor incidence and multiplicity in addition to altering expression of a variety of genes linked to initiating formation of colon tumors.¹⁷

ARACHIDONIC ACID, EICOSANOIDS, AND CYCLOOXYGENASE-2

Clinical case-control and cohort studies have shown a 40% to 50% reduction in CRC-related mortality in persons taking aspirin and other NSAIDs on a regular basis compared with those not taking these agents. The exact mechanism for cancer protection with these agents is unknown, but it might relate to altered synthesis of arachidonic acid metabolites (eicosanoids) including prostaglandins, thromboxanes, leukotrienes, and hydroxy-eicosatetraenoic acids. These compounds modulate a number of signal transduction pathways that affect cellular adhesion, growth, and differentiation. COX (or prostaglandin-endoperoxide synthase) oxidizes arachidonic acid to prostaglandin G₂, reduces prostaglandin G₂ to prostaglandin H₂, and is the key enzyme responsible for production of prostaglandins and other eicosanoids.

This enzyme exists in two isoforms: COX-1 and COX-2. COX-1, the constitutive form of the enzyme, is present in most tissues and is involved in the physiologic production of prostaglandins to maintain normal homeostasis. COX-2 is induced by cytokines, mitogens, and growth factors, and its level has been shown to be elevated in both murine and human CRCs.^18–22 Expression of COX-2 is increased markedly in 85% to 95% of CRCs and in experimental models of CRC. COX-2 inhibition prevents cancer from developing during the initiation and the promotion and progression stages of carcinogenesis.²¹ Knockout of COX-2 results in suppression of intestinal polyposis in animal models of familial adenomatous polyposis (FAP).¹⁹ 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a prostaglandin-degrading enzyme that is lost in human colon cancers and has been shown to be a physiologic antagonist of the prostaglandin-synthesizing activity of COX-2.²² It has been speculated that NSAIDs might reduce formation of colon tumors by inhibiting prostaglandin-mediated proliferation, but other evidence suggests that part of their effect might result from inducing apoptosis. Overexpression of COX-2 has been demonstrated to decrease apoptosis, whereas inhibition of COX-2 leads to an increase in apoptosis.

One potential mechanism by which NSAIDs can induce apoptosis is through elevating the prostaglandin precursor arachidonic acid. Increases in arachidonic acid after NSAID inhibition of COX stimulate conversion of sphingomyelin to ceramide, which is a mediator of apoptosis. NSAIDs also might inhibit the activation of genes by the nuclear hormone receptor peroxisome-proliferator-activated receptor δ (PPARδ) by disrupting the ability of this receptor to bind DNA. PPARδ expression is elevated in CRCs and repressed by the APC gene product, which is altered in CRC cells. The inhibition of PPARδ function enhances the ability of NSAIDs to induce apoptosis in colon cancer cells. PPARδ activates a variety of genes, including those involved in cellular growth and differentiation after exposure to a variety of ligands, such as eicosanoids. COX-2 inhibition could prevent production of these ligands and thereby prevent activation of PPARδ.

Other potential mechanisms by which COX-2 inhibition might affect tumor formation include alterations of cell adhesion to extracellular matrix proteins, inhibition of tumor neovascularization (angiogenesis), and reduction in carcinogen activation. A study using the Apc^Δ716 mouse, an animal model of FAP, demonstrated that treatment with the COX-2 specific inhibitor rofecoxib (Vioxx) was associated with a significant dose-dependent reduction in size and number of polyps as well as alterations in polyp morphology. COX-2 inhibition was associated with decreased levels of vascular endothelial growth factor (VEGF) and with lower rates of DNA replication.²⁰

In summary, environment and diet might affect the genesis of CRC, but their exact roles remain unclear. Their complex nature renders definition of the influence of individual environmental and dietary components difficult.

CHEMOPREVENTION

Chemoprevention refers to the use of natural or synthetic agents to reverse, suppress, or prevent progression or recurrence of cancer^23,24; this is a cornerstone of primary prevention. Data on chemoprevention of CRC come from studies in laboratory animals (see earlier), observational epidemiologic studies, case-control studies, and randomized controlled trials (RCTs). Because the natural history of CRC is protracted, clinical RCTs often have concentrated on preventing colorectal adenomas, which represent a form of intraepithelial neoplasia and are the precursors to carcinoma. The duration of the studies required, sample sizes necessary, cost, and ethical considerations make the use of cancer as an end point impractical. This difficulty has led to an increasing use of surrogate biomarkers to study chemoprevention of CRCs,²⁵ with the hope that use of such markers will lead to shorter, smaller, and less expensive trials.²⁶ To be valid, however, such biomarkers need to accurately represent the events involved in the process of carcinogenesis. When an intervention such as a chemopreventive agent is tested, there should be a clear relationship among the agent, modulation of the biomarker, and the development of cancer. Surrogate end points for cancer ideally should be validated in the context of clinical studies that use cancer as the ultimate end point. This is a difficult task because these are the very trials that such markers are designed to complement or replace.

There has been interest in the use of magnifying endoscopy to study aberrant crypt foci of the colon as possible markers in chemoprevention trials.²⁴ These foci consist of large, thick crypts that can be detected by chromoendoscopy using agents such as methylene blue or indigo carmine (Fig. 123-4). Aberrant crypt foci, particularly large crypts with dysplastic features, are thought to be precursors of adenomas in the colon. Standardization of techniques to identify and quantify these lesions is crucial to the successful interpretation of data from these trials, because it is unclear whether these lesions, which appear to be precursors to neoplasia in animal models, play a similar role in the human colon.²⁷ Studies suggest that the majority of aberrant crypt foci in the human colon may be hyperplastic rather than dysplastic (see Chapter 122).

Figure 123-4. A-C, Three magnification views of aberrant crypt foci at different magnifications. Aberrant crypt foci consist of large thick crypt, and are thought to be precursors of adenomas in the colon.

The potential benefit of low-fat, high-fiber diets based on descriptive epidemiology and case-control studies already has been discussed, but current data from prospective human trials are thus far equivocal or negative. Two large RCTs examined the effects of fiber supplementation on recurrence of adenomas. The Polyp Prevention Trial¹¹ randomized 2079 subjects with a history of colorectal adenomas to receive counseling together with a low-fat, high-fiber diet rich in fruits and vegetables or to receive their usual diet alone. The incidence of recurrent adenomas at one and four years, as determined by colonoscopy, was similar in both groups. In a study conducted by the Phoenix Colon Cancer Prevention Physicians’ Network,¹⁰ 1429 patients with a history of colorectal adenoma were randomized to receive 2.0 g or 13.5 g of supplemental wheat bran per day. Colonoscopy failed to show a difference in the incidence of recurrent adenomas at a median follow-up of 34 to 36 months.

A large body of observational and laboratory studies suggests a role for dietary calcium supplementation in chemoprevention. A prospective double-blind placebo-controlled trial showed that supplemental calcium (3000 mg of calcium carbonate per day, equivalent to 1200 mg of elemental calcium) reduced the incidence and number of recurrent adenomas in subjects chosen for a recent history of such lesions.²⁶ The effect of calcium was modest: 19% reduction in recurrence of adenomas and 24% reduction in the number of adenomas over three years, independent of age, sex, or dietary intake of calcium, fat, or fiber. The protective effect of calcium supplementation on the risk of colorectal adenoma recurrence extended up to five years after cessation of active treatment, even in the absence of continued supplementation.²⁸ Analysis of subjects’ serum vitamin D status suggested that calcium supplementation and vitamin D status appear to act together to reduce the risk of adenoma recurrence.²⁹ The results of a Japanese study, the Fukuoka Colorectal Cancer Study,³⁰ support the joint action of calcium and vitamin D in preventing CRC. Human trials using antioxidant vitamins A, C, and E have provided equivocal results, and current data do not support their routine use for colon cancer prevention in average-risk persons.

Folic acid and its metabolites play an important role in DNA synthesis, strand integrity, and methylation. Epidemiologic studies have found a lower incidence of CRC among those with high compared with low dietary intake of folate.³¹ This protective effect also was suggested by the Nurses’ Health Study, in which high doses of folate (as part of multivitamin supplementation) given over several years were protective against CRC. A large prospective RCT^32,33 failed, however, to demonstrate a protective effect of 1 mg/day of folate supplementation on recurrence of adenoma compared with placebo and suggested that folate supplementation in persons with prior adenomas actually might increase adenoma risk. Analysis of baseline dietary and serum folate levels in these subjects supports the idea that although moderate doses of folate may be protective compared with deficiency, at some point of sufficiency, supplementation provides no additional benefit.³⁴ The lack of efficacy of folate (0.5 mg/day) supplementation to prevent adenoma recurrence also was found in another RCT.³⁵

Epidemiologic, case-control, and prospective cohort trials suggest a protective effect against the development of CRC in women taking hormone (estrogen) replacement therapy. It has been postulated that estrogen might protect against colon cancer by decreasing production of secondary bile acids, by decreasing levels of insulin-like growth factor-1, or through as yet undetermined direct effects on colonic mucosal epithelial cells. Data from the Women’s Health Initiative demonstrated that short-term use of estrogen plus progestin was associated with a decreased risk of CRC; however, CRCs in women who took estrogen plus progestin were diagnosed at a more advanced stage than those in women who took placebo.³⁶

Cigarette smoking has been associated with incidence of and mortality from CRC in observational studies,³⁷ but the long-term effects of smoking cessation on CRC have not been studied.

The most promising results for CRC prevention come from trials using aspirin and NSAIDs. Case-control and cohort studies have suggested that the risk for development of adenoma and carcinoma may be reduced substantially (40% to 50%) among aspirin and NSAID users, compared with controls.^6,24,38 A prospective cohort study among male health professionals demonstrated that persons who take aspirin more than twice per week were at lower risk for CRC (relative risk [RR], 0.68) than were controls. An RCT that assessed the effect of low-dose aspirin in an average-risk population demonstrated no significant reduction in the number of CRC cases during the first six years of follow-up; longer follow-up may be necessary to demonstrate a significant effect of aspirin on development of cancer, because the Nurses’ Health Study demonstrated that the benefits of aspirin might not be evident until after at least a decade of regular aspirin consumption.⁶ Three prospective adenoma prevention trials (see later) now provide compelling evidence that aspirin use reduces the risk of developing colorectal adenoma in persons with a personal history of adenoma or carcinoma.^39–41