LINUS PAULING INSTITUTE RESEARCH REPORT

Gary Merrill

Cancer Chemoprotection by Selenium and Thioredoxin

Gary Merrill, Ph.D.
Professor of Biochemistry & Biophysics
Oregon State University

Selenium is an essential trace element necessary to maintain good health. Laboratory animals reared in the absence of selenium show retarded growth, and farm animals raised on selenium-poor crops develop white muscle disease. Humans living in isolated regions with insufficient selenium in the food chain show susceptibility to cardiomyopathies. Pathologies reversed by nutritionally adequate levels of selenium are classified as selenium deficiency diseases. In modern societies there is sufficient selenium in the normal diet to prevent outright selenium deficiency disease. Nevertheless, accumulating evidence suggests that supplementing the diet with supranutritional levels of selenium has additional health benefits (see figure below). In laboratory animals, a high selenium diet results in a significant reduction in the frequency of several types of cancers. Selenium may also have cancer preventive activity in humans. An oft-cited clinical study published by Dr. Larry Clark in 1998 found that in patients who had been treated for skin cancer and given yeast-derived organic selenium (200 µg selenium per day) or a placebo for several years, selenium had no significant effect on skin cancer reoccurrence, but showed a remarkably large effect on the occurrence of lung cancer (46% reduction), colon-rectum cancer (58% reduction), and prostate cancer (63% reduction). Clinical studies to further explore these findings are under way.

The molecular basis for the cancer-preventive activity of selenium is not known. One idea is that selenium boosts the activity of enzymes that contain the unusual amino acid selenocysteine. Such selenoproteins are rare. Mammalian genomes may encode about twenty selenoproteins, of which about half have actually been biochemically identified. All of the selenoproteins thus far characterized seem to have a role in protecting the cell against oxidants or maintaining redox homeostasis. Selenium is chemically similar to sulphur and, like sulphur, can accept or donate electrons during oxidation and reduction reactions. When selenocysteine is removed from proteins, their activity decreases dramatically. Given that most, if not all, selenoproteins have a role in combating oxidative stress, and that the selenocysteine is required for protein activity, a reasonable hypothesis for the cancer- preventive activity of dietary selenium is that it ensures that the selenoproteins are present at levels sufficient to prevent or reverse the damage imposed by reactive oxygen and nitrogen species. One problem with this hypothesis is that animals and humans that have been given supranutritional levels of selenium and show lower cancer rates generally do not have elevated selenoprotein levels. However, in support of the hypothesis, supplemental selenium has been shown to increase the levels of certain selenoproteins in cultured cells, and it is possible that in vivo, synthesis of selenoproteins in certain cells and tissues undergoing oxidative stress may be important for cell homeostasis and preservation of genome integrity.

One of the selenoproteins induced by pharmacological concentrations of selenium in cell culture is thioredoxin reductase. As its name implies, thioredoxin reductase reduces thioredoxin—a small protein that has multiple enzymatic and regulatory roles in the cell. Reduced thioredoxin supplies the electrons required by enzymes like ribonucleotide reductase, which synthesizes DNA precursors, and peroxiredoxins, which decompose potentially cytotoxic hydroperoxides. Reduced thioredoxin is also capable of protecting other proteins from oxidative inactivation. In proteins, the sulphur-containing amino acid cysteine is the residue most susceptible to oxidation. Most of the cysteines in intracellular proteins are in the reduced "thiol" form. However, as a result of reaction with reactive oxygen species, the cysteines of certain proteins can become oxidized to the disulfide state. Oxidation often distorts the protein and prevents it from carrying out its normal function in the cell. Reduced thioredoxin can reduce the disulfide bond in these oxidized proteins and thereby restore them to their active state. In the process, thioredoxin itself becomes oxidized and must be reduced by thioredoxin reductase before it can again serve as an electron donor. In the absence of thioredoxin reductase or in the presence of oxidative conditions there may not be sufficient reduced thioredoxin to meet all the demands of the cell, and proteins containing oxidation-prone cysteines may accumulate in the inactive disulfide state. Recent evidence suggests that one of the proteins susceptible to oxidative inactivation in mammalian cells is the tumor suppressor protein p53.

It has been hypothesized that cells cannot become cancerous if they have an intact tumor suppressor p53 system. Mutations in the p53 gene are detected in most cancers, and in those cancers that lack a p53 gene mutation, mutations in other genes that control p53 activity are often detected. p53 functions as a tumor suppressor by turning on other genes in response to stresses, such as DNA damage or oncogene activation.

The target genes induced by p53 encode proteins that either block cell proliferation or trigger programmed cell death. In this way, damaged or abnormal cells that are at risk of becoming cancerous are either given time to repair the damage or are eliminated from the body. Because of its importance as a tumor suppressor, p53 has been the object of intense study for many years. The protein is present at low levels in a latent state in most normal cells. In response to stresses like DNA damage or oncogene activation, the p53 protein undergoes modifications that result in its stabilization, accumulation, and activation as a transcription factor that stimulates the synthesis of RNA from DNA. The p53 modification most frequently studied is the attachment of phosphate groups to amino acids, such as serine, threonine, and tyrosine, in proteins. Such phosphorylation reactions have been shown to affect p53 stability and activity. More recently, pharmacological evidence in mammalian cells and genetic evidence in yeast suggests that p53 is subject to another type of modification—the reversible oxidation of critical cysteine thiols to disulfides.

The yeast Saccharomyces cerevisiae has been a remarkably potent model for understanding how the eukaryotic cells of higher organisms carry out basic biological processes. One of the many advantages of yeast is the relative ease with which genes can be added, deleted, or replaced in the yeast genome. By introducing the p53 gene into yeast, we were able to show that human p53 is able to activate targeted genes in wild-type yeast. However, we and others discovered that yeast with mutations in the thioredoxin reductase gene have severely inhibited p53 activity. To find out if p53 similarly requires thioredoxin reductase in mammalian cells, we needed to develop a means of inhibiting thioredoxin reductase activity in mammalian cells. Through the support of a Linus Pauling Institute pilot project grant, we investigated several ways to reduce thioredoxin reductase activity in human breast cancer cells. One approach was to introduce a gene encoding an abnormal form of thioredoxin reductase that would interfere with the functionality of the normal endogenous thioredoxin reductase. A second approach was to introduce a gene that inhibited the synthesis of thioredoxin reductase by affecting RNA. Both approaches reproducibly inhibited thioredoxin reductase activity, and, importantly, resulted in a proportional decrease in the activity of p53. These new results confirm that p53 is dependent on thioredoxin reductase in mammalian cells and justify further investigation of the mechanism and consequences of thioredoxin reductase dependence.

Since non-vascularized tumors containing a normal p53 gene rarely grow larger than the size of a grain of rice, p53 may be activated by the abnormally low oxygen levels in the interior of these tumors. P53 activation then limits tumor growth by triggering cell cycle arrest or cell suicide. Conversely, once blood vessels develop within a tumor, the resulting increase in oxygen levels may lead to oxidative inactivation of p53 and resumption of tumor growth. In this scenario, induction of selenoenzymes like thioredoxin reductase by dietary selenium may help keep p53 in its reduced and active state. To test the link between selenium, thioredoxin, p53, and tumor suppression, we are now conducting studies in mice, thanks to support from a Linus Pauling Institute pilot project grant.

Last updated May, 2004


Micronutrient Research for Optimum Health


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