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Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant
Medical Hypotheses (2006) 67, 318–322
http://intl.elsevierhealth.com/journals/mehy
Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant E.N. Drake
*
Rocky Mountain Selenium, Inc., 2101 Ridge Road, Estes Park, Rocky Mountain, CO 80517, United States Received 16 January 2006; accepted 18 January 2006
Summary Although the average daily dietary selenium (Se) intake in the United States is consistently above the adult RDA of 55 lg Se/day, supranutritional supplements of 200 lg Se/day have been shown to provide chemopreventive benefits against several cancers, particularly prostate cancer. The hypothesis herein contends that selenium compounds with the greatest anticarcinogenic potency are likely to be sodium selenite with Se in the +4 oxidation state and methylseleninic acid. These compounds exert their cancer chemopreventive effects by directly oxidizing critical thiol-containing cellular substrates, and are more effective than the more frequently preferred (used) supplements of selenomethionine and Se–methylselenocysteine that lack oxidation capability. Selenate (+6 Se) the immediate precursor of selenite (+4 Se) can be metabolically reduced, and although less potent than the +4 Se compounds cited above, appears to be a more effective anticarcinogen than organic forms of dietary selenium. Apoptosis, an important, Se-induced anticarcinogenic mechanism, is accomplished by the direct oxidation of vicinal sulfhydryl groups in cysteine clusters within the catalytic domains of cellular enzymes (e.g., protein kinase C), and by the production of CH3Se, which reacts with O2 to generate superoxide and other reactive oxygen species (ROS). Activated oncogenes ‘‘prime’’ cells for Se-induced prooxidative apoptosis thereby providing the needed margin for ‘‘killing’’ cancer cells while leaving normal, healthy cells unharmed. Selenoethers, such as selenomethionine and Se–methylselenocysteine are not oxidizing agents, and first, must be converted to methylselenol (CH3Se) that can be directly oxidized to methylseleninic acid. The addition of methioninase, to selenomethionine, or b-lyase to Se–methylselenocysteine, rapidly produces significant amounts of methylselenol, which may be oxidized to methylseleninic acid or may react with O2 to produce superoxide and ROS, resulting in anticarcinogenic activities comparable to selenite or methylseleninic acid. The relatively large amounts of selenomethionine or Se–methylselenocysteine needed to produce apoptosis in cancer cells compared with selenite or methylseleninic acid are a probable consequence of low tissue levels of the required enzymes. Even though many studies have consistently shown that selenomethionine is an ineffective anticarcinogen at doses corresponding to those currently allowed by the FDA, it has been chosen as the Se intervention agent in the 32,500-man (phase III), NCI-funded SELECT trial, which tests the effectiveness of dietary supplements of dietary supplements of Se and tocopherol, individually or in combination, in the prevention of prostate cancer. In 2013, when the data are in, the value of using Se supplements for cancer chemoprevention is likely to be underestimated. c 2006 Elsevier Ltd. All rights reserved.
* Tel.: +1 970 586 1946; fax: +1 970 577 1064. E-mail address: [email protected].
0306-9877/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2006.01.058
Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant
Introduction The most succinct summary of the chemopreventive potential of selenium (Se) was presented by Meuillet, Stratton, Cherukuri, et al. [1] in their 2004 review of the chemoprevention of prostate cancer with Se. These authors conclude: ‘‘there may not be [a] more extensive body of evidence for a cancer prevention potential of [a] dietary component nutrient than there is for Se’’. For many years, those studying the relationship between Se and cancer presumed that Se was exerting its effects as an antioxidant [2]. As a component of glutathione peroxidase (GPx), a widely recognized biochemical function of Se is the removal of reactive oxygen species (ROS) [3]. Although low Se status has been shown to be associated with reduced GPx activity [4], the observation that anticarcinogenic effects continued to increase after GPx had reached optimal (controlled) levels was an early indication that Se might well exert some of its anticancer activity by other mechanisms [5]. The fact that ROS sometime produce cellular DNA damage and ‘‘oxidative stress’’ continues to contribute to the thinking that Se’s anticarcinogenic properties must somehow be explained in terms of its role as an antioxidant. The persistence of such reasoning is particularly enigmatic in view of the long-established knowledge that many chemical forms of Se produce ROS in cells [6]. This paper presents evidence that prooxidative rather than antioxidative properties of Se compounds best account for their observed anticancer effects.
319
3. are expected to produce high steady-state levels of selenide anions (R-Se) and associated ROS; 4. trigger apoptosis in mutated cells; and 5. are not removed from the metabolic Se pool by incorporation into proteins. The naturally occurring inorganic Se compound, sodium selenite and the metabolite methylseleninic acid tend to more completely satisfy the above criteria, and are more effective anticarcinogens than are the organic Se compounds that lack oxidation ability. In 1996, the 10-year, randomized, double-blind, placebo-controlled, clinical trial conducted by the Nutritional Prevention of Cancer (NPC) Group at the University of Arizona [7] demonstrated that subjects receiving daily nutritional supplements of 200 lg of Se in the form of Se-enriched yeast reduced their risk of prostate cancer by some 63%. The early analytical data [8] indicating that the predominant form of Se in the Se-enriched yeast was selenomethionine, presumably, provided a logical basis for choosing selenomethionine as the Se intervention agent in the 12-year, phase III, NCI-funded clinical trial (SELECT) for the prevention of prostate cancer that began in 2001 and will continue until 2013. However, more recent analytical data obtained using actual Se-enriched yeast samples from the NPC study [9] have shown that some samples of supplements used in the trial may have contained as little as 27% selenomethionine. Studies published both before and after the beginning of the SELECT trial provided ample evidence that a chemical form other than selenomethionine was responsible for the impressive chemoprevention of prostate cancer.
The hypothesis Ros and apoptosis The premise of the present paper is that the most effective dietary selenium anticarcinogen presently approved by the FDA is selenite, which inactivates critical thiol-containing enzymes by oxidation and produces ROS such that Se-induced apoptosis can be triggered in mutated but not normal cells. Contrary to prevailing thought, there is much evidence supporting the contention that selenium exerts its cancer chemopreventive activity by functioning as a biochemical oxidizing agent. Se compounds with Se in +4 oxidation states are more effective anticarcinogens that: 1. exert their effects in small doses; 2. directly oxidize critical cellular substrates (enzymes);
Reduction of O2 produces superoxide, peroxides, and hydroxyl free radicals, each of which have various essential cellular functions including the oxidation of fatty acids and alcohols, hydroxylation reactions, conversion of 12-HPETE to 12-HETE, the synthesis of thyroxine, and the implementation of phagocytosis [3]. However, excessive cellular levels of these ROS may produce oxidative damage (mutations of nucleic acids, etc.) and are limited by antioxidants, such as tocopherols, ascorbate, reduced glutathione, and the selenium enzyme, glutathione peroxidase. Reductions in amounts of antioxidants or increases in ROS predispose cells to ‘‘oxidative stress’’. ROS have been shown to play an important role in triggering apoptosis [6]. Apoptosis is the
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genetically controlled cell suicide that preferentially occurs in abnormal (damaged) cells, providing a non-inflammatory mechanism for ridding multicellular organisms of dangerous or unneeded cells. Cells with activated oncogenes initiate apoptosis in response to stimuli that only arrest growth in normal cells [10]. Cells with active oncogenes are, therefore, ‘‘primed’’ for apoptosis, and allow for the destruction of premalignant or malignant cells while leaving normal, healthy cells intact. Some cancer chemotherapeutic compounds rely on the fact that cancer cells undergo apoptosis more readily than normal cells.
Discussion
3
Sodium selenite with Se in the +4 oxidation state reacts directly with cysteine clusters in the catalytic subunits of enzymes, such as protein kinase C, (PKC) oxidizing the sulfhydryl groups to disulfide linkages, thereby inactivating the enzyme [11]. Inactivation of PKC has been shown to induce apoptosis in cancer cells [12]. Since selenite oxidation of vicinal sulfhydryl groups is also associated with the production of both superoxide and peroxide [13], apoptosis may be triggered by either or enzyme inactivation or ROS production. If the carcinostatic activity of Se does, in fact, depend upon the oxidation of some critical substrate, it is ironic that volatile methylselenol (CH3SeH) with selenium in its lowest oxidation state is generally regarded as one of the most effective chemical forms of Se as a chemopreventive agent [14]. Indeed, methylselenol has no effect on the activity of PKC. The excellent work of Gopalakrishna and Gundimeda [11], however, has shown that arachidonate hydroperoxide oxidizes methylselenol to non-volatile methylseleninic acid. The proposed reaction is: 2 C19 H31 –CO3 H+CH3 SeH!2 C19 H31 –CO2 H+CH3 SeO2 H Methylseleninic acid thus produced then inactivates PKC in exactly the same manner as selenite, by oxidizing the sulfhydryl groups in the cysteine clusters in the catalytic domain of the enzyme. Presumably, the reaction would be: R
R SH SH + CH3SeO2H
2 R’
in PKC regenerates methylselenol by reduction of the methylseleninic acid. If excess amounts of methylseleninic acid are present, it also prevents the activation of PKC by destroying the four zinc fingers required for the binding of either diaceylglycerol or phorbol esters to the regulatory domain of PKC. In addition to its oxidation of enzymes, selenite is reduced by glutathione (GSH) producing GS–SeH, GS–Se–SG, and other species that are ultimately converted metabolically to CH3Se when supranutritional doses of selenite are ingested. CH3Se may either be oxidized to methylseleninic acid (CH3SeO2H) or react with oxygen to produce the free radical CH3Se and ROS. The latter reaction is: CH Se þ O ! CH Se þ O
S 2
S + CH3SeH + 2 H2O R’
Methylseleninic acid is essentially behaving as a catalyst since the oxidation of the sulfhydryl groups
2
3
2
Although both selenomethionine and Se–methylselenocysteine are considered to be preferred anticarcinogens, neither can function directly as an oxidizing agent, and both require enzymatic conversion to some selenium compound (perhaps methylselenol) that can undergo facile and direct oxidation to an effective anticarcinogen. To date, most direct comparisons of the antitumor activity of these selenoamino acids with that of either selenite or methylseleninic acid indicate that either selenomethionine or Se–methylselenocysteine in concentrations ranges of 50–200 lmol/l are required to produce effects equivalent to those of selenite or methylseleninic acid at very much lower (1–10 lmol/l) levels [15–18]. Se–methylselenocysteine is thought to offer the most promise as an anticarcinogen because it is the immediate precursor of methylselenol and does not produce large amounts of H2Se [19]. However, the conversion of Se–methylselenocysteine to methylselenol requires the enzyme b-lyase, which is not known to be ubiquitous in vivo in humans. Although one in vivo study in mice [20] reports that equal concentrations of Se–methylselenocysteine and methylseleninic acid have essentially the same anticarcinogenic activity, the b-lyase in some mouse tissues has reported to be 800 times greater than that in human tissues [21]. The higher Se– methylselenocysteine concentrations required to produce antitumor activity in in vitro studies is presumed to be a consequence of insufficient amounts of b-lyase in tumor-cell cultures. Selenomethionine is unique among all biological selenium compounds in that it is incorporated into proteins in place of methionine. Unlike selenocysteine, there is no codon specific for selenomethionine so that methionine and selenomethionine are inserted into proteins based upon availability [22]. Sequestering some of the selenomethionine
Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant in proteins removes it from the metabolic selenium pool, so that less is available for conversion to methylselenol and chemoprevention of cancer. In the presence of the methioninase enzyme, selenomethionine can be converted directly into methylselenol [23]. However, there is currently no evidence that this enzyme is present in human tissues in significant amounts, and the high concentrations of selenomethionine required for anticarcinogenic activity amounts to prima facie evidence that tissue levels of any required enzymes are too small for appreciable catalytic activity. Both selenomethionine and Se–methylselenocysteine produce ROS in the presence of sufficient levels of methioninase and b-lyase, respectively. These findings are consistent with early evidence [24] that significant amounts of selenide anions (RSe), in these cases CH3Se, readily produce ROS. If tissue levels of such enzymes are too low, very small amounts of ROS, CH3Se (methylselenol), and methylseleninic acid are produced so that the anticarcinogenic effects of these selenoethers are necessarily diminished.
Existing data and future studies The preponderance of evidence suggests that the most frequently used dietary selenium supplement, selenomethionine, is least likely among currently approved Se supplements to produce impressive reductions in the risk of prostate, colorectal, lung, breast or any other classification of cancer. As early as 1980, Greeder and Milner [25] demonstrated that supplements of inorganic forms of selenium, such as sodium selenite, sodium selenate, or selenium dioxide completely prevented Ehrlich ascites tumors in rats injected with 500,000 live tumor cells, while all of the animals receiving identical doses of selenomethionine developed tumors. Thompson [26] reported that 5 part-per-million supplements of sodium selenite were 8.3 times more effective in inhibiting DMBAinduced mammary tumors in rats than were the same supplemental doses of selenomethionine. In their comparison of the abilities of sodium selenite and selenomethionine to inhibit DMBA-induced breast cancers in rats, Ip and Hays [22] reported that in spite of higher blood, liver, kidney, and muscle levels of selenomethionine, in all cases and at all doses, sodium selenite produced larger percentages of cancer inhibitions. Others [27] have found that selenomethionine supplements of 10 or 15 parts per million had no effect on the incidence or multiplicity of azoxymethane-induced colon tu-
321
mors in rats. More recently [28], the relative anticarcinogenic effectiveness of sodium selenate, selenomethionine, Se–methylselenocysteine, and selenized yeast supplements were compared by administering two different and equivalent doses of each supplement separately to nude mice with one million PC-3 prostate cancer cells injected directly into their prostate glands. Sodium selenate, but none of the organic selenium supplements, retarded the growth of prostate tumors and decreased the number of retroperitoneal lymph nodes in these hormone refractory prostate cancer cells. The question concerning the most effective chemical form of selenium to use for the chemoprevention of cancers will only be answered by double-blind, human intervention trials comparing sodium selenite and selenomethionine dietary supplements at different supranutritional doses with placebos. Selenomethionine is currently being used as the Se intervention agent in the ongoing SELECT trial in the United States and is regarded as sufficiently safe. However, the safety record for sodium selenite is less frequently acknowledged. The fiveyear, trial [29] involving 20,847 subjects receiving15 part-per-million sodium selenite-enriched salt as an intervention agent in Qidong County, China, is excellent evidence for the position that sodium selenite is also appropriate for use in large-scale clinical trials in the United States. Subjects receiving sodium selenite-enriched salt experienced a 43% reduction in primary liver cancer relative to 109,624 adjacent residents serving as a control group with no incidents of toxicity. Moreover, sodium selenite has been sold as a dietary supplement in the United States without reports of toxicity for more than two decades. Faced with the uncertain composition of the Seenriched yeast supplements used in the NPC trial and the compelling evidence above, it is unlikely that 200 lg supplements of selenomethionine, the Se intervention agent chosen for the SELECT trial, will be shown to significantly reduce the risk of prostate cancer. Such a result could contribute to the erroneous conclusion that dietary selenium supplements are of no value in the chemoprevention of cancer.
Acknowledgement The author expresses his gratitude to Dr. Julian E. Spallholz, Texas Tech University, whose comments and suggestions were enormously helpful in writing the paper.
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References [1] Meuillet E, Stratton S, Cherurukuri DP, Goulet A-C, Kagey J, Porterfield B, et al. Chemoprevention of prostate cancer with selenium: an update on current clinical trials and preclinical findings. J Cell Biochem 2004;91:443–58. [2] Shamberger RJ, Tytko SA, Willis CE. Antioxidants and cancer part IV: selenium and age-adjusted human cancer mortality. Arch Environ Health 1976;24:231–5. [3] Neve J. Physiological and nutritional importance of selenium. Experientia 1991;47:187–93. [4] Levander OA. A global view of human selenium nutrition. A Rev Nutr 1987;7:227–50. [5] Vernie LN. Selenium in carcinogenesis. Biochim Biophys Acta 1984;78:203–17. [6] Stewart MS, Sallholz JE, Neldner KH, Pence BC. Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis. Free Radical Biol Med 1999;26:42–8. [7] Clark LC, Combs GF, Turnbull BW, Slate EH, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. J Am Med Assn 1996;276:1957–63. [8] Bird SM, Uden PC, Tyson JF, Block E, Denoyer E. Speciation of selenoamino acids and organoselenium compounds in selenium-enriched yeast using high-performance liquid chromatography-inductively coupled mass spectrometry. Anal Atomic Spec 1997;12:785–8. [9] Larson EH, Hansen M, Paulin H, Moesgaard S, Reid M, Rayman M. Speciation and bioavailability of selenium in yeast-based intervention agents used in cancer chemoprevention studies. J O A C Int 2004;87:225–32. [10] Lowe SW. The role of p53 in apoptosis. In: Sluyser M, editor. Apoptosis in normal development and cancer. London: Taylor & Francis Ltd.; 1996. p. 97–125. [11] Gopalakrishna R, Gundimeda U. Antioxidant regulation of protein kinase C in cancer prevention. J Nutr 2002;132:3819–S3823. [12] Jarvis WD, Grant S. Protein kinase C targeting in antineoplastic treatment strategies. Inves New Drugs 1999;17:227–40. [13] Spallholz JE. On the nature of selenium toxicity and carcinostatic activity. Free Radical Biol Med 1994;17:45–64. [14] Ganther HE. Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis 1999;20:1657–66. [15] Sinha R, Said TK, Medina D. Organic and inorganic selenium compounds inhibit mouse mammary cell growth in vitro by different cellular pathways. Cancer Lett 1996;107:277–84.
Drake [16] Yeo J-K, Cha S-D, Cho C-H, Kim S-P, Cho J-W, Baek W-K, et al. Se–methylselenocysteine induces apoptosis through caspace activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells. Cancer Lett 2002;182:83–92. [17] Mentor DG, Sabichi AL, Lippman SM. Selenium effects on prostate cell growth. Cancer Epidemiol Biomarkers Prev 2000;9:1171–82. [18] Sinha R, Kiley SC, Lu JX, Thompson HJ, Moraes R, Jaken S, et al. Effects of methylselenocysteine on PKC activity, cdk2 phosphorylation and gad gene expression in synchronized mouse mammary epithelial tumor cells. Cancer Lett 1999;146:135–45. [19] Lu J, Pei H, Ip C, lisk DJ, Ganther H, Thompson HJ. Effect of an aqueous extract of selenium-enriched garlic on in vitro markers and in vivo efficacy in cancer prevention. Carcinogenesis 1996;17:1903–7. [20] Ip C, Thpmpson HJ, Zhu Z, Ganther H. In vitro and in vivo Studies of methylseleninic acid: evidence that a monomethylated metabolite is critical for cancer chemoprevention. Cancer Res 2000;60:2882–6. [21] Rooseboom M, Vermeulen NPE, Andreadou I, Commandeur JNM. Evaluation of the kinetics of b-elimination reactions of selenocysteine Se-conjugates in human renal cytosol: possible implications for the use as kidney selective prodrugs. J Pharmacol Exp Ther 2000;294:762–9. [22] Ip C, Hayes C. Tissue selenium levels in selenium-supplemented rats and their relevance in mammary cancer protection. Carcinogenesis 1989;10:921–5. [23] Spallholz JE, Palace VP, Reid TW. Methioninase and selenomethoinine but not Se–methylselenocysteine generate methylselenol and superoxide in an in vitro chemiluminescent assay: implications for the nutritional carcinostatic activity of selenoamino acids. Biochem Pharmacol 2004;67:547–54. [24] Chaudiere J, Courtin O, Leclaire J. Glutathione oxidase activity of selenocystamine: a mechanistic study. Arch Biochem Biophys 1992;296:328–36. [25] Greeder GA, Milner JA. Factors influencing the inhibitory effect of selenium on mice inoculated with Ehrlich ascites tumor cells. Science 1980;209:825–7. [26] Thompson HJ. Selenium as an anticarcinogen. J Agric Food Chem 1984;32:422–5. [27] Reddy BS, Hirose Y, Lubet RA, Steele VE, Kelloff GJ, Rao CV. Lack of chemopreventive efficacy of DL-selenomethionine in colon carcinogenesis. Int J Mol Med 2000;5:327–30. [28] Corcoran NM, Najdovska M, Costello AJ. Inorganic selenium retards progression of experimental hormone refractory prostate cancer. J Urology 2004;171:907–10. [29] Yu S-Y, Zhu Y-J, Li W-G, Huang Q-S, Zhi-Huang C, Zhang QN, et al. A preliminary report on the intervention trials of primary liver cancer in high-risk populations with nutritional supplementation of selenium in China. Biol Trace Elem Res 1991;29:289–94.
http://intl.elsevierhealth.com/journals/mehy
Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant E.N. Drake
*
Rocky Mountain Selenium, Inc., 2101 Ridge Road, Estes Park, Rocky Mountain, CO 80517, United States Received 16 January 2006; accepted 18 January 2006
Summary Although the average daily dietary selenium (Se) intake in the United States is consistently above the adult RDA of 55 lg Se/day, supranutritional supplements of 200 lg Se/day have been shown to provide chemopreventive benefits against several cancers, particularly prostate cancer. The hypothesis herein contends that selenium compounds with the greatest anticarcinogenic potency are likely to be sodium selenite with Se in the +4 oxidation state and methylseleninic acid. These compounds exert their cancer chemopreventive effects by directly oxidizing critical thiol-containing cellular substrates, and are more effective than the more frequently preferred (used) supplements of selenomethionine and Se–methylselenocysteine that lack oxidation capability. Selenate (+6 Se) the immediate precursor of selenite (+4 Se) can be metabolically reduced, and although less potent than the +4 Se compounds cited above, appears to be a more effective anticarcinogen than organic forms of dietary selenium. Apoptosis, an important, Se-induced anticarcinogenic mechanism, is accomplished by the direct oxidation of vicinal sulfhydryl groups in cysteine clusters within the catalytic domains of cellular enzymes (e.g., protein kinase C), and by the production of CH3Se, which reacts with O2 to generate superoxide and other reactive oxygen species (ROS). Activated oncogenes ‘‘prime’’ cells for Se-induced prooxidative apoptosis thereby providing the needed margin for ‘‘killing’’ cancer cells while leaving normal, healthy cells unharmed. Selenoethers, such as selenomethionine and Se–methylselenocysteine are not oxidizing agents, and first, must be converted to methylselenol (CH3Se) that can be directly oxidized to methylseleninic acid. The addition of methioninase, to selenomethionine, or b-lyase to Se–methylselenocysteine, rapidly produces significant amounts of methylselenol, which may be oxidized to methylseleninic acid or may react with O2 to produce superoxide and ROS, resulting in anticarcinogenic activities comparable to selenite or methylseleninic acid. The relatively large amounts of selenomethionine or Se–methylselenocysteine needed to produce apoptosis in cancer cells compared with selenite or methylseleninic acid are a probable consequence of low tissue levels of the required enzymes. Even though many studies have consistently shown that selenomethionine is an ineffective anticarcinogen at doses corresponding to those currently allowed by the FDA, it has been chosen as the Se intervention agent in the 32,500-man (phase III), NCI-funded SELECT trial, which tests the effectiveness of dietary supplements of dietary supplements of Se and tocopherol, individually or in combination, in the prevention of prostate cancer. In 2013, when the data are in, the value of using Se supplements for cancer chemoprevention is likely to be underestimated. c 2006 Elsevier Ltd. All rights reserved.
* Tel.: +1 970 586 1946; fax: +1 970 577 1064. E-mail address: [email protected].
0306-9877/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2006.01.058
Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant
Introduction The most succinct summary of the chemopreventive potential of selenium (Se) was presented by Meuillet, Stratton, Cherukuri, et al. [1] in their 2004 review of the chemoprevention of prostate cancer with Se. These authors conclude: ‘‘there may not be [a] more extensive body of evidence for a cancer prevention potential of [a] dietary component nutrient than there is for Se’’. For many years, those studying the relationship between Se and cancer presumed that Se was exerting its effects as an antioxidant [2]. As a component of glutathione peroxidase (GPx), a widely recognized biochemical function of Se is the removal of reactive oxygen species (ROS) [3]. Although low Se status has been shown to be associated with reduced GPx activity [4], the observation that anticarcinogenic effects continued to increase after GPx had reached optimal (controlled) levels was an early indication that Se might well exert some of its anticancer activity by other mechanisms [5]. The fact that ROS sometime produce cellular DNA damage and ‘‘oxidative stress’’ continues to contribute to the thinking that Se’s anticarcinogenic properties must somehow be explained in terms of its role as an antioxidant. The persistence of such reasoning is particularly enigmatic in view of the long-established knowledge that many chemical forms of Se produce ROS in cells [6]. This paper presents evidence that prooxidative rather than antioxidative properties of Se compounds best account for their observed anticancer effects.
319
3. are expected to produce high steady-state levels of selenide anions (R-Se) and associated ROS; 4. trigger apoptosis in mutated cells; and 5. are not removed from the metabolic Se pool by incorporation into proteins. The naturally occurring inorganic Se compound, sodium selenite and the metabolite methylseleninic acid tend to more completely satisfy the above criteria, and are more effective anticarcinogens than are the organic Se compounds that lack oxidation ability. In 1996, the 10-year, randomized, double-blind, placebo-controlled, clinical trial conducted by the Nutritional Prevention of Cancer (NPC) Group at the University of Arizona [7] demonstrated that subjects receiving daily nutritional supplements of 200 lg of Se in the form of Se-enriched yeast reduced their risk of prostate cancer by some 63%. The early analytical data [8] indicating that the predominant form of Se in the Se-enriched yeast was selenomethionine, presumably, provided a logical basis for choosing selenomethionine as the Se intervention agent in the 12-year, phase III, NCI-funded clinical trial (SELECT) for the prevention of prostate cancer that began in 2001 and will continue until 2013. However, more recent analytical data obtained using actual Se-enriched yeast samples from the NPC study [9] have shown that some samples of supplements used in the trial may have contained as little as 27% selenomethionine. Studies published both before and after the beginning of the SELECT trial provided ample evidence that a chemical form other than selenomethionine was responsible for the impressive chemoprevention of prostate cancer.
The hypothesis Ros and apoptosis The premise of the present paper is that the most effective dietary selenium anticarcinogen presently approved by the FDA is selenite, which inactivates critical thiol-containing enzymes by oxidation and produces ROS such that Se-induced apoptosis can be triggered in mutated but not normal cells. Contrary to prevailing thought, there is much evidence supporting the contention that selenium exerts its cancer chemopreventive activity by functioning as a biochemical oxidizing agent. Se compounds with Se in +4 oxidation states are more effective anticarcinogens that: 1. exert their effects in small doses; 2. directly oxidize critical cellular substrates (enzymes);
Reduction of O2 produces superoxide, peroxides, and hydroxyl free radicals, each of which have various essential cellular functions including the oxidation of fatty acids and alcohols, hydroxylation reactions, conversion of 12-HPETE to 12-HETE, the synthesis of thyroxine, and the implementation of phagocytosis [3]. However, excessive cellular levels of these ROS may produce oxidative damage (mutations of nucleic acids, etc.) and are limited by antioxidants, such as tocopherols, ascorbate, reduced glutathione, and the selenium enzyme, glutathione peroxidase. Reductions in amounts of antioxidants or increases in ROS predispose cells to ‘‘oxidative stress’’. ROS have been shown to play an important role in triggering apoptosis [6]. Apoptosis is the
320
Drake
genetically controlled cell suicide that preferentially occurs in abnormal (damaged) cells, providing a non-inflammatory mechanism for ridding multicellular organisms of dangerous or unneeded cells. Cells with activated oncogenes initiate apoptosis in response to stimuli that only arrest growth in normal cells [10]. Cells with active oncogenes are, therefore, ‘‘primed’’ for apoptosis, and allow for the destruction of premalignant or malignant cells while leaving normal, healthy cells intact. Some cancer chemotherapeutic compounds rely on the fact that cancer cells undergo apoptosis more readily than normal cells.
Discussion
3
Sodium selenite with Se in the +4 oxidation state reacts directly with cysteine clusters in the catalytic subunits of enzymes, such as protein kinase C, (PKC) oxidizing the sulfhydryl groups to disulfide linkages, thereby inactivating the enzyme [11]. Inactivation of PKC has been shown to induce apoptosis in cancer cells [12]. Since selenite oxidation of vicinal sulfhydryl groups is also associated with the production of both superoxide and peroxide [13], apoptosis may be triggered by either or enzyme inactivation or ROS production. If the carcinostatic activity of Se does, in fact, depend upon the oxidation of some critical substrate, it is ironic that volatile methylselenol (CH3SeH) with selenium in its lowest oxidation state is generally regarded as one of the most effective chemical forms of Se as a chemopreventive agent [14]. Indeed, methylselenol has no effect on the activity of PKC. The excellent work of Gopalakrishna and Gundimeda [11], however, has shown that arachidonate hydroperoxide oxidizes methylselenol to non-volatile methylseleninic acid. The proposed reaction is: 2 C19 H31 –CO3 H+CH3 SeH!2 C19 H31 –CO2 H+CH3 SeO2 H Methylseleninic acid thus produced then inactivates PKC in exactly the same manner as selenite, by oxidizing the sulfhydryl groups in the cysteine clusters in the catalytic domain of the enzyme. Presumably, the reaction would be: R
R SH SH + CH3SeO2H
2 R’
in PKC regenerates methylselenol by reduction of the methylseleninic acid. If excess amounts of methylseleninic acid are present, it also prevents the activation of PKC by destroying the four zinc fingers required for the binding of either diaceylglycerol or phorbol esters to the regulatory domain of PKC. In addition to its oxidation of enzymes, selenite is reduced by glutathione (GSH) producing GS–SeH, GS–Se–SG, and other species that are ultimately converted metabolically to CH3Se when supranutritional doses of selenite are ingested. CH3Se may either be oxidized to methylseleninic acid (CH3SeO2H) or react with oxygen to produce the free radical CH3Se and ROS. The latter reaction is: CH Se þ O ! CH Se þ O
S 2
S + CH3SeH + 2 H2O R’
Methylseleninic acid is essentially behaving as a catalyst since the oxidation of the sulfhydryl groups
2
3
2
Although both selenomethionine and Se–methylselenocysteine are considered to be preferred anticarcinogens, neither can function directly as an oxidizing agent, and both require enzymatic conversion to some selenium compound (perhaps methylselenol) that can undergo facile and direct oxidation to an effective anticarcinogen. To date, most direct comparisons of the antitumor activity of these selenoamino acids with that of either selenite or methylseleninic acid indicate that either selenomethionine or Se–methylselenocysteine in concentrations ranges of 50–200 lmol/l are required to produce effects equivalent to those of selenite or methylseleninic acid at very much lower (1–10 lmol/l) levels [15–18]. Se–methylselenocysteine is thought to offer the most promise as an anticarcinogen because it is the immediate precursor of methylselenol and does not produce large amounts of H2Se [19]. However, the conversion of Se–methylselenocysteine to methylselenol requires the enzyme b-lyase, which is not known to be ubiquitous in vivo in humans. Although one in vivo study in mice [20] reports that equal concentrations of Se–methylselenocysteine and methylseleninic acid have essentially the same anticarcinogenic activity, the b-lyase in some mouse tissues has reported to be 800 times greater than that in human tissues [21]. The higher Se– methylselenocysteine concentrations required to produce antitumor activity in in vitro studies is presumed to be a consequence of insufficient amounts of b-lyase in tumor-cell cultures. Selenomethionine is unique among all biological selenium compounds in that it is incorporated into proteins in place of methionine. Unlike selenocysteine, there is no codon specific for selenomethionine so that methionine and selenomethionine are inserted into proteins based upon availability [22]. Sequestering some of the selenomethionine
Cancer chemoprevention: Selenium as a prooxidant, not an antioxidant in proteins removes it from the metabolic selenium pool, so that less is available for conversion to methylselenol and chemoprevention of cancer. In the presence of the methioninase enzyme, selenomethionine can be converted directly into methylselenol [23]. However, there is currently no evidence that this enzyme is present in human tissues in significant amounts, and the high concentrations of selenomethionine required for anticarcinogenic activity amounts to prima facie evidence that tissue levels of any required enzymes are too small for appreciable catalytic activity. Both selenomethionine and Se–methylselenocysteine produce ROS in the presence of sufficient levels of methioninase and b-lyase, respectively. These findings are consistent with early evidence [24] that significant amounts of selenide anions (RSe), in these cases CH3Se, readily produce ROS. If tissue levels of such enzymes are too low, very small amounts of ROS, CH3Se (methylselenol), and methylseleninic acid are produced so that the anticarcinogenic effects of these selenoethers are necessarily diminished.
Existing data and future studies The preponderance of evidence suggests that the most frequently used dietary selenium supplement, selenomethionine, is least likely among currently approved Se supplements to produce impressive reductions in the risk of prostate, colorectal, lung, breast or any other classification of cancer. As early as 1980, Greeder and Milner [25] demonstrated that supplements of inorganic forms of selenium, such as sodium selenite, sodium selenate, or selenium dioxide completely prevented Ehrlich ascites tumors in rats injected with 500,000 live tumor cells, while all of the animals receiving identical doses of selenomethionine developed tumors. Thompson [26] reported that 5 part-per-million supplements of sodium selenite were 8.3 times more effective in inhibiting DMBAinduced mammary tumors in rats than were the same supplemental doses of selenomethionine. In their comparison of the abilities of sodium selenite and selenomethionine to inhibit DMBA-induced breast cancers in rats, Ip and Hays [22] reported that in spite of higher blood, liver, kidney, and muscle levels of selenomethionine, in all cases and at all doses, sodium selenite produced larger percentages of cancer inhibitions. Others [27] have found that selenomethionine supplements of 10 or 15 parts per million had no effect on the incidence or multiplicity of azoxymethane-induced colon tu-
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mors in rats. More recently [28], the relative anticarcinogenic effectiveness of sodium selenate, selenomethionine, Se–methylselenocysteine, and selenized yeast supplements were compared by administering two different and equivalent doses of each supplement separately to nude mice with one million PC-3 prostate cancer cells injected directly into their prostate glands. Sodium selenate, but none of the organic selenium supplements, retarded the growth of prostate tumors and decreased the number of retroperitoneal lymph nodes in these hormone refractory prostate cancer cells. The question concerning the most effective chemical form of selenium to use for the chemoprevention of cancers will only be answered by double-blind, human intervention trials comparing sodium selenite and selenomethionine dietary supplements at different supranutritional doses with placebos. Selenomethionine is currently being used as the Se intervention agent in the ongoing SELECT trial in the United States and is regarded as sufficiently safe. However, the safety record for sodium selenite is less frequently acknowledged. The fiveyear, trial [29] involving 20,847 subjects receiving15 part-per-million sodium selenite-enriched salt as an intervention agent in Qidong County, China, is excellent evidence for the position that sodium selenite is also appropriate for use in large-scale clinical trials in the United States. Subjects receiving sodium selenite-enriched salt experienced a 43% reduction in primary liver cancer relative to 109,624 adjacent residents serving as a control group with no incidents of toxicity. Moreover, sodium selenite has been sold as a dietary supplement in the United States without reports of toxicity for more than two decades. Faced with the uncertain composition of the Seenriched yeast supplements used in the NPC trial and the compelling evidence above, it is unlikely that 200 lg supplements of selenomethionine, the Se intervention agent chosen for the SELECT trial, will be shown to significantly reduce the risk of prostate cancer. Such a result could contribute to the erroneous conclusion that dietary selenium supplements are of no value in the chemoprevention of cancer.
Acknowledgement The author expresses his gratitude to Dr. Julian E. Spallholz, Texas Tech University, whose comments and suggestions were enormously helpful in writing the paper.
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