Unifying Mechanism for Nutrients as Anticancer Agents : Electron Transfer , Reactive Oxygen Species and Oxidative Stress

A recent article deals with various nutrients in relation to bactericidal action. The present article focuses on a unifying mode of action for the nutrients, namely, resveratrol, epigallocatechin, polyene-ß-carotene, polyene lycopene, piperine, curcumin, genistein, luteolin, sulforaphane and pomegranate extract. The mechanism is based on electron transfer, reactive oxygen species and oxidative stress, which comprises an extension of earlier reports involving agents. Most of the compounds are precursors of electron transfer quinones, whereas others fit into the polyene category. The nutrients are better known as antioxidants. The dichotomy is addressed.


Introduction
Anticancer drugs are often synthetic agents originally derived from plants.Recently an article reported anticancer activity by diet nutrients, namely, resveratrol, epigallocatechin, polyene-ß-carotene, polyene lycopene, piperine, curcumin, genistein, luteolin, sulforaphane and pomegranate extract (Scarpa & Ninfali, 2015).Mode of action was attributed to cell signaling involving cancer stem cells.In 2007, a unifying mechanism for anticancer action was proposed as discussed below (Kovacic, 2007).The nutrients fit into the unifying mechanism which has been widely applied previously as set forth as follows (Kovacic & Somanathan, 2010).
"The preponderance of bioactive substances, usually as the metabolites, incorporate ET functionalities.We believe these ET-metabolites play an important role in physiological responses.The main group include quinones (or phenolic precursors), metal complexes (or complexors), aromatic nitro compounds (or reduced hydroxylamine and nitroso derivatives), and conjugated imines (or iminium species).Resultant redox cycling is illustrated in Scheme 1.In vivo redox cycling with oxygen can occur, giving rise to oxidative stress (OS) through generation of reactive oxygen species (ROS), such as hydrogen peroxide, hydroperoxides, alkyl peroxides, and diverse radicals (hydroxyl, alkoxyl, hydroperoxyl, and superoxide) (Scheme 1).Cellular and mitochondrial enzymes can also perform catalytically in the reduction of O 2 .Scheme 1. Redox cycling with superoxide and ROS formation.
It is important to recognize that mode of action in the bio domain is often involved with many physiological actions and is multifaceted.In addition to the ET-ROS-OS approach, other aspects may pertain, such as, enzyme inhibition, allosteric effects, receptor binding, metabolism and physical factors.A specific example involves protein binding by quinones in which protein and nucleophiles, such as amino or thiol, effect conjugate addition." As indicated above, focus is on ROS-OS in relation to mechanism.However, most attention in the literature is paid to AO action in relation to physiological action, but is of little importance in the present case.An example is discussed with ß-carotene.

ROS and Cancer
In recent years, a large body of literature has emerged documenting the link between ROS, reactive nitrogen species (RNS), OS and ET and cancer (Liou & Storz, 2010;Sullivan & Chandel, 2014;Haliwell, 2001).
Reactive oxygen species are radicals, ions or molecules that have unpaired electrons.They can be classified into two groups: free oxygen radicals and non-radical ROS (Table 1).Among these, superoxide, hydrogen peroxide and hydroxyl radicals are the most potent and well studied species.The roles of reactive oxygen species in tumorigenesis , prevention, and therapy has been discussed in more recent reviews (Liou & Storz, 2010;Sullivan & Chandel, 2014;Haliwell, 2001;Kovacic & Jacintho, 2001a;Gupta, Hevia, Patchva, Park, Koh, & Aggarwal, 2012).1. Reactive oxygen and nitrogen species A variety of enzymatic and non-enzymatic sources of ROS exists in the biological system.Enzymes within the cell are primary sources of ROS/RNS.Superoxide is produced by one electron reduction of oxygen by several enzymes, such as NAD(P)H oxidase, xanthine oxidase and cytochrome P450 within the cell.In addition to these, a number of external mediators also contribute to the ROS generation, such as ionizing radiation.Heavy metals, like Hg, Pb, Cd, Cr, and Cu, metal complexes, nano particles, cigarette smoke, pollutants from automobiles, fossil burning furnaces, various drugs and certain types of other chemical compounds (Scheme 1) play a role.In cancer affected cells, high levels of ROS can result from mitochondrial dysfunction, high metabolic activity, cellular signaling, peroxisome activity, oncogene activity, oxidase activity, cyclooxygenase and lipoxigenase activity (Liou & Storz, 2010;Sullivan & Chandel, 2014;Haliwell, 2001).

Scheme 1. Generation of ROS
In mitochondria, superoxide ROS is produced as a natural by-product of electron transport chain activity (Cui, Reichner, Mateo, & Albino, 1994).The superoxide leaks through the mitochondrial permeability transition pore in the outer membrane into the cytoplasm, where it is dismutated into hydrogen peroxide by MnSOD or in the cytosol by Cu/ZnSOD.The diffusible hydrogen peroxide also serves as a second messenger, and may cross cellular membranes through members of the aquaporin family (Scarpoa & Ninfali, 2015).Cancers arise from sites of chronic irritation, or inflammation and play a critical role in tumor progression (Liou & Storz, 2010).Within the cancer cells, macrophages produce ROS which kill the tumor cells.This production of superoxide, hydroxyl radical, hydrogen peroxide, nitric oxide and peroxynitrite radicals contributes to tumor cell apoptosis (Liou & Storz, 2010;Cui, Reichner, Mateo, & Albino, 1994).
A 2012 review deals with ROS as initiators of cancer and also as therapy in destroying the cells (Cui, Reichner, Mateo, & Albino, 1994).The review highlights: a) ROS play an important role in the initiation and progression of As a result of these accumulated data, a number of pro-oxidant and antioxidant based anticancer agents have been developed.Many of these approved drugs do show promise in suppressing cancer growth.However, a majority of the chemotherapeutic drugs shrink tumor size, but often fail to eradicate tumors.

Genistein
Genistein (Figure 2), an isoflavone, is the active ingredient in soya-rich food.

Figure 2. Genistein
Studies show that genistein and high soya-food consumption have been associated with reduced risk of breast and other forms of cancer among Asian Americans (Ziegler, Hoover, Pike et al., 1993;Iwasaki, Inoue, Otani et al., 2008;Verheus, Van Gils, Keinan-Boker et al., 2007;Barnes, 1995).Long term, low dose genistein consumption decreases stem cell population and sensitizes inflammatory breast cancer cell lines to radiation (Sims-Moutada, Opdenaker, Davis, & Wu, 2015).A report deals with genistein as a dietary phytochemical target in human prostate cancer stem cells (Zhang, Li, Jiao et al., 2012).The phenol suppressed tumorsphere and colony formation of prostate cancer cells by inhibiting Hedgehog-Gli1 pathway.Genistein inhibited breast cancer cell growth, proliferation and promoted apoptosis (Fan, Fan, Wang et al., 2013).Genistein decreased breast cancer stem cells by inhibiting cancer stem cells by down regulating Hedgehog-Gli1 signaling pathway.Gli1 expression was inhibited, resulting in the attenuation of cancer stem like properties in gastric cancer cells (Yu, Shin, Lee et al., 2014).In addition, genistein suppresses the cell invasive capacity that is required for tumor growth and metastasis.
A study showed inhibition by the phenol of β-catenin -mediated WNT signaling through gene expression by demethylating its silenced promoter in colon cancer cell lines (Zhang & Chen, 2011).A similar study with renal cancer cells also showed inhibition of Wnt-signaling, thus effecting the proliferation and renewal of cancer stem cells (Hirata, Ueno, Nakajima et al., 2013).Data showed genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, epithelial-mesemchymal transition, cancer stem cell phenotype, and formation of pancreatospheres in pancreatic cancer cells (Bao, Wang, Ali et al., 2011;Bao, Wang, Ali et al., 2011).A novel isoflavone NV-128, significantly targeted mitochondria to induce cell death in chemo-resistant ovarian cancer stem cells (Alvero, Montagna, Holmberg et al., 2011).The phenol protects hematopoietic stem cells against granulocyte colony-stimulating factor (G-CSF)-induced DNA damage (Souza, Silva, Calloway et al., 2014).There is ample precedent for metabolism entailing phenolic oxidation followed by conversion to catechol or hydroquinone type with subsequent oxidation to quinone followed by generation of ROS-OS.
O HO

OH
O OH

Luteolin
Luteolin, 3',4',5,7-tetrahydoxyflavone (Figure 3), is a common flavonoid that exists in many types of plants including fruits, vegetables, and medicinal herbs.Plants rich in luteolin have been used in Chinese traditional medicine for treating diseases, such as hypertension, inflammatory disorders, and cancer.Having multiple biological effects, the phenol functions as an antioxidant and as well as a pro-oxidant biochemically (Lin, Anxin Shi, Wang, & Shen, 2008).
Luteolin inhibits the hypoxia-induced EMT in malignant melanoma cells both in vitro and in vivo via the regulation of β3 integrin, suggesting action as a potential anticancer chemo preventive and chemotherapeutic agent (Ruan, Liu, Zhang et al., 2012).Luteolin induces apoptosis in multidrug resistant cancer cells via ROS generation, DNA damage, activation of p53, NF-kB signaling pathways, activation of p38 pathway and depletion of anti-apoptic proteins (Rao, Satelli, Moridani et al., 2011).Glioma is one of the most common malignant tumors affecting the central nervous system.Drug screening using curcurmin, luteolin, chrysin and apigenin, showed suppression of the tumor cells (Feng, Zhou, Liu, & Tao, 2012).
Scheme 3. Formation of sulforaphane from glucosinolate
The peel of pomegranate possesses a higher content of polyphenols.The fruit contains large amounts of ellagic acid and its derivatives (Figure 4), along with punicalagin (Figure 6), a large polyphenol being the major constituent, possesses >50% of the antioxidant activity of pomegranate juice.Pomegranate also ontains other polyphenols, such as anthocyanins, cyanidin (Figure 5), caffeic acid, coumaric acid and flavonols.Ellagic acid is metabolized by the colon microflora to form urolithins A and B that circulate in the blood stream reaching various organs, playing a role as antioxidants, anti-inflammatory and anticancerous agents (Sudhakar, Venugopal, & Karthikaeyan, 2015).Four recent reviews deals with the role of pomegranate juice in breast cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, prostate cancer and human larynx epidermal carcinoma (Bhandari, 2012;Sudhakar, Venugopal & Karthikaeyan, 2015;Tuurini, Ferruzzi, & Filmognari, 2015).Mechanistically, as discussed in prior sections, the catechol can act as o-quinone precursusor leading to ET-ROS-OS as part of the unifying theme.

Resveratrol (RSV)
There is considerable literature that documents RSV (Figure 7) as a pro-oxidant, which is in accord with the unifying theme of ET-ROS-OS (Kovacic & Somanathan, 2010).Compounds related to RSV display oxidative properties (Miura, Muraoka, & Fujimoto, 2002).Conditions are important for DNA oxidation leading to apoptosis (Athar, Back, Kopelovich et al., 2009).A study revealed formation of superoxide (Ahmad, Clement, & Pervaiz, 2003).In the dark, AO properties prevail whereas under light exposure, oxidative effects are predominant (Gadacha, ben-Attia, Bonnfont et al., 2009).Another rationale exists for the pro-oxidant effects based on complex formation with heavier metals capable of ET (see Introduction).This route is more favorable for catechol types.
Figure 7. Resveratrol Relevant research literature has been published in the last five years.RSV is similar to chemotherapeutic drugs in relation to DNA damage (Colin, Limagne, Ragot et al., 2014).The insult was the result of large scale generation of ROS.The results are rationalized in the Introduction involving oxidation of RSV to a catechol type followed by oxidative conversion to an o-quinone leading to ET and ROS-OS.Exposure to RSV and histone deactylase inhibitors resulted in prolonged ROS generation with subsequent DNA cleavage (Yasen, Chen, Hock et al., 2012).A free radical scavenger hindered production of ROS, DNA damage and apoptosis, indicating an important role for oxidative insult.
RSV together with UVA produces pronounced OS in mitochondria which leads to apoptosis in keratinocytes (Yar, Menevse, Dogan et al., 2012).These findings may have practical application in treatment of skin cancer.The polyphenol is known to act as an antitumor agent and in chemoprevention (Stocco, Toledo, Salvador et al., 2012).High concentrations were needed to induce death of cancer cells.Exposure to RSV resulted in apoptosis and DNA damage in human gastric cancer cells via ROS (Wang, Li, Meng, & Jia, 2012).Exposure of the cells to SOD or catalase countered apoptosis by destroying ROS.In a report on colorectal cancer, RSV interacted with mitochondria resulting in enhancement of ROS and lipid peroxidation (Zhou, Chen, Yang et al., 2014).Combination of RSV with 5-flurouracil resulted in enhanced OS.Arsenic trioxide is a powerful anticancer drug for treatment of leukemia (Gu, Chen, Jiang, & Zhang, 2015).The combination of RSV and arsenite increased apoptotic cancer cell death through generation of OS.A report deals with OS in eye cells of diabetic rats involving an increase in OS (Boyer, Jandova, Janda et al., 2012).Protein carbonyl content was increased and GSH levels were decreased.We suggest a result of ROS-OS.

Epigallocatechin gallate (EGCG)
EGCG (Fig. 8 ) is a polyphenol prevalent in green tea (Scarpa & Ninfali, 2015).Clinical trials comprise the phenol alone or together with platinum (Pt) drugs involving synergism against prostate and colon cancers.One mode of action entails induction of apoptosis in tumor cells and animal models, which can result from OS (Halliwell & Gutteridge, 1999).Increase in ROS occurs during gallate-induced apoptosis of hepatic cancer cells (Li, Nie, Yu, & Xie, 2009).The compound enhanced the production of radicals, both ROS and RNS (Jeong, Park, Cho et al., 2015).A study was made of the drug effect on Leishmania (Inacio, Gervazoni, Canto-Cavalheiro et al., 2014).Results suggest a mode of action involving ROS.The effect was reversed by catalase, providing evidence for ROS involvement.
In relation to the ET-ROS-OS unifying mechanism, the catechol portion can act as precursor of an o-quinone which is an ET agent capable of inducing ROS-OS which plays a role in cancer destruction.Piperine (Figure 9) is an alkaloid which produces the pungency of pepper.Various mechanisms have been advanced in connection with bioactivity.The compound activates ions associated with pain-sensing nerve cells (McNamara, Randall, & Gunthorpe, 2005), and also inhibits enzymes involved with metabolism of xenobiotics (Bhardwaj, Glaser, Becquemont et al., 2002;Srinivasan, 2007), of drugs (Atal, Dubey, & Singh, 1985;Reen, Jamwal & Taneja, 1993) and of glucuronidation (Singh, Dubey, & Ata, 1986).A molecular mechanism is proposed for killing of breast cancer stem cells involving inhibition of Wnt signaling (Kakaral, Brenner, Khakaya et (Yaffe, Doucette, Walsh, & Hoskin, 2013;Moorthy & Kathiresan, 2013).The AO N-acetylcysteine decreased apoptosis in the treated cells, demonstrating that the induced cytotoxicity was mediated by ROS, at least in part.
The unifying mechanism of ET-ROS-OS can also be applied by two methods.In one case, the polyene benzenoid structure in conjugation with amide may participate in ET.In the other instance, the catechol ether portion undergoes oxidative dealkylation to form a substituted catechol with subsequent oxidation to ET o-quinone.

Polyene-Lycopene
Lycopene (Figure 10), possessing a red color, is found in tomatoes, watermelon and pink grapefruit (Scarpa & Ninfali, 2015).Research demonstrates that vegetables with high amounts of the polyene decrease various cancers, such as prostate, cervic and digestive systems (Dow, 2005).A mixture of the compound with selenium and vitamin E inhibits prostate cancers with increase in survival.Growth of lung cancer cells is suppressed.Lycopene from tomatoes may act against prostate cancer, although the evidence is not conclusive (Choi & Lee, 2015).Also, mitochondrial dysfunction occurred.A similar report deals with action as a bacteriacide involving the hydroxyl radical which damages E.coli DNA.Cell filamentation also took place (W. Lee & D. G. Lee, 2014).
There are numerous reports of AO action displayed by lycopene with several examples provided herein.A study indicated possible alleviation of OS (Chen, Song, & Zhang, 2013).Administration of the polyene provided protection from mitochondrial dysfunction and OS (Snadhir, Mehrotra, & Kamboj, 2010).The compound is a powerful radical scavenger and its AO properties are involved in many health effects (Kujawska, Ewertowska, Adamska et al., 2014).A tomato paste suppressed OS in rats.Lycopene pretreatment suppressed ROS generation and increased survival (Kim, Han, Lee et al., 2015).
A 2006 book provides rationale for the ability of certain compounds to act as both pro-and anti-oxidants (Kovacic & Somanathan, 2006).
Relevant information concerning mechanism is relation to amphotericin B is presented in the section on β-carotene.11) (Scarpa & Ninfali, 2015).An orange-red pigment, occurs in carrots, pumpkins and sweet potatoes.The polyene reduces the risk of breast cancer in women before menopause.β-Carotene was found to exacerbate UV carcinogenesis in mice (Black & Gerguis, 2003).A mode of action was suggested for the repair of the β-carotene radical cation, a potent oxidizing entity formed by reaction with oxidizing species.Useful information is provided by electron affinity (EA) studies on amphotericin B (Figure 12) (Kovacic & Cooksy, 2012).The EAs are found to be 1.30 and 1.36 eV for the conformers.These values are considerably higher than those prominent ET functionalities, such as quinones (0.54-0.64 eV) and aromatic nitro compounds (0.59 eV for dinitrophenol).The EA values should be more favorable for lycopene having eleven conjugates and β-carotene with nine versus seven for amphotericin B.

Conclusion
Nutrients display various beneficial and harmful effects with focus on anticancer action.The ET-ROS-OS scheme serves as unifying mechanism.Polyenes and ET quinone metabolites appear to play roles as ET agents.Evidence indicates involvement of nutrients as AOs in some actions.The unifying mode of action adds to prior studies.

Figure
Figure 8. Epigallocatechin gallate Evidence revealed increased generation of ROS in piperine-treated cancer cells