The summary of the effects of flavonoids in breast cancer.
Breast cancer is the most prevalent cancer type among women. Despite recent progress in early detection and therapeutic strategies, the rate of mortality is increasing. Anti-estrogens or aromatase inhibitors are preferred to treat the women diagnosed with estrogen-receptor (ER) positive tumors. However, breast tumors usually show intra-tumoral heterogeneity with ER-positive and -negative cells. The advanced breast cancer cells lose the estrogen responsiveness and become aggressive by developing new strategies for rapid proliferation such as mutations in cell cycle machinery. New promising drugs are still being investigating against these types of tumors especially to overcome acquired resistance against chemotherapeutic drugs; however, a successful treatment for metastatic tumors is still unclear. Flavonoids, with various pharmacological activities, are plant or fungus secondary metabolites present in human diet. In plants, beside their role in pigmentation, they may also act as messengers, regulators and cell cycle inhibitors. Therefore, they are being tested in ovarian, cervical as well as breast cancer. Due to the positive correlation between flavonoids-rich diet and lower risk of cancer, flavonoids are referred as chemopreventive agents. The current chapter emphasizes the therapeutic potential of flavonoids and their synthetic analogues as anti-cancer agents in breast cancer providing new insights into the molecular mechanisms.
- breast cancer
The use of natural and dietary agents for cancer chemoprevention and therapy received attention for their health benefits. As consumption of fruits or vegetables has been associated with a reduced risk of human cancers especially breast cancer , dietary flavonoids, found particularly in these alimentary groups with more than 5000 polyphenolic compounds, have been identified as potential cancer-preventive components [2, 3]. Polyphenols can be divided into ten different classes based on their chemical structure . Flavonoids, phenolic acids, stilbenes, and lignans are the most abundant polyphenols in plants. Polyphenols, mainly flavonoids, possess a number of functions including pollination, pollen tube growth, resorption of minerals, and tolerance to abiotic stress . Flavonoids represented greater attention with the decreased incidence of cancer and cardiovascular diseases in Mediterranean population, which was associated with vegetables, fruits, and red wine consumption. Therefore, they have been under investigation for their therapeutic significance in the protection of human health for decades. Flavonoids are one of the common components in the human diet and generally are present as O-glycosides with sugars bound at C3 position .
Breast cancer is the leading cause of cancer death among women worldwide. Despite the presence of new promising advances in therapeutics, the breast cancer mortality rate is still increasing. Recent reports suggest that breast cancer prognosis is lower in countries consuming a healthy, plant-based diet . The possible cause to this scenario has been suggested as flavonoids in fruits and vegetables. Epidemiologic investigations showed that flavonoids exhibit important effects on cancer chemoprevention and chemotherapy. They have been shown to interact with different genes and enzymes including those playing role in antiproliferation, cell cycle arrest, apoptosis, angiogenesis, and multidrug resistance. Therefore, this chapter focuses on the chemopreventive and chemotherapeutical roles of flavonoids in the treatment of breast cancer .
2. Structure, classification and metabolism in humans
The chemical structure of flavonoids is based on a C15 skeleton with a chromane ring bearing a second aromatic ring B in position 2, 3, or 4 (Figure 1).
Flavonoids are subdivided into different groups based on the nature of C3 element: flavones, flavonols, flavanones, flavanols, anthocyanins, and isoflavones (Figure 2).
Flavonoids participate light-dependent phase of photosynthesis , and they catalyze electron transport. They have been shown to be synthesized from phenylalanine and tyrosine, the aromatic amino acids, with acetates . First, aromatic amino acids are converted to cinnamic acid and parahydroxycinnamic acid, respectively, by phenylalanine and tyrosine ammonia lyase enzymes . Then, parahydroxycinnamic acid accumulates with acetate units to give rise to cinnamoyl, which is the derivative of caffeic acid and chlorogenic acid. Cinnamoyl, then, is converted to ortho-hydroxyacetophenone with a benzaldehyde derivative generating flavonones. If ortho-hydroxyacetophenone condenses with a benzoic acid derivative, flavones are formed. Anthocyanins are naturally occurring glycosides of flavylium (2-phenyl-1-benzopyrylium) ions substituted by hydroxyl and methoxyl groups. Biotransformation of flavonoids occurs in the gut and various secondary metabolites are produced as well such as phenolic acids, lignins, lignans, and stilbenes .
Flavonoids, mainly flavanols and quercetin glucosides, are absorbed from the small intestine, while quercetin, quercetin galactoside, and many others are not . Those absorbed by the intestine have been shown to be transported through membrane and use both ATP-dependent pumps and ATP-independent transporters . Following absorption, they are metabolized
3. Flavonoid-rich food and medicinal plants
The plant extracts have been used as folk remedies against various health problems, including metabolic diseases, cancer, and neurodegenerative disorders. According to in vitro and in vivo studies, a number of plant species have antiproliferative and antitumoral role in breast cancer pathogenesis. In addition, plants which have higher amount of flavonoids are accepted as chemopreventive agents. According to the United States Department of Agriculture (USDA) database, the six subclasses of flavonoids are listed for 506 food items. According to the database, flavonols (quercetin, kaempferol, myricetin, isorhamnetin), flavones (luteolin, apigenin), flavanones (hesperetin, naringenin, eriodictyol), flavan-3-ols ((+)-catechin, (+)-gallocatechin (GC), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin 3-gallate, (−)-epigallocatechin 3-gallate, theaflavin, theaflavin 3-gallate, theaflavin 3′-gallate, theaflavin 3,3′ digallate, thearubigins), anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin), and isoflavones (genistein, daidzein, glycitein) are listed.
Generally, these dietary compounds are known with their antioxidant, anti-inflammatory, and anticarcinogenic effects. According to the Seven Countries Study report, the average consumption of quercetin, kaempferol, myricetin, luteolin, and apigenin in composite food samples have ranged from 6 mg/day in Finland to 64 mg/day in Japan, with intermediate intakes in the United States (13 mg/day), Italy (27 mg/day), and the Netherlands (33 mg/day). In a similar study report, average flavonoid intake in Hungarian population was lower compared to Dutch, Danish, and Finnish citizens. The intake of five flavonoids in 17 different diets was estimated. When diet types were compared to each other according to flavonoid consumption ratio, it was shown that South African diet is the lowest flavonoid consumed diet type as 1–9 mg/day consumption. In contrary, Scandinavian diet in correlation with population-based study outcomes was the higher flavonoid intake diet type (75–81 mg/day).
In addition dietary origin of the flavonoids varied between countries. While tea is the main dietary source of flavanoids in Japan by 95% and the Netherlands by 64%, alcoholic beverages such as famous resveratrol based popularity of red wine and beer in Italy by 46%. The vegetables and fruits are the most common dietary sources of Scandinavian countries such as Finland by 100%. Similar ratio was also observed in the United States by 80%. In Australia, tea is the major source of flavonoid, and flavan-3-ols are 75% of whole intake. Therefore, it is important to evaluate the chemopreventive and chemotherapeutic potential of flavonoids in breast cancer disease.
In this section, it is aimed to discuss potential molecular mechanism of above-listed flavanoids in breast cancer studies.
Quercetin is a natural dietary flavonoid which exerts antioxidant, anti-inflammatory and anticancer properties. Quercetin is found in barks of many plants, fruits, and vegetables. It is one of the well-established grape polyphenols like other members, resveratrol, naringenin, and catechin, can exert antitumoral, antioxidant, anti-angiogenic properties and modify selectively estrogen-receptor (ER). According to a recent study, it is found that quercetin at IC50 value (37 μM) modulated Twist and p38 MAPK signaling, which lead to apoptosis in MCF-7 and MDA-MB-231 breast cancer cells . In addition it is well documented that quercetin exerts its therapeutic effect through modulating different cellular targets. According to the previous study, it was shown that quercetin induced p21 CDK inhibitor with a concomitant decrease of phosphorylation of retinoblastoma (Rb), which inhibits the G1/S cell cycle progression by trapping E2F1. A low dose of quercetin induced mild DNA damage and Chk2 activation, which is the main regulator of p21 expression by quercetin. In addition, quercetin downregulated the cyclin B1 and CDK1, essential components of G2/M cell cycle progression. Inhibition of the recruitment of key transcription factor NF-Y to cyclin B1 gene promoter by quercetin led to transcriptional inhibition SKBR3, MDA-MB-453, and MDA-MB-231 cells [22, 23].
Similar to previous findings, MCF-7 breast cancer cells were exposed to the increasing concentrations of quercetin; consequently, cell viability ratios were decreased, and apoptosis was triggered. Following exposure of cells to moderate cytotoxic dose of quercetin for 48 h, cells undergo apoptosis due to activation of caspases. In addition, quercetin mediates the disruption of Bcl-2/Bax ratio in MCF-7 cells .
A dietary flavonoid, kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), is found in edible plants such as kale, beans, endive, tea, broccoli, cabbage, tomato, and grapes, is commonly used in traditional medicine (e.g.,
The use of plant derivatives, which exert biological functions, has gained importance in recent years. Myricetin (3,5,7,3′,4′,5′-hexahydroxyflavone cannabiscetin) is a natural flavonol, which has a unique hydrophobic chemical structure found in different varieties of fruits, vegetables, tea, berries, etc. . The dietary intake of myricetin from our foods is about 0.98–1.1 mg per day, which is quite higher than some other flavonols . Recent studies showed that myricetin is an antioxidant and it possesses cytoprotective, anticarcinogenic, antiviral, and antimicrobial effects .
Isorhamnetin is one of the important flavanols found in
In recent years, chemopreventive potential of fruits, vegetables, and medicinal herbs such as tea due to ingredients rich in phytochemicals that act as an antioxidant become an important agent. One of the polyphenolic flavonoids silymarin that is isolated from milk thistle (
Luteolin (3′,4′,5,7-tetrahydroxyflavone) which belongs to flavonoids is a heat-stable and nontoxic compound. It is found in vegetables and fruits such as celery, parsley, broccoli, onion leaves, carrots, peppers, cabbages, apple skins, and mignonette and chrysanthemum flowers. As well as other flavanoids, cardiovascular protection, immune system stimulation, antioxidant, anti-inflammatory, and anticarcinogenesis capacities of luteolin have been shown in previous studies . Luteolin exerts its molecular effect
Apigenin is known as the phytoestrogen, used in postmenapausal symptom treatment, and presented in various plant species. Although it is a nontoxic and non-mutagenic plant derivative, it exerts antitumoral activity in different types of cancers and induces oxidative stress in breast cancer cells . However, there are contradictory reports showing that apigenin might stimulate cell proliferation in ERα-positive MCF-7 and T47D cells, but not effective in ERα-negative MDA-MB-435 breast cancer cells . The molecular mechanism of apigenin- induced apoptotic cell death was caspase-dependent, mitochondria  and NF-κB, STAT signaling-mediated . Moreover, apigenin inhibited cell growth, metastasis, and invasion in breast cancer cells
It was shown that flavanone-rich diet mediated 0.1 –100 μM physiologically achievable concentration in the plasma. One of the mostly known flavanones is naringenin, which is especially abundant in the Mediterranean diet, rich for consumption of grapes, tomato, and citrus. Naringenin was shown with anticancerous effect in various cancer cells. According to in vitro studies, it was shown that naringenin modulated NF-κB to induce apoptosis in the cells. Naringenin was effective in MCF-7 ERα+/ERβ + cell line, but not in ER-independent SKBR-3 (ERα−/ERβ−) cell line [52–55].
Similar to naringenin, eriodictyol has promising therapeutic effects in cancer cells. Eriodictyol, a flavanone, activated Nrf2 and induced phase II proteins to exert its antioxidant effects [59, 60]. However, there are less studies to evaluate the molecular mechanism of eriodictyol compared to naringenin.
Hesperetin is also a promising flavanone and induced cell cycle arrest at G1 phase. According to the previous study, hesperetin regulated CDK4 and p21 (Cip1) in MCF-7 cells and led to block of cell cycle. Hesperetin is also known with its apoptotic effect in breast cancer cells without effecting normal mammary epithelial cells. It was shown that hesperetin induced apoptosis in dose- and time-dependent manner in MCF-7 cells through triggering ROS generation. Pretreatment of NAC prevented hesperetin-induced apoptosis, which is under control of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple-negative breast cancer MDA-MB-231 cells
The most important member of flavan-3-ols (catechins) is abundantly present in green and black tea, red wine and chocolate. Catechins, which are generally found in green tea, comprise epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (CG), epicatechin gallate (ECG), gallocatechin gallate (GCG), and catechin (C). Although green tea is a favorable catechin source, it is required to design more bioavailable structures to treat various cancer types, including breast cancer. The detailed investigation for EGCG was established in different studies. According to xenograft model studies, EGCG with tamoxifen has potential in ER-negative breast cancer models. The MDA-MB-231-mediated tumor volume was decreased following 25 mg/kg treatment of EGCG and/or EGCG + tamoxifen in athymic nude female mice model [65–68]. The potentiation of green tea catechins is generally acted on mTOR and EGFR pathways. Similar to these findings, studies indicated that EGCG produces anticancer effect by modulating the activity of MAPKs, IGF/IGF-1 receptor, Akt, NF-κB, and HIF-1α [69–73]. Although catechins have multiple molecular targets in the cells, it is required to improve their structural properties to achieve powerful treatment results.
Anthocyanins confer the bright red, blue, and purple colors to plants such as berries, grapes, and apples. Anthocyanidins lack the sugar component of the anthocyanin . Six anthocyanidins occurred most commonly in nature are pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin. It has been suggested that the consumption of cyanidin lowers the risk of cardiovascular disease, diabetes, and cancer due to the antioxidant and anti-inflammatory activities . The phenolic structure is responsible for the antioxidant activity such as the ability to scavenge superoxide (O2·−), singlet oxygen (‘O2), peroxide (ROO−), hydrogen peroxide (H2O2), and hydroxyl radical (OH·), members of ROS , in in vitro cell lines including colon, liver, and breast cancer cells .
The anticancer effect of cyanidin-rich extracts of different plant has been shown in MCF-7 ERα (+), MDA-MB-231 ER α (−), and MDA-MB-453 ER α (−) breast cancer cell lines. Moreover, apoptotic induction in MDA-MB-453 cells through the intrinsic pathway of apoptosis by activating caspase cascade, cleaving poly (ADP-ribose) polymerase (PARP), depolarizing mitochondrial membrane potential, and releasing cytochrome C has been shown . In addition, in the same study, 100 mg/kg/day oral administration of cyanidin has been shown to reduce tumor growth and angiogenesis by affecting the expression of angiogenic factors MMP-9, MMP-2, and cell/extracellular matrix (ECM) interaction in nude mice bearing MDA-MB-453 cell xenografts . Furthermore, inhibition of proliferation and cell cycle arrest were induced in MCF-7 human breast cancer cells after the treatment of bilberry extract, which contains high amount of cyanidin . The same study also compared the effect of cyanidin with a well-known antioxidant Trolox, a vitamin E analog, and showed that cyaniding induced apoptosis and cell cycle arrest as much as Trolox . In another study, pycnogenol, derived from pine bark, which contains high amounts of procyanidins, has been shown to induce cell death in breast cancer cells (derived from human fibrocystic mammary tissue) but not in normal human mammary MCF-10A cells .
Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% cases of breast cancer. Therefore, HER2-targeted therapies have been evaluated in recent years. In Liu et al.’s  study, cyanidin-3-glucoside, extracted from black rice, inhibited phospho-HER2 and phospho-AKT and induced apoptosis both in vitro and in vivo HER2-positive breast cancer cells and tissues. Another study also revealed that anthocyanidin-rich extracts from berries and grapes have been shown to exhibit proapoptotic effects in multiple cell types in vitro . They induce apoptosis through both intrinsic (mitochondrial) and extrinsic (FAS) pathways.
Delphinidin is a member of anthocyanins mainly found in pomegranate extract and found in many dietary supplements as complementary cancer medicine. A recent study showed that delphinidin treatment inhibited cell proliferation and induced apoptosis in ER-positive, triple-negative, and HER2-overexpressing breast cancer cell lines without any toxic effect in normal breast epithelial cells . In addition, the same study also indicated that MAPK signaling was inhibited in both triple-negative and ER-negative breast cancer cells but not in MCF-10A normal epithelial cells.
Breast cancer cells overexpressing p65, the unit of NF-κB responsible from cell survival and proliferation, underwent apoptosis following delphinidin treatment. The possible explanation to this process was shown as the inhibition of phosphatidylinositol 3,4,5-trisphosphate (PI3K)-dependent phosphorylation of AKT in vitro and inhibition of the activation of NF-κB in vivo . The same study also pointed out that miR-27a and miR-155 were able to inhibit PI3K and NF-κB and responsible from the anti-inflammatory and cytotoxic activity of delphinidin in MDA-MB-231 breast cancer cell line . Delphinidin has been also shown to inhibit hepatocyte growth factor (HGF)-mediated tyrosyl phosphorylation of focal adhesion kinase (FAK), Src, paxillin, Gab-1, and GRB-2, which are inducers of cell proliferation upon phosphorylation by growth factor signaling. Delphinidin, in the same study, was found to repress Ras-ERK MAPKs and PI3K/AKT/mTOR/p70S6K pathways . The compound also has anti-angiogenic and anti-invasive properties by decreasing MMP-9 activity in ER+ MCF-7 cells. Im et al. showed that delphinidin inhibited MMP-9 transcription by blocking NF-κB through MAPK signaling pathways .
Pelargonidin, a subclass of anthocyanin with estrogenic activity, was tested in MCF-7 breast cancer cells. The cytotoxic dose (5 μg/ml) of strawberry extract containing pelargonidin-3-O-glucoside caused 50% decrease in cell proliferation . A study performed in breast cancer tissue of rats showed that pelargonidin could inhibit the synthesis of cytochrome c p450 family 1 subfamily A member 1 (CYP1A1) enzyme which converts estradiol into 2-hydroxy-estradiol that can cause DNA damage . The inhibition of the estrogenic activity by 55% was also indicated following pelargonidin containing pomegranate seed oil in ER+ MCF-7 cells. On the other hand, pelargonidin treatment induced apoptosis in both MCF-7 and MDA-MB-231 (ER-). Seventy-five percent inhibition of invasion across a Matrigel was also observed in MCF-7 cells at 10 μg/ml pomegranate seed oil concentration. Studies suggest that further investigations on chemopreventive and therapeutic applications of pelargonidin should be performed against human breast cancer .
Daidzein, is one of the isoflavonoid present in various plants and herbs such as soybeans, tofu, kwao krua (
Soybean is one of the dietary components, which contains phytoestrogens and genistein acting as a chemopreventive agent against various cancer cells such as prostate and breast cancer. As a predominant isoflavone, genistein inhibits growth and proliferation of ER-positive and ER-negative breast cancer cells by inhibiting receptor-associated tyrosine kinase (RTK) signaling . Genistein inhibited cell proliferation, growth, invasion, and metastasis and acted as anticarcinogenic and anti-angiogenic compound on breast cancer in vitro and in vivo models . The molecular mechanism of anticarcinogenic effect of genistein on breast cancer cells is due to DNA topoisomerase, 5-reductase enzyme inhibition, suppressing the NF-κB, Akt and MAPK signaling pathways . Genistein is one of the flavonoids that has been shown to effect on chronic diseases such as atherosclerosis and hereditary hemorrhagic telangiectasia. The molecular target of genistein was reported to enhance the action of transforming growth factor-β (TGF-β) . Like other plant secondary metabolites (tocopherols, curcumin), flavonoids reported to regulate VEGF in breast tumors in vivo and in vitro studies. The anti-angiogenic, anticarcinogenic effect of genistein target VEGF receptor-2 (VEGFR-2) mediated PI3K/Akt/mTOR signaling pathway . Angiogenesis is the formation of new blood vessels and sprouting of circulation by activation of VEGF family member, VEGF-A, leading to endothelial cell proliferation, migration, and destruction of matrix metalloproteins. Although the anti-angiogenic effect of isoflavonoids has been reported in various studies, the exact molecular inhibition mechanism has not been clarified yet. One of the anti-angiogenic effects of genistein is the inhibition of VEGF and its receptor secretion. Ten to fifty micrometer genistein prevented basal VEFG expression both in breast cancer and human umbilical vein endothelial cells (HUVECs) . Moreover, under hypoxia conditions genistein has been shown to induce both VEGF downregulation and inhibition of hypoxia-inducible factor 1 (HIF-1) activation. The anti-angiogenic effect of genistein has been accelerated with curcumin-combined treatment in HUVEC cells by VEGFR-1 and VEGFR-2 downregulation . According to in vivo experiments such as xenografts, chick chorioallantoic membrane or zebra fish experimental models showed the reduction of microvessel density due to genistein treatment mediated by plasminogen activator inhibitor-1, endostatin, angiostatin, and thrombospondin-1 activation. Pretreatment with genistein leads to the reduction of MMP-2, MMP-3, MMP-13, and MMP-15 mRNA expression and VEGF-mediated plasminogen activator (PA) and PAI1 expression blockage. However, no significant effect has been determined on MMP-2 and MMP-9 activity. Antiproliferative and anti-angiogenic effect of genistein is also shown by inhibition of cadherin, integrin V, connexin 43 mRNA expression, and genistein (40 mmol/L), or daidzein (110 μm/L) treatment suppresses epidermal growth factor (EGF) and insulin-like growth factor (IGF-I) . NF-κB signaling pathway plays an important role in not only angiogenesis but also cell growth apoptosis, inflammation, and invasion. Thus, genistein treatment inhibited MMP-9 by NF-κB nuclear translocation-induced NF-κB signaling inactivation . In addition, genistein induced cell proliferation suppression by acting on MAPKs such as ERK-1/2, c-Jun N-terminal kinases (JNK), and p38 dephosphorylation. In order to clarify the anti-angiogenic effect of flavonoids, genistein, one of the major catalytic enzymes of prostaglandin production [cyclooxygenase-2 COX-2], associated VEGF production was investigated . COX isoenzyme catalyzes the production of prostaglandins, VEGF production, and angiogenesis induction. In MCF-7 breast cancer cells, genistein alone or combined treatment with capsaicin leads anti-angiogenic and anticarcinogenic effect
In summary, flavonoids can potentially contribute to breast cancer prevention and treatment either by antioxidant or apoptotic activity (Table 1). Previous studies highlighted that plant-derived flavonoids are promising when their bioavailability is increased to provide better therapeutic approach in the treatment of disease. However, elucidation of their molecular targets in cell type-specific manner may increase their potential therapeutic effects. Noteworthy that consumption of dietary flavonoids in diet types might be advised to control disease and poor prognosis.
|The effect caused by flavonoid treatment in breast cancer models|