Plant flavonoids in cancer chemoprevention: role in genome stability

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ScienceDirect Journal of Nutritional Biochemistry 45 (2017) 1 – 14

REVIEWS: CURRENT TOPICS

Plant flavonoids in cancer chemoprevention: role in genome stability Vazhappilly Cijo George a , Graham Dellaire b, H.P. Vasantha Rupasinghe a, b,⁎ a

Department of Plant, Food, and Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia, Canada b Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada

Received 3 July 2016; received in revised form 27 August 2016; accepted 14 November 2016

Abstract Carcinogenesis is a multistage process that involves a series of events comprising of genetic and epigenetic changes leading to the initiation, promotion and progression of cancer. Chemoprevention is referred to as the use of nontoxic natural compounds, synthetic chemicals or their combinations to intervene in multistage carcinogenesis. Chemoprevention through diet modification, i.e., increased consumption of plant-based food, has emerged as a most promising and potentially cost-effective approach to reducing the risk of cancer. Flavonoids are naturally occurring polyphenols that are ubiquitous in plant-based food such as fruits, vegetables and teas as well as in most medicinal plants. Over 10,000 flavonoids have been characterized over the last few decades. Flavonoids comprise of several subclasses including flavonols, flavan-3-ols, anthocyanins, flavanones, flavones, isoflavones and proanthocyanidins. This review describes the most efficacious plant flavonoids, including luteolin, epigallocatechin gallate, quercetin, apigenin and chrysin; their hormetic effects; and the molecular basis of how these flavonoids contribute to the chemoprevention with a focus on protection against DNA damage caused by various carcinogenic factors. The present knowledge on the role of flavonoids in chemoprevention can be used in developing effective dietary strategies and natural health products targeted for cancer chemoprevention. © 2016 Elsevier Inc. All rights reserved. Keywords: Cancer; Flavonoids; Chemoprevention; Dietary antioxidants; DNA damage signaling and repair; Polyphenols; DNA damage detection; Genome stability

1. Introduction The term cancer can be described as a set of complex processes involving impaired cells death, unlimited cell proliferation and temporal–spatial changes in cell physiology that often leads to malignant tumor formation resulting in invasion of distant tissues to form metastasis [1]. Multistage carcinogenesis is a widely accepted hypothesis in the development of cancers and is operationally divided into three stages, namely, initiation, promotion and progression [2,3]. Carcinogenesis may result from extensive DNA damage, often caused by exposure to a variety of exogenous and endogenous agents including ultraviolet radiation (UVR), ionizing radiations (IRs), mutagenic chemicals, environmental agents, therapeutic agents or diagnostic imaging. DNA damage as a term encapsulates both frank single and double-stranded DNA breaks, as well as stable modifications to nitrogen bases in DNA or its sugar-phosphate backbone, caused by external (e.g., IR) or internal sources [e.g., reactive oxygen

species (ROS) generated during oxidative metabolism], which impact the cell by disrupting gene function and/or impairing transcription, DNA replication and cell proliferation [4]. Maintaining genomic integrity is therefore crucial for the organism since it is a key feature in the maintenance of cell function and inappropriate DNA repair is associated with both the initiation and progression of cancer [5]. Failure in proper DNA protection and DNA repair mechanisms, decrease in cellular defenses, malfunctions in cell cycle checkpoints and aberrant inflammatory signaling can contribute to poor genomic stability and provide an “Achilles heel” exploited by many cancer therapeutics [6]. As such, the differences in the DNA damage response between normal and cancer cells often underlie the utility of DNA damaging agents in cancer treatment [7]. DNA damage occurring during “S” phase of cell cycle, when DNA is replicated, was considered as the most lethal DNA damage [8] and, given the uncontrolled proliferation of cancer cells, may explain why DNA-damaging agents can be so effective in targeting cancers.

Abbreviations: ATM, ataxia telangiectasia mutated; ATR, ATM-Rad3-related; BRCA, breast cancer proteins; CHK, checkpoint kinases; DDR, DNA damage response; DNA-PK, DNA-protein kinases; DRI, dose reduction index; DSBs, double strand breaks; EGCG, epigallocatechin-3-gallate; H2O2, hydrogen peroxide; HDAC, histone deacetylase; HR, homologous recombination; IF, immunofluorescence; IR, ionizing radiation; MDC1, mediators of DNA damage checkpoint 1; mTOR, the mechanistic target of rapamycin; NHEJ, nonhomologous end joining; PI, propidium iodide; PI3K, phosphatidylinositol-3 kinases; RAD51/52, radiation-induced assembly; ROS, reactive oxygen species; SSBs, single-strand breaks; UVR, ultraviolet radiation. ⁎ Corresponding author. Tel.: +1 902 893 6623; fax: +1 902 893 1404. E-mail address: [email protected] (H.P.V. Rupasinghe). http://dx.doi.org/10.1016/j.jnutbio.2016.11.007 0955-2863/© 2016 Elsevier Inc. All rights reserved.

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2. Cellular DNA-damaging agents Cellular DNA can be damaged by cytotoxic or genotoxic agents with different mechanism of action. Some of the classical groups of DNA damage inducers used in chemotherapy are alkylating agents, platinum drugs, antimetabolites, topoisomerase inhibitors and forms of ionizing radiation [9]. Alkylating agents like the nitrogen mustards, nitrosoureas, aziridine compounds, alkyl sulphonates and triazine compounds are electrophiles that covalently transfer alkyl groups onto the DNA bases, disrupting the DNA helix shape [10]. Cisplatin and its analogs, carboplatin and oxaliplatin, are DNA-intrastrand crosslinking agents, and these platinum-based drugs are often used to treat testicular or ovarian cancers [11]. The fluoropyrimidine 5-fluorouracil, an antimetabolite having similar structures that are related to nucleotide metabolites, induces DNA damage by either inhibiting biosynthetic processes or being incorporated into nucleic acids such as DNA and RNA [12]. Camptothecin, a plant alkaloid, is a topoisomerase inhibitor widely used to treat colorectal, ovarian and lung cancers that targets DNA-TOP1 (type 1 topoisomerase) cleavage complexes, blocking religation and resulting in the accumulation of transient single-strand breaks (SSBs) [13]. A summary of factors which causes DNA damage is depicted in Fig. 1. 3. DNA damage response (DDR) The DDR is a complex cascade of molecular and cellular events that is necessary to eliminate lethal and tumorigenic mutations caused by different genotoxic stress including carcinogens produced by physical or chemical sources. This signaling mechanism regulates cell proliferation, cell cycle and apoptotic induction [14]. Failure in proper DNA damage response mechanisms may result in improper DNA repair which can drive to tumorigenesis and can affect sensitivity to genotoxic chemotherapy [10]. DNA repair can be initiated by various enzymes that modify the DNA and nuclear damage by activating polymerases, topoisomerases, ligases, kinases, phosphatases and glycosylases [15]. DDR is commonly activated in early neoplastic lesions and likely protects against malignancy [16]. 4. Signal transduction mechanisms in DNA damage DNA double-strand breaks (DSBs) are well regarded as the most lethal lesions among all types of damages and possess the greatest challenge to human beings. DSBs can be caused by UV, radiotherapy, dysfunctional telomeres or genotoxic agents, and the breaks they induce can activate phosphatidylinositol-3 kinases (PI3K) including ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR) or DNA-dependent protein kinases (DNA-PK) that serve as the pinnacle step in DNA damage signaling (Fig. 2). DNA-PK is a multicomponent complex consisting of the DNA-PK catalytic subunit (DNA-PKcs) and the Lupus Ku autoantigen protein heterodimer (Ku80 and Ku70) [17]. Although there is some cross talk in downstream targets of ATM and ATR, these kinases are activated by ionizing irradiation or ultraviolet light/hydroxyurea, respectively [18]. While DNA-PK regulates a small group of proteins involved in DNA DSB end joining, it also cooperates with ATR and ATM to phosphorylate proteins involved in the DNA damage checkpoints [19]. Thus, protein phosphorylation plays a crucial role in DNA damage signaling, activating over 700 proteins in response to DNA damage which in turn counteract genotoxic stresses by regulating protein–protein interactions and other posttranslational modifications [20]. However, the functional significance of many of these proteins is unclear and remains an area of intense research. DNA is packaged within chromatin, and the reversible acetylation of proteins within chromatin-like histone H3 and H4 can affect DNA repair by a number of mechanisms including altering the association of DNA repair factors with damaged DNA and the modulation of the

relaxation (or condensation) state of chromatin that may be important in regulating physical access of the DNA repair machinery to the break within chromatin [7,21]. Acetylation is mediated by histone acetyltransferases (or HATs), and deacetylation is mediated by histone deacetylases (HDACs); the balance of HAT and HDAC activities is often perturbed in cancers. Thus, histone acetylation could serve as a therapeutic target for cancer treatment [22]. For example, HDAC inhibitors have the potential to interfere with DNA repair and chromatin relaxation mechanisms, potentially sensitizing cancer cells to DNA damage [23]. Another critical posttranslational modification of chromatin is phosphorylation, and one of the earliest events during DNA DSB repair is the phosphorylation of Ser139 on the specialized histone H2AX called, which is then referred to as γ-H2AX. H2AX is one of the heteromorphous variants of family of at least eight protein species of the nucleosome core histone H2A [24]. Cytologically, γ-H2AX forms punctate structures in the nucleus known as DNA repair foci, which result from the spread of γ-H2AX along chromatin surrounding the DNA break up to 1–2 megabases of DNA via the action of ATM. These repair foci serve as platforms for the assembly and recruitment of other DNA repair factors, including mediators of DNA damage checkpoint 1 (MDC1) to initiate the DNA damage response [25]. Once initiated, the DNA damage signal is amplified by checkpoint kinases 1 and 2 (CHK1 and CHK2) that are activated by phosphorylation by ATM and ATR, and a number of effector proteins, including breast cancer antigens 1 and 2 (BRCA1, BRCA2), p53 and murine double minute [26]. Chk1 and Chk2 can phosphorylate DNA repair proteins like BRCA2 and are involved in cell cycle checkpoint control by phosphorylating proteins such as p53 [27]. BRCA1 and BRCA2 are involved in recruiting the repair protein RAD51 to sites of DNA damage to facilitate DSB repair by homologous recombination (HR) [28–30]. The p53 is phosphorylated by Chk1 and Chk2 in response to DNA damage and in turn regulates cell fate decisions including cell cycle arrest and apoptosis by inducing expression of protein such as p21 and B-cell CLL/lymphoma 2 (BCL2), respectively [7,31]. Highlighting the intimate link between DNA repair and cancer, germline or acquired mutations in several DNA repair and signaling factors including BRCA1, BRCA2, ATM, p53 and CHK2 can contribute to the development of cancers affecting multiple organs including breast and ovary, lungs, pancreas and blood (i.e., leukemias) [32–35]. Other common syndromes associated with increased cancer susceptibility include Seckel syndrome (mutated ATR), radiosensitive severe combined immunodeficiency disease and ligase IV (LIG4) syndrome (mutated LIG4) [36]. After DNA has been repaired, chromatin must be restored to its original state before damage to allow efficient transcription and replication of DNA. Some of the first steps in chromatin restoration include the dephosphorylation of γ-H2AX by the phosphatases PP4 and PP2A, the proteasomal degradation of MDC1 within repair foci, and deacetylation of H3 or H4 lysines by HDAC [37]. Other proteins like the histone chaperones chromatin assembly factor 1 and antisilencing factor 1 also play a crucial role in restoring chromatin structure and cell cycle progression [38]. Failure of these mechanisms may result in epigenetic alterations and thus cause genomic instabilities and its associated diseases [39]. Epigenetic alterations in DNA, such as methylation, and of chromatin, such as modification of histones by acetylation, methylation, and phosphorylation, are increasingly being recognized for their role in health [36,40]. Most of the diseases involving dysregulation of epigenetics are marked with common characteristics such as developmental defects, immunodeficiency, neurological degeneration and cancer predisposition [17]. DNA methylation of cytosine (5methylcytosine) is a very common epigenetic mechanism involved in controlling DNA structure, chromosome stability, the mobility of viral DNA-repeated elements (transposons, retrotransposons), gene imprinting and gene expression [41]. In tumor tissues, tumor suppressor

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UV rays IR rays Gamma rays X-rays Microwaves Radio waves CT scan Mammography Etoposide Teniposide . Actinomycin-D Mitomycin-C Daunorubicin Epirubicin

Thiopurines Ciclosporin Sirolimus Azathioprine Cyclosporine

Radiations

Chemotherapeutic drugs

5-Fluorouracil (5-FU) 6-Mercaptopurine Capecitabine Cytarabine Floxuridine Fludarabine Gemcitabine Hydroxyurea

DNA damaging agents

Immunosuppressive and genotoxic drugs

Environmental and chemical toxins

Cisplatin Dacarbazine Doxorubicin Epirubicin Idarubicin Ifosfamide Irinotecan Lomustine Mitomycin C Temozolomide Topotecan

Acrylamide Asbestos Silica Titanium dioxide Arsenite Bisphenol A Quinones Benomyl

Vanadium Arsenic Lead Nickel Cobalt Cadmium Chromium

Fig. 1. Examples of various factors causing DNA DSBs in mammalian systems.

genes are often inactivated epigenetically by methylation of cytosine when compared with normal tissue [42]. 5. Pathways of DNA repair are lesion specific DNA repair mechanisms are very specific for many types of lesions. Mismatch repair facilitates the replacement of incorrect bases with correct bases. Base excision repair enables to carry small chemical alterations of DNA bases through excision of damaged nitrogen bases [43]. Lesions caused by pyrimidine dimers and intrastrand crosslinks are rectified by removing an oligonucleotide containing the damaged bases through a process called nucleotide excision repair (NER) [44]. SSBs and DSBs are processed by SSB repair and nonhomologous end joining (NHEJ)/HR mechanism, respectively. Among these mechanisms, NHEJ promotes the potentially inaccurate regulation of DSBs, while HR restores the genomic sequence of the broken DNA ends by employing sister chromatids as a template for repairing DNA [15]. Lupus Ku autoantigen protein p70 (Ku70) binds to DNA DSBs and activates the DNA-PKcs to form DNA-PK holoenzyme which is required for NHEJ pathway of DNA repair [45]. 6. Methods to detect DNA damage There are few traditional methods used to detect DNA damage including neutral sucrose gradients and pulse-field gel electrophoresis which are laborious, are time consuming and have low sensitivity [46]. Recent research involved much more precise techniques to quantify the DNA damage, and one such common method is single-cell gel electrophoresis (comet assay) that appears to be an attractive method to measure the kinetics of the process including excision followed by resynthesis and ligation of fragments [47,48]. Flavonoids have been

studied extensively for their mechanism of action on DNA damage and repair mechanism on various models by using comet assay [49]. As a sensitive measurement of DNA DSBs, phosphorylated histone H2AX protein (γ-H2AX) can be measured by immunofluorescence (IF) analysis by using antibodies against γ-H2AX [50]. p53-binding protein 1 (53BP1), MDC1 and PP2A are the other nuclear foci that can detect DNA damage by IF analysis [51]. Double-stranded DNA fragments can also be labeled with terminal deoxynucleotidyl transferase and detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using fluorescence microscopy [52]. In addition, carcinogen-induced cell death can be measured through multiparameter flow cytometry for apoptosis or necrosis cell death [annexin V/propidium iodide (PI), cell cycle analysis (PI) or cytosolic reactive oxygen species (dichlorofluorescein diacetate)] [53]. Various methods used to detect and measure DNA damage and its associated proteins are summarized in Tables 1 and 2. 7. Cancer chemotherapy Treatment methods for cancers normally involve surgery, radiation therapy, immunotherapy, targeted therapy and chemotherapy with drugs that can kill cancer cells. Chemotherapy for cancer control is based on the presumption that cancer develops through a multistep process. Chemopreventive agents function as either blocking agents, which act immediately before or during the initiation of carcinogenesis by chemical carcinogens, or suppressing agents, which act after initiation during the prolonged stages of promotion and progression [54]. Development of chemopreventive drugs requires specific and/or multiple molecular and cellular targets to act on in order to control proliferation/growth of cancerous cells. Dose selection of chemotherapeutic drug is critical for therapy since some of the anticancer drugs

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Pro-carcinogens

Exogenous and endogenous factors

2

1

Carcinogens

DNA damaging agents

Antioxidant defence & Phase I/II enzymes ROS

Modification of histone proteins A

Activation of damage Signalling proteins 3

4

5

H2A

H2B

H4

H3

DSBs A

Activation of DNA repair

p53 ATM ATR

Failure in DNA repair

P

γ-H2AX

1

DNA-PK

CHK1

CHK2

BRCA1

BRCA2

7

Activation of DNA repair mechanisms

RAD51/52

Epigenetic alterations of un-repaired DNA; somatic gene mutations ; genomic instability

HDAC CAF1

Restoring chromatin structure

Normal cells

Cancer cells

Uncontrolled cell cycle

Metastatic cancer cells

NHEJ/HR

Apoptosis & cell death

Cell survival

Genomic stability restored

Fig. 2. Cellular mechanisms of flavonoids on DNA damage and repair. Different DNA-damaging agents may form DSBs followed by chromatin modifications in order to initiate damage signaling proteins and repair mechanisms. These effects can be modulated by flavonoids which inhibit carcinogenesis process by multiple mechanisms including acting as antioxidants, induction of antioxidant defense and phase I/II enzymes, inhibition of DNA damaging proteins, stimulating repair mechanisms in normal cells and restoring genomic integrity in normal cells. Failure of repair mechanisms may result in the uncontrolled cell cycle, somatic mutations (chromosomal abnormalities, telomerase shortening etc.) and malignant tumor formation. Selected examples of flavonoids that are known to regulate carcinogenesis in normal cells: luteolin (1), apigenin (2), EGCG (3), quercetin (4), fisetin (5) and epicatechin (7). Abbreviations: P, phosphorylation; A, acetylation.

possess systemic toxic effects when used at higher concentrations. In the process of increasing efficacy for chemotherapeutic drugs, increasing dosage may have a reversible pharmacological effect to be cytostatic rather than cytotoxic [55]. Combination of two drugs, often indexed as dose reduction index (DRI), is important in systemic

toxicity of drugs. Lower DRI values (N1) may be a good choice as it may protect normal cells and selectively destroys cancer cells [56]. However, a good chemotherapeutic drug at therapeutic dosages ideally targets specific pathways which are aberrant in cancer cells relative to normal cells.

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Table 1 Methods to detect DNA damage and DNA repair proteins Method

Uses

Mechanism

Reference

TUNEL assay Fluorescent microscopy Comet assay/single cell gel electrophoresis Flow cytometry/FACS Western blotting

To detect DNA fragmented; apoptosis To detect DNA damage response proteins To detect and quantify DNA tail damage level To detect DNA damage related proteins To detect DNA damage related proteins DNA repair mechanisms To detect and quantify DNA damage related proteins

Labeling DNA using terminal transferase Antibody staining for γ-H2AX protein Agarose trapped cell method Antibody staining for ATM and γ-H2AX Antibody staining for ATM, γ-H2AX, and p38 DNA-PK, KU80/70, RAP1 Specific primers for DNA-PK, ATM and ATR

[149] [150] [49] [151] [152,153]

RT-qPCR

[154]

FACS, fluorescence-activated cell sorting; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

7.1. Dietary flavonoids in cancer chemoprevention Traditional knowledge and scientific reports demonstrate that medicinal plants are rich sources of biologically active compounds that can be used for the treatment of various diseases including some type of cancers. The first attempt to develop a chemotherapeutic drug began in 1940s with nitrogen mustards and antifolate drugs [57]. Research on flavonoids showed major developments in anticancer drug discoveries with potential to destroy cancer cells through apoptotic induction [58,59]. Plants have provided an endless supply of secondary metabolites or phytochemicals that are increasingly exploited against various cancers [60,61]. For example, properties of plant polyphenols including chemical diversity, structural complexity, affordability, lack of substantial toxic effects and inherent biological activities have made them attractive candidates for new therapies [62]. The efficacy of flavonoids to act on a target molecule in chemoprevention highly depends on their absorption, metabolism, distribution and availability at the site of action in the body [63,64]. Bioavailability of flavonoids is generally low and varies depends on their structure, subclass, molecular weight, glycosylation and esterification. For an instant, intake of dietary flavonoid quercetin by humans (250–500 mg) may result in b1% dose (unaltered form) that eventually reaches the circulating system with a peak in plasma concentrations in the lower nanomolar range (~20 nM) [65,66]. More often, flavonoids get transformed to conjugate metabolites such as O-glucuronides, sulfate esters and O-methyl esters by phase II and hydrolyzing enzymes in the small intestine, liver and colon [67,68]. Quercetin glycosides were found to be better absorbed than its counterpart aglycone with observed higher plasma concentrations after being transformed initially in either deglycosylation processes at the intestinal level or carrier-mediated transport [69,70]. These low doses of flavonoids may not interfere with cell cycle process in normal cells. However, some compounds like 3-hydroxyflavone, luteolin and apigenin when treated with high doses (N50 μM) showed considerable cytotoxicity with increased ROS generation [71]. They may even disturb the cellular integrity by inducing cell death mechanisms in normal cells. It is becoming clear that

many external signals including those that stimulate growth, such as growth factors, and those that inhibit growth, such as DNA-damaging agents, control cell proliferations through arresting cell cycle at various phases of cell growth [72]. Current research focuses on modulating effects of these flavonoids on cellular signaling pathways involved in DNA damage and repair, which becomes a novel approach in chemoprevention. 7.2. Flavonoids and DNA damage Studies over a decade from cell cultures, animal and human populations showed the potential of flavonoids in human and animal health benefits. Since they are abundantly present in our diet, such as fruits, vegetables, teas and wine with strong antioxidative potentials, estrogenic regulatory and antimicrobial activities, they can be exploited against many diseases including cancers [73,74]. They may inhibit several points in cancer progression including invasion, metastasis, angiogenesis, apoptotic mechanisms and cell cycle arrest. Some of the classical examples of flavonoids and their mechanism of actions are discussed below (Figs. 3 and 4; Tables 3 and 4). 7.2.1. Apigenin Apigenin, a flavone widely found in many medicinal plants including Lycopodium clavatum L. (club moss) and some vegetables such as Petroselinum crispum L. (parsley) and Apium graveolens L. (celery), has been reported to have beneficial effect against UV-Binduced DNA damage and inflammatory diseases [75,76]. Recent in vitro and in vivo studies have shown that apigenin possesses antiproliferative properties with induction of cell cycle arrest and apoptosis in cancer models. Apigenin has been reported for DNA protective activity in skin cells and mice against UV-B-induced ROS and DNA damage by removing cyclobutane rings, inhibition of ROS generation and down-regulation of NF-κB [76]. In human diploid fibroblast and bladder cancer T-24 cells, apigenin showed significant ROS scavenging potentials along with cell cycle arrest at G1 phase by modulating CDK2 kinases [77–79]. Apigenin reduces the risk of

Table 2 Methods to measure DNA adducts Method

Description

Models used

Reference

HPLC Mass spectrometry

Use to quantify total guanine or ribo- or deoxyribonucleotides to determine DNA adducts Involves derivatization of samples, structural identification of adducts, preparation can use gas chromatography, ionization and electrospray DNA is digested into nucleotides that are labeled at the 5′ end with 32P mediated by polynucleotide kinase and adducts are separated by chromatographic or electrophoretic methods Uses antisera recognizing benzo[a]pyrenediol-epoxide-modified DNA (BPDE-I-DNA) and color change of fluorescence endpoint detection Uses multiple antisera to stain tissues, cell sections or colocalization with nuclei and detect by fluorescence microscopy Antisera specific for DNA adducts are used with various end points for measurements (chemiluminescence or radioactivity) Uses affinity chromatography and HPLC to isolate tetrahydrotetrols adducts and determined by fluorescence spectroscopy

Human lung Mouse lung, liver, and kidney

[155] [156]

Cynomolgus monkeys liver

[157]

Colorectal adenoma cells

[158,159]

Mice liver

[160,161]

Human peripheral lung

[162]

Human lung

[163]

32

P-postlabeling

ELISA IHC Immunochemical assays Fluorescence spectroscopy

HPLC, high-performance liquid chromatography; TLC, thin layer chromatography; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry.

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ovarian cancers as demonstrated by a population study conducted in ovarian cancer patients [80]. Apigenin possess protective effect in endothelial cells from lipopolysaccharide (LPS)-induced inflammation. Apigenin reduces ROS production and caspase-3 expression levels and thereby keeps DNA damage in check [81]. In cancer cells, apigenin induces DNA damage/apoptosis by activating ATM and H2AX phosphorylation with down-regulation of cell cycle controlling and DNA repair genes [82]. Apigenin was also reported to modulate DNA damage by inhibiting casein kinase 2, a regulator of cell proliferation, mediator of the DNA damage response and NF-κB activation in malignant glioma cells [83].

lipogenic enzyme overexpressed in many human cancers [59]. The chemopreventive effect and associated mechanisms of luteolin in the JB6 P+ neoplastic mouse cell line and the SKH-1 hairless mouse models were described by Byun et al. [92]. Luteolin has been shown to delay or block the development of cancer cells both in vitro and in vivo, protect DNA from carcinogenic stimulus and induce cell cycle arrest and apoptosis via intrinsic and extrinsic signaling pathways [93]. Additionally, luteolin also induces apoptosis in multidrug-resistant cancer cells by ROS generation, DNA damage initiation, activation of ATR/Chk2/p53 signaling, inhibition of NF-kB signaling, activation of p38 and depletion of antiapoptotic proteins [94].

7.2.2. Epigallocatechin-3-gallate (EGCG) An antioxidant potent flavan-3-ol, EGCG, protects DNA damage and initiates repair mechanisms in various cancer models. EGCG is the most abundant catechin in green tea and is a traditional antioxidative free radical scavenger [84]. EGCG was found to be protective in skin cells after irradiation using an X-ray linear accelerator, with overexpression of heme oxygenase-1 (HO-1). EGCG reduces phosphorylation of H2AX foci in these HaCaT keratinocytes [85]. EGCG showed protection against UVR-induced DNA damage in human peripheral blood samples isolated from adult human volunteers before and after drinking 540 ml of green tea [86]. Furthermore, EGCG was also reported to possess radiomodulatory effects on pBR322 plasmid DNA and murine splenocytes against gamma-radiationinduced DNA damage [87].

7.2.4. Quercetin Quercetin glycosides, the flavonols abundant in apples, onion and garlic, were reported to induce DNA damage in cancer cells [95]. Comparatively, although quercetin has similar structure to kaempferol and luteolin, it was reported to cause more damage to normal mammalian DNA [96]. However, quercetin was also reported to protect DNA damage induced by hydrogen peroxide (H2O2) challenge in Caco-2 human epithelial colorectal adenocarcinoma cells by enhancing DNA repair mechanisms through modulation of DNA repair enzymes [97]. Intraperitoneal administration of quercetin in rats showed protection against radiation-induced DNA damage in kidney and bladder tissues [98]. Its free radical scavenging and antioxidant properties attenuate DNA protection by reducing myeloperoxidase and caspase-3 activities in rats. Quercetin metabolite quercetin-3-Oglucuronide was found to have significant inhibitory effect on noradrenaline binding to α2-adrenergic receptor by suppressing DNA damage induced by treatment with 4-hydroxyestradiol and noradrenaline in MCF-10A normal human breast cancer cells [99]. Quercetin also reduces γ-H2AX and p53 phosphorylation in HT1080 human fibrosarcoma cells [100].

7.2.3. Luteolin Luteolin, a flavone found in Salvia tomentosa Mill. (Balsamic sage) and many other plants, protects SSBs induced by oxidative stress in PC12 rat pheochromocytoma cells [88]. Luteolin was reported for its apoptotic potentials in human lung squamous carcinoma CH27 cells with higher DNA damage and “S” phase cell cycle arrest [89]. Luteolin activates intrinsic apoptotic pathways by inducing DNA damage and p53 in many cancer cells [90,91]. Luteolin induces apoptosis in prostate and breast cancer cells by inhibiting fatty acid synthase, a key

7.2.5. Chrysin Flavonoid, chrysin abundant in Passiflora incarnate L. (Maypop) and Oroxylum indicum L. (midnight horrow), was reported to have

Plant Polyphenols

Flavonoids

Mode of actions

Examples

Flavones

Apigenin 2 Luteolin 1 Chrysin Orientin Baicalein Acacetin

ROS scavenging, Cell cycle arrest, Induce apoptosis.

Flavonols

Rutin Quercetin 4 Kaempferol 8 Myricetin Fisetin 5 Isorhamnetin

Regulate γ-H2AX, Blocking agent, Caspase activity.

Isoflavones

Daidzein 9 Genistein 10 Biochanin A Formononetin Barbigerone

Inhibit ROS generation, Inhibit p53 expression.

Flavanones

Naringenin 12 Hesperetin 11 Eriodictyol Butin Poncirin Sakuranin

ROS scavenging, Induce apoptosis, Inhibit DNA damage by reducing COX-2.

Flavan-3-ols

EGCG 3 Epicatechin 7 Catechin Proanthocyanidins Thearubigins

ROS scavenging, Cell cycle arrest, Induce DNA-PK expression, Inhibit γ-H2AX.

Anthocyanidins

Cyanidin Malvidin 13 Capensinidin Rosinidin Hirsutidin Delphinidin

Suppress ROS, Increase glutathione levels, Inhibit DNA adduct formation.

Fig. 3. Classification of plant flavonoids. Different subclasses of flavonoids with selected examples and their commonly reported mode of actions in relation to DNA damage prevention.

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Apigenin

Quercetin

Daidzein

Naringenin

EGCG

Luteolin

Fisetin

Genistein

Hesperetin

Malvidin

Fig. 4. The structure of major plant flavonoids that are involved in chemoprevention.

protective effect against methylmercury (MeHg)-induced genotoxicity as evidenced by studies conducted using 2-month-old male Wistar rats. The level of glutathione (GSH) in blood was restored in animals administrated with chrysin and inhibits DNA fragmentation induced by methylmercury (MeHg+) [101]. Chrysin reduces the disturbances of redox status, named superoxide dismutase, catalase, glutathione peroxidase and GSH in liver, kidney and brain tissues of rats treated with D-galactose [102]. In another study, chrysin protects free-radical-mediated oxidative stress induced by N ω-nitro-Lariginine methyl ester in male albino rats [103]. 7.2.6. Daidzein and genistein Daidzein and genistein are isoflavones found in plants including Glycine max L. (soybean) and Pueraria mirifica L. (Kwao Krua) and were reported for their DNA photoprotective effect against UVBirradiation-induced DNA damage in skin fibroblasts. They reduce cyclooxygenase-2 and DNA-damage-inducible (Gadd45) genes effectively in treated skin samples. However, the synergistic effect of genistein and daidzein when used in combination provided much more photoprotective effects than single compound treatment [104]. 7.2.7. Malvidin Among dietary anthocyanins, malvidin-3-O-glucoside is one of the major constituents, in particular, in red wine [77]. Malvidin-3-O-glucoside protects endothelial cells against peroxynitrite-promoted apoptotic death by inhibiting ROS production and apoptotic proteins. Treatment with malvidin-3-O-glucoside also enhances proapoptotic protein expression in endothelial cells [105]. Malvidin was also found to protect DNA damage induced by chemotherapeutic drugs cyclophosphamide, procarbazine and cisplatin in mice. A dose–response protective effect of malvidin was observed in 30-min pretreated mice against cyclophosphamide-induced chromosomal damage [106]. 7.2.8. Citrus flavanones Hesperetin, a flavanone abundant in citrus fruits including oranges and grapefruit, as well as tomatoes and cherries, have antiinflammatory, antioxidant, anticarcinogenic and neuroprotective effects [107]. It protects doxorubicin-induced oxidative stress in rats by reducing DNA fragmentation as evident from the comet and TUNEL assays [108]. Dietary polyphenols like ellagic acid, genistein, emodin and guggulsterone have also been proved to be involved with the regulation of cell cycle and apoptosis in various cancer cells [109]. Another flavanone, naringenin, present in orange peel, was found to have the potential to induce DNA damage and apoptosis in HaCaT immortalized keratinocytes and in various other cancer cells [110].

7.3. Flavonoid-rich extracts and DNA damage protection Plant crude extracts rich with flavonoids (quercetin, quercitrin, isoquercitrin and rutin) isolated from the leaves of Scutia buxifolia L. protect from chromosome damage in cultured human lymphocytes by retaining mitotic index and genomic stability against H2O2-induced toxicity [111] (Table 5). An antioxidant-rich plant named Gynostemma pentaphylla (Southern Ginseng) was found to protect human endothelial cells from cholesterol-induced DNA damage by inhibiting excessive ROS production during atherosclerosis. It also inhibits the phosphorylation of H2AX and other PI3K proteins and thereby rendered DNA protection [112]. Flavonoid-rich extracts from rhizomes of Podophyllum hexandrum L. (Himalayan May Apple) were found to protect DNA damage and initiate DNA repair mechanisms in isolated human blood leukocytes. The active components were found to have potentials to inhibit H2AX and P53BP1 phosphorylation during DNA DSBs. It also up-regulates DNA-PKcs and Ku80 to facilitate DNA protection against radiation or assist in DNA repair mechanisms [113]. Withania somnifera L. (Ashwagandha), a commonly used herb in Ayurveda, was recently reported to protect brain DNA damage induced by H2O2 oxidative stress in glioblastoma and neuroblastoma cells through down-regulation of γ-H2AX and DSBs proteins such as Rad51, 53BP1 etc.[114]. An ethanolic extract of antioxidant-rich plant Nigella sativa L. was reported to protect radiation-induced DNA damage in Swiss albino mice. Orally fed mice with ethanolic extract of N. sativa showed significant protection against oxidative injury to spleen and liver as measured by lipid peroxidation and the activity of antioxidant enzymes [115]. Extracts of Crataegus pinnatifida L. (Chinese hawberry) pollen were reported to protect from DNA damage in response to H2O2-induced damage in mice lymphocytes. It also exhibits high total phenolic content (17.7±1.0 mg gallic acid equivalent/g), total flavonoid content (8.0±1.0 mg rutin/g) and strong free radical scavenging activity which might have rendered for the observed protective effects [116]. 7.3.1. Apoptosis induction by flavonoids in cancer cells Induction of apoptosis in cancer cells by flavonoids is associated with their ability to inhibit fatty acid synthase activity [59]. Methanolic extracts of Gracilaria tenuistipitata, a genus of red algae, were reported to have anticancer activity in oral squamous cell carcinoma. The extract induces apoptotic cell death by increasing DNA damage, ROS induction and mitochondrial depolarization. It also increases phosphorylation of γ-H2AX and DSBs, thus enforcing cancer cell to death [117]. Aqueous extract of Fagonia cretica L. (Virgin's Mantle), used widely as herbal treatment for breast cancer and fever, also induces apoptosis in MCF-7

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Table 3 In vitro mode of action of major flavonoids on DNA protection caused by various carcinogens Flavonoid

Experimental model

Carcinogen

Mode of action(s)

Reference

Baicalein

PAM 212 (murine keratinocyte) HaCaT (immortalized keratinocytes)

UV radiation UV-B

[164] [165]

Fisetin

V79-4 (Chinese hamster lung fibroblasts)

γ-Irradiation

V79-4 (Chinese hamster lung fibroblasts) 451Lu (human melanoma cells)

H2O2 UV-B

RWPE-1 (human prostate epithelial), LNCaP, PC-3 & DU145 (prostate cancer cells) BAECs (bovine aortic endothelial cells) HaCaT (immortalized keratinocytes) LN18 & U87 (malignant glioma cells)

H2O2

Inhibition of ROS and toll-like receptor-4 Inhibition of apoptosis-regulatory genes (p53-p21 and c-fos) Inhibition of apoptosis and restore mitochondrial membrane potential Inhibits ROS generation by increasing GSH levels Decreases phosphorylation of Akt, mTOR & p70S6K Decreases ROS and inhibits apoptosis

Apigenin

Quercetin

Human lymphocytes

Luteolin

Caco-2 (human epithelial colorectal adenocarcinoma cells) PC 12 (neuronal cells)

EGCG

HaCaT (Immortalized keratinocytes) Human peripheral blood cells

Malvidin

HUVEC (human umbilical vein endothelial cells)

Kaempferol HL-60 (human leukemia cells)

LPS UV-B γ-Irradiation

Decreases caspase-3 and ROS production Protects DNA damage by activating NER Inhibits the activation of casein kinase 2 and γH2AX Benzo(a)pyrene (B(a)P) Decreases oxidative DNA damage by inhibiting BPDE-DNA adduct formation Increases repair activity H2O2 Photosensitizer compound Protects DNA SSBs by increased expression of (Ro 19-8022) OGG1 repair gene UV-R Protects skin by HO-1 overexpression UV-R Photoprotective effects by decreasing DNA fragmentation Peroxynitrite Protects endothelial cells by inhibiting ROS production and apoptotic proteins γ-Irradiation Inhibits ROS production and cell cycle arrest

and MDA-MB-231 breast cancer cells by inducing γ-H2AX, DNA damage, cell cycle arrest and p53 expression. In the absence of p53, F. cretica extract induces apoptosis by FOXO3a (transcription factor) expression [118]. A schematic representation of different flavonoids involved in apoptotic regulation is shown in Fig. 5. 7.3.2. Regulation of mTOR signaling by flavonoids and other polyphenols The mammalian target of rapamycin (mTOR) is a serine/threonine kinase signaling protein that regulates various intracellular and extracellular mechanisms including cell growth, proliferation, apoptosis and cancer (Fig. 6). Expression levels of mTOR were found to be deregulated in most of the cancer cells, resulting in cell proliferation [119]. Plant polyphenols play a crucial role in inhibiting various kinases including mTOR signaling cascades, which are considered as a therapeutic strategy in cancer prevention. Piceatannol is one such polyphenol present in grapes, berries, peanuts and sugar cane; has natural inhibiting effect on tyrosine and serine/threonine kinases; and was found to regulate mTOR signaling in prostate cancer cells with damaging DNA and apoptosis [119,120]. It also inhibits DU145 and CaP prostate cancer cell proliferations and arrest cell cycle by suppressing

[166] [167] [123] [168] [81] [76] [83] [169] [170] [88] [85] [86] [105] [171]

CDK activities [121]. Recent research showed that piceatannol rendered protection against H2O2-induced DNA damage but was not involved in DNA repair mechanisms in Burkitt's lymphoma (Raji) and normal human epidermal keratinocytes (NHEK) [28,122]. Fisetin, a flavonol, was also found to inhibit AKT/mTOR signaling in human multiple myeloma and melanoma cells, thereby regulating cell proliferation [123]. Treatment with fisetin on A549 lung carcinoma cells reduced colony formation with decreased protein expression of PI3K and inhibited Akt or mTOR pathway [124]. Phytochemicals such as boswellic acid, butein and capsaicin have been reported to inhibit PI3K, Akt or mTOR signaling in different cancer cells and are discussed in detail elsewhere [125]. 7.4. Flavonoid-rich diet and DNA damage Increased production of ROS and defective antioxidants/DNA repair mechanisms can damage cellular macromolecules; can lead to changes in chromosome instability, genetic mutation and/or modulation of cell growth; and may result in cancer. ROS like superoxide radicals, H2O2, hydroxyl radicals, singlet oxygen, peroxyl radical,

Table 4 In vivo mode of action of major flavonoids on DNA protection caused by various carcinogens Flavonoid

Experimental model

Carcinogen

Mode of action(s)

Reference

Apigenin Orientin Baicalin

Swiss albino mice Balb C mice Balb C mice

UV-B γ-Irradiation UV-B

[76] [172] [173]

Female C57BL/6 mice

UV-B

Quercetin, cannabiscitrin, kaempferol Quercetin

Kunming mice C57BL/6 mice Sprague–Dawley rats

γ-Irradiation Nonalcoholic steatohepatitis IR radiations

Chrysin

Male Wistar rats

Methylmercury

Protects DNA damage by stimulating NER genes Enhances repair mechanisms by decreasing DNA fragmentation Protects DNA damage by inhibiting cyclobutane pyrimidine dimers (CPDs) Protects photoaging by inhibiting interstitial collagenase (MMP-1 and MMP-3) and γ-H2AX proteins Protects from DNA injury by inhibiting ROS generation Decreases liver DNA damage Protects from irradiation-induced oxidative organ injury by ROS and caspase-3 inhibition Protects genotoxicity by reducing DNA fragmentation and increasing GSH levels

[174] [175] [176] [98] [101]

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Table 5 Flavonoids-rich plant extracts in protection of DNA from various carcinogens Plant name

Major flavonoids (and phytochemicals)

Scutia buxifolia

Quercetin, quercitrin, isoquercitrin, rutin Human lymphocytes

Experimental model

Carcinogen or DNA Mode of action(s) damaging agent H2O2

Gynostemma Kaempferol pentaphyllum Podophyllum hexandrum Quercetin, kaempferol, rutin

HUVEC (human umbilical vein endothelial cells) Human blood leukocytes

Withania somnifera

Quercetin

Nigella sativa

Damascenine, thymoquinone, dithymoquinone, Ketohexosefuranosides

C6 (rat glioblastoma), IMR32 H2O2 (human neuroblastoma) Swiss female mice γ-Irradiation

Crataegus pinnatifida Desmostachya bipinnata Hippophae rhamnoides Armoracia rusticana Vaccinium vitis-idaea

Quercetin Kaempferol, isorhamnetin, quercetin, Kaempferol, quercetin Anthocyanins, flavan-3-ols, flavonols

Mice lymphocytes Yeast cells Swiss albino mice Human lymphocytes WI-38 cells (human fibroblast)

peroxynitrite and nitric oxide can be formed from mitochondria, peroxisomes, inflammatory cell activation, exogenous sources including environmental agents, pharmaceuticals and industrial chemicals. Antioxidant-rich diet helps to check the ROS production and to prevent cell and tissue damages [126]. A large number of plantderived flavonoids have been studied as new sources of natural antioxidants which showed the potential to mitigate DNA damage induced by ROS in various cells [63,127]. The best-described property of flavonoids is their antioxidant capacity. Since these phenolic compounds can delay, inhibit or prevent the oxidation by scavenging free radicals and reduce oxidative stress. Flavonoid-rich fruit extract derived from Punica granatum L. (pomegranate) was reported to inhibit brain DNA damage against cerebral ischemia/reperfusion injury [128] and blood mononuclear DNA damage in rats [129]. Resveratrol is a polyphenolic compound present in plants including grapes, red wine, nuts, berries and other foods with anti-inflammatory, antioxidant and anticancer properties [130]. Antioxidant properties of resveratrol were modulated by upregulating expression of ROS scavengers and phase II enzymes such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, selenophosphate synthase 2, thioredoxin reductase and NAD(P)H:quinone oxidoreductase-1 [131]. During pregnancy, consumption of resveratrol protects mother and fetus from the toxicity induced by environmental pollutants [132]. Berries are among the most widely consumed fruits and are abundant in antioxidant phytochemicals, especially anthocyanins, which occur along with other classes of phenolic compounds including ellagitannins, flavan-3-ols, procyanidins, flavonols and hydroxybenzoate derivatives. It was found to be effective against cardiovascular disorders, advancing-age-induced oxidative stress, inflammatory responses and diverse degenerative diseases [133]. Components of inedible berries can scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair. Strawberry bioactive compounds are widely known to be powerful antioxidants, and extracts were found to be protective in nature against skin damage induced by H2O2 by improving mitochondrial functionality [134]. Haskap (Lonicera caerulea L.) is a cool climate berry crop which is abundant in polyphenols such as anthocyanins (cyanidin-3-O-glucoside, cyanidin-3,5-diglucoside, peonidin-3-Oglucoside, cyanidin-3-O-rutinoside), hydroxycinnamic acids (chlorogenic acid, 3,5-dicaffeoylquinic acid, neochlorogenic acid), flavonols

Cholesterol γ-Irradiation

H2O2

Reference

Protects chromosome damage by restoring [111] mitotic index and chromosomal instability Inhibits ROS generation and H2AX phosphorylation [112] Protects DNA DSBs by inhibiting γ-H2AX, DNA-PKcs and Ku80 proteins Neuroprotective potentials by inhibiting ROS, γ-H2AX and MAP2 levels Prevention of radiation damage by ROS inhibition

Cytoprotection by ROS scavenging and inhibits DNA fragmentation DNA protection by ROS scavenging H2O2 UV Scavenge ROS, protect genomic and mitochondrial damage Reduces oxidative damage by ROS inhibition H2O2 2-Amidinopropane Inhibits ROS and oxidative stress dihydrochloride

[113] [114] [115] [116] [177] [178] [179] [180]

(quercetin-3-O-rutinoside, quercetin-3-O-glucoside) and flavan-3-ols (proanthocyanidins, catechins) [135,136]. These diverse components and antioxidative properties of haskap could be used as a source for natural health products [137]. Oral administration in rats with anthocyanin-rich haskap extracts showed radioprotective properties [138] and defense against the UVB rays via modulation of antioxidant enzyme activity and reduction of DNA damage in SKH-1 mice [139]. Haskap anthocyanins also protect the oxidation of red blood cell membrane induced by UVB irradiation [140]. Recent research showed that methanolic extract of haskap possesses anti-inflammatory activities which are polyphenols-dependent [136]. Kiwifruit, a rich source of vitamin C and other antioxidants, was found to be protective against oxidative DNA damage in in vitro as well as in vivo tests. Consumption of kiwifruit induces resistance to DNA oxidative damage by H2O2 in human lymphocytes [47]. In rats, it induces base-excision repair mechanisms after oral intake for 3 weeks [141]. A study conducted among Korean smokers fed with green vegetable drink (Angelica-keiskei-based juice) supplementation for 8 weeks showed protection in peripheral lymphocytes DNA damage [142], indicating that green vegetable drink exerts cancer-protective effects. Furthermore, a study with six of Taiwan's indigenous purpleleaved vegetables which are rich in flavonoids, anthocyanidins and flavonols protects DNA of lymphocytes against H2O2 toxicity [143]. These examples further support the notion that dietary polyphenols play a significant role in DNA protection from various external and internal stimulants. 8. Hormetic effects of dietary flavonoids and polyphenols When considering phytochemicals as nutraceuticals in chemoprevention or cancer therapy, it is important to consider that many flavonoids and polyphenols have hormetic effects and in high enough doses are potentially toxic. Hormesis is defined as a biphasic dose– response phenomenon characterized by low-dose beneficial or stimulatory effects and high-dose detrimental or inhibitory effects of a compound on a biological process or phenotype, often depicted as a U-shaped dose–response curve [144]. The U-shaped, biphasic response arises from the benefits of adaptation to a cellular stress at low intensity or levels, and as the stressor increases (e.g., increased levels of a dietary flavonoid), the adaptive response no longer compensates for the detrimental effects of the stressor, and the treatment is damaging. To illustrate this concept, we can use the example of the

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3

Flavonoids

Growth Factors

14

15

Flavonoids Death Receptors

FAS FADD

PI3K

2 4

P

P

ROS

Akt

P

P

Bad

Procaspase-8

p21

Bcl-2 Bcl-x

p53

2

5 mTOR Bax

Cytochrome-C Apaf-1

1

16

15 Caspase-8

ERK 1/2 p38 MAPK Caspase-9

NF-κ κB

Extrinsic pathway

6 JNK1

Intrinsic pathway

Caspase-3

Apoptosis

Cell Death

Fig. 5. Schematic illustration of flavonoids in apoptotic pathways in cancer cells. The different modes of action of flavonoids in extrinsic and intrinsic pathways which ultimately lead to cell death. Examples of flavonoid mediators: luteolin (1), apigenin (2), EGCG (3), quercetin (4), fisetin (5), chrysin (6), epicatechin (7), silibinin (14), EGC (15) and myricetin (16).

Extrinsic factors

Intrinsic factors

DNA damaging agents

Oxidative damage

P mTOR complex C2

PI3K

P

5

17

18

19

Akt

Cell survival Fig. 6. Graphical representation of the influence of polyphenols in mTOR signaling in cancer cells. Expression levels of mTOR signaling activate continuous cell division and may lead to metastatic conditions. Flavonoids and other phytochemicals inhibit this process by dephosphorylation of mTOR complex formation associated with PI3K and Akt proteins, thereby promoting apoptotic cell death. Examples of polyphenol mediators: fisetin (5), piceatannol (17), caffeic acid (18) and butein (19).

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dietary flavonoid resveratrol, which has shown biphasic effects in a number of animal models of human disease including gastric lesions, ischemic stroke, osteoporosis and both Alzheimer's and cardiovascular disease [145]. For example, at low dose, resveratrol can promote cardiac health by promoting the expression of prosurvival and antiapoptotic proteins that can improve postischemic ventricular recovery and reduce infarction size following heart attack, whereas the opposite is true for high-dose resveratrol, which promotes cardiomyocyte apoptosis and increases infarct size [146]. With respect to cancer, resveratrol has the striking feature of promoting growth of many cancers of varying histological origin and stage at low dose but exhibiting proapoptotic effects on cancer cells at elevated doses [144]. Resveratrol is more the rule than the exception among dietary flavonoids and polyphenols, and many others including genistein, apigenin, luteolin and kaempferol have all been demonstrated to show hormetic effects by promoting cancer growth at low dose and inhibiting growth at high dose [147]. Similarly, the cancerpreventative effects of quercetin are also dose dependent, being primarily an antioxidant at low doses and a pro-oxidant capable of promoting genomic instability at high doses [148]. Furthermore, the combinatorial activity of multiple dietary flavonoids or polyphenols could be strongly additive or synergistic, causing a shift to detrimental or toxic effects on biological processes at relatively low doses not expected to be toxic. These combinatorial hormetic effects should not be taken lightly and may lead to adverse effects in individuals taking health supplements, and are one possible confounding factor in clinical trials where patients enrolled may be concurrently supplementing with over-the-counter nutraceuticals that alter drug clearance, absorption and/or cellular responses. Natural does not equate to safe at all doses and combinations; thus, chemopreventive effects of flavonoids in relation to dose require further investigations. 9. Conclusions and future directions Human cancers, caused by invariable DNA damage, have posed an increasing global health care concern for over a decade. Various modern treatment methods like targeted therapies have their own limitations including cancer cells developing resistance against anticancer drugs or relapse of cancer after treatment. Hence, there is a need to develop an effective method to treat the uncontrolled cell growth which is often driven by modifications of base pairs in DNA. Studies discussed in this review give an insight on how dietary agents, especially flavonoids, help to reduce DNA damage and thereby prevent mutations and consequently restore genomic stability. A wealth of scientific evidence is available to demonstrate that flavonoids protect from DNA damage induced by various toxic agents. However, there is a lack of knowledge regarding the effects of flavonoid-rich diet in modulation of DNA damage prevention/DNA repair mechanisms in healthy cells. Setting an optimum concentration of flavonoids is of high importance from a treatment point of view since higher concentrations may also lead to the destruction of normal cells during therapy. Selective nature of some flavonoids to protect normal cells and induce cell death mechanisms in cancer cells during chemotherapy or radiotherapy makes them an attractive agent in drug discovery process. However, more studies need to be carried out to configure the mechanism of action of this selective nature of flavonoids in order to improve understanding of various epigenetic process which may provide a more rational basis for combining specific dietary compounds and thereby enhancing efficacy in the clinical setting. Furthermore, hormetic effects of dietary flavonoids and polyphenols (and their combinations) need to be carefully characterized to prevent unintended side effects, including increasing DNA damage through mechanisms such as ROS production, inhibition or promotion of autophagy required to clear toxic biomolecules and/or down-

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