Quercetin: Prooxidant Effect and Apoptosis in Cancer

Chapter 9

Quercetin: Prooxidant Effect and Apoptosis in Cancer Paola G. Mateus, Vanessa G. Wolf, Maiara S. Borges and Valdecir F. Ximenes1 UNESP—Sa˜o Paulo State University, Bauru, SP, Brazil 1 Corresponding author: e-mail: [email protected]

Chapter Outline Molecular Oxygen, Spin Restriction, and Physiological Generation of ROS 265 Molecular Oxygen, Autoxidation of Polyphenols, and Generation of ROS 268 Polyphenols as Cocatalyst in Peroxidase-Mediated Oxidation 269 Chemistry of Polyphenols 270 Quercetin as a Source of ROS 271 Apoptosis 275 Apoptosis and Cancer 277 Oxidation Therapy and Induction of Apoptosis as an Anticancer Strategy 278

Induction of Apoptosis in Cancer Cells by Quercetin: Involvement of Prooxidant Activity 279 Leukemia Cells 279 Hepatocellular Carcinoma Cell Lines 281 Cervical and Breast Cancer Cell Lines 282 Gastric Carcinoma and Thyroid Cancer Cell Lines 283 Concluding Remarks 283 284 Acknowledgments References 285

MOLECULAR OXYGEN, SPIN RESTRICTION, AND PHYSIOLOGICAL GENERATION OF ROS The evolution from anaerobic to aerobic respiration brought great benefits to the evolution of organisms. It made the process more energetically efficient, thus promoting the development of higher animals. In order to have an idea of this increase in efficiency, it is sufficient to compare the ATP yield in anaerobic fermentation for a glucose molecule (2 ATPs) with its complete oxidation in aerobic respiration (32 ATPs). However, the use of molecular oxygen in cellular respiration had its price. In fact, in order to use molecular oxygen as the final electron acceptor in mitochondrial respiration, it was Studies in Natural Products Chemistry, Vol. 58. https://doi.org/10.1016/B978-0-444-64056-7.00009-X © 2018 Elsevier B.V. All rights reserved.

265

266 Studies in Natural Products Chemistry

necessary to develop a mechanism by which paired electrons from coenzymes (NADH, FADH2) could be delivered, not in pairs, but one by one to molecular oxygen. This mechanism is the mitochondrial electron transport chain. The price is that a small percentage of electrons “leak” prematurely, leading to the formation of oxidizing intermediates. This mechanism is known as mitochondrial leakage and it is considered the main cellular source of oxidizing intermediates in nonpathological states [1]. It is worthy of note that around 2% of the molecular oxygen used in respiration is converted to reactive oxygen species (ROS), and mitochondrial leakage is mainly responsible for this “side effect” [2]. Although not yet totally clarified, it is known that mitochondrial electron leakage takes place in complexes I and III of the mitochondrial inner membrane. This phenomenon provokes the unielectronic reduction of molecular oxygen to superoxide anion radical (O2
 ) [3]. This primary reactive species is the precursor of other oxidizing intermediates such as hydrogen peroxide (H2O2), hydroxyl radical (HO
), hypochlorous acid (HOCl), hypobromous acid (HOBr), singlet oxygen (1O2), and peroxynitrite (ONOO) [4]. There is evidence that ROS are involved in a wide range of diseases and aging [5]. It is important to emphasize that mitochondrial leakage is considered as the main cellular source of ROS generation, but not the only one. Other examples are oxidases, enzymes involved in redox reactions [6]; peroxidases, which catalyze the formation of HOCl and HOBr [7]; and nitric oxide synthase, responsible for the production of the free radical nitric oxide (
NO), known as the endothelial relaxation factor and a precursor of the powerful oxidant ONOO, etc. [8,9]. Returning to the mitochondrial electron transport chain, it can be seen as a biological mechanism, developed in the evolution process, to make possible the use of molecular oxygen in the oxidation of nutrients. This is due to the biradical character of molecular oxygen, which prevents it from the direct interaction with molecules that have their electrons paired. This is known as spin restriction and is based on the principle of the conservation of spin angular momentum in chemical reactions [10]. In other words, it is not possible to combine two unpaired electrons (from molecular oxygen) with two paired electrons (from nutrients), and from this interaction to generate products (CO2, H2O) with their electrons also paired. The principle of spin restriction, applied to aerobic metabolism, can be schematized in Fig. 9.1.

O2

+

S

2

P

Spin-forbidden reaction

FIG. 9.1 Spin restriction principle applied to a chemical reaction between triplet molecular oxygen and singlet molecules (S) leading to singlet products (P). The red arrows represent electrons in the orbitals. The principle of conservation of spin angular momentum generates a kinetic barrier that prevents the reaction.

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 267

For better comprehension of this principle, one must take into account the electronic structure of molecular oxygen obtained by applying the linear combination of atomic orbitals theory. This predicts two electrons with parallel spins, occupying the two degenerate highest occupied molecular orbitals. For this reason, molecular oxygen is known as a triplet molecule, or, it has triplet multiplicity, as can be verified by applying the equation M ¼ 2S + 1, where M is the multiplicity of spin states and S is the total spin quantum number of the molecule. Applying to molecular oxygen: S ¼ 1/2 + 1/2, therefore M ¼ 3. On the other hand, nutrients, i.e., proteins, carbohydrates, and lipids, have their electrons paired, either in covalent bonds or in pairs of unbound electrons. Therefore, they have S ¼ 1/2 + (1/2) ¼ 0 and M ¼ 1 and are said to be singlet molecules. In conclusion, as schematized in Fig. 9.1, the direct interaction between molecular oxygen and nutrients is kinetically impeded, even though it is thermodynamically feasible. Returning to the electron transport chain in mitochondrial respiration, it can be identified as a mechanism to alleviate the spin restriction imposed by the use of molecular oxygen. This can be visualized by analyzing a simplified mechanism of the mitochondrial electron transport chain, where pairs of electrons from coenzymes (for instance, NADH) are transferred to oxidized ubiquinone (Q) leading to its reduced form QH2. From QH2, via intermediation of its semiquinone form (QH
), the electrons are delivered not in pairs, but one by one, to cytochromes, and finally, to molecular oxygen. It is important to note the fundamental role of the iron atoms in the heme group of cytochromes, which enable the breaking of the spin restriction for the use of molecular oxygen (Fig. 9.2). As can be seen, the total spin is maintained in each reaction; i.e., these reactions are spin allowed. It is worthy of note that the O2
 does not leave the electron transport chain until it receives three more electrons, leading to the formation of two molecules of water, except for a small fraction that leaves the chain in the process of mitochondrial leakage. To conclude this topic, it must be emphasized that spin restriction is a kinetic constraint rather than a thermodynamic one; thus, the mitochondrial electron transport chain can be seen as a catalytic process.

QH2

+

CytcFe2+

CytcFe3+

+

O2

CytcFe2+ + ·QH + H+

·−

O2

3+ + CytcFe

Spin-allowed reaction

Spin-allowed reaction

FIG. 9.2 Simplified pathway by which the mitochondrial electron transport chain enables the use of triplet molecular oxygen as the final electron acceptor. The intermediance of CytcFe3+ and QH2 bypasses the spin restriction rule because electrons are transferred one by one and not in pairs. The red arrows represent electrons in the orbitals. CytcFe2+, reduced form of cytochrome c; CytcFe3+, oxidized form of cytochrome c;
QH, semiubiquinone; QH2, reduced ubiquinone.

268 Studies in Natural Products Chemistry

As highlighted earlier, if on one hand the mitochondrial electron transport chain enabled the use of molecular oxygen in cellular respiration, on the other hand it propitiates the mitochondrial leakage, a process by which O2
 is overproduced by excessive electron leakage from the mitochondrial respiratory chain. To cope with this physiological source of ROS, organisms have developed macro- and micromolecular antioxidant defense mechanisms. In the macromolecular context, antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase are present in all aerobic organisms and are responsible for the neutralization of O2
 and H2O2 [11]. We highlight here SOD, considered an enzyme that has reached catalytic perfection, since it promotes the dismutation of O2
 with an efficiency (kcat/Km ¼ 8.0  108 M1 s1) comparable to diffusion-controlled processes [12]. From the point of view of micromolecules with antioxidant functions, the tripeptide glutathione (GSH), which is present at millimolar range in the intracellular medium [13], can be mentioned, highlighting its physiological importance as an endogenous antioxidant. In short, considering all the previous discussion, we can conclude that the generation of ROS in aerobic organisms is the price paid for using the energetically efficient aerobic metabolism. This is the oxygen paradox.

MOLECULAR OXYGEN, AUTOXIDATION OF POLYPHENOLS, AND GENERATION OF ROS The presence of molecular oxygen in the atmosphere, as well as its biradical character, not only gave rise to the formation of ROS in living organisms but also to what are conventionally called autoxidation reactions, i.e., oxidation of a chemical by its simple exposure to air. Undoubtedly, the typical demonstration of this process is food rancidity, which is particularly relevant in highlipid-content foods. More importantly, however, is the fact that such reactions are also sources of ROS and are involved in the prooxidant mechanisms triggered by polyphenols. The susceptibility of a compound to autoxidation is directly related to its oxidability, i.e., the susceptibility to oxidation, which can be evaluated by its oxidation peak potential (Epa, anodic peak potential) as usually determined in cyclic voltammetry experiments. In this sense, the susceptibility to autoxidation is usually linked to compounds with low Epa values. It is interesting to note that the same criterion is adopted to measure the antioxidant efficiency, i.e., an efficient antioxidant can also be a molecule susceptible to autoxidation. For instance, ascorbic acid, widely known for its antioxidant features, is at the same time highly susceptible to autoxidation [14]. This property of ascorbic acid is the basis for its application as an ROS generator to kill tumor cells [15]. Additional examples are the polyphenols, for which the susceptibility to autoxidation has been demonstrated and their oxidation potentials have been described: quercetin (Epa ¼ 280 mV), gallic acid

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 269

(Epa ¼ 60 mV), rutin (Epa ¼ 420 mV), epicatechin gallate (Epa ¼ 490 mV), caffeic acid (Epa ¼ 70 mV), etc. [16]. Just for comparison, we cite salicylic acid, which is much less susceptible to autoxidation and has a relatively higher Epa value (940 mV) [17]. Considering that polyphenols are molecules with singlet multiplicity, it is not surprising that their autoxidation reactions are also kinetically unfavorable due to spin restriction. This chemical property was confirmed by detecting the necessity of transition metal ions as catalysts for autoxidation of polyphenols [10]. The catalytic action of these metals is linked to the breakdown of the spin restriction, as observed in mitochondrial respiration. Fig. 9.3 shows the general mechanism by which transition metals are able to catalyze the autoxidation of polyphenols [18]. It is worthy of note that it is quite common to describe autoxidation without the presence of transition metals in the reaction medium. However, the almost ubiquitous presence of metals such as Fe, Cu, Mg, and Zn, in even a trace concentration in biological fluids and culture media, guarantees their role as catalysts in autoxidation. In summary, the autoxidation of polyphenols is one of the mechanisms by which these compounds can cause a cellular redox imbalance, i.e., leading to generation of ROS, which can be used against tumor cells, as we will show later.

POLYPHENOLS AS COCATALYST IN PEROXIDASE-MEDIATED OXIDATION Another pathway by which polyphenols can act as prooxidants in the cellular environment is through the generation of free radical intermediates (phenoxyl radicals) able to promote the oxidation of intracellular targets. The reaction involves a redox cycle, whereby phenoxyl radicals generated through the catalytic action of peroxidases are recycled, with the concomitant oxidation of the target biomolecule. In this pathway, the polyphenol can be thought of as a cocatalyst for the peroxidase-mediated oxidation of the biomolecules (Fig. 9.4).

Ph–OH + Fe2+

+

Fe3+

Ph–O· +

Fe2+ + H+

Spin-allowed reaction

O2

.− O2

Fe3+

Spin-allowed reaction

+

H2O2 FIG. 9.3 General pathway for spin-allowed autoxidation of polyphenols catalyzed by transition metals and production of ROS. The red arrows represent electrons in the orbitals. Fe2+ and Fe3+ are used as examples.

270 Studies in Natural Products Chemistry Ph–OH + H2O2

Ph–O· Peroxidase

Ph–O· + Biomolecules

Oxidized + biomolecules

Ph–OH

FIG. 9.4 General pathway for cocatalytic action of polyphenols in the oxidation of biomolecules such as GSH, NADH, and sulfhydryl residues in protein.

Galati and colleagues demonstrated this chemical property of polyphenols such as resveratrol, curcumin, caffeic acid, naringenin, and quercetin. In their studies, polyphenols were able to promote the oxidation of ascorbate, GHS, and NADH [19]. It is worthy of note that quercetin caused a 100-fold increase in the oxidation rate of ascorbate, which is an evidence of its potent prooxidant effect [19]. This chemical property of polyphenols is intrinsically related to the oxidation potential and lifetime of their radical intermediates. An example can be seen by comparing apocynin (Epa ¼ 760 mV) with protocatechuic acid (Epa ¼ 222 mV). In this case, the free radical intermediate of apocynin has a strong oxidizing power, promoting the oxidation of intracellular targets such as GSH, tocopherol, NADH, and sulfhydryl groups in proteins [20]. On the other hand, the free radicals generated by oxidation of protocatechuic acid were totally ineffective [21]. The importance of the lifetime of the transient phenoxyl radicals can be noted by comparing the prooxidant activity of esters of phenolic acids. For instance, using trolox as the target molecule, phenoxyl radicals of the heptyl ester of protocatechuic acid (Epa ¼ 266 mV) caused a 25-fold increase in the oxidation rate of trolox compared to the effect produced by the acid precursor (Epa ¼ 222 mV). This difference cannot be explained by the oxidation potential of the species. However, a correlation was found between the peak current intensity, obtained by differential pulse voltammetry experiment, and the cocatalytic effect of the radical intermediate. The increase in the current intensity can be interpreted as indicative of the longer lifetime of the transient phenoxyl radical intermediate [21]. The third pathway by which polyphenols may exert a prooxidant effect is in the formation of electrophilic quinone intermediates. These quinones can be generated during their oxidation catalyzed by peroxidases or via autoxidation. As is well known, the quinone moiety is characterized by significant electrophilic character and susceptibility to Michael reactions. They are, therefore, promoters of alkylation of proteins and DNA and act as depletors of intracellular GHS [22–24].

CHEMISTRY OF POLYPHENOLS Polyphenols are secondary metabolites with wide distribution in the plant kingdom. These phytochemicals are produced by plants in response to environmental stress or injury. The term polyphenols is used to describe aromatic compounds formed by the various biosynthetic shikimate and/or acetate

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 271

pathways, which contain one or more hydroxyl groups attached to the benzene ring [25]. According to the functional groups and number of hydroxylated aromatic rings, polyphenols can be chemically subdivided into five broad categories as follows: phenolic acids, coumarins, stilbenes, tannins, and flavonoids (Fig. 9.5) [26]. Phenolic acids are an important class of polyphenols that can be subdivided into two main categories, namely, hydroxybenzoic acids (e.g., gallic acid) and hydroxycinnamic acids (e.g., caffeic acid) [27]. Coumarins are naturally occurring metabolites, are also known as benzopyrones, and have antioxidant, antiinflammatory, and antitumor properties [28]. Some important members of this family include seselin (pyranocoumarins), 4-hydroxycoumarin (coumarins), psoralens (furanocoumarins), and warfarin (pyran-substituted coumarins) [29]. Stilbenes are secondary metabolites that contain two portions of the phenyl group connected by a two-carbon methylene bridge. These compounds are called phytoalexins. An important component of this family is resveratrol (trans-3,5,40 -trihydroxystilbene), which is found in some types of grape peel, blueberries, cranberries, and eucalyptus, and has significant antiinflammatory and antioxidant properties [26]. Tannins are generally divided into two types: hydrolysable (ellagitannins and gallotannins) and condensates (proanthocyanidins) [30]. Proanthocyanidins are a group of biologically active polyphenolic bioflavonoids that are abundant in vegetables, flowers, seeds, fruits, and peel [31]. Flavonoids represent a very diverse class of polyphenolic secondary metabolites representing the third largest group of natural products of 2-phenylchromen-4-one (flavone) [32]. Flavonoids occur in flowers, fruits, and roots and are often associated with benefits to plants, providing antioxidant and abiotic factors (wind, precipitation, temperature, and sunlight protection). Flavonoids are generally tricyclic molecules with 15 carbon atoms and 2 benzene rings (A and B), forming a C6–C3–C6 system. The skeleton may be substituted with hydroxyl, methoxy, prenyl, and/or glycosylation groups [33]. Differences in oxidation state and substitution types allow the organization of flavonoids into subgroups: chalcones, flavanones, dihydroflavonols, flavones, flavonols, flavan3,4-diols, flavan-3-ols (catechins), anthocyanidins, aurones, and isoflavonoids (Fig. 9.5) [34].

QUERCETIN AS A SOURCE OF ROS Quercetin is the main flavonoid present in the human diet, being the most characteristic representative of the subclass flavonol. Foods rich in quercetin include cocoa powder, cranberries, kale, celery, broccoli, lettuce, tomatoes (ripe red), Ginkgo biloba, and carrots [35,36]. The name quercetin comes from the Latin quercetum, meaning oak forest, and has been used since 1857. Other names for quercetin are sophoretin, meletin, xanthaurine, and quercetol [37]. Quercetin’s bright yellow color is characteristic of the aglycone form of a series of flavonoid glycosides, such as rutin and quercitrin

O O

OH

OH

OH

OH

O

HO

HO

O O

OH O

C

HO

HO

OH

HO

OH

O

OH

OH

Phenolic acids (cinnamic and hydroxy-benzoic acids)

Coumarin

Stilbenes

HO

OH OH

R2

R1

R1 OH

O

HO

HO HO

O

O

HO

OH

Flavone

OH

O

OH

Flavan-3,4-diol

lsoflavone

R1 O

O

CH R1

OH

OH

OH

HO

O

O

OH

OH OH

Chalcones

OH

OH HO

O

OH

OH HO

OH

OH

O

R2 OH

OH

OH

O

Dihydroflavonol

Flavanone R1

HO

O

R1 OH

O

OH

O

H

R1 OH

R1 OH

OH H

HO

Tannins (Gallotannins)

Anthocyanins

FIG. 9.5 General structures of polyphenols.

Aurones

Flavan-3-ol

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 273

[38]. The most abundant forms are quercetin-3-rutinoside (rutin), quercetin-3glucoside (isoquercitrin), and quercetin-3,40 -diglucoside [39]. Quercetin and many of its glycosidic derivatives have been isolated from many plants, such as Allium cepa Patrick (onion) [40], Aloe barbadensis (Aloe vera) [41], Brassica oleracea (cauliflower) [42], Euphorbia helioscopia (plant) [43], Solanum lycopersicum Manisa C-33 (tomato) [44], Malus pumila (apple) [45], Vitis vinifera (wine grape) [46], and Arontia mitschurinii Viking (chokeberry) (Table 9.1) [48]. The metabolism of the absorbed quercetin occurs mainly in the liver, while the unabsorbed part of quercetin is metabolized by microorganisms from the intestinal flora. In the intestine, quercetin is cleaved to 3,4dihydroxyphenylacetic by Eubacterium ramulus. The biotransformation of ingested quercetin occurs by glucuronidation, hydroxylation, methylation, and sulfonylation [38]. Quercetin has been extensively studied in the last 30 years and important therapeutic properties have been described, for instance, antihypertensive, hepatoprotective, antispasmodic, antiulcerogenic, antidiarrheal, sedative, antiviral, and anticarcinogenic properties [37,49]. Its structure and some of its properties are presented in Table 9.1. The biosynthesis of quercetin is summarized in Fig. 9.6. Initially, through the phenylpropanoid route, phenylalanine (1) is converted to 4-coumaryl-CoA (2) by the catalytic action of phenylalanine ammonia lyase, cinnamate-4hydroxylase, and 4-coumaril CoA ligase. Then, chalcone synthase catalyzes the combination of 4-coumaroyl-CoA with three molecules of malonyl-CoA (3), which leads to the formation of tetrahydroxy chalcone. Tetrahydroxy chalcone is converted to naringenin using the chalcone isomerase and naringenin is converted to eriodictiol by the action of enzyme flavanone-3bhydroxylase. The formed eriodictiol is converted to dihydroquercetin by the action of flavanone-3b-hydroxylase, which is finally converted to quercetin by flavonol synthase [34,50]. Hajji and coworkers demonstrated the susceptibility of quercetin to autoxidation, in either acid or neutral medium, leading to H2O2 production. The autoxidation was accelerated by the presence of transition metals in the following order: Cu1+ > Cu2+ ≫ Fe2+ [51]. These findings reinforce the importance of transition metals as catalysts through spin restriction alleviation, as discussed previously. Similarly, Zivanovic and coworkers demonstrated the catalytic effect of Mg2+ and Ca2+ on rutin autoxidation, which is a quercetin glycoside. In this study, the most pronounced effect obtained using Ca2+ compared to Mg2+ was explained by the strong bonding of the latter causing stabilization of the reaction intermediates [52]. Sahu and coworkers demonstrated the degradation of DNA and lipid peroxidation by autoxidation caused by the incubation of hepatocyte nucleus from rats incubated with quercetin in the presence and absence of Cu2+, and the latter caused a strong increase in the degradation process [53]. Oliveira and coworkers highlighted the mutagenic effect of quercetin in in vitro experiments and called attention to its

TABLE 9.1 Physicochemical Properties of Quercetin [37,47]

Chemical Structure/Name OH 3′ 4′

2′ HO

8 7 A

C

1 O

1′ 6′

B 3

6 4

5 OH

5′

2

OH

O

2-(3,4-Dihydroxyphenyl)-3,5,7trihydroxychromen-4-one

OH

Molecular Weight/Formula

Mp (°C)

log P

Water Solubility (mg mL21)

302.23 (C15H10O)

314–316

1.48

0.001

Physical State

UV–Vis Max (nm)

Yellow powder

258, 360

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 275

FIG. 9.6 Representative general scheme of the quercetin biosynthetic pathway. CHI, chalcone isomerase; CHS, chalcone synthase; FHT, flavanone-3b-hydroxylase; FLS, flavonol synthase.

autoxidation and consequent generation of ROS as a mechanism responsible for this biological effect [54]. Another determining factor in the autoxidation processes is the pH of the medium, which is not different for quercetin. This property was explored by Lei and coworkers in the development of an analytical technique for determination of quercetin by means of its autoxidation in alkaline medium, and detection by electrogenerated chemiluminescence with luminol [55], and used in the study of the degradation of quercetin at alkaline pH by Raman spectroscopy [56]. It is worthy of note that the oxidation peak potential of quercetin is pH dependent, being reduced as pH increases [57], therefore favoring the autoxidation processes at alkaline pH. Quercetin can further exert its prooxidant effect via the generation of quinone intermediates capable of alkylating proteins and DNA, and by depletion of intracellular GSH, which provokes unbalance in the cellular redox status [24,58,59].

APOPTOSIS Apoptosis is a mechanism of programmed cell death, by which cells undergo self-destruction and are rapidly phagocytosed and digested by other cells. This process is extremely regulated and is accompanied by morphological and biochemical alterations that differentiate it from other mechanisms of cell death [60,61]. For instance, the apoptotic cell retracts and condenses, thereby losing

276 Studies in Natural Products Chemistry

adherence to the extracellular matrix and neighboring cells. The cytoskeleton collapses, the nuclear envelope undergoes disintegration, and the DNA undergoes condensation and fragmentation. The cell membrane forms extensions, and if the cell is large, it ruptures, forming membrane-enveloped fragments called apoptotic bodies. In addition, the apoptotic cell also undergoes chemical alterations on its surface, signaling to neighboring and phagocytic cells that it is in the process of death, causing it to be rapidly phagocytosed and digested [60–62]. All these changes differentiate apoptosis from necrosis, another cell death pathway. In necrosis, the cells swell and rupture, spreading their cellular contents onto neighboring cells, triggering a local inflammatory response. Necrosis usually occurs when cells experience acute injury, such as lack of blood supply or trauma [60,61]. Activation of apoptosis occurs through an intracellular proteolytic cascade, mediated by enzymes known as caspases. These enzymes belong to a family of specialized intracellular proteases: the cysteine protease family. Caspases are synthesized as inactive precursors, the procaspases, which are proteolytically cleaved and thus activated in response to apoptotic signals [60,63,64]. Caspases are classified as initiators or executors. The initiator caspases are responsible for triggering the activation of the caspase proteolytic cascade. When inactive, the starter procaspases (procaspases 8 and 9) are found as monomers in the cytosol. Once the apoptotic signal is triggered, these monomers interact with adapter proteins, generating protein complexes, whereby procaspases associate to form dimers. Each procaspase cleaves its partner at specific sites in the protease domain, forming the active caspases (8 or 9) [60,61,63]. Once activated, the initiating caspases cleave the executing procaspases, provoking their activation, and so on, until the final caspases cleave the target proteins, leading to cell death [64–66]. This process of cleavage and activation is called the caspase proteolytic cascade [66,67]. Among the target proteins are nuclear, cytoskeletal, cell–cell adhesion proteins, and proteins that inhibit the DNase function. The cleavage of the latter protein releases DNase, causing the degradation of chromosomal DNA [67]. Degradation of these and other cellular components leads to the morphological changes that characterize apoptosis. There are two well-described mechanisms for apoptosis activation: the extrinsic and the intrinsic, or mitochondrial, pathways. The extrinsic pathway is triggered by binding of extracellular protein signals to death receptors on the surface of the target cell. These receptors belong to the family of tumor necrosis factor (TNF) receptors, which are responsible for activating the apoptotic program [60,61,68]. An example of the activation of apoptosis by the extrinsic pathway is the activation of the Fas death receptors, which are present on the surface of the target cell, by Fas ligands present on the surface of cytotoxic lymphocytes. In some cases, the intrinsic pathway of apoptosis is recruited from the extrinsic pathway, amplifying the caspase cascade [60,61,69]. The intrinsic or mitochondrial pathway of apoptosis is activated

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 277

by signals triggered within the cell itself, usually in response to stress, such as irreparable DNA damage or metabolic disorders. In this mechanism, proteins generally present in the intermembrane space of mitochondria are released into the cytosol and are responsible for triggering the apoptotic program [60,70]. Among the proteins released into the cytosol by an apoptotic stimulus is cytochrome c, a mitochondrial intermembrane component of the electron transport chain, which develops a key role in the intrinsic pathway of apoptosis. In the presence of apoptotic stimuli, cytochrome c is released into the cytosol and binds to an adapter protein, Apaf-1 (apoptotic protease activating factor-1). This binding leads to oligomerization and formation of Apaf-1 apoptosome, which initiates the caspase cascade leading to cell death [60,61,69,71–73]. The intrinsic pathway of apoptosis is tightly controlled. The control is made, mainly, by Bcl2 family proteins, which contain proapoptotic proteins, such as Bax and Bak, and antiapoptotic proteins, such as Bcl2 and Bcl-xL. When the intrinsic pathway of apoptosis is triggered, proapoptotic proteins become active and aggregate on the surface of the outer mitochondrial membrane, forming channels that allow the release of cytochrome c and other intermembrane proteins into the cytosol. The antiapoptotic proteins exert their functions by preventing the release of the intermembrane components of the mitochondria, through their binding to the proapoptotic proteins Bax and Bak [60,61,74,75].

APOPTOSIS AND CANCER The development of cancer is a multistep process in which the normal cell undergoes mutations and progressively evolves into the neoplastic state. The pathogenesis of cancer could be explained by the need for incipient cancer cells to acquire hallmark capabilities that make them tumorigenic and, ultimately malignant. These hallmarks allow these cells to multiply in an uncontrolled way and create a tumor microenvironment that allows the support of tumor progression, and in more advanced cases, the spread of tumor to other parts of the body [76]. Apoptosis, as mentioned previously, is triggered in response to physiological stresses, and among them is the unbalance due to increased oncogenes signaling as well as irreparable DNA damage associated with cellular hyperproliferation [76–78]. Apoptosis, therefore, constitutes a natural and essential barrier to the development of cancer, since it eliminates potentially cancerous cells. Throughout the neoplastic process, however, tumor cells develop mechanisms of escape from cell death by apoptosis, remaining alive and accumulating more mutations, resistance to apoptosis being an important hallmark of cancer [76]. Studies have shown that tumors that are able to progress to state of high malignancy have as the main characteristic the attenuation of apoptosis, with a high level of resistance to antitumor therapy, since many drugs act by inducing cells death by apoptosis [60,77,78].

278 Studies in Natural Products Chemistry

The cell under normal conditions has a series of abnormality sensors, which play an essential role in tumor development [77,78]. The most important of these sensors is the p53 protein, encoded by the p53 tumor suppressor gene. When the cell undergoes genetic damage or metabolism disorder, p53 is responsible for stopping the cell cycle; hence the damage can be repaired, or cellular metabolic conditions restored. However, if the damage is irreparable, this p53 triggers the activation of the intrinsic pathway of apoptosis [66,67]. Apoptosis triggers via p53 occurs through the activation of the transcription of the genes coding for BH3-only proteins Puma and Noxa (proteins that inhibit antiapoptotic proteins), in addition to promoting the expression of proapoptotic Bax protein [60,66,67,76,79]. One of the main escape mechanisms from apoptosis by tumor cells, therefore, is the loss of p53 signaling. The gene encoding this protein is mutated in approximately 50% of human cancers, with loss of p53 function and consequent cell permanence in the cell cycle, even in the presence of DNA damage, which contributes to the accumulation of more mutations [79]. The loss of p53 function is also responsible for the resistance of certain tumors to therapy, since many anticancer drugs induce apoptosis via this pathway [60]. In addition to the loss of p53 function, tumor cells also utilize a number of other mechanisms to evade apoptosis death; for example, the overexpression of the Bcl2 and Bcl-xL antiapoptotic proteins, survival factors, downregulation of proapoptotic proteins and by blocking the extrinsic pathway of apoptosis [76].

OXIDATION THERAPY AND INDUCTION OF APOPTOSIS AS AN ANTICANCER STRATEGY The complex nature of the cancer, associated with the great capacity of tumor cells to adapt, mutate and develop resistance, makes it difficult to establish a therapy capable of eliminating the disease. Another major difficulty is finding treatments that are specific for tumor cells, and which do not cause significant damage to healthy cells, as usually occurs with the currently used antineoplastic drugs. Among the treatments based on induction of apoptosis, oxidation therapy has excelled in recent years. This therapy is based on the use of substances with prooxidant activity, which acts through the direct oxidation of target biomolecules or through the generation of ROS [21,80,81]. The induction of apoptosis by ROS can also occur indirectly, by damaging and compromising the functions of macromolecules, such as DNA, RNA, lipids, and proteins, triggering cellular signals that activate the apoptotic machinery [62]. For instance, DNA damage leads to the activation of p53, which activates proapoptotic gene transcription, provoking cell death via the intrinsic pathway of apoptosis [66,67]. Another mechanism is the attack of ROS on internal mitochondrial proteins, as well as on membrane lipids, which leads to loss of mitochondrial function, membrane depolarization, and release of cytochrome c, which activates the intrinsic pathway of apoptosis [82,83]. By acting directly on intracellular signaling pathways, ROS also lead to

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 279

apoptosis. For instance, several studies have shown that a high level of ROS causes the activation of the JNK (Jun N-terminal kinases) pathway, which triggers cell death by apoptosis, by both the extrinsic and the intrinsic pathways [84]. Among the proapoptotic proteins expressed in response to the activation of this pathway are Fas-L (extrinsic pathway of apoptosis) and Bak (apoptosis intrinsic pathway) [85]. JNK also activates the intrinsic pathway of apoptosis by direct modulation of mitochondrial pro- and antiapoptotic protein activity through phosphorylation events [84]. It has also been shown that ROS act in the extrinsic pathway of apoptosis as an upstream event for the activation of Fas, being required for activating the phosphorylation of this protein, which in turn recruits the adapter protein and procaspase 8, forming the death-inducing signaling complex (DISC) [86]. Although ROS play an important role in apoptotic signaling, it is known that cancer cells have an elevated intrinsic level of ROS when compared to normal cells. However, even at higher concentration, ROS do not lead to death of the cancer cell; but on the contrary, they serve as signals in the activation of pathways that promote increased cell proliferation, survival, DNA damage, and genetic instability, which favor the accumulation of mutations [86,87]. Indeed, the role of ROS in cancer cells depends on their concentration. A moderate accumulation of ROS is necessary for tumor development; however, if this concentration is raised to a toxic level, an increase in oxidative stress and induction of cell death can take place [86,88–90]. Thus, in order to maintain an increased but nonlethal level of ROS, tumor cells control the intracellular redox balance [87]. This is the rationalization of oxidation therapy, which is based on raising the concentration of ROS to a lethal level, provoking cell death by apoptosis as described earlier. The selectivity is based on the lower susceptibility of normal cells to the oxidation therapy. Normal cells are poorly affected because, unlike in cancer cells, the increase in ROS can be counterbalanced by the intracellular antioxidant defense systems [21,80]. Fig. 9.7 summarizes the apoptosis-signaling pathways triggered by ROS, which justified oxidation therapy as a strategy to treat cancer.

INDUCTION OF APOPTOSIS IN CANCER CELLS BY QUERCETIN: INVOLVEMENT OF PROOXIDANT ACTIVITY In this section, we present experimental evidence that quercetin can be used, alone or in combination with other drugs, as a cytotoxic agent to kill tumor cells. We also show that, even though quercetin is usually thought of as an antioxidant, its capacity to induce apoptosis is frequently correlated with its prooxidant capacity.

Leukemia Cells With the lowest survival rate among patients, acute myeloid leukemia is one of the most aggressive types of cancer. Just to highlight the relevance

280 Studies in Natural Products Chemistry

ROS

DNA damage

Activation of the JNK pathway

Damage in mitochondrial proteins and lipids

Transcription factors activation

+ p53

Loss of mitochondrial function and membrane depolarization

Proapoptotic proteins

Bak

−

Antiapoptotic proteins

Cit.c

Noxa, Puma, Bax

Activation of phosphorylation pathways

Fas-L

Fas activation

Cit.c

Intrinsic pathway

Extrinsic pathway

Apoptosis

FIG. 9.7 Apoptosis-signaling pathways triggered by ROS.

of this disease, only in 2016, the Brazilian National Cancer Institute (INCA) registered 10,070 new cases in Brazil [91]. In this scenario, numerous phytochemicals have been studied, alone or in combination with other wellestablished chemotherapy drugs, aiming to inhibit the proliferation of leukemia cells. This is the case of quercetin, for which Matsui and coworkers evaluated the activity on several leukemia cell lines, including KU812, BV-173, U937, and THP-1 [92]. The authors demonstrated that quercetin induced apoptosis in these cell strains and was characterized by rupture of the mitochondrial membrane, decrease in the membrane potential, and activation of caspase-3 and -9. In the same direction, Niu and coworkers reported the results for the incubation of HL-60 with quercetin in the range of 12.5–100 mmol L1 for a period of 48 h [93]. Again, the typical morphological characteristics of apoptosis, such as irregular and shrunken cells, cytoplasm containing hollow bubbles, released cellular contents, and nucleus condensation, were found, confirming cell death by this pathway. The activation of caspase-3 and -9, and complementary evidence, such as the decrease in Bcl-2 protein and increase in Bax expression, served as reinforcement of the confirmation of the mitochondrial pathway involvement in apoptosis provoked by quercetin. Sakao and coworkers also reported similar results for the antiproliferative effect of quercetin to HL-60 [94]. These authors also demonstrated that apoptosis was accompanied by DNA fragmentation, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP), which responds to the break in the DNA chain. The treatment of the cells with quercetin also resulted in an intracellular increase of O2
 , which may be responsible for the induction of cells to apoptosis [94]. Lee and coworkers also found an increase in the level of ROS in different leukemia cells treated

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 281

with quercetin. The acute leukemia lines tested were THP-1, U937, HL-60, and MV4-11. The concentration of quercetin used during the cell treatment ranged from 0 to 100 mmol L1 for a period of 6–24 h. All cell lines tested showed sensitivity to quercetin-induced apoptosis in a dose-dependent manner, and activation of caspase-9, caspase-8, and eventually caspase-3 was detected. There was also a significant inhibition of HL-60 tumor growth, previously xenografted in rats, accompanied by increased intracellular levels of H2O2 and O2
 and subsequent induction to apoptosis [95]. In agreement with these results, Srivastava and coworkers also verified that quercetin was able to induce apoptosis in the leukemia cell lines CEM, K562, and Nalm-6 after 48 h of treatment. Among the studied strains, Nalm-6 exhibited maximum sensitivity to quercetin, reducing cell viability at the lowest concentration used (10 mmol L1) and IC50 of 20 mmol L1. The IC50 values for K562 and CEM were 40 and 55 mmol L1, respectively. The authors also reported that the treatment with quercetin was responsible by DNA damage, which was linked to the prooxidant activity of quercetin [96].

Hepatocellular Carcinoma Cell Lines Hepatocellular carcinoma, also known as liver cancer, has great incidence in Southeast Asia, South Africa, and Japan. The investigation of quercetin as a potential cytotoxic drug for HepG2, a human liver cancer cell line, is also abundant. For instance, Granado-Serrano and coworkers have shown that this cell line is sensitive to induction of apoptosis by this flavonoid. In their studies, HepG2 cells were incubated with quercetin in the concentration range of 10–100 mmol L1 and obtained an IC50 of 87 mmol L1. The apoptotic process was confirmed by caspase-9 and -3 activation accompanied by negative regulation of the antiapoptotic proteins Bcl-xL and Bcl-xS; that is, the mitochondrial pathway was activated by quercetin. The authors also verified that quercetin, at concentrations of 10–75 mmol L1, provoked the generation of ROS after 4 h of treatment. It has also been found that after a period of 18 h of treatment the concentration of these intracellular species decreases in a dose-dependent manner [97]. In agreement with these results, Dorta evaluated the action of quercetin, from 25 to 100 mmol L1, on cell viability and induction of apoptosis in rat HepG2 cells. Again, quercetin was able to induce apoptosis and inhibit cell proliferation. The cell death process was accompanied by a decrease in the cellular energetic capacity and activation of caspase-9 and -3, again indicative of mitochondrial death [98]. In the same line of investigation, Zhao and coworkers found that quercetin, at 0–200 mmol L1, reduced the proliferation of human HepG2 with an IC50 of 24 mmol L1. Again, the apoptosis death process was verified by increased cell membrane permeability and nuclear condensation in a dose-dependent manner [99]. Chang and coworkers also evaluated the action of quercetin on HepG2 and other human hepatoma lines: HA22T/VGH cells. The results obtained by

282 Studies in Natural Products Chemistry

the authors revealed that quercetin-induced apoptosis in the hepatoma lines was accompanied by an increase in H2O2 and O2
 levels after 72 h of treatment. The authors also verified that quercetin was able to induce lipid peroxidation in these cells, evidencing its prooxidant activity. In this same study, a synergistic quercetin action with paclitaxel was verified [100]. Paclitaxel is a chemotherapeutic agent used against some tumors, including hepatic tumors. It is already well established that its mechanism of action involves the increase of EROs, such as H2O2 and O2
 , and alteration of mitochondrial permeability [101]. HA22T/VGH cells were previously treated with the flavonoid (40–80 mmol L1) and paclitaxel (100 mmol L1) was added and incubated for 48 h. It was found that quercetin increased the cytotoxic effect and induced apoptosis due to the elevation of ROS caused by quercetin [100]. Quercetin also altered the enzymatic antioxidant system in hepatoma H4IIE cells in rats. The concentration of Mn–SOD, CuZn–SOD, and glutathione peroxidase decreased after treatment of the cells with quercetin. The decrease of the antioxidant enzymes resulted in the increased level of intracellular ROS [102].

Cervical and Breast Cancer Cell Lines Cervical cancer is derived from cellular changes that may occur during persistent genital infection with the human papillomavirus. This type of tumor is considered, according to the data from INCA, the third in the list of the most frequently found in women, being behind only breast and colorectal cancer. In Brazil, among the causes of women’s death from cancer, cervical cancer is the fourth most common and 16,340 new cases are estimated for 2016. Breast cancer is considered the most common type of cancer in the female population in the world and in Brazil. The estimate of new cases for the year 2016 is 57,960 [91]. The effect of quercetin on viability of human cervical cancer (HeLa) cells was studied by Priyadarsini and coworkers. The results obtained by these authors showed that quercetin (20–100 mmol L1) significantly decreased cell survival in a dose-dependent manner and induced the tumor cells to apoptosis without affecting healthy cells. A significant increase in p53 protein expression was observed, accompanied by positive regulation of the proapoptotic proteins and downregulation of the antiapoptotic family Bcl-2. The release of cytochrome c and Apaf-1 was also verified, along with the activation of caspase-3 and -9 [103]. Similar results were obtained by Bishayee and coworkers, who verified that quercetin was able to cause conformational changes in the DNA of HeLa cells, accompanied by cytochrome c release, mitochondrial membrane depolarization, p53 overexpression, activation of caspase-3, and an increase of ROS in the culture medium [104]. This evidence highlighted the important role of the prooxidant action of this flavonoid in its antitumor activity in this cell lineage. Srivastava and coworkers induced breast cancer in

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 283

Swiss albino mice and the effect of quercetin was studied. After 10 days, the authors already observed a reduction in tumor volume as compared to untreated mice. In addition, a fivefold increase in survival of the treated species with flavonoid was observed compared to control mice. Apoptosis was established with increase in p53 level, reduction of Bcl-2 and concomitant Bcl-xL proteins with increase of Bax. Release of cytochrome c, activation of caspase-3 and -9, and DNA fragmentation were also observed. With regard to DNA, the authors verified that quercetin was able to intercalate in the nitrogenous bases, being responsible for the morphological alterations that culminated in DNA damage [96].

Gastric Carcinoma and Thyroid Cancer Cell Lines Tumors of the stomach are a type of cancer that occurs mostly in men over 70 years old, reaching the third-highest incidence of cancers in men and the fifth-highest among the female population in Brazil. The estimate of new cases for the year 2016 was 20,520, with 12,920 men and 7600 women [91]. Quercetin has been shown to be able to induce BGC-823 gastric carcinoma cells to apoptosis. Again, the typical morphological characteristics of apoptosis, such as the condensation of chromatin and nucleus and formation of apoptotic bodies, were observed. The decrease of Bcl-2 and an increase in Bax, besides the activation of caspase-3, were also observed [105]. Borska and coworkers evaluated the synergistic effect of quercetin with the chemotherapeutic agent daunorubicin on cell lines EPG85-257P and EPG85257RDB human gastric carcinoma. The EPG85-257RDB cell line is extremely resistant to the daunorubicin; hence the aim of this study was to verify whether quercetin could sensitize the cells to this chemotherapeutic agent [106]. The mechanism of action daunorubicin involves the inhibition of the synthesis of DNA due to the formation of a complex with this macromolecule via intercalation between the pairs of its nitrogenous bases and the production of ROS. The results were positive, reducing the used dose of daunorubicin and promoting the decrease of its side effects. Quercetin (10–200 mmol L1) also provided a decrease in cell proliferation and an increase in the rate of apoptosis in papillary thyroid carcinoma cells (B-CPAP) after incubation for 72 h. Activation of caspase-3 was also observed and apoptosis was confirmed by the presence of cell surface bubbles, apoptotic bodies, and DNA fragmentation [107].

CONCLUDING REMARKS In conclusion, there is substantial evidence that quercetin, at relatively low concentration, is able to trigger the apoptotic machinery in different types of tumor cells. Among the cellular effects promoted by quercetin, the loss of mitochondrial membrane potential, release of cytochrome c, and activation

284 Studies in Natural Products Chemistry

of the caspase signaling pathway are the most frequently cited in the scientific literature. The prooxidant features of quercetin are also frequently highlighted in these studies. Considering the relationship between intracellular generation of ROS and apoptosis, we propose that the prooxidant capacity of quercetin might be involved in its tumoricidal efficacy. Obviously, this is a controversial issue, since quercetin is usually thought of as an antioxidant. Indeed, there are numerous experimental demonstrations that its beneficial health properties, including tumoricidal effects, are also linked to its antioxidant features. However, considering all evidence discussed in this review, the prooxidant capacity of quercetin should not be discarded. In our opinion, quercetin should be considered as a redox-active compound, which by altering the cellular redox status, through anti- or prooxidant action, is able to promote tumoricidal effects.

ACKNOWLEDGMENTS This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq #302793/2016-0 and #440503/2014-0) and Fundac¸a˜o de Amparo à Pesquisa do Estado de Sa˜o Paulo (FAPESP # 2016/20549-5 and INCT.Bio.Nat #2014/50926-0).

ABBREVIATIONS Apaf-1 Bak Bax Bcl2 Bcl-xL B-CPAP BH3 BV-173 CEM CHI CHS DISC Epa EPG85-257P EPG85257RDB Fas-L FHT FLS HA22T/VGH HL-60 HeLa HepG2

apoptotic protease-activating factor 1 Bcl2 antagonist killer 1 Bcl2-associated X protein B-cell CLL/lymphoma 2 B-cell lymphoma-extra large papillary thyroid carcinoma cells Bcl2 homology domain 3 basophilic leukemia cell line acute lymphoblastic leukemia chalcone isomerase chalcone synthase death-inducing signaling complex anodic peak potential gastric adenocarcinoma cells stomach cancer cells Fas ligand flavanone-3b-hydroxylase flavonol synthase human hepatoma lines human promyelocytic leukemia cells cervical cancer cell human liver hepatocellular cells

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

JNK K562 KU812 MV4-11 Nalm-6 p53 Puma Q QH2 QH
2 ROS THP-1 TNF U937

9 285

Jun N-terminal kinase chronic myelocytic leukemia peripheral blood leukemia pre-B cell acute myeloid leukemia acute lymphoid leukemia derived from B cell tumor protein p53 p53-upregulated modulator of apoptosis oxidized ubiquinone reduced ubiquinone semiubiquinone reactive oxygen species acute monocytic leukemia cell tumor necrosis factor histiocytic lymphoma-derived cells

REFERENCES [1] P.S. Brookes, Free Radic. Biol. Med. 38 (2005) 12–23. [2] D. Harman, J. Gerontol. 11 (1956) 298–300. [3] M. Jastroch, A.S. Divakaruni, S. Mookerjee, J.R. Treberg, M.D. Brand, Essays Biochem. 47 (2010) 53–67. [4] D. Vara, G. Pula, Curr. Mol. Med. 14 (2014) 1103–1125. [5] J. Bereiter-Hahn, Protoplasma 251 (2014) 3–23. [6] M.V. Chuong Nguyen, B. Lardy, M.H. Paclet, F. Rousset, S. Berthier, A. Baillet, L. Grange, P. Gaudin, F. Morel, Med. Sci. (Paris) 31 (2015) 43–52. [7] A. Strzepa, K.A. Pritchard, B.N. Dittel, Cell. Immunol. 317 (2017) 1–8. [8] C. Prolo, M.N. Alvarez, R. Radi, Biofactors 40 (2014) 215–225. [9] A. G€ orlach, E.Y. Dimova, A. Petry, A. Martı´nez-Ruiz, P. Hernansanz-Agustı´n, A.P. Rolo, C.M. Palmeira, T. Kietzmann, Redox Biol. 6 (2015) 372–385. [10] D.M. Miller, G.R. Buettner, S.D. Aust, Free Radic. Biol. Med. 8 (1990) 95–108. [11] C. Vives-Bauza, A. Starkov, E. Garcia-Arumi, Methods Cell Biol. 80 (2007) 379–393. [12] J.L. Hsu, Y. Hsieh, C. Tu, D. O’Connor, H.S. Nick, D.N. Silverman, J. Biol. Chem. 271 (1996) 17687–17691. [13] Y.M. Go, D.P. Jones, Biochim. Biophys. Acta 1780 (2008) 1273–1290. [14] W.L. Boatright, Food Chem. 196 (2016) 1361–1367. [15] H.R. Molavian, A. Goldman, C.J. Phipps, M. Kohandel, B.G. Wouters, S. Sengupta, S. Sivaloganathan, Sci. Rep. 6 (2016) 27439. [16] P.E. Milbury, Methods Enzymol. 335 (2001) 16–26. [17] A. Simic, D. Manojlovic, D. Segan, M. Todorovic, Molecules 12 (2007) 2327–2340. [18] J.D. Lambert, R.J. Elias, Arch. Biochem. Biophys. 501 (2010) 65–72. [19] G. Galati, O. Sabzevari, J.X. Wilson, P.J. O’Brien, Toxicology 177 (2002) 91–104. [20] L.R. Castor, K.A. Locatelli, V.F. Ximenes, Free Radic. Biol. Med. 48 (2010) 1636–1643. [21] M.L. Zeraik, M.S. Petr^onio, D. Coelho, L.O. Regasini, D.H. Silva, L.M. da Fonseca, S.A. Machado, V.S. Bolzani, V.F. Ximenes, PLoS One 9 (2014) e110277. [22] G. Galati, A. Lin, A.M. Sultan, P.J. O’Brien, Free Radic. Biol. Med. 15 (2006) 570–580.

286 Studies in Natural Products Chemistry [23] Y.Y. Lee-Hilz, A.M. Boerboom, A.H. Westphal, W.J. Berkel, J.M. Aarts, I.M. Rietjens, Chem. Res. Toxicol. 19 (2006) 1499–1505. [24] X.W. Chen, E.S. Serag, K.B. Sneed, S.F. Zhou, Chem. Biol. Interact. 192 (2011) 161–176. [25] R.A. Vacca, D. Valenti, S. Caccamese, M. Daglia, N. Braidy, S.M. Nabavi, Neurosci. Biobehav. Rev. 71 (2016) 865–877. [26] R.K. Lall, D.N. Syed, V.M. Adhami, M.I. Khan, H. Mukhtar, Int. J. Mol. Sci. 16 (2015) 3350–3376. [27] J. Gonc¸alves, C.L. Silva, P.C. Castilho, J.S. C^amara, Microchem. J. 106 (2013) 129–138. [28] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Mol. Pharm. 4 (2007) 807–818. [29] N.S. Dighe, S.R. Patton, S.S. Dengale, D.S. Musmade, M. Shelar, V. Tambe, M.B. Hole, Scholard Res. Libr. 2 (2010) 65–71. [30] N. Balasundram, K. Sundram, S. Samman, Food Chem. 99 (2006) 191–203. [31] M.S. Pesca, F. Dal Piaz, R. Sanogo, A. Vassallo, M. Bruzual de Abreu, A. Rapisarda, A. Braca, J. Nat. Prod. 76 (2012) 29–35. [32] J. Degenhardt, T.G. K€ollner, J. Gershenzon, Phytochemistry 70 (2009) 1621–1637. [33] M.L.F. Ferreyra, S.P. Rius, P. Casati, Front. Plant Sci. 3 (2012) 1–15. [34] C.A. Hiruma-Lima, C.M. Rodrigues, V.C. Silva, W. Vilegas, N.A.J.C. Furtado, R.C.S. Veneziani, S.R. Ambrosio (Eds.), Farmacognosia, Atheneu, Rio de Janeiro, 2017, pp. 387–406 (Colec¸a˜o Farma´cia, 7). [35] A.E. Mitchell, Y.J. Hong, E. Koh, D.M. Barrett, D.E. Bryant, R.F. Denison, S. Kaffka, J. Agric. Food Chem. 55 (2007) 6154–6159. [36] S.C. Bischoff, Curr. Opin. Clin. Nutr. Metab. Care 11 (2008) 733–740. [37] G. D’Andrea, Fitoterapia 106 (2015) 256–271. [38] A. Gupta, K. Birhman, I. Raheja, S.K. Sharma, H.K. Kar, Asian Pac. J. Trop. Dis. 6 (2016) 248–252. [39] M.R. Oliveira, S.M. Nabavi, N. Braidy, W.N. Setzer, T. Ahmed, S.F. Nabavi, Biotechnol. Adv. 34 (2016) 532–549. [40] D. Caridi, V.C. Trenerry, S. Rochfort, S. Duong, D. Laugher, R. Jones, Food Chem. 105 (2007) 691–699. [41] B. Sultana, F. Anwar, Food Chem. 108 (2008) 879–884. [42] K.H. Miean, S. Mohamed, J. Agric. Food Chem. 49 (2001) 3106–3112. [43] H.P. Liu, X.F. Shi, Y.C. Zhang, Z.X. Li, L. Zhang, Z.Y. Wang, Cell Biochem. Biophys. 61 (2011) 59–64. € Tokuşog˘lu, M.K. Unal, € Z. Yıldırım, Acta Chromatogr. 13 (2003) 196–207. [44] O. [45] A. Schieber, P. Keller, R. Carle, J. Chromatogr. A 910 (2001) 265–273. [46] M. Careri, C. Corradini, L. Elviri, I. Nicoletti, I. Zagnoni, J. Agric. Food Chem. 51 (2003) 5226–5231. [47] C. Faggio, A. Sureda, S. Morabito, A.S. Silva, A. Mocan, S.F. Nabavi, S.M. Nabavi, Eur. J. Pharmacol. 807 (2017) 91–100. [48] S.H. H€akkinen, S.O. K€arenlampi, I.M. Heinonen, H.M. Mykk€anen, A.R. T€orr€onen, J. Agric. Food Chem. 47 (1999) 2274–2279. [49] F.D. Neves Costa, G.G. Leita˜o, J. Sep. Sci. 33 (2010) 336–347. [50] B. Winkel-Shirley, Plant Physiol. 126 (2001) 485–493. [51] H. El Hajji, E. Nkhili, V. Tomao, O. Dangles, Free Radic. Res. 40 (2006) 303–320. [52] S.C. Zˇivanovic, R.S. Nikolic, G.M. Nikolic, Acta Fac. Med. Naiss. 33 (2016) 163–171. [53] S.C. Sahu, M.C. Washington, Cancer Lett. 58 (1991) 75–79. [54] N.G. Oliveira, A.S. Rodrigues, T. Chaveca, J. Rueff, Mutagenesis 12 (1997) 457–462.

Quercetin: Prooxidant Effect and Apoptosis in Cancer Chapter

9 287

[55] R. Lei, X. Xu, F. Yu, N. Li, H.W. Liu, K. Li, Talanta 75 (2008) 1068–1074. [56] Z. Jurasekova, C. Domingo, J.V. Garcia-Ramos, S. Sanchez-Cortes, Phys. Chem. Chem. Phys. 16 (2014) 12802–12811. [57] A.K. Timbola, C.D. de Souza, C. Giacomelli, A. Spinelli, J. Braz. Chem. Soc. 17 (2006) 139–148. [58] H.M. Awad, M.G. Boersma, S. Boeren, H. van der Woude, J. van Zanden, P.J. van Bladeren, J. Vervoort, I.M.C.M. Rietjens, FEBS Lett. 520 (2002) 30–34. [59] A.W. Boots, J.M. Balk, A. Bast, G.R. Haenen, Biochem. Biophys. Res. Commun. 338 (2005) 923–929. [60] B. Alberts, A. Johnson, J. Lewis, D. Morgan, M. Raff, K. Roberts, P. Walter, Molecular Biology of the Cell, 6th edition, Garland Science, 2014, pp. 1464. [61] D.R. Green, Means to an End: Apoptosis and Other Cell Death Mechanisms, Cold Spring Harbor Press, 2011, pp. 220. [62] M.C. Anazetti, P.S. Melo, Metrocamp Pesquisa 1 (2007) 37–58. [63] S. Kumar, Cell Death Differ. 14 (2007) 32–43. [64] E.D. Crawford, J.A. Wells, Annu. Rev. Biochem. 80 (2011) 1055–1087. [65] R.E. Ellis, J.Y. Yuan, R.A. Horvitz, Annu. Rev. Cell Biol. 7 (1991) 663–698. [66] I. Grivicich, A. Regner, A.B.D. Rocha, Rev. Bras. Cancer 53 (2007) 335–343. [67] R.A. Weinberg, A biologia do cancer. Artmed, Porto Alegre, 2008, pp. 728. [68] I. Lavrik, A. Golks, P.H. Krammer, J. Cell Sci. 118 (2005) 265–267. [69] P.D. Mace, S.J. Riedl, Curr. Opin. Cell Biol. 22 (2010) 828–836. [70] S.W. Tait, D.R. Green, Cold Spring Harb. Perspect. Biol. 5 (2013) a008706. [71] S.W. Tait, D.R. Green, Nat. Rev. Mol. Cell Biol. 11 (2010) 621–632. [72] S. Yuan, C.W. Akey, Structure 21 (2013) 501–515. [73] X. Jiang, X. Wang, Annu. Rev. Biochem. 73 (2004) 87–106. [74] J.E. Chipuk, T. Moldoveanu, F. Llambi, M.J. Parsons, D.R. Green, Mol. Cell 37 (2010) 299–310. [75] T. Kuwana, L. Bouchier-Hayes, J.E. Chipuk, C. Bonzon, B.A. Sullivan, D.R. Green, D.D. Newmeyer, Mol. Cell 17 (2005) 525–535. [76] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646–674. [77] J.M. Adams, S. Cory, Oncogene 26 (2007) 1324–1337. [78] S.W. Lowe, E. Cepero, G. Evan, Nature 432 (2004) 307–315. [79] K.H. Vousden, Science 309 (2005) 1685–1686. [80] G. He, G. He, R. Zhou, Z. Pi, T. Zhu, L. Jiang, Y. Xie, Biochem. Biophys. Res. Commun. 469 (2016) 1075–1082. [81] J.Y.H. Ong, P.V.C. Yong, Y.M. Lim, A.S.H. Ho, Life Sci. 135 (2015) 158–164. [82] J.V. Gerasimenko, O.V. Gerasimenko, A. Palejwala, A.V. Tepikin, O.H. Petersen, A.J.M. Watson, J. Cell Sci. 115 (2002) 485–497. [83] S.W. Ryter, H.P. Kim, A. Hoetzel, J.W. Park, K. Nakahira, X. Wang, A.M. Choi, Antioxid. Redox Signal. 9 (2007) 49–89. [84] D.N. Dhanasekaran, E.P. Reddy, Oncogene 27 (2008) 6245–6251. [85] M. Fan, T.C. Chambers, Drug Resist. Updat. 4 (2001) 253–267. [86] S.C. Gupta, D. Hevia, S. Patchva, B. Park, W. Koh, B.B. Aggarwal, Antioxid. Redox Signal. 16 (2012) 1295–1322. [87] J.N. Moloney, T.G. Cotter, Semin. Cell Dev. Biol. (2017). in press. https://doi.org/10.1016/ j.semcdb.2017.05.023. [88] V. Nogueira, Y. Park, C.-C. Chen, P.-Z. Xu, M.-L. Chen, I. Tonic, T. Unterman, N. Hay, Cancer Cell 14 (2008) 458–470.

288 Studies in Natural Products Chemistry [89] D. Trachootham, Y. Zhou, H. Zhang, Y. Demizu, Z. Chen, H. Pelicano, P.J. Chiao, G. Achanta, R.B. Arlinghaus, J. Liu, P. Huang, Cancer Cell 10 (2006) 241–252. [90] P. Storz, Front. Biosci. 10 (2005) 1881–1896. [91] Ministerio da Sau´de, Instituto Nacional de Cancer (INCA), Estimativa 2016, Incid^encia de Cancer no Brasil, Rio de Janeiro, 2016. [92] J. Matsui, N. Kiyokawa, H. Takenouchi, T. Taguchi, K. Suzuki, Y. Shiozawa, M. Saito, W. Tang, Y.U. Katagiri, H. Okita, J. Fujimoto, Leuk. Res. 29 (2005) 573–581. [93] G. Niu, S. Yin, S. Xie, Y. Li, D. Nie, L. Ma, X. Wang, Y. Wu, Acta Biochim. Biophys. Sin. 43 (2011) 30–37. [94] K. Sakao, M. Fujii, D.-X. Hou, Biosci. Biotechnol. Biochem. 73 (2009) 2048–2053. [95] W.J. Lee, M. Hsiao, J.L. Yang, T.H. Tseng, C.W. Cheng, J.M. Chow, K.H. Lin, C.C. Liu, L.M. Lee, M.H. Chein, Arch. Toxicol. 89 (2015) 1103–1117. [96] S. Srivastava, R.R. Somasagara, M. Hegde, M. Nishana, S.K. Tadi, M. Srivastava, B. Choudhary, S.C. Raghavan, Sci. Rep. 6 (2016) 24049. [97] A.B. Granado-Serrano, M.A. Martı´n, L. Bravo, L. Goya, S. Ramos, J. Nutr. 136 (2006) 2715–2721. [98] D.J. Dorta, PhD thesis, Faculdade de Ci^encias Farmac^euticas, Universidade de Sa˜o Paulo, 2007. [99] P. Zhao, J.M. Mao, S.Y. Zhang, Z.Q. Zhou, Y. Tan, Y. Zhang, Oncol. Lett. 8 (2014) 765–769. [100] Y.F. Chang, C.W. Chi, J.J. Wang, Nutr. Cancer 55 (2006) 201–209. [101] G. Varbiro, B. Veres, F. Gallyas Jr., B. Sumegi, Free Radic. Biol. Med. 31 (2001) 548–558. [102] E. R€ ohrdanz, A. Bittner, Q.H. Tran-Thi, R. Kahl, Arch. Toxicol. 77 (2003) 506–510. [103] V.R. Priyadarsini, R.M. Senthil, S. Maitreyi, K. Ramalingam, D. Karunagaran, S. Nagini, Eur. J. Pharmacol. 649 (2010) 84–91. [104] K. Bishayee, S. Ghosh, A. Mukherjee, R. Sadhukhan, J. Mondal, A.R. Khuda-Bukhsh, Cell Prolif. 46 (2013) 153–163. [105] I.K. Wang, S. Lin-Shiau, J.K. Lin, Eur. J. Cancer 35 (1999) 1517–1525. [106] S. Borska, M. Chmielewska, T. Wysocka, M. Drag-Zalesinska, M. Zabel, P. Dziegiel, Food Chem. Toxicol. 50 (2012) 3375–3383. [107] E.M. Altundag, T. Kasaci, A.M. Yilmaz, B. Karademir, S. Koc¸turk, Y. Taga, A.S. Yalc¸in, J. Thyroid. Res. (2016) 9843675.