Abstract

Quercetin is a unique dietary polyphenol because it can exert biphasic dose-responses on cells depending on its concentration. Cancer preventative effects of quercetin are observed at concentrations of approximately 1–40 µM and are likely mediated by quercetin's antioxidant properties. Pro-oxidant effects are present at cellular concentrations of 40–100 µM. However, at higher concentrations, many novel pathways in addition to ROS contribute to its effects. The potent bioactivity of quercetin has led to vigorous study of this compound and revealed numerous pathways that could interact synergistically to prevent or treat cancer. The effect of intake and concentration on emerging pathways and how they may interact are discussed in this review.

INTRODUCTION

Quercetin is a dietary polyphenol that is readily found in a variety of foods and is consumed daily. The tremendous growth in the study of this bioactive compound has revealed numerous pathways that could possibly interact to prevent or treat diseases such as cancer, so a review of the recent literature utilizing this compound is pertinent. Quercetin also has a unique ability to act as an antioxidant or a pro-oxidant depending on its concentration, which is indicative of its hormetic properties.1 For the purpose of this review, hormesis is defined as a biphasic dose-response whereby low doses of quercetin result in a given effect (antioxidant properties) and higher doses result in another effect (pro-oxidant properties). Given this assumption, there are two fundamental factors that impact quercetin's bioactivity as either an oxidant or an antioxidant. First, and arguably most important, is quercetin's tissue bioavailability and digestion process. Second is the concentration and isoform or conjugate form of quercetin in the target tissue. In this review, multiple effects of quercetin are proposed that are concentration dependent; this implies a dependence on the entire metabolic process of this flavonoid. It is also proposed that multiple pathways could interact to produce synergistic effects, which again rely on the concentration of quercetin at the tissue site. Quercetin's hormetic nature makes it well-suited to use in cancer prevention efforts; these efforts would likely involve lower and long-term consumption of quercertin-containing foods, and/or supplementation or therapeutic administration in combination with conventional therapies. Although this review focuses on quercetin in relation to cancer, it is generally understood that the same pathways could be applied to other disease states as well.

CENTRAL ROLE OF REACTIVE OXYGEN SPECIES IN QUERCETIN ACTIVITY IN CANCER

The biphasic oxidation properties of quercetin are likely beneficial in cancer prevention and therapy because different concentrations of quercetin counter the transformation and growth processes of cancer.1 Malignant tumors result from uncontrolled cell growth due to mutations. Mutations are a result of DNA damage, which is commonly incurred through exposure to reactive oxygen species (ROS). Quercetin is able to donate electrons to ROS2 and thereby reduce their ability to damage cellular DNA.3 This is the primary mechanism by which quercetin exerts antioxidant and chemopreventive effects on the cell.3 Typically, this effect is seen at cellular quercetin concentrations in the range of 1–40 µM, which could likely be achieved by diet.4 However, after a tumor has formed, quercetin could continue to have beneficial anti-tumor effects at higher doses by exerting cytotoxic effects. Quercetin is able to increase oxidative stress and cytotoxicity in tumor cells, usually at concentrations greater than 40 µM; it is able to do this by becoming an ROS itself and by increasing damage or apoptotic pathways in the transformed cell.2,3 These benefits rely on ROS and the quercetin concentration to produce either anti- or pro-oxidant effects. There are various pathways and mechanisms that can interact and these are described in more detail below. In addition, because biomedical research must be translatable to real-life situations, this review begins with an assessment of the bioavailability and metabolism of quercetin. Since much of the research discussed in this review has only been conducted in vitro or in cell culture, an attempt is made here to link these studies with biologically relevant concentrations in humans.

ABSORPTION AND METABOLISM OF QUERCETIN

Quercetin is consumed daily by millions of people through nuts, teas, vegetables, and herbs in the diet.3 It is also available as a commercial dietary supplement, and it is now being included in functional foods. Quercetin is generally recognized as safe in oral dosages of 1,000 mg/day or in intravenously administered dosages of 756 mg/day.5 Up to 60% of orally ingested quercetin is absorbed,5 and the average dietary intake of quercetin is somewhere between 6 and 31 mg daily (not including supplement/intravenous use).6 Quercetin is part of the flavanol family and it is normally found in the glycosylated form.7 Digestion of most dietary quercetin, in the form of quercetin glycosides, begins in the oral cavity with some cleavage of the glycosides catalyzed by β-glycosidases (Figure 1).7 Some of quercetin's aglycoside form is absorbed in the mouth as well.7

Figure 1

Schematic of possible pathways by which quercetin is digested, absorbed, metabolized, and excreted in the human body. Typically, quercetin glycoside is ingested orally and is then probably partially digested in the oral cavity. Surplus quercetin is then digested and absorbed at multiple sites along the gastrointestinal tract. During absorption, or shortly thereafter, quercetin undergoes modification and then enters the circulatory system in a conjugate form. The circulatory system delivers quercetin to other tissues in mostly conjugated forms, and once quercetin reaches the target tissues it can likely be converted back into the parental compound.

There is some disagreement as to the exact post-oral cavity metabolism of this substrate; however, a few possibilities exist (Figure 1). It is likely that the colonic microflora hydrolyze the glycoside-form of quercetin to the more active aglycone quercetin. Once aglycosylated, the molecule becomes more lipophillic and can then be absorbed into the epithelial cells of the colon.8 Another possibility is that some of the glycosidic quercetins are absorbed directly, particularly those that are bound with glucose.8 It is also probable that colonic microflora ferment quercetin into phenolic compounds and carbon dioxide.5 Both the carbon dioxide and the phenolic compounds are then expelled from the body.5 There are yet other hypotheses, including the idea that some hydrolysis occurs in the small intestines, via both β-glycosidase and lactase phlorizen hydrolase (LPH).9 Despite differences of opinion, it is generally accepted that bioavailability depends on the location and type of sugar group attached to quercetin.8 Most likely, the digestion and absorption of quercetin occurs through a combination of the proposed pathways, depending on which form the quercetin is in at a given point in time.

Hydrolysis of quercetin by β-glycosidase results in different metabolites of quercetin depending on what the original glycoside was (i.e., where the glycosidic bond was located and what type of sugar was attached). These metabolites include not only free quercetin, but also conjugates such as glucuronides, O-methylated products, and sulfate forms.8 This conjugation of quercetin is reported to occur throughout the processes of digestion and absorption.8 In animals, it appears that quercetin and its metabolites are transported unevenly throughout the body.10 Animal studies have also shown that blood contains mostly quercetin metabolites after quercetin ingestion,8,10 whereas only the organs involved in quercetin metabolism (i.e., kidney, liver, and intestines) can contain significant amounts of free quercetin in addition to methylated forms.10 However, few studies have focused on detecting quercetin concentrations at target tissues and further research is greatly needed in this area. The findings of one study conducted in pigs indicated that the kidney, liver, and jejunum had concentrations of quercetin between approximately 2.0 and 6.0 µM/L.10 Human studies are not available to confirm this finding4; however, both human and animal studies suggest that quercetin's distribution and absorption depend on its form.4,10 Further, studies have shown that both the bioavailability and other intestinal contents can affect absorption of quercetin and its derivatives.8

The reduction-oxidation potential of a quercetin molecule is also dependent on quercetin's form. For example, non-catechol containing structures do not chelate oxidative metals as well as those that do contain catechol.8 Given the immense variability in quercetin metabolism, it is tremendously complicated to assess quercetin's direct ability to exert pro-oxidant and antioxidant affects in the body. Additionally, there are too many factors to provide a complete comprehensive review of the literature involving quercetin metabolism. Thus, this review focuses on studies that have examined oral supplementation and/or dietary intake of quercetin versus blood concentrations or tissue concentrations. Since it is conceivable that long-term consumption and chronic quercetin blood concentrations will eventually infiltrate tissues, a general assumption is made that it may be possible to achieve levels of quercetin in tissues and tumors that are somewhat near those measured in blood. Also, given that limited data are available on quercetin concentration and form in human organs or tissue and that many of the conjugate forms of quercetin are converted back to the parental compound by cellular processes, only those mechanisms involved in free quercetin action at the target tissue will be examined.11 It should be noted that when discussing the high concentrations of quercetin that would be required for therapeutic effect, it is likely that high-dose supplementation or more direct forms of administration would be required. While this is an oversimplification, the diverse nature of the polyphenols makes it necessary to focus on the mechanisms of the parental compound in order to to further understanding of its conjugates and how all factors combined will ultimately impact the outcome of using quercetin for cancer prevention and treatment in humans.

There are very few human studies that have evaluated the absorption of quercetin, and most of them were performed with low doses that could be achieved through diet. Egert et al.4 supplemented 18 men and 18 women for 2 weeks with various levels of the oral quercetin aglycone. The study participants had no difficulty absorbing dosages of up to 150 mg/day but the researchers did find variations in individual blood serum concentrations that were independent of fat mass or sex.4 The blood plasma measurements for average total quercetin levels from 50 mg/day, 100 mg/day, and 150 mg/day supplementation were 145 nmol/L, 217 nmol/L, and 380 nmol/L, respectively, after only 2 weeks of daily ingestion.4 Two well-known, specific quercetin metabolites (isorhammetin and tamarixetin) were increased to between 9 and 23 nmol/L after treatment with the various dosages of the oral quercetin aglycone.4 These findings agree with those from other human quercetin absorption studies.4,12 It should be noted, however, that the measurement of total quercetin includes all detectable conjugates, which can then be converted to free quercetin in the cell.11 Because the preventative effects of free quercetin are seen in vitro at approximately 1,000–40,000 nmol/L (1–40 µM), for antioxidant effects, it is likely that these concentrations could be achieved through diet or, more likely, dietary supplementation of quercetin.4 However, the cancer-treating pro-oxidant effects are not commonly seen until cellular concentrations reach 40,000 to above 100,000 nmol/L (40–>100 µM). There are animal studies that support the possibility of reaching higher concentrations in vivo. Silberberg et al.13 found that the combined plasma concentration in rats, after oral consumption of 45–47 mg/day for 2 weeks, was approximately 60 µM. Another possibility is to administer quercetin intravenously.14 A phase I clinical study found that individuals with a cancer diagnosis could tolerate acute serum levels of 200–400 µM.14 Although more research is needed, it appears to be physiologically possible to meet the ranges required to both prevent and potentially treat carcinogenesis with quercetin.

ANTIOXIDANT MECHANISMS OF QUERCETIN

Quercetin is able to react with ROS and chelate ROS-producing metal ions, both of which allow for decreased oxidative DNA damage.8 Preventing this DNA damage is believed to be the general mechanism by which quercetin is able to prevent tumorigenesis.8 In particular, it is known that quercetin's hydroxyl groups have electron-accepting capacity when they are in the semiquinone state and that its catechol group is the structure that confers the ability to chelate metal ions.8 The addition of sugar molecules to form quercetin glycosides can obstruct both of its antioxidant activities. Therefore, the aglycosylated form is usually of higher antioxidant potency than the glycoside form, depending on where the sugar molecule is attached.8 In this review, references to quercetin indicate the free, aglyconated form.

A recent study looking at quercetin's antioxidant mechanism in colorectal adenocarcinoma cells (Caco2) found that treatments consisting of 1 µM concentrations of quercetin led to decreased double-stranded DNA breaks, but that higher concentrations of quercetin increased double-stranded DNA breakage.15 Recall that double-stranded DNA breakage is a major source of mutagenesis and subsequent malignancies in cells.15 However, increased double-stranded breaks can also lead to increased apoptosis, as described later in this review. Congruently, this group found decreased hydrogen peroxide-induced single-strand DNA breakage in Caco2 cells pretreated with low-dose quercetin as compared to cells that were untreated.15 Additionally, it was determined that both low (1 µM) and high (100 µM) quercetin treatments led to increased expression of human 8-oxyguanine DNA glycosylase (hOGG1). The hOGG1 protein is involved in repairing DNA.15 This suggests that quercetin is able to prevent oxidative DNA damage and increase DNA repair at lower dosages.

Quercetin also has the ability to work synergistically with other antioxidant systems in the body in order to decrease oxidative stress.16 When quercetin exerts its antioxidant power, it can advance to the semiquinone or even the 0-quinone state.16,17 In these highly oxidized states, quercetin is potentially damaging to the cell and activates another antioxidant pathway involving glutathione (GSH).17 Kim et al.17 recently examined the relationship between oxidized quercetin and GSH in a human hepatoma cell line (HepG2).17 Their findings indicate that 10 and 100 µM doses of quercetin led to antioxidant affects, but that exposure to 100 µM quercetin for longer than 30 min led to pro-oxidant/pro-apoptotic effects.17 More specifically, the data indicated that quercetin is able to chelate reactive metal ions that produce ROS, react with hydrogen peroxide to reduce ROS, and use GSH-mediated reduction in order to return ROS to their reduced states.17 This cooperativity with GSH is likely one mechanism by which quercetin can protect the cell from mutagenesis. On the other hand, quercetin may be able to cause cellular damage when it is administered in a long-term high dose.

Animal models are frequently the vehicle for measuring overall antioxidant status after treatment with quercetin. Santos et al.18 fed mice 4.2 mg of quercetin daily for 3 weeks and then measured blood values of quercetin metabolites against control mice. The primary metabolites found in the blood were glucuronide sulfate conjugates of isorhamnetin at a concentration of 4.2 µM.18 The chemical structures of these conjugates inhibit some of the antioxidant capacity as compared to the parent compound. Thus, the bioactivity of quercetin conjugates is lower than that of the parent compound.18 This decrease in bioactivity was supported by the unchanged antioxidant activity when the experimental and control blood samples were compared.18 Similar null results were found in humans when serum quercetin metabolite concentrations reached 1.031 µM after onion consumption.19 Like in the murine study,18 the researchers in the human study also attributed their null results to the blood concentration probably being lower than the threshold needed to significantly change antioxidant biomarkers.19 However, different results were obtained when higher dosages, approximately 20 mg quercetin, were administered to mice intragastrically.18,20 These acutely exposed mice achieved a 13.2 µM serum concentration of quercetin metabolites, which was expectantly higher than the concentration in the previously discussed lower-dose studies.20 The higher concentration was enough to increase the antioxidant capacity of the treated mice, at 119 nmol Trolox equivalents/mL plasma, relative to the control, at 48 nmol Trolox equivalents/mL plasma.20 These studies illustrate how dependent quercetin's antioxidant capacity is on both the concentration and form of quercetin in the blood and, presumably, target tissues.

PRO-OXIDANT MECHANISMS OF QUERCETIN

As described above, quercetin is not only an antioxidant1; it can also become a pro-oxidant at high concentrations or for longer incubations at the greater concentration. The present review of the literature indicates that, in general, quercetin is able to act as a pro-oxidant at concentrations greater than 40 µM, which is in agreement with Watjen et al.1 Although cytotoxicity may not be a desirable outcome in healthy cells, it would be greatly beneficial in tumor cells. Thus, if quercetin was supplemented at high does or administered intravenously, like other chemotherapeutic drugs, it may be possible to use this pro-oxidative tendency in order to initiate apoptosis in humans with cancer. Therefore, quercetin could likely be used as an adjuvant to current chemotherapies, and if quercetin is activated (oxidized) by enzymes in tumor cells, the dose needed for the pro-oxidant or anti-tumor responses could be considerably lower.21,22 Recently discovered mechanisms by which quercetin is able to bring about advantageous cell death are discussed below.

MITOCHONDRIAL APOPTOTIC PATHWAY (P53-DEPENDENT AND -INDEPENDENT)

The mitochondrial apoptotic pathway is initiated via Bcl-2-associated X protein (Bax) and/or Bcl-2 homologous antagonist/killer (Bak) proteins that bring about an increase in the mitochondria outer-membrane pore size. This allows for cytochrome C, among other pro-apoptotic proteins, to leak out into the cytoplasm. When cytochrome C is freed into the cytoplasm, it is able to combine with apoptotic protease activating-factor 1 (APAF-1) and undergo a conformational change, thus forming the apoptosome. The apoptosome then enlists caspase-9 in order to activate the so-called executioner proteins, caspase-7 and caspase-3. Cell death is subsequently carried out by these caspase proteins (Figure 2).23 Quercetin is a known inducer of apoptosis in multiple cancer cell lines when administered in doses of 40–50 µM or greater concentrations.24,26 Larger doses of quercetin and longer exposure times lead to decreased cancer cell viability. It has been proposed that the mitochondrial-mediated cell-death pathway is a mechanism used by quercetin in order to induce apoptosis.25

Figure 2

Map of several pro-apoptotic pathways triggered by quercetin concentrations greater than 40 µM. Quercetin can generate increased cellular ROS, which then increases tumor suppressor proteins and leads to cell death via the mitochondrial pathway. Quercetin can also initiate cell death via the death domain pathways. Lastly, quercetin contributes to the inhibition of proteins that encourage proliferation. Note: Arrows do not always indicate a direct mechanism of action.

Examples of quercetin's antiproliferative effect are largely documented as being mediated through the induction of P53.24,26 This tumor-suppressor protein can activate Bax and initiate cell death.27 Recently, Tan et al.24 investigated protein expression and cell status of a human hepatocellular carcinoma cell line after treatment with 40–120 µM dosages of quercetin. Tan et al.24 found that quercetin induced increases in P53, while decreasing the antiapoptotic Survivin and Bcl-2 proteins.24 Survivin acts at the caspase level to prevent apoptosis while the ability of Bcl-2 to prevent mitochondria-directed apoptosis is dependent on the relative amounts of Bax present. Further, the researchers found amplified caspase-9 and its downstream proapoptotic substrate, caspase-3, activity.24 Given that Bcl-2 is a negative regulator of apoptosis, and that caspase-3 is a positive regulator of apoptosis,24 one can conclude that the P53-dependent induction of the mitochondria-mediated pathway is what allows quercetin to induce cell death in this cell line.24,26

In a similar study performed in human breast cancer cells (MDA-MB-231), P53 and the mitochondria-mediated cell-death mechanism were also implicated as the mechanism by which quercetin is able to induce apoptosis. Chein et al.28 observed an increase in P53, caspase-9 activation, caspase-3, cytochrome c, and apoptosis in MDA-MB-231 cells treated with 200–250 µM quercetin in vitro. In addition, this group measured and found a decrease in mitochondrial membrane potential after treatment with quercetin.28 A decrease in membrane potential would be consistent with the “leaky mitochondrion” evident in mitochondria-mediated apoptosis.28

Even in the absence of P53, quercetin is able to exert its mitochondria-mediated cell death via the presence of P63 and P73.26 Both P63 and P73 are similar enough in structure to P53 that they are able to increase transcription of Bax.26 Recently, Zhang et al.26 demonstrated the dose-dependent cytotoxicity of quercetin to human esophageal squamous cell carcinoma cell line (KYSE-510) that is p53 mutated in vitro. The direct mechanism for apoptosis was provided by the observed increased cleavage of procaspase-9 and caspase-3 after KYSE-510 cells were treated with 80 µM quercetin.26 Quercetin is therefore able to initiate apoptosis via the mitochondrial pathway involving activation of caspase-3 downstream from caspase-9, as long as a functioning p53-like protein is activatable.26 Of interest was another finding in this same experiment involving the quercetin-provoked increase in expression of p53-inducible gene 3 (PIG3).26 PIG3 is quinone oxidoreductase and is responsible for the NADP-dependent reduction of quinones, like quercetin. It is thought that PIG3 induces cell death by enzymatically upregulating ROS, but the exact mechanism has not been elucidated fully.29

SYNERGISTIC EFFECTS OF QUERCETIN IN APOPTOSIS

Higher dosages of quercetin can trigger apoptotic cascades by multiple mechanisms and via both the mitochondrial and death-domain pathways in various cell lines. The death-domain pathway involves activation of FAS receptor, then FAS-associated death domain, and subsequently caspase-8.30 Caspase-8 then induces caspase-3, which triggers cell death.30 Quercetin can work alone or in conjunction with other molecules in order to bring about cell death (Figure 2).31 Quercetin even has some ability to differentiate between normal versus malignant cells.31 Below are some examples of novel mechanisms by which quercetin can bring about tumor cell death.

In addition to elucidating quercetin's involvement in the mitochondrial pathway, Chein et al.28 found evidence that quercetin may also induce a separate, p53-independent apoptotic pathway known as the death-receptor or death-domain pathway. In MDA-MD-231 cells, Chein et al.28 found an increase in FAS and caspase-8 activation after treatment with 250 µM of quercetin. This indicates that high doses of quercetin likely provoke cell death through the cell-death-receptor pathways, in addition to the mitochondria-dependent cell-death pathway in the same cell line.

Quercetin may also work synergistically (Figure 2) with other death-domain stimulators, like tumor necrosis factor α (TNF-α)-related apoptosis-inducing ligand (TRAIL), to bring about cancerous cell death.31 Siegelin et al.31 recently found that the coadministration of 100 or 200 µM of quercetin will lead to the sensitization of glioma cells (U87-MG, A172, U251, U373, and LN229) to TRAIL and, consequently, apoptosis. Glioma cells are notoriously resistant to TRAIL-induced apoptosis, and this group found that neither quercetin nor TRAIL alone caused significant cell death in their cell lines at doses below 300 µM.31 Combinations of quercetin and TRAIL, however, produced apoptosis, as measured by the presence of cleaved poly(ADP-ribose) polymerase and flow cytometry in all examined gliomas, except U373. This is significant given that TRAIL selectively kills only cancerous cells while leaving healthy tissue alive.31,32 To confirm the death-domain-pathway activation, Sieglin et al.31 measured and found an increased active caspase-8 cleavage product in U87-MG, A172, and U251 but not in the other cell lines. This indicates the death domain pathway is an active player in the apoptosis seen in these cells. Additionally, this study measured caspase-level inhibitors of apoptosis. X-link inhibitor of apoptosis proteins (XIAP) is similar to survivin in that it is antiapoptotic and that both are upregulated in resistant glioma cells.31,32 XIAP, along with survivin, were decreased after the cells were exposed to both quercetin and TRAIL, again except in the U373 cells.31

As stated previously, both mitochondria- and death-domain-directed apoptosis has been noted to occur in the same cell line simultaneously. In addition to the classic death-receptor-mediated pathway induced by TRAIL, Sieglin et al.31 found evidence implicating the mitochondrial pathway as well. The Bcl-2-interacting domain (Bid) is involved in the mitochondrial apoptotic pathway and leads to the inhibition of the downstream caspase-inhibiting survivin and XIAP proteins.32 Upregulation of Bid was seen in U87-MG and A172, indicating that, at least in those cell lines, both types of apoptosis are involved in quercetin/TRAIL-induced cell death.31

In addition to TRAIL, quercetin has been shown to work with the estrogen receptor α (ER-α) in order to induce cytotoxicity in some cervical cancer cell lines.33 In a recent study by Galluzzo et al.,33 doses as low as 1 µM, and up to 100 µM, were shown to decrease the number of cells in the human cervix epitheloid carcinoma cell line (HeLa) that were ER-α-positive but not the number of HeLa cells that were ER-α-delete. Due to evidence from research on a related bioflavonoid, naringen, Galluzo et al.33 hypothesized the apoptosis was a result of quercetin stimulating the ER-α-P38/mitogen-activated protein kinase apoptotic pathway. Although this pathway only seems to profoundly affect certain cell lines, it leads to increased FAS (pro-death domain pathway) and increased Bax (pro-mitochondrial-cell-death pathway).34 ER-α can initiate both estrogen-triggered proliferative and pro-apoptotic pathways.33 Since quercetin is considered a phytoestrogen, it is likely that this estrogen-mimicking ability is able to prevent the ER-α from initiating prosurvival pathways by competing with estrogen for binding at the receptor. However, the non-estrogen-dependent phosphorylation of p38 it still able to proceed and initiate the proapoptotic pathways.33

Despite quercetin's ability to interact with ER-α, it preferentially favors binding to estrogen receptor β (ER-β) over ER-α.35 Sotoca et al.35 recently explored the impact that quercetin's receptor affinity has on apoptosis in breast cancer (T47D-ER-α) and osteosarcoma (U2OS-ER-α and -ER-β) cell lines. Upon administration of ascorbate-stabilized quercetin, this group saw increased quercetin-ER-β binding and apoptosis at concentrations greater than 50 µM. A proliferative effect was noted at lower concentrations. Additionally, they observed that increased presence of ER-β independently contributed to apoptosis.35 Presumably, this is due to ER-β's apoptotic abilities when bound by a ligand and to its ability to decrease ER-α's proliferative effect.36,37 ER-β is able to induce apoptosis by increasing intracellular pH via modulation of the cells' Na+/H+ exchanger, thus inducing Bax and mitochondria-directed apoptosis.36 This ability is increasingly effective when ER-α's proliferative activity is also decreased by ER-β.36 It is via this pathway that quercetin is indirectly able to increase Bax and apoptosis in some ER-β-positive cell lines.

QUERCETIN AND PROTEIN CHAPERONE INHIBITION

Quercetin promotes apoptosis by interfering with proliferation and cell maintenance pathways as discussed above (i.e., Bcl-2, survivin, and XIAP inhibition). Specifically, however, emerging science is indicating that quercetin-directed protein chaperone inhibition may play a large role in the stimulation of cell death.38,39 Protein chaperones are responsible for the correct folding and maintenance of proteins in the body. When protein chaperones are unable to perform their duties, cell functionality is decreased and cell death is plausible.39 Heat shock protein (HSP) chaperones, specifically, are unregulated in some tumor cells and initiated by ionizing radiation.39 Quercetin is able to inactivate these protein chaperones, seemingly by its ability to inhibit the kinases that aid in HSP induction (Figure 3).39 This ability is currently being explored as an anti-cancer mechanism and is discussed below.

Figure 3

Diagram of several pro-apoptotic pathways triggered by quercetin concentrations greater than 40 µM. Quercetin can initiate cell death via induction of ER stress. Quercetin can also modulate HSP activity, which leads to alterations in cell repair and proliferation. Note: Arrows do not always indicate a direct mechanism of action.

Expression of HSP70 is stimulated by radiation-induced heat in tumor cells. The heat induces the phosphorylation of heat shock transcription factor 1 (HSF1) by either of two kinases: casein kinase 2 (CK2) and calcium/calmodulin kinase II (CamKII). Once phosphorylated, these kinases activate HSF1, which catalyzes the transcription of HSP70. A novel experiment by Wang et al.39 in Jurkat cells (immortalized T-lymphocytic cells) demonstrated that quercetin is able to inhibit the kinase activity of CK2 and CamKII, subsequently decreasing HSP70 expression and increasing tumor sensitivity to radiation.39 This effect, however, was not seen in human HeLa cells.39 Confounding the potential of quercetin's ability to inhibit HSPs is also the finding in this same experiment that quercetin somehow contributes to the phosphorylation and undesired activation of HSP27.39 This finding is consistent with other findings that quercetin's actions are cell-type dependent, and Wang et al. were resolute enough to elucidate two quercetin derivatives that both inhibited HSP70 expression while not activating HSP27.39

Quercetin has also been shown to decrease HSP90 expression in prostate cells.38 HSP90 is a chaperone protein that aids in the maintenance of oncoproteins, such as human epidermal growth factor 2 (HER2) and insulin-like growth factor binding protein-2 (IGFBP-2). One can speculate that decreased HSP90 in the cell would likely lead to diminished oncoprotein functionality and subsequently decrease cancerous growth. HSP90 expression is positively correlated with the degree of aggression of prostate cancer cells and is overexpressed in malignant prostate cells.38 Aalinkeel et al.38 found that the levels of HSP90, HER2, and IGFBP-2 were reduced as the concentration (0–100 µM) of quercetin increased in prostate cancer cells (LNCaP and PC-3). Conversely, they found that quercetin treatment in healthy prostate cells did not show this affect.38 Quercetin's selective effect in this cell line may be due to its propensity to target HSP90, which is abundant in tumor cells but not in healthy cells. This feature of quercetin action, which has also been seen in other cell lines,38 makes it a viable substrate for use in anti-cancer therapy.

QUERCETIN AND ENDOPLASMIC RETICULUM STRESS

Very little research has been done on quercetin's affect on endoplasmic reticulum stress. However, a link can clearly be made. Recall that the endoplasmic reticulum is the cellular organelle responsible for the packaging and synthesis of many nutrients, among other functions. Endoplasmic reticulum stress is also known as the unfolded protein response, as an accumulation of misshapen proteins increases endoplasmic reticulum stress in cells.40 Heat shock proteins prevent endoplasmic reticulum stress by catalyzing refolding of proteins in the cell. Specifically, inhibition of HSP90 has been shown to induce endoplasmic reticulum stress and the subsequent endoplasmic reticulum stress proapoptotic pathways.41 Endoplasmic reticulum stress has been proposed to initiate mitochondria-mediated apoptosis by increasing intermitochondrial calcium concentration.41 The increased calcium concentration leads to increased recruitment of Bax,41 decreased mitochondrial membrane potential, and subsequently cytochrome C release from the mitochondria. This release triggers the activation of caspase-9 and -3, and then cell death.41 As mentioned previously, Aakinkeel et al.38 found that quercetin treatment was able to decrease HSP90 levels in the cell. Further, they found that the amount of caspase-9 and caspase-3 activity increases in a dose-dependent manner with the concentration of quercetin added (Figure 3).38 Although further research needs to be done, it is conceivable that quercetin is able to induce apoptosis via endoplasmic reticulum stress in some cell lines.

HSP70 is also involved in alleviating endoplasmic reticulum stress. As mentioned previously, quercetin decreases HSP70 expression in cells.39,40 Recently, MCF-7, T47D, and MDA-MB-435 cell lines have demonstrated that when HSP70 is inhibited by quercetin treatments of 100 µM, there is subsequent initiation of the unfolded protein response.40 This is problematic since this endoplasmic reticulum stress pathway initiates an increased expression of glucose-regulated protein 78 (GRP78).40 GRP78 functions to protect cells against chemotherapy and to increase cell survival.40,42 This provides a mechanism for cells to resist quercetin-induced apoptosis. However, it was recently established that when GRP78 is inhibited, there is increased quercetin-mediated apoptosis. This provides evidence that quercetin may be able to work cooperatively with other compounds in order to mediate cell death via the endoplasmic reticulum stress pathway.

In an indirect fashion, previous discoveries have provided researchers with more evidence that quercetin is able to involve endoplasmic reticulum stress pathways in order to decrease cell viability. Eukaryotic initiation factor-2 (eIF-2) is responsible for regulating protein synthesis in vivo.43 PKR-like endoplasmic reticulum kinase (PERK) is a protein located on the endoplasmic reticulum. PERK is responsible for the phosphorylation of the α-subunit of the eIF-2 and is activated under endoplasmic reticulum stress.43 When the eIF-2's α-subunit is phosphorylated it is unable to dissociate from another initiation factor, which essentially prevents mRNA and protein synthesis.43 Ito et al. observed that a 100 µM quercetin treatment was able to increase eIF-2 α-subunit phosphorylation and decrease protein synthesis in multiple mouse and human lymphoma and leukemia cell lines. Upon further investigation, this group found that quercetin was able to stimulate PERK activity, along with two other eIF kinases, in order to catalyze phosphorylation of eIF-2 and decrease protein synthesis.43 Decreased protein synthesis generally leads to decreased cell growth, repair, and viability. It is thought that this is further evidence that quercetin uses endoplasmic reticulum stress as a mechanism to induce eventual apoptosis and cell cycle arrest in tumors.43

INTERACTION OF PATHWAYS

Low levels of quercetin could likely be achieved in the diet for long periods without supplementation, so investigating the effects of dietary quercetin on cancer prevention is important, while investigating higher levels for therapeutic purposes would likely require supplementation or infusion during a therapy. However, because the exact cellular concentrations of quercetin and its accumulation in cells have yet to be determined, the cellular concentrations could be higher than currently supposed. Also, many of the mechanisms and pathways described in this review could be minimally activated at what would be considered low-dose exposure and then combined to act synergistically. For example, as discussed above, the mitochondrial and death-domain pathways commonly act together to induce apoptosis. Specifically, both TRAIL and mitogen-activated protein kinase pathways intersect and activate FAS, leading to cell death (Figure 2). In addition, the inhibition of multiple HSP pathways that converge to protect the cell could also result in a combinatory effect (Figure 3).

Studies on cancer prevention in mice also focus on factors other than ROS as a mechanism of cancer prevention, such as the modulation of various signaling pathways. For example, recent studies by Ma et al.,44 Moon et al.,45 and Miyamoto et al.46 indicated that quercetin at dietary concentrations inhibited proliferation and led to chemoprevention in mice. Therefore, it is also conceivable that pathways initiated by low and high doses could interact for therapeutic purposes. Many tumors outgrow their blood supply and are poorly perfused, giving way to areas of hypoxia.47 In this case, highly perfused areas of a tumor would likely achieve higher concentrations of quercetin compared to poorly perfused regions (Figure 4). Therefore, the study of quercetin requires knowledge of the effects of the conjugates, tissue concentrations, and the duration of exposure. Microenvironmental factors such as perfusion, pH, and hypoxia may also play a role.

Figure 4

Depiction of theoretical quercetin penetration/accumulation in a carcinogenic tumor. Quercetin is better able to accumulate near the well-vascularized areas of a tumor. The poorly vascularized areas of a tumor tend to have decreased perfusion resulting in micronutrient deficiencies. Additionally, in areas of decreased blood supply, there is increased interstitial tumor pressure and other microenvironmental factors that make delivery of molecules such as quercetin to these tumor regions a challenge.

CONCLUSION

This review presents some of the most recent data regarding the pathways involved in the quercetin response. It is proposed that quercetin could be used in both the prevention and treatment of cancer and that diet would likely fulfill the concentration requirements for prevention, but supplementation or another form of delivery could be necessary for therapeutic responses. Enzymatic modification of quercetin could further lower the threshold necessary for anti-tumor activity. It cannot be ruled out that greater understanding of these pathways could lead to the promotion of quercetin in conventional therapies and its use with other drugs in order to interact and produce a therapeutic effect at lower concentrations. Quercetin's ability to interact with electrons at higher concentrations plays a central role in its mechanism of action, mainly by the activation of proteins and DNA damage leading to the induction of many downstream pathways. Key challenges remain for the study of quercetin, including the determination of quercetin's activity and concentration at the tissue site, the intracellular concentration achievable, and the effect of conjugates on the pathways. Microenvironmental factors may also play a role. Further study is also needed to explain the observed discrepancies between cancer risk and quercetin-containing food intake in larger population-based studies.48 Undoubtedly, measuring any micronutrient in foodstuff is challenging because the amount differs depending on the growth conditions, varietals, and food-preparation methods to name a few.48 Studies collecting dietary data are also well characterized to have participant reporting bias that may skew outcomes.48 Given the confounding factors of epidemiological studies and that basic science research has produced ample proof that quercetin exerts anti-cancer properties, this molecular gem appears worthy of more attention and time in the limelight.

Declaration of interest

The authors have no relevant interests to declare.

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