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Department of Human Nutrition, * Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612
4To whom correspondence should be addressed. E-mail: adiamond{at}uic.edu.
ABSTRACT
Selenium has been shown to reduce cancer incidence in animal models and more recent data indicate that it may be protective in humans as well. However, little is known about the mechanism by which selenium prevents cancer. Cytosolic glutathione peroxidase (GPX1), a selenium-containing antioxidant enzyme, has been implicated in the development of cancer of the head and neck, lung, and breast, in part because of allelic loss at the GPX1 locus. The study of allelic loss at the GPX1 locus in colon cancer was investigated by examining loss of heterozygosity (LOH) in DNA extracted from both tumor and adjacent histopathologically normal tissue obtained by laser capture microdissection. Tissue samples were obtained from 53 colon cancer patients. Two highly polymorphic markers, alanine codon repeats and a proline-leucine polymorphism (198P/L) present in the GPX1 gene, were used to examine LOH at this locus. Analysis of both polymorphisms identified LOH at GPX1 in a significant percentage of colorectal cancer (42%). These results indicated that LOH at the GPX1 locus is a common event in cancer development and that GPX1 or other tightly linked genes may be involved in the etiology of this disease.
KEY WORDS: • selenium • selenoproteins • glutathione peroxidase • cancer
One of the most promising candidate chemopreventive agents is the essential trace element selenium. Decades of animal studies have consistently demonstrated that supplementing the diet of rodents with low, nontoxic amounts of selenium is effective in reducing cancer incidence in response to a broad range of carcinogens in most organ systems examined (1). In humans, several groups reported an inverse correlation between dietary intake of selenium and cancer incidence at several sites, including lung, colon, and prostate (2–6). Human supplementation studies have been few, but the reduction in cancer incidence as a consequence of consuming selenium as a supplement at levels obtainable from over-the-counter products indicated that selenium was effective in reducing cancer incidence in lung, colon, prostate, and liver (7,8). Because of this accumulative body of evidence, large chemopreventive human studies have been initiated (9). However, the mechanism by which selenium may reduce cancer incidence remains unknown.
It is likely that many of the effects of selenium are mediated through its role as a constituent of selenium-containing proteins. Twenty-five selenoproteins were discovered in the human genome (24 in the mouse) and all contain selenium as the amino acid selenocysteine (Sec)5 (10,11). Sec is incorporated cotranslationally during selenoprotein synthesis in response to in-frame UGA codons in the mRNA for these selenoproteins. Sec insertion requires dedicated translation factors including a Sec tRNA and elongation factor in addition to the RNA element in the 3'-untranslated portion of the mRNA that directs Sec incorporation in response to all in-frame UGA codons (12,13). In mammalian cells, this process is highly regulated and responsive to selenium availability, both at the levels of RNA stability and translation. Whether individual selenoproteins or selenoproteins as a group are involved in the health benefits associated with selenium remains unknown.
Selenium is an essential micronutrient shown to reduce colon cancer incidence and preneoplastic aberrant crypts foci in animal models (14–17). In human studies, data have indicated that selenium levels are inversely associated with cancer mortality and incidence. A prospective case-control study indicated a statistically significant inverse association between toenail selenium levels and the risk of colon cancer (5). A significant inverse association between selenium levels and the incidence of large adenomatous polyps, after adjustment for confounding variables in patients <60 y of age [odds ratio (OR) = 0.17, P = 0.029], was also reported (18). The association between selenium intake and colon cancer risk was recently substantiated by an analysis of pooled data from 3 independent studies (6). In human supplementation trials, selenium reduced cancer mortality and colorectal cancer incidence (7), although the statistical significance decreased with longer follow-up (19).
One mechanism by which selenium may influence cancer incidence is via its effects on selenium-containing proteins. One selenoprotein, cytoplasmic glutathione peroxidase (GPX1), is an intracellular selenium-dependent enzyme that is ubiquitously expressed and detoxifies hydrogen and lipid peroxides. GPX1 levels are particularly responsive to fluctuations in selenium levels compared with other selenoproteins. Mice null for Gpx1 and GPx2 exhibit severe ileocolitis at a young age and develop microflora-associated cancers in the lower gastrointestinal tract (20).
GPX1 is polymorphic at codon 198, resulting in either a proline or a leucine at that position, and the frequency of the leu allele is strongly associated with an increase in the risk for lung (21) and possibly breast cancer (22). The identity of the amino acid at codon 198 (proline or leucine) has functional consequences with regard to level of enzyme activity in response to increasing levels of selenium provided to cells in culture (22).
Allelic loss of chromosome regions bearing tumor suppresser genes is a key event in the evolution of epithelial and mesenchymal tumors, and this event can be detected by loss of a heterozygous marker (23). Loss of heterozygosity (LOH) occurs at the GPX1 locus during the development of several cancer types, including those occurring in lung, breast, and head and neck (22,24,25). In the case of head and neck cancers, GPX1 allelic loss was shown to occur in histopathologically normal tissue adjacent to tumors, indicating that loss at this locus may be an early event in cancer evolution (24).
Materials and methods
Laser capture microdissection.
Sections (5 µm) of the paraffin-embedded tissue, including the tumor, were stained by hematoxylin and eosin and periodic acid Schiff using standard protocols (30). Distinct neoplastic cells and normal cells (
1000 of each) were collected by laser microdissection (PixCell® IIe, Arcturus). The selected cells on the cap were transferred to a microcentrifuge tube for DNA preparation and further analysis.
Genetic analysis at the GPX1 locus. LOH at the GPX1 locus was evaluated by examining the number of trinucleotide repeats encoding alanine residues in the amino-terminus of the protein (25). Approximately 2 µL of DNA-containing solution was used in a 25 µL PCR reaction. A segment of DNA including the alanine repeat marker in exon 1 were amplified by PCR using the forward primer 5'-ATGTGTGCTGCTCGGCTA-3' and the reverse primer 5'-AGAAGGCATACACCGACTGG-3'. PCR was performed by denaturing at 95°C for 3 min followed by 65 cycles at 95°C for 20 s, 60°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 10 min. PCR amplification of alleles encoding 5, 6, or 7 alanine repeats results in products of 52, 55, or 58 bp, respectively. PCR products were labeled with [32P]ATP and band lengths were assessed by electrophoresis in an 11% polyacrylamide gel electrophoresis with 10% spreadex and visualized by exposing to X-ray film.
LOH at the GPX1 locus was also evaluated by genotyping samples to identify the nucleotide polymorphism resulting in either a leucine or proline at codon 198. The sequence containing this region was amplified using the forward primer 5'-ATCGAGCCTGACATCGAA-3' and reverse primer 5'-AAGCAGCCGGGGTAGGAG-3' and the 76-bp PCR product was digested by Apa1 (recognition sequence GGGCCC). A proline codon (CCC) at position 198 was cleaved by the enzyme but a leucine codon (CTC) was not. The digested PCR products were analyzed by electrophoresis in 4% agarose gels. Bands were visualized by ethidium bromide staining to distinguish the uncut 76-bp fragment, indicative of the leucine-encoding allele, from the 34 and 42 bp digestion products (34 bp and 42 bp visualized as one band) indicative of a proline-encoding allele. Genotyping studies at both polymorphic sequences always included a PCR reaction omitting template as a control for contamination.
Results
Genetic analyses of the GPx-1 locus in colon cancers and adjacent tissue. Laser capture microdissection was used to obtain tumor and nontumor cells from paraffin-embedded samples obtained during the resection of colon tumors. Paraffin-embedded colon cancer blocks were randomly chosen from the Gastrointestinal Cancers Tissue Bank housed in the Jesse Brown Veterans Affairs Medical Center, Chicago, IL. All tissues studied had a confirmed histopathological diagnosis of colon cancer. Standard hematoxylin and eosin-stained sections from each lesion were reviewed to verify the diagnosis. Patients included African Americans and Caucasians, 49–97 y old, whose tumors were evenly represented across Dukes stages. To obtain samples representing tumor and histopathologically appearing normal tissue from the same colon, both tumor cells and normal cell were selected by laser capture microdissection from the same slide for genetic analysis. Representative photos taken before and after laser capture microdissection are shown in Figure 1.
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Several lines of evidence now support a role for GPX1 in colon cancer risk and development. Animal studies have consistently shown a decrease in carcinogen-induced colon cancer if their diets are supplemented with selenium (1,14–17,26) and GPX1 levels respond to selenium supplementation (27). In addition, functional polymorphisms within the human GPX1 gene are associated with increased risk of lung cancer and possibly cancers of other organs (21,22). In addition to data indicating a risk of cancer associated with specific GPX1 alleles, LOH at the GPX1 locus was shown to be a common event in cancer development (25,22,24). The previously reported data on GPX1 LOH in several tumor types is now extended to colon cancer in this study, although it still remains to be established whether it is the loss of a GPX1 allele or a tightly linked gene that promotes tumorigenesis. The GPX1 gene is located at chromosome location 3p21, and lung cancers exhibiting 3p LOH have reduced GPX1 enzyme activity and compromised oxidative defense with elevated levels of the DNA oxidation product 8-hydroxydeoxyguanosine (28). LOH at this position is also associated with higher number of relapses and shorter disease-free survival for lung cancer patients (29). Conceivably, allelic loss could unmask a recessive mutation in the remaining allele or promote cancer by resulting in the reduction in cellular GPX1 activity, in attenuated antioxidant defenses, or alterations in affected signaling pathways. Collectively, these data indicate the possibility that lower levels of GPX1 are a risk factor for cancer and that the loss of 1 of 2 copies of the gene during tumor development plays a role in disease progression. In addition, these data indicate that GPX1 may be involved in the mechanism by which selenium suppresses cancer incidence, which may include stimulating the levels of GPX1 from a single remaining allele in precancerous tissue. Additional studies are required, perhaps assessing LOH at genetic markers in the vicinity of GPX1, to determine whether the loss of that gene is the consequential event in cancer etiology. Animal studies using mice that are null for GPX1 will also be useful in assessing whether the stimulation of that gene’s product is required for the cancer-suppressing effects obtained by selenium supplementation.
FOOTNOTES
1 Published in a supplement to The Journal of Nutrition. Presented as part of the International Research Conference on Food, Nutrition, and Cancer held in Washington, DC, July 14–15, 2005. This conference was organized by the American Institute for Cancer Research and the World Cancer Research Fund International and sponsored by (in alphabetical order) California Avocado Commission; California Walnut Commission; Campbell Soup Company; The Cranberry Institute; Danisco USA, Inc.; The Hormel Institute; National Fisheries Institute; The Solae Company; and United Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Ritva R. Butrum, and Helen A. Norman. Guest Editor Disclosure: R. R. Butrum and H. Norman are employed by conference sponsor American Institute for Cancer Research; V.L.W. Go, no relationships to disclose. 
2 Author Disclosure: No relationships to disclose.
3 This work is supported by NIH grant RO1CA81153 to AMD.
5 Abbreviations used: GPX1, cytoplasmic glutathione peroxidase; LOH, loss of heterozygosity; OD, odds ratio; Sec, selenocysteine.
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