Carcinogenesis is a multi-step process of genetic and epigenetic alterations leading to the progressive transformation of normal cells towards malignancy. The process of carcinogenesis can be divided into three main stages: (i) initiation, a phase in which cellular exposition to a carcinogenic agent leads to irreversible alterations, usually at the DNA level. In this phase cells react to carcinogens by the activation of enzymes involved in the metabolism of xenobiotics that, while aiming to inactivate, may generate a mutagenic compound responsible for DNA damage and mutations, thereby initiating cancer development; (ii) the tumor promotion stage is characterized by the proliferation of abnormal cells that may initiate a pre-neoplastic focus. In this phase over-activation and/or over-expression of enzymes involved in the synthesis of nucleotides and DNA (e.g., ornithine decarboxylase), as well as in the regulation of the differentiation process (DNA polymerase or topoisomerases) occur. Moreover, oxidative stress caused by the overproduction of reactive oxygen species (ROS) induces further cell damage and genome instability; (iii) progression is the final stage of carcinogenesis. It is characterized by an uncontrolled proliferation of tumor cells which also acquire the ability to invade neighboring tissues and to form metastasis at distant sites, coupled with a loss of capacity for apoptosis or senescence. Hence, metastasis is the spread of cancer cells from a primary tumor to distant sites in the cancer patient’s body. Angiogenesis is the first step of the metastatic process that leads to the formation of new blood capillaries by outgrowth or sprouting of pre-existing blood vessels. It allows the tumor to be fed and facilitates the access of tumor cells to the bloodstream. Indeed, tumor vessels are more permeable than normal ones, since tumor-associated endothelial cells are enlarged and loosely connected. Therefore, the metastatic process is the end result of a complex series of events depending on the ability of tumor cells to detach from the primary tumor, migrate, and invade connective tissues, entering the vascular or lymphatic system, through which vital organs are reached where they proliferate to form a distant metastasis. The tendency of a primary tumor to form metastasis is the hallmark of malignant cancer, and has important diagnostic, prognostic, and therapeutic implications.
3.1. Initiation Phase Inhibition by Citrus Flavonoids
In the last twenty years, there has been an increasing awareness that flavonoids and other naturally-occurring substances in plants have protective effects against environmental mutagens/carcinogens and endogenous mutagens [
23]. In support of this, there are numerous experimental findings suggesting that certain
Citrus flavonoids may exert preventive effects against DNA damage induced by a variety of carcinogens [
24]. Naringenin and rutin prevent the accumulation of ultraviolet radiation-B (UV-B)-induced DNA damage [
25] by a mechanism that may involve the ability of flavonoids to neutralize free radicals generated near DNA, promoting mutations. The radical scavenging property of flavonoids is also responsible for quercetin protective effect against mercury-induced DNA damage and oxidative stress in a human-derived liver cell line (HepG2), that seems to be due to the maintenance of redox status [
26]. Moreover, it has been observed that naringenin at low doses (10–80 μM) can stimulate DNA repair following oxidative damage in a human lymph node prostate cancer cell line (LNCaP), leading to a significant increase in the levels of several major enzymes in the DNA base excision repair pathway [
27]. In in vivo experiments, naringenin has demonstrated its capability to inhibit
N-diethylnitrosamine (NDEA)-induced hepatocarcinogenesis [
28,
29]. Naringin has been found to reduce the rate of micronuclei formed by ifosfamide in mouse blood cells [
30] and to exert protective action against DNA deterioration induced by daunorubicin in mouse hepatocytes and cardiocytes, suggesting that this flavonoid may be useful in reducing the adverse effects found in anthracycline treatments [
31]. Moreover, it accelerated the regression of pre-neoplastic lesions in rats exposed to 1,2-dimethylhydrazine (DMH) [
32]. Experiments performed using in vivo models of genotoxicity induced by cyclophosphamide show that the antioxidative activity of hesperidin (100, 200, and 400 mg/kg body weight (BW) administered by gavages for five consecutive days) may reduce the frequency of micronucleated polychromatic erythrocytes (MnPCEs) induced by chemotherapy drugs [
33]. Furthermore, in the presence of a mammalian metabolic activation system, naringin, apigenin, hesperetin, and other flavonoids (300 μg/plate) have been shown to produce antimutagenic effects against aflatoxin B1 (1 μg/plate), with an inhibition rate of more than a 70% in
Salmonella typhimurium. In this study, the structure–activity relationship analysis suggests the flavonoid configuration containing the free 5-, 7-hydroxyl group to be essential [
34].
Flavonoids may also inhibit the first phase of carcinogenesis through an increase in detoxification processes by modulating enzyme activity resulting in the decreased carcinogenicity of xenobiotics. For example, naringenin inhibits the activity of aromatase (CYP19) in Chinese hamster ovary (CHO) cells, thereby decreasing estrogen biosynthesis and inducing antiestrogenic effects, which are important in breast and prostate cancers [
35]. Quercetin has instead proven to be a potent non-competitive inhibitor of sulfotransferase 1A1, suggesting a role for potential chemopreventive agents in sulfation-induced carcinogenesis [
36]. The chemopreventive potential of diosmin, naringenin, naringin, and rutin against CYPlA2-mediated mutagenesis of heterocyclic amines produced by high temperature cooking of meat was hinted by Bear and Teel [
37]. Several reports have described the potential anti-mutagenic properties of apigenin. For instance, exposure to apigenin prior to a carcinogenic insult has been shown to offer a protective effect in both murine skin and colon cancer models [
38], as well as to prevent the genotoxic effects of benzo(α)pyrene (BP) in vivo. Indeed, Khan et al. [
39] demonstrated that apigenin (2.5 and 5 mg/kg orally) reverts BP-induced depletion in the levels of glutathione (GSH), quinone reductase (QR), and glutathione-
S-transferase (GST), while also reducing DNA strand breaks and damage. Increased GSH by apigenin also enhances endogenous defense against oxidative stress [
40]. Moreover, topical application of apigenin has been proven to reduce dimethyl benzanthracene-induced skin tumors by strongly inhibiting epidermal ornithine decarboxylase, an enzyme that plays a key role in tumor promotion [
41]. In addition, apigenin administration has been reported diminish the incidence of UV light-induced cancers and to increase tumor-free survival in vivo [
42]. Moreover, apigenin as well naringenin, suppress colon carcinogenesis in azoxymethane (AOM)-treated rats [
43].
The antigenotoxic activity of hesperidin was investigated by Nandakumar et al., [
44]. They reported that daily administration of hesperidin at a concentration of 30 mg/kg BW for 45 days prevented 7,12-dimethylbenz(α)anthracene (DMBA)-induced experimental breast cancer formation, presumably by the regulation of both phase I and phase II metabolizing enzymes, and through its strong antioxidant activity. The results also revealed that the flavanone may act both by modulating the energy reservoir of the cell and by maintaining oxidative phosphorylation. Also, the aglycone hesperetin has been reported to modulate xenobiotic-metabolizing enzymes during DMH-induced colon carcinogenesis [
45]. Tangeretin, a pentamethoxy flavone present in significant amounts in CF peel, was found to suppress DMBA-induced breast cancer in rats [
46].
Chronic inflammation is closely connected to the carcinogenic process. Indeed, nobiletin has been shown to inhibit DMBA/tetradecanoyl-13-phorbol acetate (TPA)-induced skin tumor formation by reducing the number of tumors per mouse, manifesting its potential in inflammation-associated tumorigenesis [
47]. The studies discussed above are summarized in
Table 2.
3.2. Inhibition of Tumor Development
A great number of in vitro studies have demonstrated that
Citrus flavonoids reduce the growth of several types of tumor cells in cultures. Tangeretin, nobiletin, quercetin and taxifolin have anti-proliferative effects on squamous cell carcinoma HTB43 [
48], as well as on many other tumoral cell lines. Tangeretin, a PMF present mainly in the peel of tangerine and other CF, induced apoptosis in human myeloid leukaemia HL-60 cells, without causing cytotoxicity in human peripheral blood mononuclear cells [
49,
50]. Tangeretin and nobiletin (another PMF widely found in the mandarin epicarp) also inhibited the proliferation of both human breast cancer cell lines (MDA-MB-435 and MCF-7) and a human colon cancer cell line (HT-29) in a concentration- and time-dependent manner, by blocking cell cycle progression at the G1 phase without inducing cell death [
51]. This study showed tangeretin IC
50 values of 30–40 μM for breast and colon cell lines, and slightly higher values for nobiletin, while in other reports tangeretin exhibited much greater potency [
49,
50]. However, this discrepancy could be caused by differences related to both cell type and experimental procedures. The inhibition of the activity of cyclin-dependent kinases 2 (Cdk2) and 4 (Cdk4), accompanied by an increase in Cdk inhibitors p21 and p27 seems to be the mechanism through which tangeretin arrests cell cycle progression at the G1 phase in colon adenocarcinoma COLO 205 cells [
52]. Yoshimizu et al. [
53] documented the growth-inhibitory action of nobiletin, both alone and in combination with cisplatin, in various human gastric cancer cell lines (TMK-1, MKN-45, MKN-74, and KATO-III), through the induction of apoptosis and cell cycle deregulation. Interestingly, orange peel extract (OPE) containing 30% polymethoxyflavones, such as tangeretin (19.0%), heptamethoxyflavone (15.24%), tetramethoxyflavone (13.6%), nobiletin (12.49%), hexamethoxyflavone (11.06%) and sinensitin (9.16%), inhibited tumorigenesis in Apc
(Min/+) mice by increasing apoptosis [
54]. OPE also decreased the development of hyperplastic lesions in mouse mammary glands [
55]. The reduction of mammary cancer cell growth caused by tangeretin may be related to the inhibition of mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) phosphorylation and of other proteins like adducin α and γ, protein kinase Cδ, signal transducer and activator of transcription (STAT) 1 and 3, and stress-activated protein kinase (JNK) [
56]. Tangeretin and nobiletin also inhibited the proliferation of both SH-SY5Y neuroblastoma cells [
57] and brain tumor cells [
58], reducing also invasion, migration, and adhesive properties. Moreover, it has been reported that tangeretin sensitizes cisplatin-resistant human ovarian cancer cells through the downregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (also known as Akt) signaling pathway, suggesting a potential approach for the treatment of drug-resistant cancers [
59]. Tangeretin also induced apoptosis in gastric cancer AGS cells through the activation of both extrinsic and intrinsic signaling pathways [
60]. Nobiletin and the coumarin auraptene have been reported to counteract prostate carcinogenesis both in vitro and in vivo. In particular, nobiletin inhibited the growth of several prostate cancer cell lines with IC
50 values of around 100 μM, by a mechanism involving apoptosis and cell cycle arrest at the G
0/G
1 phase, as well as inhibited development of prostate adenocarcinomas in a transgenic rat model [
61]. The preventive effects of nobiletin on prostate cancer have recently been confirmed in a study that also reported the ability of this flavonoid to reduce the risk of colon cancer [
62]. Furthermore, nobiletin reduces AOM-induced rat colon carcinogenesis [
63] and, like quercetin (100 ppm), is able to decrease preneoplastic lesions and serum levels of both leptin and insulin in an in vivo model of colon carcinogenesis, suggesting a promising role in preventing tumors associated with obesity [
64,
65]. Experiments performed using both in vitro and in vivo models showed the anti-proliferative property of nobiletin on lung cancer cells. The mechanism involves the activation of the apoptotic process and cell cycle arrest at the G2/M phase due to decreased Bcl-2 and increased Bax protein expression, both of which positively correlated with elevated expression of p53 [
66]. As reported by Ohnishi et al. [
67], nobiletin treatment suppressed HepG2 and MH1C1 hepatocarcinoma cell growth by inducing cell cycle inhibition and apoptosis, but without apparent effects in the early stages of in vivo hepatocarcinogenesis. In glioma cells, it suppresses proliferation by inhibiting Ras activity and mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) signaling cascade, probably via a Ca
2+-sensitive protein kinase C (PKC)-dependent mechanism [
68]. There are more recent results that demonstrate the ability of nobiletin to inhibit cell growth and migration via cell-cycle arrest and suppression of the MAPK and Akt pathways [
69]. In human gastric p53-mutated SNU-16 cells, nobiletin was found to be effective in inhibiting cell proliferation, inducing apoptosis, and enhancing the efficacy of 5-Fluorouracil (FU) [
70]. Its anti-cancer effects have also been demonstrated in acute myeloid leukemia cells [
71], where it was responsible for the induction of cell-cycle arrest and apoptosis. Moreover, orally administrated nobiletin inhibited colitis-associated colon carcinogenesis in AOM/dextran sulfate sodium-treated mice [
72].
Apigenin is a flavone present mainly in fruits and vegetables, and among
Citrus species it is abundant in grapefruit. It possesses anti-inflammatory and free radical scavenging activity, and as a candidate anti-cancer agent, is capable of reducing cancer cell proliferation of without affecting normal cells. It has been reported that apigenin possesses growth inhibitory properties in breast cancer, inducing apoptosis by: (i) the involvement of the caspase cascade [
73]; (ii) inhibiting STAT3 and nuclear factor kappa B (NF-κB) signaling in HER2-overexpressing breast cancer cells [
74]; (iii) reducing the activity of both PI3K and Akt kinase [
75] and regulating the p14ARF-Mdm2-p53 pathway [
76]. Apigenin is reported to exert growth inhibitory effects by increasing the stability of p53, leading to cell cycle arrest in many cancer cell lines, including rat neural and liver epithelial cells, as well as human breast, ovarian, cervical, prostate, colon, and thyroid cancers [
77]. In epidermal cells and fibroblasts reversible G2/M and G0/G1 arrest is also mediated by the inhibition of p34 (Cdc2) kinase activity [
78,
79], while in breast carcinoma the G2/M phase cell cycle arrest after apigenin treatment led to a significant decrease in cyclins (B1, D1, and A) and cyclin-dependent kinase (Cdk1 and 4) protein levels [
80]. In pancreatic cancer cell lines, apigenin caused both time- and concentration-dependent inhibition of DNA synthesis and cell proliferation through G2/M phase cell cycle arrest caused by the suppression of cyclin B-associated Cdc2 activity [
81,
82]. Moreover, in the same cell lines, it inhibited the glycogen synthase kinase-3β/NF-ĸB signaling pathway and upregulated the expression of cytokine genes, which potentially contributed to its anti-cancer properties [
83]. In addition, apigenin has been shown to induce WAF1/p21 levels, resulting in G1 phase cell cycle arrest in androgen-responsive (LNCaP) and androgen-refractory (DU145) human prostate cancer cells [
84,
85]. Indeed, the apoptosis observed in these cell lines appeared to be correlated with: (i) the alteration in Bax/Bcl-2 ratio; (ii) the down- regulation of the constitutive expression of NF-ĸB/p65; (iii) the release of cytochrome c; (iv) the induction of apoptotic protease activating factor-1 (Apaf-1), which leads to caspase activation and PARP-cleavage [
84,
85]. Apigenin-induced growth inhibition by different mechanisms has also been reported in colon [
86,
87], prostate [
88], and neuroblastoma [
89,
90] cancer cells. In endothelial cells, the anti-proliferative effect exerted by the flavanone is due to the blocking of cells in the G2/M phase, as a result of the accumulation of the hyperphosphorylated form of retinoblastoma protein [
91]. Diosmin, another important
Citrus flavone (mostly due to its venotonic activity), occurs naturally as a glycoside, and after ingestion is rapidly transformed by intestinal flora to its aglycone form, diosmetin. Diosmin has been shown to inhibit Caco-2 and HT-29 colon cancer cell growth [
92]. In the hepatocellular carcinoma HA22T cells, it inhibited cell viability, reduced cellular proliferative proteins, and induced cell cycle arrest in the G2/M phase through p53 activation and inhibition of the PI3K-Akt-mouse double minute 2 homolog (MDM2) signaling pathway. In addition, it suppressed tumor growth through protein phosphatase 2 (PP2A) activation in HA22T-implanted xeno-graft nude activation [
93]. The effectiveness of diosmin as an anti-cancer agent has also been demonstrated in DU145 prostate cancer cells, where it promotes genotoxic events and apoptotic cell death [
94]. Moreover, it has been shown that diosmin may reduce the development of esophageal cancer induced by
N-methyl-
N-amylnitrosamine (MNAN) when given during the initiation phase [
95], decreases oral carcinogenesis initiated by 4-nitroquinoline 1-oxide (4-NQO) [
96], counteracts
N-butyl-
N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary-bladder carcinogenesis [
97], and prevents AOM-induced rat colon carcinogenesis, either alone or in combination with hesperidin [
98]. In these cases [
95,
96,
97,
98], rats were fed a diet containing diosmin (1000 ppm), hesperidin (1000 ppm), or diosmin + hesperidin (900 ppm and 100 ppm, respectively), and the cancer inhibition found could be related to the suppression of the increased cell proliferation caused by the carcinogens in the affected mucous membranes.
Quercetin is a water-soluble flavonol, widely distributed in nature and the most common dietary flavonol. It represents the aglycone form of a number of other flavonoid glycosides, such as rutin and quercitin. In CFit is present mainly in lemon peel. Experimental data have shown quercetin to be a potential anti-carcinogenic agent against several human tumor cell lines, including HL-60 (promyelocytic leukemia cells), A431 (epithelial carcinoma cell line), SK-OV-3 (ovary adenocarcinoma), HeLa (cervical carcinoma) and HOS (osteosarcoma) [
99]. The inhibitory effect of quercetin on HL-60 growth may be due to the induction of apoptosis mediated by an up-regulation of pro-apoptotic Bax and post-translational modification (phosphorylation) of anti-apoptotic Bcl2 [
100]. This flavonol also demonstrated concentration-dependent anti-proliferative activity against both meningioma [
101] and colon cancer cells (CRC) [
102]. Growth inhibition of several CRC cells has been reported and numerous mechanisms explaining the in vitro anti-proliferative effect of quercetin have been proposed [
103]. Interestingly the combination of quercetin and low-frequency ultrasound selectively induced cytotoxicity in skin and prostate cancer cells, while having minimal effect on corresponding normal cell lines [
104]. Quercetin has been reported to induce cell growth inhibition in MDA-MB-231 breast cancer cells by inhibition of the F-box protein S-phase kinase-associated protein 2 (Skp2) and induction of p27 expression, thereby blocking cell cycle progression [
105]. Moreover, several reports have shown that if quercetin is associated with antineoplastic drugs it may then play a relevant role in development of chemotherapeutic combinations. For example, in human breast cancer cells, quercetin inhibits lapatinib-sensitive and -resistant breast cancer cell growth by modifying levels of factors that regulate cell cycle G2/M progression and apoptosis, such as cyclin B1, p-Cdc25c (Ser216), Chk1, caspase 3, caspase 7, and PARP [
106]. In breast cancer cells, it potentiated the antitumor effects of doxorubicin, attenuating unwanted cytotoxicity to non-tumoral cells [
107], and markedly increased the effect of adriamycin in a multidrug-resistant MCF-7 human breast cancer cell line [
108] and in MCT-15 human colon carcinoma cells [
109].
Naringin and naringenin are two of the most abundant flavanones in CF, although the amounts differ. Naringenin is the aglycone and is a metabolite of naringin (naringenin-7-neohesperoside), the main flavonoid of grapefruit. Diverse biological and pharmacological properties, including anti-carcinogenic activity, have been reported for both of these flavanones. Kanno et al. [
110] showed the anti-proliferative effect of naringenin in a range of human cancer cell lines (breast, stomach, liver, cervix, pancreas, and colon) as well as its ability to inhibit tumor growth in sarcoma S-180-implanted mice. The same authors reported that the exposure of human promyeloleukemia HL-60 cells to naringenin at concentrations up to 0.5 mM induced apoptosis via the activation of NF-κB, while a higher concentration (1 mM) reduced intracellular ATP levels, causing mitochondrial dysfunctions leading to necrosis [
111]. Naringenin-induced inhibition of colon cancer cell proliferation has also been reported by Frydoonfar et al. [
112]. A mechanism through which naringenin might cause a reduction of breast cancer growth seems to be the impairment of glucose uptake. Indeed, in MCF-7 cells, the flavanone impaired the insulin-stimulated glucose uptake, thus decreasing the availability of glucose concentration in the culture medium and inhibiting proliferation [
113]. In human leukemia THP-1 cells, naringenin exerts an anti-proliferative effect in a concentration-dependent manner, inducing apoptosis through the modulation of the Bcl-2 family, mitochondrial dysfunction, activation of caspases, and PARP degradation that correlate with inactivation of the PI3K/Akt pathway [
114]. Using the same cell line, Shi et al. [
115] have demonstrated naringenin may enhance curcumin-induced apoptosis through inhibition of the Akt and ERK pathways, and by activating the JNK and p53 pathways. In human epidermoid carcinoma A431 cells, the ability of naringenin to induce apoptotic cascade and cell cycle arrest in the G0/G1 phase has been demonstrated [
116]. Several in vitro studies have demonstrated the naringenin-induced intrinsic apoptotic pathway initiated by the caspase cascade [
111,
114,
117]. It has also been reported activation of the apoptosis extrinsic pathway, triggered by ligands binding plasma membrane death receptors. Indeed, it has been observed that naringenin enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in TRAIL-resistant A549 human lung cancer cells by the upregulation of TRAIL receptor 5 (death receptor 5, DR5, also named TRAIL-R2)) without inhibition of cell growth in human normal lung fibroblast WI-38 cells [
118]. Moreover, naringenin (50 μM) and other flavonoids, among which hesperetin and apigenin, produced a more than three-fold increase in mitoxantrone accumulation by inhibition of breast cancer resistance protein (BCRP; an ATP-binding cassette transporter conferring multidrug resistance to a number of important anti-cancer agents) in BCRP-overexpressing MCF-7 (breast cancer) and NCI-H460 (lung cancer) cells, whereas the glycoside form (naringin) had no significant effects [
119]. The presence of the 2,3-double bond in the C ring of flavonoids, as well as ring B being attached at position 2, hydroxylation at position 5, lack of hydroxylation at position 3, and hydrophobic substitution at positions 6, 7, 8, or 40, are structural properties important for potent flavonoid–BCRP interaction, and critical for potent BCRP inhibition [
120]. Some studies have suggested that naringenin also inhibits the P-glycoprotein (P-gp), thus improving antitumor activity both in vitro [
121] and in vivo [
122,
123]. Conversely, other experimental studies indicate that naringenin modulates drug efflux pathways by inhibiting the activity of multidrug resistance-associated proteins (MRPs) but not P-gp [
124]. Similarly, Zhang and collaborators [
124] have claimed that doxorubicin in combination with naringenin enhanced antitumor activity in vivo, while others have asserted that the pharmacokinetics of intravenously administered doxorubicin (the plasma concentration, biliary, and urinary clearance and tissue distribution) is not altered by pre-treatment with naringin, naringenin, and quercetin [
125]. A number of in vivo studies on the antitumor effects of naringenin have also been performed. These found that it suppresses colon carcinogenesis through the aberrant crypt stage in AOM-treated rats [
43], reduces tumor size and weight loss in
N-methyl-
N’-nitro-
N-nitrosoguanidine-induced gastric carcinogenesis [
126,
127], promotes apoptosis in cerebrally-implanted C6 glioma cells rat model [
128] and, like naringin, inhibits oral carcinogenesis [
129].
Several findings have identified naringin to be a promising chemotherapeutic agent for diverse types of cancers. Naringin (750 μM) showed an anti-proliferative effect on SiHa human cervical cancer cells through cell cycle arrest in the G2/M phase and apoptosis induction via disruption of mitochondrial transmembrane potential, and the activation of both the intrinsic and extrinsic pathways [
130]. By contrast, naringin (1 mM) induced growth inhibition and apoptosis by suppressing the NF-κB/COX-2-caspase-1 pathway on HeLa cells [
131]. Recently, the role of glycoconjugates in cancer cells has been a focus because of their regulatory effects on malignant phenotypes. A study by Yoshinaga [
132] reported naringin to suppress HeLa and A549 cell growth through the alteration of glycolipids. This effect may largely be due to the attenuation of epidermal growth factor receptor (EGFR) signaling through GM3 ganglioside accumulation. Triple-negative (ER-/PR-/HER2-) breast cancer is an aggressive cancer with poor prognosis and a lack of targeted therapies. In this kind of tumor, Li et al. [
133] demonstrated that naringin inhibited cell proliferation and promoted cell apoptosis and G1 cycle arrest. These effects were accompanied by increased p21 levels and decreased survival by modulation of the β-catenin pathway.
Moreover, 100 μM naringin resulted in a significant concentration-dependent growth inhibition of 5637 bladder cancer cells together with of cell-cycle blocking [
134]. In this cell line, the naringin-induced anti-proliferative effect seems to be linked to the activation of Ras/Raf/ERK-mediated p21WAF1 induction, which in turn leads to a decrease in the levels of cyclin D1/CDK4 and cyclin E-CDK2 complexes, causing G1-phase cell-cycle arrest [
134]. Recently, naringin has been investigated regarding its ability to induce autophagy. Several studies have reported that autophagy promotes cancer cell death in response to various anti-cancer agents on apoptosis-defective cells [
135,
136]. Accordingly, over-activation of autophagy in cancer cells has been proposed to be an important death mechanism occurring in the tumor progression phase, where apoptosis is limited [
136]. In AGS gastric adenocarcinoma cells, naringin showed autophagy-mediated growth inhibition by suppressing the PI3K/Akt/mTOR cascade through MAPKs activation [
137]. Naringin has been demonstrated to reduce glioblastoma cell proliferation by inhibiting the FAK/cyclin D1 pathway, and promoting cell apoptosis by influencing the FAK/bads pathway [
138].
Furthermore, an in vivo study documented that grapefruit pulp powder (13.7 g/kg) or isolated naringin (200 mg/kg) or limonin (200 mg/kg) protect against AOM-induced aberrant crypt foci (ACF) by suppressing proliferation and elevating apoptosis through anti-inflammatory activities, suggesting that the consumption of grapefruit or its flavonoids may help to suppress colon cancer development [
139]. Camargo et al. [
140] showed that the treatment of rats bearing Walker 256 carcinosarcoma (W256) with 25 mg/kg of naringin reduced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels and tumor growth by ~75%. Very recently, it has been proven that naringin prevent intestinal tumorigenesis in a adenomatous polyposis coli multiple intestinal neoplasia (Apc
(Min/+)) mouse model [
141].
Another important
Citrus flavanone is hesperidin (hesperetin-7-rutinoside), the principal flavonoid in sweet orange and lemon, being the glycosides form of hesperetin (free state). It is water-soluble as a glycoside conjugate due to the presence of the sugar in its structure, which on ingestion releases its aglycone hesperetin. Along with other flavonoid compounds, hesperidin has been widely reported to possess venotonic and vasculo-protective pharmacological properties, and it is effectively used as a supplement in patients suffering from blood vessel disorders including capillary fragility and excessive permeability [
142]. Both hesperidin and hesperetin have shown anti-cancer activities, although the latter exhibited higher anti-proliferative activity in vitro. Chen et al. [
143] showed hesperetin to exert stronger cytotoxic activity than hesperidin in the HL-60 human leukemia cell line. Moreover, at the same concentrations (40 and 80 μM), hesperetin induced apoptosis, while hesperidin did not. The Authors suggest that the rutinoside group at C-7 causes the reduction of apoptotic induction on HL-60 cells by hesperidin. This hypothesis is strengthened by evidence that the aglycone naringenin also induces anti-proliferative and pro-apoptotic effects, but not the glycone naringin. Furthermore, hesperetin inhibits the expression of CDK2, CDK4, and cyclin D, thus inducing cell cycle arrest in the G1 phase, which in turn reduces MCF-7 cell proliferation in a concentration-dependent manner [
144]. Moreover, hesperetin (5 to 100 μM) inhibits human colon adenocarcinoma HT-29 cellular growth and induces apoptosis via the Bax-dependent mitochondrial pathway, involving oxidant/antioxidant imbalance [
145]. It also enhances Notch1 levels, that in turn decreases the expression of the neuroendocrine tumor markers ASCL1 and CgA, causing inhibition of human gastrointestinal carcinoid (BON) cell growth [
146]. Furthermore, hesperetin exerts anti-proliferative and pro-apoptotic effects in human cervical cancer SiHa cells, via both death receptor- and mitochondria-related mechanisms [
147], while it induces ROS-mediated cell death in hepatocarcinoma cells [
148]. In the same study, the Authors showed that hesperetin significantly inhibited the growth of xenograft tumors [
148]. Hesperidin (20 mg/kg BW) suppressed cell proliferation markers, angiogenic growth factors, COX-2 mRNA expression, enhanced apoptosis, and reduced aberrant crypt foci in DMH-induced colon carcinogenesis in rats [
149,
150].
Anti-proliferative activity has also been described for the glycone hesperidin: Patil et al. [
151] found that it inhibits cell cycle progression in Panc-28 human pancreatic carcinoma cells, while Park et al. [
152] described its cytotoxic and pro-apoptotic effects on SNU-C4 human colon cancer cells. In HepG2 hepatocarcinoma cells, its ability to induce apoptosis via both mitochondrial and death receptor pathways has been demonstrated [
153], as well as the non-apoptotic programmed cell death namely paraptosis [
154]. Hesperidin also inhibits proliferation of Ramos Burkitt’s lymphoma cells and sensitizes them to doxorubicin-induced apoptosis through the inhibition of both constitutive and doxorubicin-mediated NF-κB activation in a PPARγ-independent manner [
155]. In hematopoietic malignancies, hesperidin promoted p53 accumulation and downregulated constitutive NF-κB activity in both PPARγ-dependent and -independent pathways [
156]. Induction of apoptosis by hesperidin has also been reported in human mammary carcinoma MCF-7 [
157,
158] and human cervical cancer HeLa cells [
159].
Other reports have shown that hesperidin and neohesperidin increase the sensitivity of Caco-2 cells to doxorubicin, which is consistent with decreased Pgp activity demonstrated in drug-resistant human leukaemia cells (CEM/ADR5000) at non-toxic concentrations (0.32–32 μM) [
160]. Inhibition of Pgp has also been described for hesperetin and quercetin in breast cancer resistance protein (BCRP/ABCG2)-overexpressing cell lines [
161]. Moreover, hesperidin has been reported suppress proliferation of both human breast cancer and androgen-dependent prostate cancer cells through mechanisms other than antimitotic ones, suggesting a possible interaction with androgenic receptors [
162].
Encouraging results in vivo of carcinogenesis inhibition by hesperidin have also been observed. The compound (500 ppm/kg BW) was found to inhibit 4-NQO-induced oral carcinogenesis and to decrease the number of lesions, polyamine levels in tongue tissue, and cell proliferation activity [
163]. Later, the same group reported the inhibition of 4-NQO, AOM, MNAN, and OH-BBN-initiated tumorigenesis by hesperidin alone or in combination with diosmin, as described above [
95,
97]. Moreover, when administered subcutaneously to CD-1 mice, hesperidin inhibited TPA-induced tumor promotion, although it did not inhibit DMBA-induced tumor initiation [
164]. Later, they documented the protective effect of hesperidin against the TPA-stimulated infiltration of neutrophils, suggesting its potential as a chemopreventive agent against tumor promoter-induced inflammation and hyperplasia [
165]. Daily administration of hesperetin (20 mg/kg BW)
per os for 15 weeks inhibited rat colon carcinogenesis during and after DMH initiation [
166]. Further, in rats with DMBA-induced mammary gland tumors, pretreatment with hesperetin (50 mg/kg BW/day) significantly reduced the tumor burden and the overexpression of the proliferating cell nuclear antigen (PCNA), as well as restoring the decreased Bcl-2 and increased Bax expression. By contrast, in the liver of mice treated with DMBA, at a dosage of 10 mg/kg BW, it prevented DNA fragmentation and decreased Bax expression and cleaved caspase-3, caspase-9 and PARP [
167]. This study suggests that hesperetin may act as either pro-apoptotic or anti-apoptotic agent depending on the circumstance [
167]. Attenuation of BP-induced lung cancer afforded by hesperidin supplementation (25 mg/kg BW) has also been reported [
168]. Finally, dietary administration of hesperetin at 1000 ppm and 5000 ppm significantly deterred xenograft growth in athymic mice ovariectomized and transplanted with aromatase-overexpressing MCF-7 cells, while no such effect was observed in mice treated with apigenin or naringenin. Western blot analysis indicated that cyclin D1, CDK4, and Bcl-XL were reduced in the tumors of hesperetin-treated mice, and there are also results suggesting that the flavonone reduces plasma estrogen [
169].
Didymin and poncirin are two flavanones that have been investigated less. However, studies have shown their ability to induce the extrinsic apoptosis pathway in human non-small cell lung cancer cells [
170] and gastric cancer cells [
171], respectively.
Anthocyanidins and anthocyanins occur ubiquitously in the plant kingdom and confer the bright red, blue, and purple colors to fruits and vegetables. In CF, they are found most commonly in oranges, predominantly as mixture of them. Several investigations have shown the antiproliferative effects of anthocyanidins and anthocyanins both in vitro (towards multiple cancer cell types) and in vivo [
172]. The main characteristics of the studies presented in this section are reported in
Table 3.
3.3. Inhibition of Tumor Progression: Focus on Angiogenesis and Metastatization
Both development and progression of solid neoplasms requires rapid and persistent growth of new blood vessels (neo-angiogenesis) around the cancer tissue to supply the growing tumor with nutrients and oxygen. Cancer cells can stimulate angiogenesis by secreting angiogenesis-promoting growth factors, such as the vascular endothelial growth factor (VEGF), the most important endothelial cell-selective mitogen in vitro. VEGF also produces a substantial increase in vascular permeability that allows tumor cells access to the bloodstream, thereby linking angiogenesis and metastases with a poor prognosis [
91].
It has been reported that some flavonoids, including naringin, apigenin, and rutin, are able to inhibit VEGF release in MDA human breast cancer cells [
173], and VEGF and transforming growth factor-β1 (TGF-β1) in the GL-15 glioblastoma cell lines [
174]. Several findings suggest that apigenin can be considered a natural anti-angiogenic compound. Indeed, it reduces VEGF transcriptional activation via hypoxia-inducible factor 1 (HIF-1) pathway in A549 lung cancer cells, and inhibits angiogenesis in the tumor tissues of nude mice [
175]. The inhibition of HIF-1 and VEGF expression has been described in different cancer cells in normoxic or hypoxic conditions [
176]. The Authors described the inhibition of tumor angiogenesis using both chicken chorioallantoic membrane and Matrigel plug assays [
176]. Apigenin-induced reduction of neo-angiogenesis in the human umbilical vein endothelial cell (HUVEC) seems to be mediated by inhibition of matrix-degrading proteases [
177]. Recently, it has been shown that apigenin may act by modulating the inflammatory cytokine IL-6/activators of transcription 3 (STAT3) (IL-6/STAT3) signaling pathways in HUVEC cells. Angiogenesis inhibition resulted in modulation of the activation of extracellular signal-regulated kinase-1/2 (ERK 1/2) signaling triggered by IL-6, as well as in a marked reduction in the proliferation, migration, and morphogenic differentiation of endothelial cells. These effects were coupled with reduced expression of the IL-6 signal transducing receptor-alpha (IL-6Rα) and suppression of cytokine signaling (SOCS3) protein, as well as the secretion of extracellular matrix metalloproteinase (MMP)-2 [
178].
Other
Citrus flavonoids have been evaluated for their potential anti-angiogenic capability. Lam et al. [
179] demonstrated the anti-angiogenic activity of some polymethoxylated flavonoids, including hesperetin and nobiletin, both in vitro (HUVEC cells) and in vivo (the zebrafish embryo model). The structure–activity relationship (SAR) analysis indicated that a flavonoid with a methoxylated group at the C3′ position offers stronger anti-angiogenic activity, whereas the absence of a methoxylated group at the C8 position causes lower lethal toxicity in addition to enhancing anti-angiogenic activity. Anti-angiogenic activity of nobiletin in vitro and in vivo previously reported by Kunimasa et al. [
180], gave an in-depth description of the mechanisms underlying its inhibitory action on multiple functions of the proliferation, migration, and tube formation of HUVEC cells. Wang et al. [
181] reported nobiletin to inhibit tumor growth and angiogenesis by reducing VEGF expression of K562 cells xenograft in nude mice. Moreover, quercetin inhibited tube formation in HUVEC cells and suppressed the angiogenic process in a chick chorioallantoic membrane assay [
182]. Interestingly, the flavonoid quercetin possessed strong inhibitory effects on vessel formation and on endothelial cell proliferation, and concomitantly showed strong antioxidant activity [
183].
Many studies have reported that flavonoids, many of which are abundant in the
Citrus genus, are an effective natural inhibitor of cancer invasion and metastasis [
184]. In particular, tangeretin and nobiletin appear to be able to inhibit the progression phase of carcinogenesis.
In MCF-7/6 breast cancer cells, tangeretin was found to upregulate the function of the E-cadherin/catenin complex, which consequently led to firm cell–cell adhesions and inhibited cell invasion [
185]. In brain tumor cells, nobiletin, and to a lesser extent, tangeretin, exhibited inhibitory activity on the adhesion, migration, invasion, and secretion of MMP-2/MMP-9. In glioblastoma, nobiletin inhibited human U87 and Hs683 glioma cell growth and migration by arresting cell cycle and suppressing the MAPK and Akt pathways [
69]. Naringin inhibited the invasion and migration of glioblastoma U87 MG cells by increasing the expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), thereby decreasing the expression and proteinase activity of MMP-2 and MMP-9 and enhancing the focal adhesion kinase (FAK)/MMPs pathway [
138]. Moreover, naringin inhibited cell migration and invasion of chondrosarcoma cells via vascular cell adhesion molecule 1 (VCAM-1) down-regulation by increasing miR-126 [
186], while in bladder cancer cells it downregulated the Akt and MMP-2 pathways [
187]. In an experimental model of pulmonary metastasis generated by inoculating albino Swiss mice with highly metastatic murine melanoma cells B16F10, diosmin reduced the number of metastatic nodules in the lung more effectively than tangeretin and rutin [
188]. Furthermore, oral administration of naringenin or hesperitin reduced the number of lung metastases in C57BL6/N mice inoculated with B16F10 cells, and increased survival time after tumor cell inoculation [
189]. In addition, in a breast cancer resection model that mimics clinical situations after surgery, orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Both in vitro and in vivo experimental results have further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism underlying metastasis inhibition by naringenin [
190]. Some reports have illustrated the mechanisms by which nobiletin may reduce tumor invasion and metastasis in vitro. In human fibrosarcoma HT-1080 cells stimulated with TPA, it directly inhibited the phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (MEK), thereby suppressing either the sequential phosphorylation of extracellular regulated kinases (ERK) and the expression of MMP [
191]. MMP-1 and -9 expression were suppressed by nobiletin in fibrosarcoma cells with an associated increase in tissue inhibitors of MMPs [
192]. Additionally, MMP-7 was down-regulated in colorectal cells [
193], while MMP-2 in human nasopharyngeal carcinoma cells [
194]. Nobiletin exerts antimetastatic effects on human breast cancer cells [
195] through the down-regulation of both CXC chemokine receptor type 4 (CXCR4) and MMP-9 via a mechanism involving NF-κB inhibition and MAPKs activation. Minagawa et al. [
196] showed that pro-MMP-9 activity was inhibited by nobiletin in gastric cell lines, and reported a significant reduction in the peritoneal dissemination of stomach cancer nodules when the polymethoxylated flavone was administered subcutaneously to severe combined immune deficient (SCID) mice. Moreover, nobiletin has been shown to reduce adhesion, invasion, and migration of highly metastatic human gastric adenocarcinoma AGS cells by inhibiting the activation of FAK and PI3K/Akt signals, which in turn downregulates MMP-2 and -9 expression and activity [
197]. Finally, nobiletin inhibited the epithelial–mesenchymal transition of human non-small cell lung cancer cells by antagonizing the TGF-β1/Smad3 signaling pathway, thus prohibiting the growth of metastatic nodules in the lungs of nude mice [
198].
Treatment of MDA-MB-231 breast tumor cells with apigenin (ranging from 2.5 to 10 μg/mL) led to a partial decrease in urokinase-plasminogen activator (uPA) expression and completely inhibited phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion [
199]. Apigenin also inhibited hepatocyte growth factor (HGF)-induced migration and invasion and decreased HGF-stimulated integrin β4 and Akt phosphorylation in MDA-MB-231 cells. It also inhibited HGF-promoted metastasis in nude mice and in chick embryos [
200]. In prostate cancer, the motility and invasion of PC3-M cells were inhibited by apigenin through a FAK/Src signaling mechanism [
201]. In ovarian cancer, it inhibited FAK-mediated migration and invasion of A2780 cells, and repressed spontaneous metastasis formation on the ovaries of nude mice following inoculation with A2780 cells [
202]. In cervical cancer, apigenin inhibited the motility and invasiveness of HeLa cells [
203]. Moreover, its administration significantly decreased the incidence of cancer metastasis in AOM-induced intestinal adenocarcinoma in rats [
204]. Noh et al. [
205] further reported that this flavone inhibited PMA-induced migration and invasion of human cervical carcinoma Caski cell line via the suppression of p38 MAPK-dependent MMP-9 expression. Finally, intraperitoneal administration of apigenin and quercetin into syngeneic mice injected with B16-BL6 melanoma cells resulted in a significant delay in tumor growth and lungs metastases, with flavonoids being more effective than tamoxifen [
206].
Over the last decade, there has been extensive researches into the potential anti-invasive role of quercetin. In breast cancer, the invasive activity of PMA-induced MCF-7 cells was blocked by the flavonol by reducing MMP-9 expression and by blocking activation of the protein kinase C (PKC)/ERK/AP-1 signaling cascade [
207]. In MDA-MB-231 cells the anti-invasive effect was mediated by inhibiting MMP-3 activity [
208]. In PC-3 prostate cancer cells, quercetin (50 and 100 μM for 24 h) decreased MMP-2/MMP-9 expression [
209] and downregulated the mRNA of uPA, uPA receptor (uPA-R), EGF, and EGF receptor (EGF-R), thereby inhibiting invasion and migration [
210]. In human glioblastoma U87 cells, quercetin blocked PMA-induced migration and invasion by inhibiting ERK-dependent COX-2 activation and MMP-9 activity [
211], while in the DAOY medulloblastoma cell line, it reduced both Met-induced cell migration and HGF-mediated Akt activation [
212]. Moreover, quercetin decreased the invasiveness of A431 epidermal cancer cells by increasing EGF-depressed E-cadherin, by down-regulating both epithelial–mesenchymal transition (EMT) markers and MMP-9, leading to the restoration of cell–cell junctions [
213]. In addition, it inhibited cell–matrix adhesion, migration, and invasion of HeLa cells [
214] and inhibited the motility and invasion of murine melanoma B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway [
215]. The administration of quercetin to DMBA-induced mammary carcinoma rats has been reported to significantly decrease both tissue type plasminogen activator (t-PA) and u-PA [
216]. Lastly, didymin was observed to suppress phthalate-mediated breast cancer cell proliferation, migration, and invasion, suggesting that it is capable of preventing phthalate ester-associated cancer aggravation [
217].
Table 4 summarizes the essential features of the studies on the anti-angiogenic and anti-metastatic activity of
Citrus flavonoids.