The Hallmarks of Flavonoids in Cancer

Flavonoids represent an important group of bioactive compounds derived from plant-based foods and beverages with known biological activity in cells. From the modulation of inflammation to the inhibition of cell proliferation, flavonoids have been described as important therapeutic adjuvants against several diseases, including diabetes, arteriosclerosis, neurological disorders, and cancer. Cancer is a complex and multifactor disease that has been studied for years however, its prevention is still one of the best known and efficient factors impacting the epidemiology of the disease. In the molecular and cellular context, some of the mechanisms underlying the oncogenesis and the progression of the disease are understood, known as the hallmarks of cancer. In this text, we review important molecular signaling pathways, including inflammation, immunity, redox metabolism, cell growth, autophagy, apoptosis, and cell cycle, and analyze the known mechanisms of action of flavonoids in cancer. The current literature provides enough evidence supporting that flavonoids may be important adjuvants in cancer therapy, highlighting the importance of healthy and balanced diets to prevent the onset and progression of the disease.


Introduction
Flavonoids represent the largest group of polyphenols found in plant-based foods, including fruits, vegetables, grains, and herbs, as well as in beverages such as tea, wine, and juices [1]. In plants, flavonoids play the role of secondary metabolites, acting as protectors against biotic and abiotic threats, particularly in the defense against ultraviolet radiation and pathogen action. Additionally, they also actively participate in odor, flavor, and color determination in several species [2,3]. The concentration of flavonoids in food is related to several factors, including the variety of the phylum, order, family, and/or species of the plant, as well as the characteristics related to plantings, such as the type of soil, the climatic conditions of the region and the level of maturation of the food. Flavonoids concentration and composition also vary depending on the different parts of the plant. Leaves and the peels of fruits are commonly rich sources of flavonoids due to increased susceptibility to stress [4][5][6].
These compounds are composed of fifteen carbon atoms in their chemical structure (Table 1), presenting two benzene rings (A and B) connected through a heterocyclic ring containing oxygen (C). Flavonoids can be subdivided into flavones, isoflavones, flavanones, flavonols, anthocyanidins, and flavans. These classes differ from one another according rich in phenolic compounds suggests that flavonoids can protect against the development of several types of diseases, including cancer [15][16][17]. However, further studies in humans are needed since most of them are carried out in vitro or in animals. The development of these studies takes place through clinical trials that are classified as the gold standard in the evaluation of therapeutic and preventive actions in health [18]. Currently, several clinical studies are being developed considering flavonoids in cancer therapy (Table 2). Those who reached the final stage showed promising results in the use of these compounds as an auxiliary therapy in the treatment or prevention of various types of cancer. Therefore, more studies are necessary to obtain answers that have not yet been clarified regarding this subject.  Bioavailability is a complex process and depends on several factors, including stability of the digestive system (adequate concentration of enzymes and pH), the composition of the food matrix, and the initial concentration of the compound [27,28]. Most flavonoids are ingested in the usual diet in the form of glycosides, which have one or more sugar molecules linked to phenolic groups or the C-3 hydroxyl group [28]. Normally, these compounds can be absorbed in two ways: the first and simplest is related to the forms of aglycones, which are absorbed directly in the small intestine; the second involves substances found in their glycosylated form, which undergo metabolic reactions of biotransformation (phase I), including oxidation, reduction, and hydrolysis reactions. These compounds are affected by the action of Lactase-Phlorizin Hydrolase (LPH) or Cytosolic β-Glucosidase (CBG), present in intestinal epithelial cells, causing the separation and release of the phenolic compound in an aglycone form. After absorption, the compounds are metabolized by enzymes in the intestinal or liver cells that promote conjugation reactions with methyl, sulfate, and glucuronic acid groups (phase II). These conjugated metabolites are then transported to the bloodstream or returned to the digestive system through enterohepatic recirculation. The absorption of the phenolic compound may be low in the upper gastrointestinal tract. The compounds that were not absorbed in the small intestine pass into the large intestine, where they are metabolized by bacteria present in the microbiota and then can be absorbed or excreted in the feces [29][30][31]. Once absorbed, the flavonoids may have their systemic or local effects on different cell types and biological processes.
It is worth mentioning that there are limitations regarding the use of flavonoids, mainly due to their low bioavailability and toxicity in the human body. Regarding the bioavailability process, the concentration of flavonoids in food does not necessarily correspond directly to the actual amount absorbed and metabolized in the body, which can lead to a reduction of the beneficial effect on health [32,33]. As for toxicity, when flavonoids are ingested in the form of supplements in exorbitant amountss, they can induce pro-oxidant activity, mitochondrial toxicity (potential apoptosis-inducing properties), and interactions with metabolic enzymes [34,35]. Currently, the development of nanoformulations is an alternative to reduce these limitations [36], however, studies targeting methods to improve bioavailability, as well as the concentrations that avoid flavonoid toxicity, should be further investigated.
In this context, this review aims to clarify how flavonoids act in the modulation of several biological signaling pathways and defensive systems involved in cancer, including inflammation and immunity, redox metabolism, cell growth, autophagy, apoptosis, and cell cycle (Figure 1). Considering the great number of studies in this area, here we present detailed figures demonstrating the participation of different classes of flavonoids in several steps of those signaling pathways. We also highlight the state-of-art research of these compounds, presenting in-depth tables containing molecular mechanisms, compound concentrations, incubation conditions, and experimental models regarding recent in vitro and in vivo studies. We believe that the compilation of this information brought by this review may greatly contribute to future studies in the area. Flavonoids significantly alter the biological processes involved in cancer, including inflammation and immunity, redox metabolism, cell growth, autophagy, apoptosis, and cell cycle. Signaling pathways related to these biological processes, which include Mitogen-Activated Protein Kinases (MAPK), Nuclear Factor-κB (NF-kB), Nod-Like Receptor Pyrin domain containing 3 (NLRP3) inflammasome, Signal Transducer and Activator of Transcription 3 (STAT3) pathway, Warburg effect, Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Hypoxia-Inducible Factor 1 alpha (HIF1α), mechanistic Target Of Rapamycin (mTOR), Ras, macroautophagy, cell cycle checkpoints, intrinsic and extrinsic apoptotic pathways, and senescence, are modulated by flavonoids in cancer and highlighted in this figure. Original figure made for this review using the Biorender software.

Flavonoids, Inflammation and Immunity
Inflammation has been classified as one of the hallmarks of cancer and is involved in the progression of most types of cancer [14]. Innate and adaptive inflammatory responses are mainly mediated by immune system cells, such as macrophages [37]. Thus, in this section, the immunomodulatory and anti-inflammatory properties of flavonoids will be explored by focusing on specific signaling pathways such as Nuclear Factor-κB (NF-κB), Mitogen-Activated Protein Kinases (MAPK), Nod-Like Receptor Pyrin domain containing 3 (NLRP3) inflammasome, and Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT). A summary of the actions of flavonoids in inflammation and immunity is presented in Table 3.
Some studies showed that Quercetin ameliorates the inflammatory process in cancer cells [55,67]. Quercetin presented chemopreventive effects against metastatic cell lines of the human salivary gland, since it increases the expression of IκB-α, an inhibitor of NF-κB activation, and reduces translocation of NF-κB to the nucleus [67]. In HeLa cells, Quercetin reduced IκB-α and IKK-β phosphorylation, NF-κB members (p50 and p65), and cyclin-D1 expression [55].
A study showed that Quercetin reduced the production of nickel-induced cytokines, such as IL-1β, IL-6, TNF-α, and IL-10, in lung cancer cell lines. In addition, Quercetin suppressed the expression of TLR4 and Myd88, in addition to reducing the phosphorylation of IKK-β and IκB-α, the nuclear expression of p65 (NF-κB), and the expression of MMP9 in A549 cells exposed to nickel. Thus, Quercetin presents potential preventive effects in lung cancer [56].
p38 is a serine/threonine kinase activated by direct phosphorylation of MKK3 and MKK6 and contributes to AP-1 activation through phosphorylation and activation of Activating Transcription Factor 2 (ATF2) and Ternary Complex Factors (TCFs) [79,80]. p38 has a key role in inflammatory diseases, and its role in cancer is contradictory [81]. p38 may be considered a tumor suppressor, however, some studies also provide evidence for an oncogenic potential related to its pro-inflammatory properties, capacity to regulate epithelial-mesenchymal transition, and angiogenesis [82][83][84][85]. A study showed that Kaempferol reverses LPS-induced inflammation through inhibition of c-fos and decreases the activity of the AP-1 complex in RAW 264.7 macrophages [60]. Luteolin is another flavonoid that possesses an impact on the MAPK pathway. Kang et al. (2010) showed that Luteolin can reduce phosphorylation of JNKs (JNK1, and JNK2) and inflammatory cytokines (TNF-α, IL-8, IL-6) induced by PMA and A23198 in HMC-1 cells [63]. Naringenin inhibits p38, MMP2, and MMP9 activity, blocking migration in glioblastoma cells [86]. Delphinidin is also able to block PMA-induced JNK and p38 MAPK pathways in MCF-7 cells, reducing PMA-induced breast cancer cell invasion. Vitexin, a derivative of Apigenin, also has anti-inflammatory activity through modulation of the MAPK pathway [87]. Rosa et al. (2016) showed that Vitexin reduces neutrophil migration and decreases pro-inflammatory mediators, such as TNF-α, IL-1β, and Nitric Oxide (NO) releases in the peritoneal cavity of LPS-treated mice. In addition, vitexin can reduce TNF-α, IL-1β, NO, Prostaglandin E2 (PGE2) levels and increase IL-10 release in LPS-treated RAW 264.7 cells. Mechanistically, Vitexin inhibits the phosphorylation of p38 and JNK, which explains its described anti-inflammatory effects [87].

The Inflammasome Pathway
Inflammasomes are multimeric protein complexes that are part of the innate immune system. The most studied inflammasome complex is the NLRP3, which consists of the NLRP3 sensor, the Apoptosis-associated Speck-like protein containing CARD (ASC) adapter, and the pro-caspase 1. The NLRP3 sensor has a Pyrin Domain (PYD) in its Nterminal, a central domain of nucleotide-binding and oligomerization (NATCH), and a Leucine-Rich Repeats (LRR) in its C-terminal [88]. The NLRP3 complex is activated by at least two signals. The first involves NF-κB signaling activation, which upregulates pro-IL-1β, pro-IL-18, and NLRP3 protein levels. The second involves extracellular ATP mediated-P2X7 receptor activation, K + efflux, and a set of inflammation-inducing stimuli, such as Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular Patterns (DAMPs) [89,90]. The activation of the NRLP3 inflammasome complex occurs by the oligomerization of NLRP3 mediated by the homotypic interaction between NATCH domains. NLRP3 also interacts with the ASC, an adapter protein, through PYD domains. ASC then recruits cysteine protease pro-caspase-1 through CARD-domain interactions, resulting in autocatalysis and activation of caspase 1 [88]. Pro-IL-1β and pro-IL-18, potent pro-inflammatory cytokines, can be cleaved into biologically active forms of IL-1β and IL-18 by caspase 1, promoting pyroptosis, a form of cell death programmed by inflammation ( Figure 2) [91].
The NLRP3 inflammasome is involved with several inflammatory-based diseases, including cancer [92]. Studies have shown that NLRP3 increased expression and/or activity in several types of cancer, such as melanoma [93], head and neck squamous cell carcinoma [94], lung squamous cell carcinoma [95], pancreatic [96], and bladder cancer [97]. Thus, NLRP3 has been investigated as a potential and attractive therapeutic target. Figure 2 illustrates the activation of the NLRP3 inflammasome and summarizes the point actions of flavonoids in this pathway.
Ulcerative colitis (UC), a chronic and inflammatory bowel disease, is an essential factor of colorectal cancer [98]. NLRP3 mediates Dextran Sodium Sulfate (DSS)-induced ulcerative colitis in mice [99]. A study showed that Genistein can inhibit NLRP3 activation and protect DSS-treated mice from ulcerative colitis [100]. In vivo, Genistein also suppressed the production of IL-1β, caspase-1, and the protein level of NLRP3. In macrophages, Genistein inhibited the NLRP3 inflammasome by activating the G-protein coupled bile acid receptor 1 (TGR5)-cAMP signaling pathway, which signals for ubiquitination and degradation of NLRP3 [100]. Thus, Genistein may be an important candidate in the prevention or treatment of ulcerative colitis and colorectal cancer.
Another study showed that Gen-27 is a potent inhibitor of LPS-induced inflammation in RAW264.7 cells, through inhibition of NLRP3 and NF-κB signaling pathways [101]. Gen-27 inhibited nitrite and nitric oxide levels, suppressed the release of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-18, and decreased the expression of COX-2 and iNOS. In addition, Gen-27 decreased caspase 1 activity, NLRP3 protein levels, and NF-κB (p65) transcriptional activity [101]. Genistein is a promising therapeutic targeting the prevention and therapy of diseases associated with inflammation. Apigenin also reduced LPS-induced inflammation by several mechanisms in human THP-1-derived macrophages [64]. One of them involves inhibition of NLRP3 inflammasome, causing inhibition of caspase 1 activity and IL-1β production [64].

The STAT Family Pathway
Members of the STAT family are signal transducers and activators of transcription [102]. Cytokines such as interleukins, interferons, and peptides hormones can bind to cell surface receptors leading to their homo or heterodimerization. This event causes activation of JAK proteins, which phosphorylate tyrosine residues in the tail of the receptor, thereby creating docking sites for the STAT-receptor interaction. Then, JAK phosphorylates STAT, which is released from the receptor and interacts with other STAT proteins through SH2-domains ( Figure 2). JAK-STAT signaling is one of the main pathways in the conversion of the cytokine signal to the response of gene expression, coordinating the proliferation and differentiation of immune cells. STAT1, STAT2, STAT3, STAT4, and STAT6 proteins have pro-or anti-inflammatory properties and are activated by cytokines in inflammation processes [103]. STAT3 is the most studied member of the STAT family. Various cytokines such as IL-11 [104], IL-23 [105], IL-21 [106], IL-6 [107], IL-17 [108], genes related to Epithelial-Mesenchymal Transition (EMT), such as TWIST, MMP2, and MMP9 [109], and angiogenesis such as VEGF [109] are upregulated by STAT3 and reinforce the oncogenic role of this protein.
In leukemia cells, HL60, and TF1 cells, Apigenin exerts a negative regulation in the JAK/STAT pathway. In both cell lines, Apigenin reduces JAK2 and STAT3 phosphorylation, and in TF1 cells it also reduces STAT5 phosphorylation [112]. In the same study, Apigenin also impaired phosphorylation and activation of Src, an activator of STATs, in both cell lines [112]. Cao et al. (2016) showed the role of Apigenin in the regulation of STAT3 in melanoma B16F10 cell lung metastasis [113]. Apigenin enhances the immune response in these cells by inhibiting STAT3 and its direct target, VEGF, which has an immunosuppressive function. Apigenin also downregulates other STAT3 target genes like MMP2, MMP9, and TWIST1, thereby reducing cell migration and invasion [113].
The ethanolic extract of black raspberries has two abundant phytochemicals metabolites upon ingestion called Cyanidin-3-rutinoside and quercitin-3-rutinoside. Both reduced the phosphorylation of STAT3 in peripheral blood mononuclear cells treated with IL-6 [115]. In conclusion, flavonoids can reduce several inflammatory markers such as cytokines, chemokines, inflammatory enzymes, and proteins related to migration and invasion in cancer through modulation of NF-kB and JAK/STAT3 signaling, AP-1 complex, and NLRP3 inflammasome.

Flavonoids and Redox Metabolism
Reactive Oxygen Species (ROS) are among the most important mutagenic factors that occur naturally in the body and cause genetic instability within cells. Such instabilities can generate harmless or malignant mutations, which can eventually lead to cancer. It is known that malignant changes at the genomic level are the main pathological driving force of carcinogenesis, thus being frequently associated with oxidative stress [120]. Multiple biochemical reactions where oxygen is metabolized can lead to the generation of toxic reactive intermediates that can damage DNA [121]. Adaptive changes, which increase over long periods cumulatively, must occur within a cell for the malignant transformation. On the other hand, there are several examples of how genetic mutations (inherited or acquired) lead to increased ROS production, which in turn is associated with DNA damage and contributes to the malignant transformation [122,123]. Therefore, cancer cells depend on and adapt to this highly unstable and mutagenic environment, further highlighting the crucial role of oxidative stress in cancer [124].
The metabolic changes involved in carcinogenesis contribute to a high degree of oxidative stress in the tumor environment. However, oxidative stress defenses are adapted in cancer, enabling cancer cells to survive [124]. This is associated with the fact that most cancer cells no longer use the complete oxidative phosphorylation to generate ATP, but instead glycolysis, which is known as the Warburg effect. Such an effect has much influence not only on the energetics of cells but also on the redox system, the resilience, and the adaptation of cancer. Several molecular changes are known to relate the metabolic adaptations of cancer cells and the redox balance.

Metabolic Alterations in Cancer
In the Warburg effect, the M2 isoform of Pyruvate Kinase (PKM2), although less enzymatically efficient compared to the M1 isoform, is more commonly found in tumors due to its promotion via Myc Proto-Oncogene Protein C (c-Myc) [125]. The c-Myc oncoprotein affects the splicing of Pyruvate Kinase (PK) mRNA through the positive regulation of the Polypyrimidine Tract Binding protein (PTB) and heterogeneous nuclear Ribonucleoproteins (hnRNPs) A1 and A2, leading to the predominant production of PKM2 [126]. The less efficient PKM2 is advantageous for cell proliferation, as it enables the entry of carbohydrate metabolites from glycolysis into alternative pathways to produce macromolecules and Nicotinamide Adenine Dinucleotide Phosphate (NADPH), which are necessary for tumor growth and the support of altered redox balance [127]. This is accomplished since PKM2 shifts metabolic precursors from glycolysis to the pentose phosphate pathway to produce NADPH and ribose. Isocitrate Dehydrogenase Dependent on NADP 1 (IDH1), IDH2, and Malic Enzyme 1 (ME1) also contribute to the production of NADPH [128]. Myc increases glutamine uptake and glutaminolysis, leading to de novo synthesis of Glutathione (GSH). Therefore, Myc contributes to the production of NADPH, promoting the expression of PKM2. Together, NADPH and GSH control the increased levels of ROS driven by the increased proliferation of cancer cells [128].
With the influence of the Warburg effect, GSH plays a central role in the cellular antioxidant defense system in cancer. GSH participation in cellular metabolic redox processes and elimination of ROS is present in all existing aerobic organisms [129]. GSH is a non-enzymatic antioxidant that is intracellularly synthesized from cysteine, glycine, and glutamate and is highly abundant in all major cell compartments, such as cytosol, nuclei, and mitochondria [124]. At the cell nucleus, GSH protects sulfhydryl groups of proteins essential for DNA repair and gene expression. GSH antioxidant properties are also manifested in the direct elimination of hydroxyl radicals and singlet oxygen (O 2− ), hydrogen peroxide (H 2 O 2 ), lipid peroxides, and even 4-Hydroxynonenal (HNE), in conjunction with the enzymatic action of Glutathione Peroxidase (GPx) and Glutathione Transferases (GSTs). GSH is also involved in the reductive regeneration of important antioxidants, including water-soluble vitamin C and lipid-soluble vitamin E [129]. The oxidized form of GSH is Glutathione Disulfide (GSSG), formed by the oxidation of two GSH molecules. GSSG is generally formed during the reduction of organic hydroperoxides and inorganic peroxides such as H 2 O 2 in enzymatic reactions catalyzed by GPx or Peroxiredoxins (PRXs). GSSG can be reduced back to GSH in the tandem enzymatic action of Glutathione Reductase (GR) and the reducing equivalent NADPH + H + . Therefore, the GSH:GSSG ratio is considered an important indicator of redox balance in cells, where a higher ratio means less oxidative stress [130].
Another important factor for redox metabolism in cancer is NRF2, which is one of the main antioxidant transcription factors and regulates positively the expression of various antioxidant and detoxifying molecules [139]. When ROS levels are low, NRF2 binds to Kelch-like ECH-Associated Protein 1 (KEAP1), which triggers the degradation of NRF2. Under oxidative stress, p53 is activated and stimulates the expression of p21 [128]. p21 prevents KEAP1-NRF2 interaction and preserves NRF2, increasing antioxidant protection [139], through the nuclear heterodimerization with Musculoaponeurotic Fibrosarcoma Proteins (MAFs) [140]. The loss of p53 in a cancer cell inactivates this redox maintenance mechanism: as p21 is not activated, NRF2 continues to be degraded, antioxidant proteins are not expressed, and the redox balance is lost [128]. It may be possible to explore mutations of loss of p53 function or other tumor suppressor genes by applying additional oxidative stress. In the absence of redox maintenance pathways that are supported by these tumor suppressors, malignant cells can be selectively killed [141].

Flavonoids and Oxidative Stress in Cancer
The best-described property of almost all groups of flavonoids is their ability to act as antioxidants [142]. The antioxidant activity of flavonoids has already been shown to depend on the organization of functional groups on the nuclear structure of the molecule [7]. The configuration, substitution, and the total number of hydroxyl groups substantially influence the antioxidant activity, such as radical scavenging and metal ion chelation capacity [143]. The hydroxyl configuration of the B ring is the most significant determinant for the elimination of ROS and Reactive Nitrogen Species (RNS) because it donates hydrogen and electron to hydroxyl, peroxyl, and peroxynitrite radicals, stabilizing the flavonoid radical [7].
In healthy tissues, the antioxidant mechanisms of flavonoids may include suppression of ROS formation by inhibiting enzymes such as GST or by chelating trace elements involved in the generation of free radicals [144]; direct neutralization of ROS; and positive regulation or protection of antioxidant defenses [145]. In cancer, such mechanisms of action are also observed, however, the countless metabolic differences in cancer cells significantly affect the oxidative balance of the cell. Therefore, it is necessary to understand how the oxidative balance is affected in a cancer cell to understand the action of flavonoids in this context.
There is growing evidence of the importance of flavonoids in modulating the carcinogenic pathways associated with glucose metabolism that indirectly affect the redox balance of the cancer cell. Flavonoids target the activity of certain enzymes involved in aerobic glycolysis, the expression of transporters responsible for glucose uptake, the modulation of HIF1 in normoxic conditions, and several other factors associated with the Warburg phenotype [146].
Several inhibitory effects of flavonoids in cancer were analyzed in vitro through evaluations of the enzymatic activity of PKM2, as shown in Figure 3. Among these flavonoids, Taxifolin, Apigenin, Catechin gallate, and Epicatechin are the most effective in inhibiting PKM2 activity in in vitro experiments [147,148]. Additionally, Quercetin significantly decreased the level of glycolysis-related proteins, including PKM2, by modulating the protein kinase B (AKT) and mechanistic Target Of Rapamycin (mTOR) pathway in vivo [149]. Quercetin also reduced the level of PKM2 in the colon mucosa of F344 rats, pointing to a chemopreventive role of this flavonoid [150]. In the context of the Warburg effect, Kaempferol reduced the mRNA levels of the Glucose Transporter 1 (GLUT1) and prevented the uptake of Monocarboxylate Transporter 1 (MCT1), leading to the accumulation of extracellular lactate in breast cancer [151]. Epicatechin stimulated mitochondrial respiration in vitro in pancreatic cancer cells [152] and can act indirectly on the Warburg effect via MAPK/ERK2/Cytochrome c Oxidase (COX) [153]. In addition, flavonoids such as Icariside II, aspalatin, baicalein, and hesperetin are effective in important molecules in the Warburg effect, such as IDH1, IDH2, OCT1, PDK1, and NOX2 [154][155][156][157][158].
Flavonoids have shown direct effects on glutathione metabolism in cancer models. GSH and GST levels were increased after treatment with Luteolin in colorectal cancer models [159,160]. Apigenin treatment increased the expression of genes encoding phase II enzymes, blocking the NADPH oxidase complex and, consequently, downstream target inflammatory genes, which leads to increased NRF2 expression and nuclear translocation [161]. In addition to the proteins involved in cell growth and the Warburg effect, flavonoids have been shown to stabilize important oxidative factors such as HIF1 and NRF2, as already mentioned above [162].
The modulation of NRF2 expression for cancer therapy is well studied, both for cancer types that have it overexpressed, and for those that express it less. [163,164]. The class of NRF2-inducing compounds helps in the detoxification of carcinogens and environmental mutagens ( Figure 3). They can also decrease the levels of ROS in tumor cells and make them susceptible to therapy [165]. Tamoxifen-resistant MCF7 breast cancer cells showed a response to a combination of EGCG and siRNA against NRF2 [166]. A similar mechanism has been observed in cervical cancer, where EGCG increased cisplatin activity and induced apoptosis [167]. Luteolin can potently inhibit NRF2 in A549 NSCLC cells, increasing their sensitivity to various anticancer drugs, as well as in a xenographic model of nude athymic mice [168]. Quercetin also directly interacts with NRF2: as part of the NAD(P)H Quinone Dehydrogenase 1 (NQO1) induction process, Quercetin binds to NRF2 protein and increases its half-life four times [169,170]. Apigenin inhibited the NRF2/ARE/CAT pathway in doxorubicin-resistant hepatocarcinoma (BEL-7402/ADM and HepG2) [171,172]. Apigenin inhibited NRF2, at the mRNA level, and detoxification enzymes in phase 2, at protein and mRNA levels, in BEL-7402/ADM cells [172], and sensitized doxorubicin-resistant (DOX-resistant) BEL-7402/ADM cells to doxorubicin (DOX), reducing the IC50 value of DOX treatment [173]. Myricetin activated NRF2 by regulating KEAP1 interaction, also decreasing NRF2 ubiquitination and increasing Heme Oxygenase-1 (HO-1) levels in hepatocarcinoma [174]. Epicatechin treatment has been shown to increase NRF2 phosphorylation and nuclear translocation also in hepatocarcinoma cell culture [175]. Taxifolin also inhibited TPA-induced colon carcinogenesis in albino Swiss mice through the epigenetic induction of NRF2 [66,176]. Genistein reduced the methylation level of the KEAP1 promoter region, leading to increased mRNA expression, thereby effectively inhibiting NRF2 transcription to the nucleus [177]. In this context, Vitexin increased p21 expression and decreased CDK1 in melanoma cell lines and a mice model, suppressing melanoma cell growth through DNA damage by increasing ROS levels [178]. Regarding HIF1, many flavonoids were also effective in the anticancer context, as shown in Figure 3. For example, Quercetin inhibited the accumulation of HIF1α, as well as its synthesis under hypoxia conditions and in a concentration-dependent manner in several cell lines, including LNCaP prostate cancer cells, SKBR3 breast cancer cells, and CX-1 colon cancer cells [179,180]. Interestingly, Quercetin increased the accumulation of HIF1α in healthy cells, showing improvement in the therapeutic index of DOX through its opposite effects on HIF1α in healthy and cancer cells [181]. EGCG also significantly suppressed the accumulation of HIF1α protein in gastric cancer cells but did not affect the expression of HIF-1α mRNA [182]. The mechanism linked to the HIF1 inhibitory properties of EGCG is explained by its interference with the PI3K/AKT/mTOR pathway and its translation function [183]. Luteolin suppressed HIF1 activation in M2-like tumor-associated macrophages under hypoxia conditions [184]. Kaempferol showed strong inhibitory effects on HIF1 activity in Huh7 hepatocellular carcinoma cells by relocating HIF1 into the cytoplasm due to inactivation of p44/p42 MAPK, which decreased cell viability under hypoxia conditions [185]. In addition to these flavonoids, Apigenin also downregulated hypoxia-responsive genes, such as HIF1α, GLUT-1, and VEGF, in human pancreatic cancer cells [186]. Genistein sensitized liver cancer cells to apoptosis, directly regulating HIF1α, inactivating GLUT1 and Hexokinase 2 (HK2) to suppress aerobic glycolysis [187].
When observing associated redox pathways, it is possible to identify several effects of flavonoids on cancer (Table 4). In vivo studies have indicated that Naringenin can suppress the early stage of colon cancer by attenuating levels of iNOS and COX-2 in mice injected with carcinogen [188,189]. Luteolin also inhibited iNOS and COX-2 [190]. The treatment of murine hepatomas with Daidzein and/or Genistein led to increased expression of Quinone Reductase (QR) mRNA and its activity, as well as greater NRF/ARE binding capacity [191]. Apigenin and Luteolin treatments in osteosarcoma models showed Forkhead box protein O1 (FOXO1) translocation and reduced Glucose-6-Phosphatase (G6Pc) mRNA levels, as well as Phosphoenolpyruvate Carboxykinase (PEPCK), CAT, and SOD [192,193]. Kaempferol triggered the generation of ROS and apoptosis by reducing the concentrations of thioredoxin and proinflammatory cytokines, also increasing SOD activity in glioblastoma cells [194]. On the other hand, in a colon cancer model, Kaempferol blocked the generation of ROS, causing the cell cycle to arrest in G1 and G2/M and affecting cell migration [195,196]. In stomach cancer, Kaempferol reduced the expression levels of COX-2, p-AKT, and p-ERK, which are involved in cell proliferation and cell cycle arrest [197]. Inflammation can also be inhibited by anthocyanins (Delphinidin, Cyanidin, and Petunidin) through the PI3K/AKT and NF-κB pathways, suppressing the expression of COX-2 and iNOS and regulating the expression of antioxidant enzymes from phase II via NFR2/ARE signaling [198,199]. Vitexin also decreased ROS levels and increased GSH and SOD levels in pheochromocytoma cells [200]. Down-regulates of HIF1α and GLUT-1 mRNA expression. Represses hypoxia-mediated induction of GLUT-1 expression.

Flavonoids and Cell Growth Signaling
In mammalian cells, growth signaling is necessary from embryogenesis to adult cells, ensuring adequate functions in cells [203]. Hanahan and Weinberg described that one of the main and fundamental characteristics of cancer cells is to sustain a chronic proliferation state [14]. In normal cells, there is a fine regulation of the extracellular growth stimulus, while cancer cells develop metabolic autonomy to support chronic growth signaling [203]. Cancer cells enabling signals are based in large part on growth factors that bind cellsurface receptors, typically the Tyrosine Kinase Receptors (TKRs) [14]. These receptors are messengers of pathways that control tumor progression, including the AKT/mTOR and Ras/ERK signaling. In this topic we explore one of the hallmarks of cancer, focusing on what are the main targets of cell proliferation in which flavonoid compounds can act. A summary of the mechanisms of action of flavonoids in cell growth signaling is shown in Table 5.

The AKT/mTOR Pathway
In tyrosine kinase signaling cascades, insulin and other growth factors, such as IGF, bind to their receptors to promote the recruitment of Insulin Receptor Substrate 1 (IRS1) and the production of Phosphatidylinositol (3,4,5)-triphosphate (PIP3) through the activation of Phosphoinositide-3-Kinase (PI3K) [204]. PI3Ks catalyzes the conversion of PIP2 to the second messenger PIP3 and its reaction is reversed by the Phosphatase and Tensin Homolog (PTEN) (Figure 4) [205]. The PIP2 conversion recruits and activates the AKT, which phosphorylates the Tuberous Sclerosis Complex 2 (TSC2), dissociating the TSC1/2 protein complex and promoting mTOR activation [204].
Most flavonoids can inhibit mTOR signaling early on. Vitexin [206,207], Taxifolin [208], and Eriodyctiol [209] decreased p-PI3K in human lung cancer cells, as Daidzein [210,211] in human breast and ovarian cancer cell lines, and Delphinidin [212] in human lung cancer cells. Some of these compounds decreased p-PI3K while increasing PTEN expression, such as Kaempferol in hepatocarcinoma [213] and cervical cancer cells [214] and EGCG in pancreatic cancer cells [215], reinforcing the inhibition of mTOR signaling by these two axis. Remarkably, Taxifolin can bind to EGFR and PI3K and decrease their activities in murine epidermal JB6 P+ cells [216]. mTOR has the same catalytic subunits in two distinct complexes (mTORC1 and 2), which respond differently to rapamycin: while complex 1 has most functions inhibited, complex 2 is insensitive to the acute treatment [217]. Since its discovery, the mTOR pathway has been suggested as a central mechanism that converges different signaling pathways to coordinate cell growth [218]. Thus, mTOR acts as a sensor of energy, nutrient availability, and growth factors to orchestrate assertively catabolism and anabolism [218]. Overactivation of mTOR is commonly reported in cancer and is extensively associated with poor prognosis [219,220]. The sensitivity to mTOR inhibition by rapamycin may vary by several orders of exposure, indicating an intrinsic resistance to mTOR inhibition in different cancer types [221].
When active, mTORC1 phosphorylates substrates that control the production of proteins, lipids, nucleotides, and ATP, initiating anabolic programming while limiting the autophagic process [218]. During protein synthesis and translation initiation, mTOR mainly phosphorylates Eukaryotic Initiation Factor 4E-Binding Proteins (4EBPs) and p70 S6 Kinase 1 (S6K1), leading to the release of Eukaryotic Translation Initiation Factor 4E (eIF4E) by 4E-BP1 and Ribosomal Protein S6 (S6/RPS6) phosphorylation by S6K1 (Figure 4) [218,222,223]. The flavonoids Quercetin [224], Apigenin [225], and Luteolin [226] decreased 4E-BP1 phosphorylation in breast, keratinocytes, and lung cancer cells, respectively, presumably limiting protein translation. Notably, Quercetin at 15 µM decreased p-4EBP1, p-S6K1, p-AKT in breast cancer cell culture and decreased tumor growth after 13 weeks of treatment in the MDA-MB-231 xenograft model, highlighting that flavonoid as a rationally approach against breast tumor progression [224]. In the AKT/mTOR pathway, one of the primary stages of signaling is the conversion of PIP2 to PIP3. This conversion can be inhibited by some flavonoids by inhibiting their converter, PI3K, or by stimulating the reverse conversion of PIP3 to PIP2 through PTEN, known as a tumor suppressor. Although some compounds stimulate AKT, probably through a feedback loop, most flavonoid compounds can inhibit AKT, preventing the activation of mTOR and, consequently, its effectors. The compounds directly inhibit mTOR, in addition to inhibiting 4EBP1/2 and S6K1, proteins that control especially the translation initiation and ribosomal and mRNA biogenesis. The compounds also stimulate p53, which targets SESTRIN 1/2, to inhibit the AKT/mTOR pathway by stimulating AMPK. mTOR signaling also controls lipid synthesis and angiogenesis, which can be both inhibited by flavonoids. These inhibitory regulations direct the cells to inhibition of cancer progression. Epi: Epigallocatechin, Kae: Kaempferol; Myr: Myricetin; Que: Quercetin; Api: Apigenin; Gen: Genistein; Dai: Daidzein; Lut: Luteolin; Nar: Naringenin; Tax: Taxifolin; Eri: Eriodictyol; Vit: Vitexin. Original figure made for this review using the Biorender software.  In JAR cells, increases p-ERK1/2, p-AKT, and p-S6K1, but decreases p-S6. In JEG cells, increases p-ERK1/2, p-S6K1, but decreases p-S6 and p-AKT   Since cell growth demands lipid synthesis, mTOR also coordinates the lipid synthesis through two main processes: activation of Sterol Regulatory Element-Binding Proteins 1/2 (SREBP1 and 2) and Peroxisome Proliferator-Activated Receptor-γ (PPARγ) (Figure 4) [227]. Luteolin significantly reduced the mRNA levels of SREBP1 and SREBP2, decreasing SREBP1 protein expression in JAR and JEG-3 Human Placental Choriocarcinoma Cells [228]. Furthermore, in C6 rat glioma cells, Quercetin inhibited cholesterol and fatty acid synthesis, decreasing the expression of SREBP1, SREBP2, and ChREBP, a transcription factor that regulates genes involved in lipogenesis [229].
AKT, an oncogene, is highly expressed in human cancers and can be considered the central and convergent point of several growth signaling pathways, including mTOR [230]. AKT is capable of phosphorylating several downstream effectors, such as apoptotic proteins, transcription factors, and other oncogenes [231]. Most of the compounds with anticancer activity decrease AKT activation as the main molecular mode of action. Quercetin suppressed breast cancer progression by decreasing AKT/mTOR pathway, inducing autophagy, in vitro and in vivo [149], and inhibited AKT-mediated activation of mTOR and its effectors in hepatocellular carcinoma [232], prostate [233], and breast cancer cells [234]. Similarly, in concentrations ranging between 25-100 µM, Kaempferol decreased AKT phosphorylation in hepatocarcinoma [213], leukemia [235], endometrial cancer [236], renal carcinoma [237], and human lung cancer cells [238].

The Ras/ERK Pathway
In addition to AKT, one of the most important pathways that control cell growth is the Ras/ERK signaling pathway, responsible for alterations in cell morphology, differentiation, and neoplastic transformation [264]. The gain-of-function mutations in members of the Rat Sarcoma viruses (Ras) family are one of the most common genetic alterations in cancer [265], which leads to prolonged activation of Ras signaling [264]. The first Ras effector pathway identified was the RAF/MEK/ERK axis and the second best-characterized Ras effector is PI3K, cross-talking with the mTOR/AKT pathway (Figure 4) [265,266]. The first evidence of the interaction between Ras and PI3K was achieved by detecting PI3K activity in Ras immunoprecipitation in transformed cells [267].
Different strategies that can inhibit these pathways are widely considered therapeutic interventions in cancer [268]. Flavonoids appear to synergistically inhibit both Ras and mTOR signaling, such as Apigenin in melanoma cell lines [244], Daidzein [269] in human prostate cancer cell lines, Delphinidin [212,263] in lung and ovarian cancer cells, and EGCG in colorectal cells in vitro and in vivo [250]. Studies reported that Myricetin can decrease both AKT and ERK 1 and 2 phosphorylation (also known as Mitogenactivated protein kinase 2, MAPK2, and Mitogen-Activated Protein Kinase 1, MAPK1, respectively) [135,242,270,271]. Myricetin can reduce the phosphorylation of AKT while decreasing the activation of Ras, RAF proto-oncogene serine/threonine-protein kinase (Raf), and ERK in human glioblastoma cells [242]. Myricetin also showed to be a potent Mitogen-Activated Protein Kinase Kinase 1 (MEK1) inhibitor, leading to decreased ERK phosphorylation and, consequently, impairing the neoplastic transformation of mouse epidermal cell lines [135]. Thus, flavonoids are highlighted as important compounds in adjuvant cancer therapy, presenting potent inhibition activity against cancer cell growth and proliferation.

Flavonoids and Autophagy
Autophagy is a cell survival pathway that is composed of 3 routes: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy is the most studied one and a catabolic process, responsible for the degradation of organelles within a vesicle derived from the smooth endoplasmatic reticulum and mediated by lysosomal hydrolytic enzymes. This pathway is activated to ensure the maintenance of homeostasis in cases of starvation, dysfunctional organelles, and stress compounds such as ROS inducers. Macroautophagy is also modulated in cases of cell proliferation, differentiation and directly interacts with the apoptotic pathway [281].
As shown in Figure 5, the macroautophagy pathway starts by the formation of the Unc-51 Like Autophagy Activating Kinase (ULK1) complex, that activates by phosphorylation the Beclin1 complex, a type of Phosphoinositide 3 Kinase 3 Complex (PI3K3C), leading to the production of Phosphoinositol 3 Phosphate (PIP3) from Phosphoinositol 2 Phosphate (PIP2). The PIP3 accumulation recruits WD-repeat protein Interacting with Phosphoinositides (Wipi) and some autophagy-related genes (Atgs) proteins for the formation of a single-layer pre-autophagosome. In the next step, there is the nucleation of the pre-autophagosome, generating a double layer membrane. This occurs when the Microtubule-associated protein 1A/1B-light chain 3 (LC3-I) protein is lipidated to the form LC3-II, binding to the autophagosome by autophagy receptors such as sequestosome-1 (p62). Lastly, the lysosomes fuse with the autophagosome, generating the autolysosome, which digest the content by Cathepsins enzymes action [282][283][284].
Currently, the macroautophagy pathway has been intensively studied in oncology, since it is related to some important pathways that frequently present mutations in tumoral models, related to proliferation (mTOR and ERK) and survival and DNA repair (Bcl-2, p53). In addition, the dysregulation of autophagy is related to some of the hallmarks proposed by Weinberg and Hanahan in 2011 [14,285], and other relevant characteristics to tumors as presented below: -Autophagy and genome instability and mutation: autophagy acts as a clearance pathway that removes dysfunctional organelles. These organelles can lead to the accumulation of toxic compounds, such as ROS, that increase genomic instability, promoting mutations [286,287]. -Autophagy and sustaining proliferative signaling: the regulation of this process by some molecules such as the accumulation of Adenosine Monophosphate (AMP) and the depletion of Adenosine Triphosphate (ATP) leads to the activation of antiproliferative pathways, for example, the Liver Kinase B1 (LKB1) and AMP-activated Kinase (AMPK) pathways. These proteins reduce the rate of cell proliferation and also intensify the autophagy process. In some types of cancer with driver mutation in PTEN and LKB1, there is a decrease of autophagy to maintain high levels of proliferation [288,289].
-Autophagy and deregulation of cellular energetics: some types of cancer promote reprogramming of cellular bioenergetics known as the Warburg effect, where autophagy plays a critical role to protect the cell against high levels of ROS and lactate, helping cell survival [290,291]. -Autophagy and resistance to cell death: Beclin1 protein interacts with Bcl-2 protein, inhibiting the autophagy process. Thus, when a cancer cell receives a death stimulus such as chemotherapy, the interaction between Beclin1 and Bcl-2 proteins is lost and the cell presents high levels of anti-apoptotic and autophagy activities, turning it resistant to apoptosis [292,293]. -Autophagy and induction angiogenesis: when the supply of O 2 and nutrients are low, autophagy plays an important role in cell survival. However, some types of cancer can purposefully decrease autophagy pathways to promote tumoral angiogenesis [294,295]. -Autophagy and activation of invasion and metastasis: low levels of autophagy are related to EMT with the association of inflammation and macrophages in the tumor. Cancer cells can then detach from the primary tumor region, promoting the metastasis process and producing an invasive tumoral colony. Tumoral colonies can benefit from autophagy since they can avoid the anoikis process, a type of apoptosis due to the loss of contact from the extracellular matrix. Autophagy also plays an important role to overcome starvation and hypoxia until the metastatic cells invade a new region [296,297]. -Autophagy and chemoresistance: some types of cancer have mutations on proliferative pathways such as p53 and MAPK and are known as "autophagy addicted". The scenario of intense proliferation can be associated with a cellular metabolic disturbance due to the preference for glycolytic metabolism. In this case, autophagy plays a crucial role in tumor progression, protecting the cell against the damage of super proliferation and generating energy substrates through the degradation of organelles and proteins. Thereby, when the autophagy pathway is activated, the cells often present anti-apoptotic activity caused by Beclin1 interaction with Bcl-2, through the BH3 domain, which can lead to chemotherapeutic resistance [292,298]. -Autophagy and senescence: evidence points out that autophagy may correlate with the senescence process. CDKs (Cyclin-dependent-kinase) and cyclins control important pathways in proliferation, such as mTOR and AMPK, that can regulate autophagy. Thus, the deregulation of this system can slow the senescence process [299,300].
In this sense, it is important to highlight the autophagy process in oncology studies. According to some authors, autophagy inhibitors such as chloroquine, 3-Methyladenine (3-MA), and bafilomycin can increase the cancer sensitivity to chemotherapy and can be associated with therapies [301]. Other ways to modulate the autophagy process in cancer studies were demonstrated by using natural compounds. The literature points to strong evidence of the potential of flavonoids in stimulating autophagy in cancer. Table 6 describes the flavonoids that present solid evidence regarding the modulation of the autophagy process. Studies describe Quercetin, Apigenin, Luteolin, Kaempferol, Myricetin, Delphinidin, Naringin, Catechin, and Genistein as flavonoids targeting autophagy [32].
Currently, some authors have reported that bioactive compounds such as flavonoids present strong anticancer properties associated with chemotherapy treatment in cancer models [313,332]. Flavonoids, as already mentioned, can increase the canonical autophagy pathway and modulate non-canonical autophagy pathways, through the inhibition of proliferative routes (mTOR, Ras), hypoxia, and cytokines routes (STAT3). In addition, flavonoids may increase toxicity when associated with chemotherapeutic treatment. Ultimately this overactivation of autophagy may lead cells to a cytotoxic autophagy process, which is known as autophagic cell death or type II cell death [333]. Figure 5. Flavonoids modulate autophagy pathways in cancer. Macroautophagy begins by activation of ULK complex in the endoplasmic reticulum that leads to Beclin1 phosphorylation this permits the activation of PI3KC3 complex that converts PIP2 to PIP3 promoting an accumulation of phospholipid, this accumulation recruits Wipi proteins and some autophagy-related genes (Atg 5,12 and 16) for the formation of the single-layer pre-autophagosome (in blue), the autophagy receptor p62 interacts with the autophagy target and binds to active LC3-II forming a mature double membrane (in purple) that is fused with lysosomes that will digest the autophagosome content with cathepsin enzyme. Flavonoids can stimulate macroautophagy, through the increase in expression of Beclin1, p62, LC3-II, and some Atgs. Kae: Kaempferol, Myr: Myricetin, Que: Quercetin, Api: Apigenin, Gen: Genistein, Del: Delphinidin, Lut: Luteolin, Nar: Naringin, Epi: Epigallocatechin. Original figure made for this review using the Biorender software.   Finally, it is worth mentioning the duality of autophagy in cancer. Firstly, this pathway can act to prevent cancer, through the removal of dysfunctional organelles, decrease of inflammation, removal of toxic compounds such as ROS, protection of DNA against damage and mutations, and maintenance of homeostasis. However, the autophagy pathway can be purposely modulated by cancer cells for the acquisition of hallmarks, becoming more aggressive and resistant to therapies and leading to a worse prognosis of the disease. Thus, the autophagy pathway can support tumorigenesis and its modulation may be carefully considered for cancer treatment [334,335].

Flavonoids, Apoptosis, and Cell Cycle
Apoptosis, a type of programmed cell death, occurs during early development and, in adults, it is used to eliminate from the body cells that have been damaged beyond repair. For this reason, apoptosis also plays an important role in preventing cancer [336]. Cells under apoptosis present a series of distinct changes in their morphology, such as loss of cell attachment, cytoplasmic contraction, and DNA fragmentation. In addition, a class of proteins is recruited, called caspases, which can be activated through the extrinsic or intrinsic apoptotic pathways [337].
Intrinsic apoptosis occurs inside the mitochondria, where its outer membrane is permeabilized and leads to cytochrome c release into the cytoplasm. The cytochrome c releasing is stimulated by pro-apoptotic proteins such as Bcl-2-Like Protein 4 (BAK) and Bcl-2 Homologous Antagonist/killer (BAX), leading to caspase 3 activation and apoptosome formation. Bcl-X L, Bcl-2, and Induced Myeloid Leukemia Cell Differentiation Protein (Mcl-1) are proteins of the intrinsic apoptosis pathway that can be inhibited ( Figure 6) [338].

Apoptotic Pathway
In the cancer context, apoptosis is critical, and the regulation of pro-apoptotic and anti-apoptotic proteins is well studied. Among the modulators of the apoptotic pathway are the flavonoids, which have been shown to activate pro-apoptotic processes in cancer cell lines and animal models [340]. A summary of these actions of flavonoids in apoptosis is described in Table 7.
Other pro-apoptotic proteins were described as upregulated by flavonoids, such as Caspase 9, BAK, BH3 Interacting-domain Death Agonist (BID), and BAX. Increased levels of BAX, for example, were observed in cancer cells treated with Myricetin, Vitexin, Quercetin, Apigenin, Genistein, Daidzein, Luteolin, Naringenin, and Taxifolin ( Figure 6) [55,206,[344][345][346][347][348][349][350]. The effects of flavonoids in pro-apoptotic proteins were not only detected in cancer cell cultures but also in athymic nude mice models, where 23 days of treatment with 0.2-0.4 mg/kg of Genistein was able to significantly increase the levels of BAX and BAK [348]. The downregulation of anti-apoptotic proteins has also been observed in the literature. The levels of Bcl-2 were lower in renal carcinoma, human colon cancer, prostate cancer, human leukemia, and hepatocellular carcinoma cells treated with Epigallocatechin, Myricetin, Apigenin, Genistein, Daidzein, respectively, compared with control cells (Figure 6) [346][347][348]351,352]. p53, a key protein related to several pathways involved in cancer, is also found modulated by flavonoids. In several types of cancer, p53 is mutated, losing its function and becoming inactivated [137]. When inactivated, apoptosis is blocked and the cell cycle is facilitated, leading to the uncontrolled proliferation and growth of cells. In leukemia cells (Nalm6) treated with 20 µM of Quercetin for 24 h, the total content of p53 was significantly increased [353]. The same occurred in ATL cells from adult T-cell leukemia treated with 50-100 µM of Apigenin for 16 h [354] and in HeLa cells treated with Cyanidins for 24 h (Figure 6) [342]. These collected data strongly support the evidence of the influence of flavonoids on cancer cell apoptosis, acting as isolated treatments or combined with other therapies.

Cell Cycle Control
The cell presents a series of events, in a cycle, that prepare the cell for its division and duplication to produce daughter cells [337]. The cell cycle has four steps-named G1, S, G2, and M-in which the cell increases in size, duplicates its genetic material, prepares for division, and divides, respectively. Each step is ordered and programmed [355]. Several molecules stimulate the cell cycle, including the cyclins, proteins that promote the activation and binding to Cyclin Dependable Kinases (CDKs) ( Table 7), both working as protein complexes that orchestrate the progression and maintenance of the cell cycle. On the other hand, there are specific points in the cell cycle, called checkpoints, that stop the cell cycle to analyze the integrity of DNA, aiming at the production of daughter cells with correct genetic material [356]. In several types of cancer, the fine control of these checkpoints is lost, and cell proliferation is unlimited. For this reason, the search for molecules that promote cell cycle arrest is a promising therapeutic approach against cancer [357].
Flavonoids regulate cell growth and cell cycle progression by modulating the expression of these cell cycle regulatory proteins (Cyclins and CDKs) [337]. For instance, the treatment with Kaempferol downregulated the expression of cyclin D, E, and A in Human colon cancer cells (HT-29) [196]. Other flavonoids have been found to downregulate cyclins such as Daidzein, Cyanidin, Luteolin, and Taxifolin ( Figure 6) [196,342,344,358].
Some flavonoids have been reported to mediate G2/M cell cycle arrest via the upregulation of the expression of Cyclin-Dependent Kinase Inhibitor 1 (p21). p21 is a protein that is activated by p53 and promotes the inhibition of cyclin B and E. HeLa cells, derived from cervical cancer, presented lower p21 expression when treated with Quercetin for 24 h [55]. The same was observed in a human colon cancer cell (HCT116) treated with Genistein [359], breast cancer cells (MDA-MB-231) under 10-30 µM of Luteolin [344], and colorectal cancer (HCT116) treated with Taxifolin for 24 h [350].

DNA Damage and Repair
The DNA is constantly damaged by exogenous and endogenous sources such as genotoxic chemicals, ultraviolet (UV), ionizing radiation, and reactive oxygen and nitrogen species [360]. The resulted damage in DNA can be double or single-strand breaks, base oxidation, deamination, interstrand crosslinks, or adduct formation. For each of these damages, there are specific cascades of molecules able to repair the DNA and to protect the cells from replicating with wrong genetic information [360]. DNA damage is one of the major causes for cancer initiation and progression due to genetic alterations, which may lead to loss of tumor suppression or increase of oncogenes [360].
Cancer treatments based on this pathway aim to decrease the expression of proteins that stimulate DNA repair, leading the cells to death when this treatment is combined with chemotherapy that induces DNA damage. Kuo et al. (2016) treated breast cancer cells MDA-MB-231 with flavonoids and observed decreased DNA repair pathway [360]. These cells were submitted to UV radiation and treated with different concentrations of Catechin and Epigallocatechin. The treatments with 5-10 µM of Catechin and 10-40 µM of Epigallocatechin were able to significantly reduce the phosphorylation of Checkpoint Kinase 1 (CHK1), a protein involved in DNA repair signaling. The same study submitted MDA-MB-231 cells to pretreatment with 10 µM or 50 µM of flavonoids followed by exposure to 10 µM cisplatin for 6 h to induce CHK1 and Checkpoint Kinase 2 (CHK2) phosphorylation. At 50 µM the flavonoids Kaempferol, Genistein, Naringenin, and Epigallocatechin were effective to reduce the levels of CHK1 and CHK2 phosphorylated ( Figure 6) [360].

Cell Senescence
Cellular senescence is described as the irreversible arrest of cell proliferation and this phenomenon can be observed in several types of cells. The senescence occurs due to the gradual loss of telomere length after each cell division, and for this reason, this process is closely involved with the aging of cells [361]. As senescence leads to cell cycle arrest, at least in part, this process is linked to cancer suppression, being regulated by two major pathways: the p53/p21; and the cyclin-dependent kinase inhibitor 2A and Retinoblastoma-associated protein (p16INK1a/pRB), which are considered senescence markers [362]. Indeed, cells with mutations in p53 or p16INK4a present the inability to respond to certain senescenceinducing signals, which greatly increase cancer susceptibility [363].
Besides the inability to proliferate, the senescent cells have the feature of secreting several proinflammatory cytokines, chemokines, growth factors, and proteases, a process termed senescence-associated secretory phenotype (SASP) [362]. Although the SASP cells can attract immune cells to promote the clearance of the tumor, these senescent cells are also able to create a microenvironment that stimulates the cancer cells growth and metastasis, including the release of Stromelysin3 (MMP3) and VEGF, which are cancer inductors of metastasis and angiogenesis, respectively [361].
Studies already reported the effect of flavonoids in eliminating senescence cells (a process called senolytic effect) and consequently impairing the tumor progression [364]. Prosenescence therapy was also recently proposed as an anti-cancer strategy and has been shown to effectively inhibit cancer. Özsoy et al. (2020) treated colon adenocarcinoma cell lines (Colo-320 and Colo-741) with Quercetin for 48h and showed a higher immunoreactivity to p16 [365]. The treatment with ECGC was able to induce senescence in U251 human glioblastoma cells, culminating in the shortening of telomere and cell cycle arrest [366]. Lastly, Banerjee et al. (2015) showed the effectiveness of apigenin in improving the activity of β-galactosidase in HCT-15 cells, leading the cell to senescence [367] (Figure 6).

Conclusions
In conclusion, flavonoids are important natural compounds with broad effects described in several signaling pathways related to cancer. Flavonoids can inhibit inflammation and increase immunity, modulating the NF-κB, MAPK, inflammasome, and JAK-STAT pathways, which are usually altered in cancer. In addition, flavonoids inhibit growth signaling pathways, such as the mTOR and Ras oncogenic pathways, and, at the same time, activate apoptosis and cause cell cycle arrest. In the case of autophagy and oxidative stress, which flavonoids can also modulate, both have a dual relationship with cancer depending on the stage of the disease. Thus, future studies must cautiously address these pathways in this context. Although more studies are needed to explore details on how flavonoids exert their mechanism of action in cells, the increasing amount of evidence strongly suggests the potential use of flavonoids as anti-cancer bioactive compounds.

Conflicts of Interest:
The authors declare no potential conflict of interest.