Flavonoids in oral cancer prevention and therapy.

Review article 517

Flavonoids in oral cancer prevention and therapy Daniele Maggionia, Luisa Biffia,b, Gabriella Nicolinia and Werner Garavelloa,b Oral cancer, representing all the malignancies arising in the oral cavity, is the eighth most diffused neoplasm worldwide. Despite therapeutic improvements, its survival rate has not changed significantly over the past few decades, with a 5-year survival rate slightly above 50%. In this context, a search for new therapeutic strategies is mandatory. Flavonoids, polyphenolic compounds derived from plants, have a broad spectrum of biological activities, including antioxidant and anticancer. They have been proved to counteract the growth of several types of cancer through multiple mechanisms including the inhibition of cell cycle progression, apoptosis induction, and the modulation of intracellular pathways. Because of their multiple biological activities and their safe toxicological profile, flavonoids have been studied widely in the last decade as potential leads for anticancer therapy. Several studies have reported different flavonoid effects according to cancer cell type. In the

Introduction Oral cavity cancer is a significant component of the global cancer burden (Petersen, 2009) as ∼ 264 000 cases of oral cavity cancer are diagnosed each year worldwide (Hashibe and Sturgis, 2013). In a recent analysis of the global cancer diffusion on the basis of GLOBOCAN 2008 data, cancer of the lips and oral cavity is the eighth most common cancer worldwide by incidence in men (Jemal et al., 2011). Smoking and alcohol consumption have been identified as the major risk factors (Jemal et al., 2011). Indeed, about 65% of oral cavity cancers are attributed to smoking (Hashibe et al., 2009). Moreover, large-scale epidemiological investigations have documented a synergistic effect of tobacco and alcohol drinking (Blot et al., 1988; Hashibe et al., 2009): the attributable risk of these two factors in the population has been globally (men and women) estimated to be about 74% (Hashibe et al., 2009; Anantharaman et al., 2011). A relationship between oral cancer and other unsafe habits, such as betel quid (Nair et al., 2004) or mate beverage consumption (Dasanayake et al., 2010), and periodontal disease has also been established (Meyer et al., 2008). Despite general improvements achieved in cancer therapy, overall oral cancer survival has not increased significantly over the past few decades. Moreover, the toxicity usually associated with current chemotherapeutics provides a further incentive to search for the development of new anticancer strategies. Research has recently been focused on dietary habits that have been 0959-8278 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

present review, therefore, we have evaluated the data available on the effect of flavonoids on oral cancer, with the aim of identifying the molecular mechanisms underlying their potential anticancer properties. European Journal of Cancer Prevention 24:517–528 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. European Journal of Cancer Prevention 2015, 24:517–528 Keywords: chemoprevention, flavonoids, oral cancer Departments of aSurgery and Translational Medicine and bOtorhinolaryngology, Head and Neck Surgery, San Gerardo Hospital, University of Milan-Bicocca, Monza, Italy Correspondence to Daniele Maggioni, PhD, Department of Surgery and Traslational Medicine, University of Milan-Bicocca, Via Cadore 48, 20900 Monza (MB), Italy Tel: + 39 0264488124; fax: + 39 0264488253; e-mail: [email protected] Received 16 July 2014 Accepted 30 October 2014

shown to play a key role in the development and prevention of oral cancer (Garavello et al., 2009): studies have shown that a poor diet with infrequent fruit and vegetable consumption is related to an increased risk of oral cancer (Lagiou et al., 2009), whereas a high intake of fruits and vegetables seems to reduce the risk of oral cancer (Garavello et al., 2009). In particular, natural compounds, such as flavonoids, have attracted attention because of their multiple pharmacological activities and their nontoxic profiles (Ravishankar et al., 2013). Flavonoids are polyphenol phenyl benzopyran derivatives consisting of a 15-carbon basic skeleton sharing a common structure based on a three-carbon bridge between two phenyl groups cyclized with oxygen (C6-C3-C6). More than 4000 flavonoids have been identified and they are ubiquitously found in plants, where they are involved in disease resistance, and are widely present in vegetables, fruits, and plant-derived beverages (Corradini et al., 2011). According to their hydroxylation pattern and variations in the chromane ring, flavonoids can be further classified into subgroups: flavanones, flavones, flavonols, isoflavons, and flavan-3-ols (Tsao, 2010) (Fig. 1). They have been shown to have a wide range of biological and pharmacological activities, even at nontoxic concentrations (Miean and Mohamed, 2001). Recent findings have supported the role of flavonoids in the prevention of neurodegenerative and cardiovascular diseases and of cancer (Tsao, 2010). Over the past two DOI: 10.1097/CEJ.0000000000000109

Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.

518 European Journal of Cancer Prevention 2015, Vol 24 No 6

Fig. 1

Flavonoids B

O A C

Classification Flavonols O

Flavones

Flavanones

O

O

O

O

O

O

OH

Kaempferol Quercetin

Isoflavones

Apigenin Baicalein Flavopidirol Luteolin Sylibinin

Hesperetin Naringenin

Daizdenin Genistein

Flavan-3-ols O

O

OH

Epicatechin Epigallocatechin Gallocatechin

The basic structure of flavonoids and their classification as flavonols, flavones, flavanones, isoflavones, and flavan-3-ols.

decades, the interest in the potential antitumoral effects of such compounds has been growing continuously. Indeed, the number of published papers on flavonoids and cancer has grown from a few articles in the mid-1990s to almost 1000 in 2012 (http://www.pubmed.gov). Although their strong antioxidant property is generally referred to as the most important anticancer effect of flavonoids, several in-vitro and in-vivo experiments have identified their ability to modulate cell signaling pathways, thus affecting multiple biological aspects, such as cell proliferation, cell migration, and apoptosis (Kandaswami et al., 2005; Corradini et al., 2011). Several lines of evidence have strongly highlighted a correlation between dietary flavonoid consumption and a reduced risk of cancer (Ren et al., 2003). Two case–control studies carried out in the north of Italy from 1992 to 2000 pointed out a significant inverse correlation between flavanone and flavanol intake and laryngeal and esophageal cancer risk (Garavello et al., 2007; Rossi et al., 2007).

attempt to highlight and provide a general vision of the molecular mechanisms underlying their putative anticancer action.

Flavonols Flavonols represent the major subclass of flavonoids and are characterized by a hydroxyl group at the thirdposition of the chromane ring. They are widespread in higher plants, where they are found especially in the leaves. Among these, the most diffused and studied flavonols are quercetin and kaempferol. Quercetin

Quercetin (3.5.7.3′.4′-pentahdroxyflavone) is the most important and one of the most abundant molecules in the class of flavonoids (Haghiac and Walle, 2005). Many often-consumed foods contain quercetin – that is, onions, apples, tea berries, wine, and many others (Hertog et al., 1993; Lamson and Brignall, 2000).

Despite the growing evidence supporting the role of flavonoids in oral cancer prevention, very few data are available on the mechanisms of action of the different flavonoids in oral cancer. Flavonoid-based chemoprevention might be particularly useful in oral squamous cell carcinoma (SCC) as it is characterized by a defined progression from premalignant oral epithelial transformations to invasive cancer, and flavonoids may potentially affect both tumor initiation and progression.

The antioxidant, anti-inflammatory, antiproliferative, and apoptotic properties of quercetin have been investigated widely (Lamson and Brignall, 2000; Hirpara et al., 2009). Unfortunately, the health benefits of quercetin are limited by its very low absorption rate and its bioavailability (Lamson and Brignall, 2000). However, Walle et al. (2005) reported that dietary flavonoid glucosides may be hydrolyzed in the oral cavity by both bacteria and shed epithelial cells to deliver the biologically active aglycones at the surface of the epithelial cells (Walle et al., 2005).

Therefore, the aim of this review is to summarize the data available on flavonoid effects in oral cancer in an

The antiproliferative effects of quercetin have been shown in different cancer cell lines. In particular, Haghiac


Flavonoids in oral cancer prevention Maggioni et al. 519

and Walle (2005) analyzed the antiproliferative effect of quercetin on the oral SCC line SCC-9. They found that quercetin inhibited oral cancer cell proliferation in an irreversible dose-dependent and time-dependent manner. Similar results were obtained in other oral cancer cell lines: several groups used SCC-25 taken from tongue SCC and confirmed that quercetin has a dose-dependent growth-inhibitory effect (Chen et al., 2013). Elattar and Virji (2000), however, reported a biphasic effect of quercetin depending on its concentration: stimulating cell growth at doses between 1 and 10 mmol/l, while inhibiting cell proliferation at higher concentrations. Human SCC HSC-3 and TW-206 were used by Huang et al. (2013), confirming a dose-dependent growthinhibitory effect. Also, SCC-1483 from the retromolar trigone have been used with similar results (Kang et al., 2010). Cell cycle progression impairment and apoptosis seem to underlie the quercetin-induced cell growth inhibition in oral cancer cell lines. Indeed, both G0/G1 and S phase blockage have been reported following quercetin exposure in SCC-9 and SCC-25 cells (Haghiac and Walle, 2005; Chen et al., 2013). In addition, the authors found that thymidylate synthase (TS), a key-S phase enzyme, commonly overexpressed in oral cancer cells, was inhibited by quercetin at the protein level, whereas TS mRNA levels were not affected. They suggest that TS downregulation occurs at the translational level and that this is presumably linked to the apoptotic effect of quercetin in SCC-9 cells (Haghiac and Walle, 2005). Furthermore, quercetin treatment increased the expression of p21 and induced G2/M phase arrest through Fork-head Box protein O 1 (FOXO1) in HSC-3 and TW-206 cells (Huang et al., 2013). The proapoptotic effect of quercetin on oral cancer cell lines has also been investigated, analyzing different apoptotic pathways. Chen et al. (2013) found that quercetin-treated SCC-25 cells had an increased Bax/Bcl-2 ratio. The modulation of the Bax/Bcl-2 ratio seems to be cell type specific as it has not been confirmed in HSC-3 cells (Huang et al., 2013). Moreover, in contrast to the above-mentioned study, flow cytometry studies of the SCC-25 cell cycle distribution showed that quercetin induced mainly G1-phase arrest with a dose-dependent cyclin D1 decrease and increased expression of p21cip1 protein (Chen et al., 2012). Quercetin determined apoptosis with a caspase-3-dependent mechanism even on SCC-1483 (from the retromolar trigone zone) and SCCQLL1 (a squamous cell line originating from metastatic lymph nodes of oral cavity cancer) (Kang et al., 2010). Aberrant epidermal growth factor receptor (EGFR) signaling is often associated with a poor prognosis in SCC of the head and neck. Huang et al. (2013) found that quercetin treatment on HSC-3 cells, an EGFR-overexpressing oral cancer cell line, could serve an apoptotic function,

increasing FOXO1 protein levels that suppressed EGFR/Akt activation. Quercetin-induced FOXO1 activation led to increased levels of FasL and cleaved caspase-3, suggesting the activation of the extrinsic apoptotic pathway. Moreover, quercetin has been proven to be effective in preventing human tongue carcinoma SAS cell migration by inhibiting phosphoinositol 3 kinase and mitogen activated protein kinases (MAPKs) signaling pathways and by reducing matrix metalloproteinase 2 (MMP-2) and metalloprotease 9 (MMP-9) expression (Lai et al., 2013). Consistent with the in-vitro results, the chemotherapeutic potential of quercetin has also been confirmed by the in-vivo model. Indeed, quercetin pretreatment or simultaneous administration prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis, significantly reducing the incidence of both papillomas and tumors (Balasubramanian and Govindasamy, 1996). Conversely, post-treatment quercetin administration to DMBA-painted hamsters resulted in a significant tumor growth delay (Priyadarsini et al., 2011). Moreover, Makita and colleagues (1996) reported the chemopreventive potential of quercetin in a rat model of 4-nitroquinoline1-oxide-induced carcinogenesis, mainly related to the inhibition of cell proliferation in both the initiation and the postinitiation phases of carcinogenesis. Investigations have also been carried out into the potential therapeutic role of quercetin in combination with cisplatin, a chemotherapeutic drug used widely for patients with advanced oral cancer. Chen and colleagues reported that the p38 MAPK-heat shock protein 27 (Hsp27) apoptotic axis plays an essential role in the development of chemoresistance of oral cancer cells to cisplatin and that quercetin could induce apoptosis in these cells by suppressing the expression of p-Hsp27. The authors used quercetin as a coadjuvant of cisplatin in both in-vitro and in-vivo models, and they found that the combination could reduce tumor growth and decrease drug resistance (Chen et al., 2012). Kaempferol

Kaempferol (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1benzopyran-4-one) is a flavonol found in many edible plants (e.g. tea, broccoli, cabbage, beans, and grapes) and in plants or botanical products commonly used in traditional medicine (e.g. Ginkgo biloba, Tilia spp., Sophora japonica, and propolis). It has been shown that kaempferol, like quercetin, effectively inhibits cell proliferation and it induces apoptosis in a caspase-3-dependent manner on SCC, SCC-1483, SCC-QLL1, and SCC-24 cells (Kang et al., 2010). Lin et al. (2013) investigated the potential antimetastatic effect of kaempferol in human tongue SCC, SCC-4 cells. No cytotoxic effect on SCC-4 cell viability was found



Table 1

The most relevant studies on flavonols and oral cancer

Compound

Cell lines

Quercetin

SCC-9

Quercetin

SCC-9

Quercetin

SCC-25

Quercetin Quercetin

SCC-25 TW-206 HSC-3

Quercetin Quercetin

SCC-1483 SCC-QLL1 SCC-25

Quercetin

SAS

Effect Dose-dependent and irreversible inhibition of cell proliferation Dose-dependent and irreversible inhibition of cell proliferation 48 h treatment induces necrosis 72 h treatment apoptosis Dose-dependent inhibition of cell proliferation Inhibition of resistance to cisplatin Proapoptotic effect Inhibition of cell proliferation Dose-dependent inhibition of cell proliferation at doses comprised between 1 and 100 μmol/l Proapoptotic effect Dose-dependent inhibition of cell proliferation proapoptotic effect Inhibition of epithelial to mesenchymal transition reduction of resistance to cisplatin Inhibition of cell migration

Mode of action

References Walle et al. (2005)

G0/G1 and S phase blockage Apoptosis induction through inhibition of thymidylate synthase expression

Haghiac and Walle (2005)

G1 phase blockage Decreased cyclin D1 expression Increased p21 expression Apoptosis induction through increased Bax/Bcl-2 ratio Inhibition of DNA synthesis G2/M phase arrest FOXO1 activation

Chen et al. (2013)

Elattar and Virji (2000) Huang et al. (2013)

Caspase-3-dependent mechanism

Kang et al. (2010)

Promotion of apoptosis through suppression of pHSP-27 Inhibition PI3K and MAPK pathways, leading to reduced expression of MMP-2 and MMP-9

Chen et al. (2012) Lai et al. (2013)

MAPK, mitogen activated protein kinases; MMP-2, matrix metalloproteinase 2; PI3K, phosphoinositol 3 kinase; SCC, squamous cell carcinoma.

following kaempeferol treatment at various concentrations (0–100 μmol/l). In contrast, the suppression of oral SCC cell migration and invasion through the downregulation of MMP-2 expression and extracellular regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner was reported (Lin et al., 2013). Moreover, it was shown that kaempferol decreased the nuclear translocation of activator protein-1, the transcription factor involved in MMP-2 gene expression regulation (Lin et al., 2013). The most relevant studies on flavonols and oral cancer are summarized in Table 1.

Flavones Baicalein

Baicalein (5,6,7-trihydrixyflavone) is a flavonoid derived from the root of Scutellaria baicalensis Georgi, a medicinal plant used traditionally in Chinese herbal medicine. Many studies have shown that baicalein has antioxidant (Gao et al., 1999), anti-inflammatory (Lin and Shieh, 1996), and antimicrobial (Lu et al., 2011) properties. Baicalein has also shown antiproliferative properties in different cancer cell lines (Li-Weber, 2009; Donald et al., 2012; Huang et al., 2012; Zhang et al., 2013). Nevertheless, there have been only a few studies on the anticancer properties of baicalein on oral cancer cells. Lin et al. (2007) investigated the effect of baicalein treatment on SCC-4 human tongue cancer cells. They found that baicalein induced apoptosis on SCC-4 cells in a Ca2 + associated mitochondrial and caspase-3-dependent manner. In particular, they observed an increase in the levels of apoptosis-associated proteins such as p53 and BAX, and a reduction in the level of Bcl-2 (Lin et al., 2007). On human oral squamous carcinoma HSC-3 cells, baicalein inhibited cell proliferation in a dose-dependent and time-dependent manner and it induced G1-phase arrest

by decreasing levels of cyclin D1, phosphorylated Rb, and CDK4 and by activating aryl hydrocarbon receptor (AhR), which had been reported to play a role in regulating the cell cycle. The authors suggested that the hyperphosphorylation of Rb in baicalein-treated HSC-3 cells could be mediated both by its agonistic activity on AhR and by its facilitation of cyclin D1 degradation (Cheng et al., 2012). In the study by Yang et al. (2008a, 2008b), no cytotoxic effect of baicalein concentrations up to 100 μmol/l was reported. The authors also evaluated the antimetastatic activity of baicalein: dose-dependent inhibition of in-vitro cell migration was observed. In particular, at a dose of 100 μmol/l, baicalein reduced the invasion ability of SCC-4 cells by 35.8% in comparison with untreated controls (P < 0.001). It also led to a reduction in MMP-2 and urokinase-plasminogen activator (u-PA) activities, enzymes that are known to be involved in cancer invasion and metastatization. Furthermore, the authors found that baicalein could inhibit the invasive and migratory ability of SCC-4 cells also in an in-vivo model (Yang et al., 2008a, 2008b). Apigenin

Apigenin (4′,5,7-trihydroxyflavanone) is a naturally occurring plant flavone. It is abundant in common fruits such as grapefruit and oranges, and in plant-derived beverages, and vegetables and herbs such as parsley, onions, tea, chamomile, wheat sprouts, and some spices. Apigenin is commonly present in red wine and beer brewed from natural ingredients (Shukla and Gupta, 2010). It has been shown to possess anti-inflammatory (Funakoshi-Tago et al., 2011), antioxidant (Woodman and Chan, 2004), and anticancer properties (Shukla and Gupta, 2010). However, very few data are available on the effect of apigenin on oral cancer.



It has been shown by Walle et al. (2007) that the treatment of human SCC, SCC-9 cells with apigenin resulted in cell proliferation inhibition and G2/M phase arrest. Consistent with these data, the apigenin-negative modulation of cell cycle progression has also been reported by Maggioni et al. (2013), who reported both G0/G1 and G2/M phase blockage through CDK1 inactivation, induced by apigenin in SCC-25 cells. Apigenin’s ability to induce apoptosis has been shown in several cell lines derived from tongue SCC, such as SCC-25 cells (Chan et al., 2012; Maggioni et al., 2013), CAL-27, and SCC-15 cells (Masuelli et al., 2011). Chan et al. (2012) showed that apigenin inhibited SCC-25 cell growth, reporting that apigenin induced a cell cycle arrest in the G2/M phase. In the same study, the authors also found that apigenin induced apoptosis in SCC-25 cells by different pathways: by upregulating tumor necrosis factor receptor and tumor necrosis factor-related apoptosisinducing ligand receptor and by downregulating Bcl-2, both leading to caspase-mediated cell death. Moreover, a synergistic effect of apigenin with 5-FU and cisplatin in inhibiting SCC-25 cell proliferation was found (Chan et al., 2012). As described above for baicalein, the antimetastatic activity of apigenin on SCC-4 was also evaluated (Yang et al., 2008a, 2008b). The authors observed that apigenin inhibited the invasion and migration of SCC-4 cells in a dose-dependent manner. In particular, at a dose of 100 μmol/l, apigenin reduced the invasion ability of SCC-4 cells to 55.5% of that of controls (P < 0.001). It also led to a significant reduction (P < 0.05) in MMP-2 and u-PA activities (Yang et al., 2008a, 2008b). At least two studies have indicated apigenin as a chemoprotective agent against oral carcinogenesis induced by DMBA in hamsters (Silvan et al., 2011; Gómez-García et al., 2013). In the study by Silvan et al., the authors induced oral carcinogenesis by a topical application of 0.5% DMBA on hamster left buccal pouches three times a week for 14 weeks. A group of induced hamsters were administered apigenin orally at a dose of 2.5 mg/kg body weight/day starting a week before exposure to DMBA. Cancer formation was observed in 100% of DMBA-onlytreated animals, whereas in apigenin–DMBA doubleexposed hamsters, no tumors could be observed, but only hyperplasia and mild dysplasia. The results suggest that oral administration of apigenin could prevent, or at least reduce, tumor incidence. In addition, in DMBAtreated animals, there was an altered status of phase I (cytochromes P450 and b5) and phase II (glutathione-Stransferase, glutathione reductase, reduced glutathione, and DT-diophorase) detoxification agents, suggesting the accumulation of toxic metabolites during DMBAinduced oral carcinogenesis. Oral administration of apigenin reversed both phase I and II detoxification agent status to an almost normal range, suggesting that apigenin

enhances the process of conjugation and elimination of carcinogenic metabolites. A similar trend could be observed for phase I and II detoxification agents in buccal mucosa, indicating that apigenin inhibits the activation of DMBA. Eventually, oral administration of apigenin restored the lipid peroxidation and the antioxidant status in DMBA-induced hamsters. In the authors’ opinion, apigenin improved the antioxidant defensive mechanism by scavenging reactive oxygen species generated during DMBA-induced oral carcinogenesis (Silvan et al., 2011). Luteolin

Luteolin, 3′,4′,5,7-tetrahydroxyflavone, is a common dietary flavonoid that can be found widely in the plant kingdom. Vegetables and fruits such as thyme, celery, parsley, broccoli, onion leaves, carrots, peppers, cabbages, apple skins, and chrysanthemum flowers are luteolin rich (Lin et al., 2008). The average luteolin human daily intake is ∼ 16 mg/day (Hertog et al., 1993). As with other flavonoids, luteolin has been shown in many preclinical studies to have numerous pharmacological properties including anticancer activities (Kim et al., 2013). Nevertheless, only one study is available on luteolin’s effect on oral cancer. Yang et al. (2008a, 2008b) examined the chemotherapeutic effects of luteolin on SCC-4 oral cancer cells. Luteolin decreased the viability of SCC-4 in a dose-dependent and time-dependent manner, causing a significant increase in the population of dead cells. The flow cytometry analysis of the cell cycle profile indicated that luteolin induced a dosedependent accumulation in the G0/G1 phase. The authors also found that the effect of luteolin on cell cycle progression involved the cell cycle regulatory molecules as luteolin treatment downmodulated cyclin-dependent kinases (CDKs) 2, 4, and 6 and cyclin D3. Moreover, luteolin caused a significant decrease in pRb, which is a key G1-to-S phase regulator. Furthermore, 48 h luteolin treatment caused apoptosis through a modulation of the Bax/Bcl-2 balance, which led to an increase in cleaved caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP). In addition, luteolin (5 or 10 μmol/l) significantly enhanced the cytotoxic effects of paclitaxel (0.3 nmol/l) on SCC-4 cells (Yang et al., 2008a, 2008b). Finally, the antitumor effect of luteolin and palitaxel was evaluated in an in-vivo model using a nude mouse xenograft injected with subcutaneous SCC-4 cancer cells. The daily administration of 3 or 5 mg/kg body weight of luteolin and 1 mg/kg body weight of paclitaxel by an intraperitoneal injection showed a synergistic inhibitory effect on tumor growth (Yang et al., 2008a, 2008b). Flavopiridol

Flavopiridol (5,7-dihydroxy-8-(4-N-methyl-2-hydroxypyridyl)-6′-chloroflavone hydrochloride) is a semisynthetic



flavonoid derived from rohitukine, a plant indigenous to India. In a primary screening promoted by the Developmental Therapeutics Program of the US National Cancer Institute (NCI), flavopiridol was identified as a suitable antineoplastic drug (Bates et al., 1995) as it showed antiproliferative and cytotoxic effects in both in-vitro and in-vivo models (Sedlacek et al., 1996; Drees et al., 1997). These biological effects correlate with its ability to strongly inhibit CDKs 1, 2, and 4 which are known to govern cell cycle progression (Kelland, 2000). It has also been shown that it can induce G1/S or G2/M cell cycle arrest through CDK1 and CDK2 inhibition; in addition, decreases in cyclin D1 expression and apoptosis following flavopiridol treatment have been described (Senderowicz, 2003). The NCI 60 cell line panel of the above-described NIH screening did not include cells derived from head and neck squamous cell carcinomas (HNSCC). However, Patel et al. (1998) reported that HNSCC cell (HN4, HN8, HN12, and HN30) proliferation was inhibited by flavopiridol in the nanomolar range. In particular, 300 nmol/l flavopiridol led to an ∼ 80% reduction in cellular thymidine uptake (indicating antiproliferative effects). A cell cycle profile analysis showed that flavopiridol caused an increase in the sub-G1 cell fraction, which was suggestive of apoptosis. A concomitant ability to alter the phosphorylation activity on CDC2 and CDK2 and to reduce Table 2

Mihara et al. (2003) investigated the ability of flavopiridol to inhibit the growth of five different oral squamous cancer cell lines (OSCC). In particular, they used human HSC-2, HSC-3, SCC-15, SCC-25, and the laryngeal SCC-derived Ca9–22 cell lines; they confirmed that, even on OSCC cells, flavopiridol inhibited cell proliferation in a dose-dependent and time-dependent manner. The maximal effect was obtained between 300 and 500 nmol/l, with a growth inhibition ranging between 95.2 and 98.7% at 72 h compared with untreated controls (Mihara et al., 2003). Flavopiridol treatment on OSCC decreased CDK1, CDK4, and p34 CDK2 (CDK2-inactive form), whereas it upregulated the CDK2 active form (p33CDK2). Moreover, flavopiridol treatment on OSCC cells resulted in the downregulation of cyclin A, B, and D1 expression, which usually creates complexes with CDKs that allow cell cycle progression. In addition, flavopiridol induced an increased expression of two Bcl-x proteins (Bcl-xS and Bcl-xL), whereas Bcl-2 and Bax expression did not change (Mihara et al., 2003). Silibinin

Silibinin or silybinis is a popular dietary supplement isolated from the milk thistle, Silybum marianum Gaertn

The most relevant studies on flavones and oral cancer

Compound

Cell lines

Apigenin Apigenin

SCC-9 SCC-25

Apigenin Apigenin

CAL-27 SCC-15 SCC-25

Apigenin

SCC-4

Effect

Mode of action

References

Inhibition of cell proliferation Inhibition of cell proliferation Proapoptotic effect Proapoptotic effect

G2/M phase blockage G0/G1 and G2/M phase blockage through CDK1 inactivation

Walle et al. (2007) Maggioni et al. (2013)

Caspase-3-dependent apoptosis reduced activation of ERK1/2

Masuelli et al. (2011)

Proapoptotic effect Inhibition of cell proliferation Synergistic effect with 5-FU and cisplatin Inhibition of cell migration and invasion

TNF-R and TRAIL-R upregulation Downregulation of Bcl-2 G2/M phase blockage Decreasing MMP-2 and u-PA

Chen et al. (2012)

Baicalein

SCC-4

Proapoptotic effect

Baicalein

HSC-3

Baicalein

SCC-4

Luteolin

SCC-4

Flavopiridol

HSC-2 HSC-3 SCC-15 SCC-25 Ca9–22 SCC-4

Dose-dependent and time-dependent inhibition of cell proliferation Inhibition of cell migration Inhibition of cell migration in in-vivo model Dose-dependent and time-dependent inhibition of cell proliferation Proapoptotic effect Synergistic cytotoxic effect with paclitaxel Dose-dependent and time-dependent inhibition of cell proliferation Proapoptotic effect

Silibinin

cyclin D1 protein expression was considered to be responsible for the effects of flavopiridol on cell cycle progression and apoptosis (Patel et al., 1998).

Any inhibitory effect on cell proliferation Inhibition of cell invasion and motility in a dosedependent manner

2+

Ca -associated mitochondrial and caspase-3-dependent decreased MMP expression G1 phase blockage by decreasing cyclin D1, pRb, and CDK4/ activating AhR Decreased MMP-2 and u-PA expression and activity G0/G1 phase blockage CDK2, 4,6 cyclin D3, and pRb downregulation Modulation of Bax/Bcl-2 balance

Yang et al. (2008a), (2008b) Lin et al. (2007) Cheng et al. (2012) Yang et al. (2008a), (2008b) Yang et al. (2008a), (2008b)

Downregulation of CDK1, 2, 4, CAK, cyclin A/B/D1 Increased sub-G1 cell population Upregulation of Bcl-xS and Bcl-xL

Mihara et al. (2003)

Reduction of MMP-2 and u-PA activities upregulation of TIMP-2 and PAI

Haghiac and Walle (2005)

AhR, aryl hydrocarbon receptor; ERK1/2, extracellular regulated kinase 1/2; MMP-2, matrix metalloproteinase 2; PAI, plasminogen activator inhibitor; SCC, squamous cell carcinoma; TIMP-2, tissue inhibitor of metalloproteinase-2; TNF-R, tumor necrosis factor receptor; TRAIL-2, tumor necrosis factor-related apoptosis-inducing ligand receptor; u-PA, urokinase-plasminogen activator.



(Family Asteraceae), known to have anticancer properties. Indeed, it has been shown that silibinin causes significant growth inhibition through G0/G1 or G2 arrest (Agarwal et al., 2003) and induces cellular apoptosis by activation of the caspase cascade in different cancer cells (Mallikarjuna et al., 2004). On human tongue cancer SCC-4 cells, silibinin 24 h treatment (0–100 μmol/l) failed to exert any inhibitory effect on cell proliferation. However, it could significantly reduce the invasion and motility of SCC-4 cells in a dose-dependent manner. It has also been shown that silibinin strongly reduces MMP-2 and u-PA activities in a dose-dependent manner, with only 14.1 and 4.3% protease activity remaining, respectively, after 50 and 100 μmol/l silibilin treatment (Chen et al., 2006). Furthermore, immunoblotting analysis has shown that the tissue inhibitor of metalloproteinase-2 (TIMP-2) and plasminogen activator inhibitor-1 (PAI-1) protein levels are gradually increased by silibinin. Moreover, the suppression of MMP-2, u-PA, and membrane type-1-matrix metalloproteinase (MT1-MMP), activator of MMP-2, activities, and the enhancement of TIMP-2 or PAI-1 are consistent with the results of RT-PCR analysis indicating that silibinin affects MMP-2, MT1-MMP, u-PA, TIMP-2, and PAI-1 expression at transcriptional levels. The authors also found that silibinin inhibits the activation of ERK1/2, resulting in a marked decrease in the nuclear levels and binding activities of NF-kB, c-Jun, and c-Fos, whereas it has no effect on p38 MAPK and Akt activity (Chen et al., 2006). The most relevant studies on flavones and oral cancer are summarized in Table 2.

Flavanones The effect of flavanones such as naringenin and hesperetin in oral cancer has been addressed only in a study by Miller et al. (2008) that evaluated the chemopreventive activity of six citrus flavonones in a hamster model of dimethylbenz[a]anthracene-induced carcinogenesis. Data from this study showed that naringenin significantly lowered the number of tumors, suggesting that this flavanone might prevent the development of cancer (Miller et al., 2008).

Isoflavones Genistein

Genistein is a soy-derived isoflavone, a well-known inhibitor of protein tyrosine kinases and a chemopreventive agent that can suppress tumorigenesis by multiple pathways (Polkowski and Mazurek, 2000; Szkudelska et al., 2007). It has been estimated that, in humans consuming a diet with soy plasma, genistein concentrations may even reach 2.4 μmol/l (Adlercreutz et al., 1993). One of the most important advantages of genistein is its low toxicity in comparison with current chemotherapeutics (Okazaki et al., 2002). The first in-vitro report on the effects of genistein on a human head and neck cell

line was in 1999 (Alhasan et al., 1999), and showed that genistein inhibited the proliferation of human tonguederived HN4 SCC cells. In particular, genistein-treated HN4 SCC cells underwent morphological changes suggesting growth arrest, cell differentiation, and eventual death. A flow cytometric analysis confirmed that genistein induced S/G2M phase cycle arrest. Moreover, genistein determined apoptosis in HN4 SCC cells as the appearance of apoptotic bodies and DNA fragmentation could be observed in treated cells. No toxicity could be observed on genistein-treated normal keratinocytes (Alhasan et al., 1999). A year later, Elattar and Virji (2000) carried out a study to compare the potency of three different plant phenolic compounds (curcumin, genistein, and quercetin) with that of cisplatin on SCC-25 cell growth; they concluded that genistein and quercetin exerted a biphasic effect on cell growth (Elattar and Virji, 2000). Ye and colleagues (2004) also carried out a study on the effect of genistein on SCC-25. As with Elattar and Virji (2000), they investigated the anticancer activity of genistein in comparison with other agents: indomethacin (a nonselective COX inhibitor), colecoxib (a COX-2 specific inhibitor), and baicalein (see above). They found that all four compounds could inhibit the growth of SCC-25 cells, genistein showing a weaker effect in comparison with indomethacin and baicalein (Ye et al., 2004). According to Alhasan et al. (1999), genistein induced a significant G2/M phase arrest in SCC-25 cells. The treatment with 10 μmol/l genistein significantly decreased the percentage of the proliferating cell nuclear antigen-positive SCC-25 cells from 62 to 40% (P < 0.01), without inducing apoptosis. In partial disagreement with the above-reported data, Johnson et al. (2010) tested the effect of three isoflavones, including genistein, on human tongue oral cancer SCC-15 and SCC-25 cells. Their results showed that genistein could decrease the SCC-25 cell survival in a concentration-dependent manner at concentrations higher than 20 μmol/l. Seventy-two-hour treatment with genistein 50 and 100 μmol/l decreased the expression of total ERK (the enzyme involved in controlling cell growth) and phosphorylated ERK levels. In addition, genistein was found to reduce Akt expression and phosphorylation. The results of this study suggest that genistein exerts an antiproliferative effect by modulating MAPK and AKT pathways (Johnson et al., 2010). Genistein also significantly reduced HSC-3 cell invasion in an in-vitro assay and, in fact, genistein treatment could reduce MMP-2 activity. Furthermore, northern blot analysis indicated that genistein downregulated vascular endothelial growth factor mRNA compared with the untreated controls, whereas it did not affect basic fibroblast growth factor or MMP-2 mRNA (Myoung et al., 2003). In addition, in an in-vivo model, a single



Table 3

The most relevant studies on genistein and oral cancer

Compound

Cell lines

Genistein

HN4

Genistein

SCC-25

Genistein Genistein

SCC-25 SCC-15 SCC-25 HSC-3

Genistein

Effect Growth arrest Cell differentiation Eventual death Biphasic action: stimulation of cell growth at doses below 10 mmol/l Inhibition of cell proliferation at concentrations above 10 mmol/l Weak inhibition of cell proliferation Inhibition of cell proliferation in a dose-dependent manner (>20 μmol/l) Inhibition of cell invasion Antiangiogenic activity

Mode of action

References

S/G2M phase blockage

Alhasan et al. (1999)

Inhibition of DNA synthesis

Elattar and Virji (2000)

G2/M phase arrest, downregulation of PGE2 Downregulation of ERK/pERK and Akt/pAkt after 72 h treatment Downregulation of VEGF Downregulation of MMP-2 and its activated form

Ye et al. (2004) Johnson et al. (2010) Myoung et al. (2003)

ERK, extracellular regulated kinase; MMP-2, matrix metalloproteinase 2; SCC, squamous cell carcinoma; VEGF, vascular endothelial growth factor.

administration of genistein 0.5 mg/kg intraperitoneally in HSC-3 tumor-bearing Balb/c mice reduced the levels of CD31 expression, indicating a lower blood vessel density. However, no significant differences were found between treated and untreated animals in tumor volume or in distant metastasis (Myoung et al., 2003). In a subsequent study, the anticancer potential of genistein in combination with cetuximab was evaluated (Park et al., 2010a, 2010b). The combined treatment of cetuximab (0–400 μmol/l) and genistein (0–50 μmol/l) reduced the expression of several key downstream molecules involved in EGFR signaling pathways (including pEGFR and pAKT) further than cetuximab alone in HSC-3 cells. The apoptotic indices were significantly higher in double-treated HSC-3 cells than after single-agent exposure. In the in-vivo model, the combined therapy significantly inhibited tumor growth and the number of proliferating cells after 4 weeks of treatment compared with the untreated controls. Moreover, the combination of cetuximab and genistein showed the lowest microvessel densities. Although the cetuximab–genistein combination appeared to be a promising cancer therapy, the authors asserted that it had considerable limitations, the overriding one being that not all oral cancer cell lines were responsive (Park et al., 2010a, 2010b). The most relevant studies on isoflavones and oral cancer are summarized in Table 3.

derived cell lines such as SCC-4, SCC-9, SCC-25, HSC-3, and OSC-2 and the oral mucosa carcinoma derived OC-2 cells (Fan, 1992; Khafif et al., 1998; Elattar and Virji, 2000; Masuda et al., 2003; Weisburg et al., 2004; Hsu et al., 2005; Koh et al., 2011). The effectiveness of catechins in these studies varies according to cell type; however, almost all studies report a strong inhibition of cell proliferation for EGCG doses above 100 μmol/l. Hsu et al. (2005) highlighted the involvement of p21/Waf1 in the EGCG-induced cell cycle arrest. In particular, a G0/G1 blockage of the cell cycle, accompanied by decreased cyclin D1 levels, has been observed following EGCG exposure in both primary cultures of normal epithelial cells and in cancer cell lines (Khafif et al., 1998; Elattar and Virji, 2000; Masuda et al., 2001; Masuda et al., 2003; Weisburg et al., 2004; Hsu et al., 2005). However, some reports have suggested a selective action of catechins toward tumoral cells (Weisburg et al., 2004; Babich et al., 2005; Gonzalez de Mejia et al., 2005) as IC50 are usually three- to four-fold lower in cancer cells than in normal fibroblasts (Babich et al., 2005). EGCG has been shown to induce apoptotic cell death (Ishino et al., 1999; Hsu et al., 2001; Hsu et al., 2005) with a mechanism involving the inactivation of AKT (Park et al., 2010a, 2010b; Koh et al., 2011) and the alteration of the Bcl-2/ bax ratio (Mohan et al., 2007).

Flavanols Flavonoids include the important class of flavanols, comprising catechins and epicatechins, the former representing the transconfiguration isomer and the latter representing the cis-configuration isomer (Tsao, 2010). They are a class of polyphenolic compounds found in fruits such as grapes and blueberries, and highly present in tea leaves. The most diffused catechins are epigalloccatechin-3-gallate (EGCG), epigallocatechin, and epichatechin (Tsao, 2010); EGCG, being the most diffused, accounts for more than 30% of tea leaves’ dry extract (Yang et al., 2002). Several beneficial properties have been attributed to tea catechins, including oral cancer prevention (Lee et al., 2004). Several lines of evidence have shown a dose-dependent growthinhibitory effect of EGCG on different HNSCC-

A very important role in catechin-induced cell cycle arrest and apoptosis in oral cancer cells is played by p57, a cyclin-dependent kinase and apoptosis inhibitor, as EGCG treatment induces a marked increase in p57 expression in normal cells, but not in tumoral cells. It has been hypothesized that p57 plays a protective role in normal cells, whereas in cancer cells lacking p57, EGCG induces apoptosis through caspase-3 activation (Hsu et al., 2001). In addition, one putative mechanism underlying EGCG cytotoxicity is the pro-oxidant effect in cancer cells. In fact, Yamamoto et al. (2003) reported a marked increase in reactive oxygen species levels in cancer cells, whereas they observed a decreased redox potential in normal cells; these data are consistent with the reports of Weisburg et al. (2004) and Mohan et al. (2007).



Catechins might also stimulate tumor cell recognition by the immune system as it has been reported that EGCG treatment reduces the levels of 2,3-dioxygenase, an immunomodulatory protein, which is often expressed by cancer cells and associated with immune escape (Cheng et al., 2010). Apart from growth inhibition, catechins may also counteract the spread of oral cancer. In fact, EGCG has showed an interesting ability to prevent epithelial to mesenchymal transition, a key process for cancer cells to invade surrounding tissues, leading to tumor spread and metastasis. Several studies have shown catechins’ inhibition of cell motility and migration in vitro through modulation of paxillin and FAK activation (Park et al., 2010a; Chang et al., 2012; Hwang et al., 2013). They also inhibit MMP and u-PA expression and activation: for Table 4

Cell lines

EGCC

HeLa KB 3T3 Oral cancer Oral leucoplakia-derived cell lines HSC-2 HSG SCC-25

EGCC

OSCC cell lines

EGCC

Normal keratinocyte SCC-25 OSC-2 NHOK SCC-25 Oral keratinocyte Oral cancer cell line Normal gingival Keratinocyte CAL-27 HSC-2 OSC-2 Normal oral fibroblasts HSC-2 NHOK SCC-25 OC-3 OEC-M1 OC-2 cells Tongue SCC CAL-2 NHEK OSC-2 OSC-4 SCC-9 SCC-25 HSC-3 SGT adenocarcinoma cells

EGCC

EGCC

EGCC EGCC EGCC

EGCC EGCC EGCC

EGCC EGCC EGCC

EGCC

EGCC EGCC

EGCC EGCC

These promising in-vitro abilities have also been confirmed in some in-vivo studies: Azuine and Bhide (1994) reported that orally administered catechins reduced methyl-(acetoxymethil)-nitrosamine-induced oral cancer in Syrian golden hamsters. These data are consistent with a report by Ko et al. (2007), who showed that administration of EGCG markedly reduced oral cancer incidence in N-methyl-N-benzylnitrosamine-exposed hamsters. Similarly, prolonged oral administration (1.2 mg/body/day) significantly inhibited oral xenograft tumor growth in Balb/c nude mice. Of note in this study was the selective effect of EGCG toward telomerase-

The most relevant studies on flavanols and oral cancer

Compound Catechin

instance, Kato et al. (2008) reported that EGCG reduces methylation levels of reversion-inducing cystein rich with kazal motif (RECK) protein, a key molecule in the regulation of MMP-2 and MMP-9 expression.

HSC-3 SAS Ca-922 Human Kb Murine OSCC SCC-4 S-G cells

Effect

Mode of action

References

Time-dependent and dose-dependent inhibition of cell growth

Inhibition of DNA synthesis

Fan (1992)

Inhibition of cell proliferation

G1 phase arrest

Khafif et al. (1998)

Apoptosis

Activation of caspase pathway

Ishino et al. (1999)

Inhibition of proliferation

DNA synthesis inhibition

Inhibition of cell proliferation and cell motility Apoptosis in malignant cells

Reduction of invavopoda formation through modulation of FAK-paxillin signaling Increase of p57 (CDK1 inhibitor) protein in normal keratinocytes

Elattar and Virji (2000) Hwang et al. (2013)

Inhibition of cell proliferation Inhibition of proliferation

Induction of differentiation through increased IGFBP-5 expression Reduction of Her-2 phosphorylation

Inhibition of cell proliferation

Increase ROS levels in tumoral cells

Weisburg et al. (2004)

Inhibition of cell proliferation Inhibition of cell proliferation apoptosis

Increase of p21-WAF Oxidative stress induction in tumoral cells

Hsu et al. (2005) Babich et al. (2005)

Inhibition of cell invasion in vitro

Suppression of MMP-13 expression

Chiang et al. (2006)

Inhibition of cell migration Inhibition of cell proliferation apoptosis

Inhibition of MMP-2/9 and u-PA activities Reduction of Bcl-2/Bax ratio

Ho et al. (2007) Mohan et al. (2007)

Inhibition of cell proliferation apoptosis induction

Increase of p57 expression Increased ROS levels in tumoral cells

Yamamoto et al. (2007)

Inhibition of cell migration

Downregulation of MMP-2 and MMP-9 through RECK methylation

Kato et al. (2008)

Inhibition of cell migration

Reduced expression of integrin B1 dowregulation of FAK, AKT, and ERK1/2 Upregulation of IDO

Park et al. (2010a)

Reduction of immunescape ability

Apoptosis and inhibition of cell migration Inhibition of cell migration

Downregulation of ERK1/2 and AKT activation of p38 MAPK Reduced activity of MMP and u-PA FAK and SRC pathways downregulation

Hsu et al. (2001)

Lin et al. (2002) Masuda et al. (2003)

Cheng et al. (2010)

Koh et al. (2011) Chang et al. (2012)

ERK1/2, extracellular regulated kinase 1/2; IDO, 2,3-dioxygenase; MAPK, mitogen activated protein kinases; MMP-2, matrix metalloproteinase 2; PI3K, phosphoinositol 3 kinase; RECK, reversion-inducing cystein rich with kazal motif; ROS, reactive oxygen species; SCC, squamous cell carcinoma; u-PA, urokinase-plasminogen activator.



dependent cancers (Naasani et al., 2003). The most relevant studies on flavanols and oral cancer are summarized in Table 4. Conclusion

The data reported in this review highlight the growing interest in the use of flavonoids as anticancer agents. In particular, their safe toxicology profile and the possibility of being included in the diet make them attractive candidates for cancer prevention and therapy. The results of preclinical studies on flavonoids suggest that not only may they help to develop new natural product-based medicines to reduce the risk of cancer, but flavonoids might also be a potential therapeutic option. Indeed, the current therapeutic approaches to cancer are usually characterized by high toxicity, whereas the low toxicity associated with flavonoids makes them interesting candidates as new anticancer drugs. Moreover, in the present study, the various mechanisms underlying the potential anticancer activity of flavonoids in oral cancer have been pointed out. Two principal flavonoid biological activities may be highlighted: an antioxidant ability that may play a pivotal role in cancer prevention and a cytostatic or cytotoxic one that may support the potential chemotherapeutic use of flavonoids. Furthermore, flavonoids have been found to affect various aspects of cancer development and progression. The combination, therefore, of different flavonoids might significantly improve the therapeutic outcome as mechanisms might overlap and be synergic. In addition, flavonoids might have interesting effects if used in combination with current anticancer agents as they might increase the chemotherapeutic effect and also counteract, because of their antioxidant properties, the adverse side effects of some chemotherapeutic agents. To date, few studies have addressed the topic of the combined use of different flavonoids or even with antineoplastic agents. However, some flavonoids, such as flavopiridol and genistein, have been used in combination with docetaxel and cisplatin, respectively, with interesting preclinical results. In fact, the combination of flavopiridol and docetaxel has also been evaluated in a phase I and phase II clinical trial (Tan et al., 2004). The poor survival rate related to SCC of the oral cavity is mainly because of lymph node metastasis and locoregional spread; flavonoids and, in particular, EGCC, which has been proven to exert a strong antimigratory effect, might be particularly useful in counteracting oral cancer development. Despite the growing mass of data on the biological activities of flavonoids, the current challenge is to prove their pharmacological activities. Indeed, although their multiple anticancer properties have been shown, their main drawback remains their poor availability. In fact, the majority of such compounds are hydrophilic and they hardly enter the cell; thus, the achievement of

pharmacologically relevant doses is still an ongoing challenge. However, new technologies have been evaluated in recent years to improve the bioavailability of flavonoids: for instance, nanotechnology has been used successfully to increase water solubility and the in-vivo efficiency of silybin (Wang et al., 2014). Therefore, new studies aimed at evaluating the potential efficacy of optimized flavonoids are required.

Acknowledgements The authors are grateful to Dr E. Genton for language assistance. Conflicts of interest

There are no conflicts of interest.

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