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Simultaneous Activation of Nrf2 and Elevation of Dietary and Endogenous Antioxidant Chemicals for Cancer Prevention in Humans Kedar N. Prasad PhD

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Antioxidant Research Institute, Premier Micronutrient Corporation, Novato, California Published online: 07 Jul 2015.

Click for updates To cite this article: Kedar N. Prasad PhD (2015): Simultaneous Activation of Nrf2 and Elevation of Dietary and Endogenous Antioxidant Chemicals for Cancer Prevention in Humans, Journal of the American College of Nutrition, DOI: 10.1080/07315724.2014.1003419 To link to this article: http://dx.doi.org/10.1080/07315724.2014.1003419

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Review

Simultaneous Activation of Nrf2 and Elevation of Dietary and Endogenous Antioxidant Chemicals for Cancer Prevention in Humans Kedar N. Prasad, PhD Antioxidant Research Institute, Premier Micronutrient Corporation, Novato, California

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Key words: oxidative stress, chronic inflammation, antioxidant enzymes, reactive oxygen species Despite extensive studies in cancer prevention, the incidence of cancer is increasing. We review studies that have identified several biochemical and genetic defects as well as potential carcinogens in the diet, environmental factors, and lifestyle-related habits. Two of the biochemical abnormalities increased oxidative stress and chronic inflammation, and chronic exposure to carcinogens and mutagens play a significant role in the initiation of multistage carcinogenesis. Therefore, attenuation of these biochemical defects may be useful in reducing the incidence of cancer. Activation of the transcriptional factor called nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which enhances the levels of antioxidant enzymes and phase-2-detoxifying enzymes by complex mechanisms, may be one of the ways to reduce oxidative stress and chronic inflammation. Antioxidant enzymes destroy free radicals by catalysis, whereas phase-2detoxifying enzymes remove potential carcinogens by converting them to harmless compounds for elimination from the body. However, increasing the levels of antioxidant enzymes by activating Nrf2 may not be sufficient to decrease oxidative stress and chronic inflammation optimally, because antioxidant chemicals, which are decreased in a high oxidative environment, must also be elevated. This review discusses the regulation of activation of Nrf2 and proposes a hypothesis that an elevation of the levels of antioxidant enzymes and dietary and endogenous antioxidant chemicals simultaneously may reduce the incidence of cancer by decreasing oxidative stress and chronic inflammation. The levels of antioxidant chemicals can be increased by supplementation, but increasing the levels of antioxidant enzymes requires activation of Nrf2 by reactive oxygen species (ROS)-dependent and-independent mechanisms. Several phytochemicals and antioxidant chemicals that activate Nrf2 have been identified. This review also describes clinical studies on antioxidants in cancer prevention that have produced inconsistent results. It discusses the possible reasons for the inconsistent results and proposes criteria that should be included in the experimental designs of future clinical studies to obtain consistent results.

Key teaching points:  Reducing oxidative stress and chronic inflammation optimally requires an elevation of the levels of antioxidant enzymes and phase-2-detoxifying enzymes as well as dietary and endogenous antioxidant chemicals.  How the levels of antioxidant enzymes and phase-2-detoxifying enzymes are regulated by a nuclear transcriptional factor Nrf2.  How the activation and transcription of Nrf2 is regulated.  Identification of antioxidants that activate Nrf2 by ROS-dependent and-independent mechanisms, those that destroy free radicals by scavenging, and those that exhibit both functions.  Possible reasons for the inconsistent results produced by the previous clinical studies on antioxidants in cancer prevention.  The criteria that should be included in the experimental designs of future clinical studies on antioxidants in cancer prevention in high-risk populations to obtain consistent results.

Address correspondence to: Kedar N. Prasad, PhD, Antioxidant Research Institute, Premier Micronutrient Corporation (PMC), 14/Galli Drive, Suite 200, Novato, CA 94949. E-mail: [email protected].

Journal of the American College of Nutrition, Vol. 0, No. 0, 1–10 (2015) Ó American College of Nutrition Published by Taylor & Francis Group, LLC 1

Activation of Nrf2 and Elevation of Antioxidants for Cancer Prevention

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INTRODUCTION Despite extensive basic and clinical research in cancer prevention, the incidence of cancer is increasing. Current cancer prevention strategies include consumption of a diet rich in antioxidants, low in fat, and high in fiber, together with a reduced exposure to potential environment-and lifestyle-related mutagens and carcinogens. These recommendations, although useful, have not decreased the incidence of cancer. As a matter of fact, the incidence of cancer increased from about 1.1/million new cases in 2002 to 1.64/million in 2012, an increase of 49% in 10 years [1]. This could have been due to the fact that human behavior with respect to diet, lifestyle, and exposure to environmental carcinogens is difficult to change. Consequently, increased oxidative stress and chronic inflammation that contribute to the risk of cancer continued to exist [2–10]. Intervention studies mostly with 1 or 2 antioxidants in high-risk populations produced inconsistent results, varying from no effects, to some beneficial effects, to harmful effects [11–17]. The exact reasons for these inconsistent results are unknown. The question arises of how to reduce oxidative stress and chronic inflammation optimally. The levels of antioxidant enzymes and phase-2-detoxifying enzymes play an important role in reducing oxidative stress and elimination of potential carcinogens from the body. The transcription factor called nuclear factor (erythroidderived 2)-like 2 (Nrf2), when activated by the reactive oxygen species (ROS)-dependent and-independent mechanisms, migrates from the cytoplasm to the nucleus, where it binds with the antioxidant response elements (ARE) in order to enhance the transcription of target genes coding for antioxidant enzymes and phase-2-detoxifying enzymes. An elevation of antioxidant enzymes by activating Nrf2 alone may not be sufficient to decrease the levels of oxidative stress and chronic inflammation optimally. This may be due to the fact that elevated levels of oxidative stress and chronic inflammation may decrease dietary and endogenous antioxidant chemicals; therefore, their levels must also be increased in order to reduce these biochemical defects optimally. In addition, antioxidant enzymes and chemicals destroy free radicals in part by different mechanisms. Antioxidant enzymes destroy free radicals by catalysis, whereas antioxidant chemicals destroy them by scavenging. The levels of antioxidant chemicals can be increased by an oral supplementation, but increasing the levels of antioxidant enzymes is complex and normally requires activation of Nrf2 by ROS-dependent mechanisms [18–20]; however, it appears that Nrf2 becomes unresponsive to ROS during chronic oxidative stress [21–23], suggesting that activation of Nrf2 by ROS-independent mechanisms exists.

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This review discusses the regulation of activation of Nrf2 and proposes the hypothesis that an elevation of the levels of antioxidant enzymes as well as the levels of dietary and endogenous antioxidant chemicals simultaneously may reduce the incidence of cancer by decreasing oxidative stress and chronic inflammation optimally. This review also describes the clinical studies on antioxidant and cancer prevention in high-risk populations that have produced inconsistent results. It discusses the possible reasons for such results and proposes the criteria that should be included in the experimental designs of future clinical studies on antioxidants in cancer prevention to obtain consistent results.

NRF2 IN CANCER The nuclear transcriptional factor, Nrf2 belongs to the Cap’N’Collar (CNC) family that contains a conserved basic leucine zipper (bZIP) transcriptional factor [24]. The role of Nrf2 when associated with the Keap 1 (Kelch-like erythroidcell-derived protein with CNS homology (ECH) associated protein 1)-signaling pathway in cancer prevention appears to be different from that in cancer cell survival, proliferation, and treatment. Activation of Nrf2 in normal cells may contribute to the prevention of cancer, whereas amplified expression of Nrf2, due to its increased transcriptional activity, contributes to the survival and growth of several types of tumor as well as resistance to cancer therapy [24–26]. Amplification of expression of Nrf2 may occur due to a mutation in Nrf2 or the Keap1 gene found in cancer cells [27,28]. The mutated Keap1 showed reduced affinity to Nrf2, resulting in constitutive activation of Nrf2 in cancer cells, whereas mutated Nrf2 impairs its recognition by Keap1-Cul3 E3 ligase, resulting in the stabilization of Nrf2 and increased translocation to the nucleus where it activates target genes that code for antioxidant enzymes and phase-2-detoxifying enzymes. It has been demonstrated that Nrf2 regulates Keap1 levels by controlling its transcription, whereas Keap1 regulates Nrf2 levels by controlling its degradation by proteasome [29]. The levels of Nrf2 are also regulated epigenetically by methylation of CpG (cytosine-phosphate-guanosine) and acetylation of histone3. Hypermethylation of CpG [30] and hyperacetylation of histone3 [31] increase the expression of Nrf2, whereas hypomethylation of CpG and hypoacetylation of histone3 decrease it. Therefore, any agent that can prevent hypermethylation of CpG or hyperacetylation of histone3 in normal cells would be of chemopreventive value. Immediate early response-3 (IER-3) gene, a multifunctional stress response gene, also plays a role in the regulation of Nrf2 activity. Genetic deletion of IER-3 gene increases the Nrf2 activation, whereas overexpression of IER-3 decreases it [32]. As expected, IER-3-deficient human colonocytes exhibited

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Activation of Nrf2 and Elevation of Antioxidants for Cancer Prevention reduced levels of ROS due to antioxidant protection via the Nrf2/ARE pathway.

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Regulation of Activation of Nrf2 In order to understand the role of Nrf2 in cancer prevention, it is essential to review briefly the regulation of Nrf2 activation in normal cells. Normally, Nrf2 is bound to Keap1, which acts as an inhibitor of Nrf2 and is located in the cytoplasm [33]. Keap1 protein serves as an adaptor to link Nrf2 to the ubiquitin ligase CuI-Rbx1 complex for degradation by proteasomes, and it maintains the steady levels of Nrf2 in the cytoplasm [34]. In addition, ubiquitin-specific peptidase 15 (USP15), a deubiquitinating enzyme, plays a role in regulating degradation of Nrf2 [24]. USP15 can deubiquitinate Keap1, stabilize the Keap1-Cul3-E3 ligase complex, and increase its E3 ligase activity, which leads to degradation of Nrf2 [35]. Keap1 acts as a sensor for ROS/electrophilic stress. In response to increased ROS, Nrf2 dissociates itself from the Keap1CuI-Rbx1 complex and translocates into the nucleus, where it heterodimerizes with a small Maf protein and binds with ARE, which increases expression of target genes coding for antioxidant enzymes and phase-2-detoxifying enzymes. Activated Nrf2 is translocated from the cytoplasm to the nucleus; however, accumulation of Nrf2 in the nucleus may not be sufficient to increase the expression of antioxidant genes. Nrf2 must bind with ARE for increasing the expression of antioxidant genes. This binding ability of Nrf2 is impaired in aged rats, and this defect is corrected by supplementation with alpha-lipoic acid, an endogenous antioxidant [36]. It is not known whether the binding ability of Nrf2 with ARE is reduced during increased oxidative stress and chronic inflammation which precede cancer development. It appears that Nrf2 responds to ROS generated during acute and chronic oxidative stress differently. For example, acute oxidative stress during strenuous exercise activates Nrf2, which translocates itself from the cytoplasm to the nucleus, where it binds with ARE to up-regulate antioxidant genes. However, during chronic oxidative stress commonly observed in high-risk populations to develop chronic diseases such as cancer, the Nrf2/ARE pathway becomes unresponsive to ROS. Several plantderived phytochemicals that can activate Nrf2 have been identified. Some examples include sulforaphane, curcumin, resveratrol, epigallocatechin-3-gallate, carestol, kahweol, cinnamonyl-based compounds, zerumbone, garlic sulfur compounds, lycopene and carnosol [24,37,38]. Some of them activate Nrf2 without ROS stimulation. Thus, Nrf2 is activated by ROS-dependent and-independent mechanisms. An activation of Nrf2 by ROS-

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independent mechanisms is essential for cancer prevention, because increased levels of oxidative stress in the presence of Nrf2 may exist in high-risk populations. The ROS-independent mechanisms of activation of Nrf2 may include prevention of hypermethylation of CpG and/or hyperacetylation of histone3 as well as reduction of the expression of the IER-3 gene. The presence of Nrf2 is essential for cancer prevention. Mice genetically deficient in Nrf2 become very susceptible to chemical-induced cancer and less responsive to chemopreventive agents [37]. One of the actions of cancer-preventive agents may involve prevention of hypermethylation of CpG and hyperacetylation of histone3 [9,39].

Antioxidant Chemicals that Activate Nrf2, Scavenge Free Radicals, or Perform Both Functions All antioxidants directly scavenge varying levels of free radicals; however, some can activate Nrf2 by ROS-independent and some by ROS-dependent mechanisms, while others exhibit both functions. They are described here. 1. Free radical scavenging antioxidants: All dietary and endogenous antioxidant chemicals reduce varying levels of oxidative stress by directly scavenging free radicals. Some examples are dietary antioxidants, such as vitamin A, betacarotene, vitamin C, and vitamin E, and polyphenolic compounds, and endogenous antioxidants, such as glutathione, alpha-lipoic acid, and coenzyme Q10. 2. Antioxidants activating Nrf2 by a ROS-independent mechanism: Some examples are organosulfur compound sulforaphane, found in cruciferous vegetables; kavalactones, found in Kava shrubs; Puerarin, a major flavonoid from the root of Pueraria lobata [40–42]; genistein; vitamin E [43]; coenzyme Q10 [44], which activate Nrf2 by a ROS-independent mechanism. 3. Antioxidants scavenging free radicals as well as activating Nrf2 by a ROS-independent mechanism: Some examples are vitamin E [43], alpha-lipoic acid [36], curcumin [45], resveratrol [46,47], omega-3-fatty acids[48,49], and NAC [50]. 4. Antioxidant activating Nrf2 by a ROS-dependent mechanism: L-carnitine activates Nrf2 by a ROS-dependent mechanism [51], probably by generating transient ROS. A combination of selected antioxidants from the above groups may reduce oxidative stress optimally. Activation of Nrf2 suppresses inflammation [52,53]. Some individual antioxidants from the above groups have been shown to reduce chronic inflammation [54–61]. Thus, a combination of selected antioxidants used for reducing oxidative stress may also decrease chronic inflammation optimally.

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CLINICAL STUDIES ON ANTIOXIDANTS IN CANCER PREVENTION Clinical studies on the effect of orally supplemented antioxidants in cancer prevention among high-risk populations are discussed here. The epidemiologic studies on the relationship between dietary antioxidants and vitamins and cancer risk are not included, because they have been reviewed previously [2]. Studies on the effects of antioxidants on the recurrence of primary tumor after completion of cancer therapy are also not included, because they are more pertinent to cancer treatment rather than cancer prevention.

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Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study In the Finnish Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC) study, 29,133 male heavy tobacco smokers aged 50–69 years were recruited between May/1, 1993 and April/30, 1999. They were randomized to receive 50 mg of alpha-tocopherol, 20 mg of beta-carotene, both agents, or a respective placebo daily orally for 5–8 years. The results showed that supplementation with beta-carotene increased the incidence of lung cancer by 18%, but had no effect on other cancers. Supplementation with alpha tocopherol had no effect on lung cancer, but it reduced the incidence of prostate cancer. The group receiving both beta-carotene and alpha-tocopherol had fewer incidences of prostate cancer than those who did not [11]. Subsequent study on the ATBC population showed an increase in the incidence of lung, prostate, and stomach cancer among those who were taking beta-carotene alone. On the other hand, supplementation with alpha-tocopherol reduced the incidence of prostate and colorectal cancers, but it increased the incidence of stomach cancer. In contrast to these results of intervention studies on beta-carotene supplementation alone on lung cancer, men who had higher levels of serum beta-carotene and consumed higher amounts of dietary beta-carotene exhibited a lower incidence of lung cancer [12]. A further study of the ATBC population revealed that supplementation with alpha-tocopherol or beta-carotene alone did not prevent lung cancer; however, beta-carotene supplementation increased the risk of lung cancer. The increase in lung cancer was most pronounced among those who were heavy smokers and alcohol drinkers [13]. Another study of the ATBC population showed that higher serum levels of alpha-tocopherol and gamma-tocopherol were associated with reduced prostate cancer risk, and this association was stronger in the vitamin E-supplemented group than in those not receiving a vitamin E supplement [62]. It was further demonstrated that serum concentration of vitamin E and not the dietary vitamin E was inversely proportion to the risk of prostate cancer [14]. Additional studies on the ATBC population revealed that the excess lung cancer risk after beta-carotene treatment or reduction in

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prostate cancer after treatment with alpha-tocopherol disappeared 4–6 years after ending the intervention [63]. A subcohort population of 25,563/men from this study was followed for 18 years. The results showed that neither alpha-tocopherol nor beta-carotene had a significant effect on cancer incidence. Alpha-tocopherol treatment continued to show modest reduction in prostate cancer incidence. Alpha-tocopherol treatment also decreased prostate cancer mortality, whereas beta carotene supplementation increased it [15].

U.S. Male Physicians’ Health Study II Beginning in 1997, a total of 22,071 U.S. male physicians aged 50 years or older were recruited for this study. They were randomized to receive 400/IU of vitamin E orally every other day, 500 mg of vitamin C orally every day, or their respective placebos. The treatment period ended in 2007, and the observational period continued through June 2011. The results showed that during the 10-year follow-up, neither vitamin E nor vitamin C had any effect on the risk of total cancers, prostate cancer, or other site-specific cancers [16]. This observation on the effect of vitamin E supplementation on prostate cancer risk is in sharp contrast to the findings with the ATBC population, which exhibited a reduction in prostate cancer incidence after vitamin E supplementation. Plasma levels of lycopene were strongly associated with a decreased risk of prostate cancer in men with low plasma levels of lycopene, and supplementation with carotene was associated with a reduced risk of prostate cancer [17]. In contrast to supplementation with a single antioxidant, supplementation with a commercial preparation of multivitamin which lacked endogenous antioxidants for a period of 9.2 years showed a modest reduction in the incidence of total cancer during a follow-up period of 11.2 years, but had no significant effect on the prostate cancer incidence [64]. The addition of endogenous antioxidants could have provided a more pronounced reduction in cancer incidence.

Beta-Carotene Trial among U.S. Physicians For this study, 22,071 healthy male physicians aged 40–84 years (11% were smokers and 39% were former smokers) were recruited in 1982 and randomly assigned to receive 50 mg of beta-carotene or a placebo. The results showed that supplementation with beta-carotene for a period of 12–13 years had no effect on cancer incidence or death from all causes [65,66].

Selenium and Vitamin E Cancer Prevention Trial For the Selenium and Vitamin E Cancer Prevention Trial (SELECT), a total of 35,533 healthy men (aged 50 years or older for black men and 55 years or older for all others) from 427 study sites in the United States, Canada, and Puerto Rico were

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Activation of Nrf2 and Elevation of Antioxidants for Cancer Prevention recruited. They were randomized between August 22, 2001 and June 24 2004. The criteria of eligibility included prostate-specific antigen (PSA) of 4.0 ng/mL or less, and no evidence of prostate cancer as confirmed by a digital rectal examination. 34,887/men were assigned to 1 of 4 treatment groups: 8752/men received oral selenium (as L-selenomethionine, 200/mcg/day); 8737/men received vitamin E (as all rac-a-tocopheryl acetate); 8702/men received selenium and vitamin E; and 8696/men received a corresponding placebo, and analysis was performed. The follow-up period for analyzing the incidence of prostate cancer was 7– 12 years. The results showed that neither selenium nor vitamin E, alone or together, prevented prostate cancer; however, a nonsignificant increase in prostate cancer risk was observed in men treated with vitamin E alone [67]. A longer follow-up of men in the SELECT revealed that supplementation with vitamin E significantly increased the risk of prostate cancer by about 17% [68]. The subsequent follow-up of the population of SELECT showed that selenium supplementation did not prevent prostate cancer in men with low levels of toenail selenium; however, it increased the risk of high-grade prostate cancer in men with high levels of selenium. Vitamin E supplementation also increased the risk of prostate cancer in men with low levels of toenail selenium [69]. The population of SELECT was analyzed for the risk of cataract. The results showed that daily oral supplementation of selenium and/or vitamin E did not significantly reduce the incidence of age-related cataracts [70]. The plasma levels of a-tocopherol and g-tocopherol among a sub-cohort of 3,211/ men derived from the SELECT showed that neither a-tocopherol nor g-tocopherol levels were associated with the risk of prostate cancer; however, supplementation with selenium in men with high plasma levels of a-tocopherol increased the risk of prostate cancer [71].

Women’s Antioxidant Cardiovascular Study A total of 8171/women were recruited for the Women’s Antioxidant Cardiovascular Study. They were randomized to receive 500 mg of vitamin C orally daily and 600/IU of alphatocopherol and 50 mg of beta-carotene orally every other day, or placebos individually or in combination. However, only 7627/women who were free of cancer before randomization were followed for an average of 9.4 years to compare the incidence of cancer and cancer mortality between the antioxidant and the placebo groups. The results showed that daily oral supplementation of vitamin C, vitamin E, and beta-carotene individually or in combination had no significant effect on overall cancer incidence or cancer-related mortality [72].

Beta-Carotene and Retinol Efficacy Trial A total of 18,314/men and women with high risk of developing lung cancer were randomized to receive a combination

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of 30 mg of beta-carotene and 25,000/IU of retinyl palmitate or placebo. The study was stopped after 21/months because of an increase in lung cancer by 28% and mortality by 17% [73].

Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial In this study of 29,361 men during 8 years of follow-up, there was no association found between prostate cancer risk and dietary or supplemented vitamin E, vitamin C, or beta-carotene. However, among smokers, an inverse association between prostate cancer and increasing dose and duration of vitamin E was observed. In addition, supplementation with beta-carotene at a dose of at least 2 mg/mL was associated with decreased prostate cancer risk among men with low dietary intake of beta-carotene [74]. A further analysis of results showed that high serum beta-carotene levels were associated with increased risk of prostate cancer [74]; however, serum levels of lycopene and other carotenoids were not associated with this form of cancer [75].

Cancer Prevention Trial in Linxian General Population The people of Linxian County, China have very high rates of esophageal and stomach cancer and have low dietary intake of several micronutrients. In 1985, 29,584 adults aged 40–69 from 4 communes were recruited for this trial. They were randomized to receive retinol and zinc, riboflavin and niacin, vitamin C and molybdenum or beta carotene, vitamin E, and selenium. The doses ranged from one to two times the U.S. recommended daily allowance (RDA) values. The incidence of cancer and mortality were determined between March 1986 and May 1991. The results showed that the incidence of cancer and total mortality were reduced in the group receiving a combination of beta-carotene, vitamin E, and selenium. The pattern of decreased incidence of cancer was noted 1–2 years after supplementation. Other groups who received retinol and zinc, riboflavin and niacin or vitamin C, and molybdenum did not show any significant change in cancer incidence or mortality [76]. A follow-up period of 10 years revealed that reduction in gastric cancer incidence and mortality were evident in individuals younger than 55 years; however, in individuals aged over 55 years, an increase of 14% in esophageal cancer was observed [77]. In addition, an increase in total and strokerelated mortality in the group who received vitamin A and zinc was noted, while a decrease in stroke-related mortality was found in the group who received vitamin C and molybdenum. This trial had no placebo group; therefore, the results cannot be assessed correctly. Furthermore, the rationale for selecting micronutrients for each group was not provided. Since the people of Linxian were considered deficient in micronutrients due to poor dietary intake, they could have increased oxidative

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Activation of Nrf2 and Elevation of Antioxidants for Cancer Prevention stress. Micronutrients used in this trial cannot decrease oxidative stress optimally.

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Vitamin D3 Supplementation and Cancer Risk Daily oral intake of 1000 IU of vitamin D3 or serum level of 33 ng/mL of vitamin D3 has been associated with a 50% reduction in colorectal cancer [78]. This was not confirmed in a large randomized, double-blind, and placebo-controlled trial. A total of 36,282 postmenopausal women from 40 Women’s Health Initiative Centers were recruited to evaluate the role of vitamin D3 and calcium in the risk of colorectal cancer and breast cancer. They were randomized to receive vitamin D3 200 IU and calcium 500 mg or a placebo twice a day for an average of 7 years. The results showed that supplementation with vitamin D3 and calcium had no effect on the incidence of colorectal cancer [79] or breast cancer [80].

Supplementation with Folic acid on Cancer Risk A review of several studies showed that folic acid supplementation had no impact on the incidence of site-specific cancers during the first 5 years of treatment [81].

Problems Associated with the Use of 1 or 2 Antioxidants or Vitamins in High-Risk Populations The studies discussed above clearly demonstrate that supplementation with one or two antioxidants in high-risk populations produces inconsistent results, varying from no effect, to some reduction in cancer incidence, to increase in cancer incidence, even for the same cancer type and same antioxidant. There could be are several reasons for these inconsistent results. Some of them are described here. 1. High-risk populations such as heavy tobacco smokers have high levels of oxidative stress and chronic inflammation [82–85]. In heavy smokers, administered beta-carotene or other single antioxidant would be oxidized under such an oxidative environment. The levels of oxidized antioxidant would gradually increase, which may further enhance the levels of oxidative environment in the body, which may account for the increased risk of cancers among high-risk populations. 2. One or 2 antioxidants cannot elevate antioxidant enzymes and all antioxidant chemicals in the body at the same time, which is required for reducing oxidative stress and chronic inflammation optimally. 3. Distribution of dietary and endogenous antioxidant chemicals differs from one organ to another; they differ from one cell to another within the same organ; they also differ from one compartment to another within the same cell. Therefore, supplementation with 1 or 2 antioxidants may not reduce oxidative stress throughout the body.

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4. A single antioxidant cannot reduce oxidative stress in both lipid and aqueous environments of the cells. 5. The gradient of oxygen pressure varies from one tissue to another. Vitamin E is a more effective scavenger of free radicals in reduced oxygen pressure, whereas vitamins A and C are more effective in higher oxygen pressure [86]. A single antioxidant cannot reduce oxidative stress under both conditions of oxygen pressure. 6. Activation of Nrf2 is required for elevation of the levels of antioxidant enzymes; however, ROS-dependent activation of Nrf2 becomes unresponsive during chronic oxidative stress. Thus, activation of Nrf2 by ROS-independent mechanisms becomes essential for reducing oxidative stress. The use of one or two antioxidants cannot achieve this goal. Further follow-up of the same clinical studies on antioxidant in cancer prevention discussed in this review would continue to yield inconsistent results and therefore may not be useful in developing public policy regarding the value of antioxidants in reducing the risk of cancer in humans.

PROPOSED CRITERIA TO BE INCLUDED IN THE EXPERIMENTAL DESIGNS OF STUDIES ON ANTIOXIDANTS IN CANCER PREVENTION The following criteria should be taken into account while designing the experiments for the clinical trials with antioxidants in cancer prevention: 1. High-risk populations, such as heavy tobacco smokers, cancer survivors and individuals with a family history of cancer, and individuals aged 50 years or older, are suitable for cancer prevention studies with antioxidants. 2. Experimental design should be randomized, doubleblinded, and placebo-controlled. 3. The number of participants in the study should be high, generally in the thousands, for a meaningful statistical analysis and conclusions. 4. Levels of markers of oxidative stress and chronic inflammation in a subset population of a smaller number (in the hundreds) before and after treatments should be measured. 5. Daily oral supplementation with a mixture of antioxidants and vitamins that can enhance the levels of antioxidant enzymes through activation of Nrf2, and the levels of dietary and endogenous antioxidant chemicals simultaneously should be included.; 6. Doses (high but safe) and dose-schedules of twice-a-day should be adopted. 7. Primary end-points, such as total cancers, site-specific cancers, and mortality are appropriate.

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Activation of Nrf2 and Elevation of Antioxidants for Cancer Prevention 8. Generally, treatment periods of 5 years and observation periods of 5–15 years are adequate.

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The experimental designs of most of the clinical studies discussed above on the role of antioxidants in cancer prevention in high-risk populations have taken into consideration only criteria 1,2, 3, 7, and 8, which may account for the inconsistent results on cancer incidence. Criteria 4, 5, and 6 were not included in the experimental designs of clinical studies.

PROPOSED MIXTURE OF DIETARY AND ENDOGENOUS ANTIOXIDANTS AND VITAMINS FOR CANCER PREVENTION I propose that a mixture of antioxidants and vitamins that would activate Nrf2 by ROS-dependent and-independent mechanisms and enhance the levels of dietary and endogenous antioxidant chemicals simultaneously may reduce the incidence of cancer by decreasing oxidative stress and chronic inflammation optimally. This mixture contains dietary antioxidants and vitamins (vitamins A, C, and E, beta-carotene, vitamin D, selenium, B-vitamins, curcumin, and resveratrol), endogenous antioxidants (alpha-lipoic acid, L-carnitine, and coenzyme Q10), and a synthetic antioxidant N-acetylcysteine (NAC) which elevates glutathione levels in the cell. In conclusion, the levels of antioxidant enzymes and phase-2-detoxifying enzymes can be increased by activation of a nuclear transcriptional factor Nrf2, whereas the levels of dietary and endogenous chemicals are elevated by oral supplementation. Enhancement of both components of the cellular defense system simultaneously is essential for optimal reduction of oxidative stress and chronic inflammation, and thereby, for decreasing the incidence of cancer in highrisk populations. Supplementation with 1 or 2 antioxidants or vitamins is unlikely to achieve the above goals. The proposed mixture of micronutrients is expected to reduce the incidence of cancer by attenuating oxidative stress and chronic inflammation optimally by increasing the levels of antioxidant enzymes through activation of Nrf2 and antioxidant chemicals through supplementation. A well-designed clinical study with the suggested mixture of micronutrients should be initiated to test the proposed hypothesis for cancer prevention.

Conflict of Interest The author is an employee of and has stocks in Premier Micronutrient Corporation.

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