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Contents lists available at ScienceDirect

Cancer Lettersjournal homepage: www.elsevier.com

Mini-review

Apigenin: A dietary flavonoid with diverse anticancer propertiesJosip Madunića, 1, Ivana Vrhovac Madunićb, 1, Goran Gajskic, Jelena Popićd, Vera Garaj Vrhovacc, ∗

a Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000, Zagreb, Croatiab Molecular Toxicology Unit, Institute for Medical Research and Occupational Health, Ksaverska Cesta 2, 10000, Zagreb, Croatiac Mutagenesis Unit, Institute for Medical Research and Occupational Health, Ksaverska Cesta 2, 10000, Zagreb, Croatiad Department of Diagnostic and Interventional Radiology, Clinical Hospital Merkur, Zajčeva 19, 10000, Zagreb, Croatia

A R T I C L E I N F O

Article history:Received 9 August 2017Received in revised form 25 October 2017Accepted 26 October 2017Available online xxx

Keywords:ApigeninAnticancer activityApoptosisCell cycle arrestSignalling pathways

A B S T R A C T

Apigenin is a natural flavonoid found in several dietary plant foods such as vegetables and fruits. A largenumber of studies conducted over the past years have shown that this particular natural compound has poten-tial antioxidant, anti-inflammatory, and anticancer properties. Therefore, apigenin has generated a great dealof interest as a possible chemotherapeutic modality due to its low intrinsic toxicity and remarkable effects onnormal versus cancerous cells, compared with other structurally related flavonoids. Here, we review its rolein anticancer research, as well as several cancer signalling pathways, including MAPK, PI3K/Akt and NF-κBpathways, and their specific role in different cancer types. Based on the available literature, the beneficial ef-fects of apigenin as a future anticancer modality are promising but they require further in vitro and in vivostudies to enable its translation from bench to bedside.

© 2017.

1. Introduction

Cancer is nowadays one of the most serious life-threating diseases,affecting people of all ages and is considered one of the leading causesof mortality and morbidity worldwide. Statistics show it is the secondmost common cause of death after cardiovascular diseases in devel-oped countries [1]. Cancer cells are characterized by mutations andgenetic instabilities which consequently lead to impaired regulationof cell cycle, uncontrolled proliferation and overcoming of apoptosisand similar checkpoint mechanisms [2]. Anticancer treatments usuallyuse compounds that target fast-dividing cells. This approach, regret-tably, has a negative side effect because normal, fast-dividing cellssuch as hair follicles and epithelial cells in the digestive system arealso affected. Furthermore, one of the aggravating circumstances isthat many cancer cells gradually develop resistance to conventionalforms of therapy [3]. Therefore, many studies in the last few yearshave focused on the development of an effective anticancer therapywhich would have little or no effect on normal cells.

In this regard, natural compounds from plants are proving to besuitable candidates for such a therapy [4]. Interfering with the processof carcinogenesis through diet or by the added digestion of naturalcompounds has been termed “chemoprevention” [5]. An increasingimportance is being given today to alternative medicine and dietaryapproach in prevention and treatment of cancer. A large number of

∗ Corresponding author.Email address: vgaraj@imi.hr (V. Garaj Vrhovac)1 These authors contributed equally to this work.

epidemiological, in vitro, in vivo and clinical studies demonstratedgrowing evidence linking increased consumption of a plant-based dietwith a reduced risk of chronic diseases such as cancer, as well asneurodegenerative, metabolic and heart diseases [6–11]. It should benoted that many epidemiological studies reported inconsistent results.This can be partly explained by the fact that those studies are basedon food questionnaires, which are not always an exact source of in-formation. Furthermore, within an abundant number of plant species,only 10% of them have been analysed as pharmacology agents. There-fore, both in vitro and in vivo research present a better way to elucidatethe beneficial effect of plant phytochemicals. Accordingly, many re-searchers have dedicated their studies to analyse a possible anticancereffect of natural compounds from vegetables and fruits.

There are many categories of plant bioactive compounds, such asalkaloids, glycosides, polyphenols, tannins, gums, resins and oils, andmany of these phytochemicals have been shown to possess low intrin-sic toxicity and exert prominent effects on cancerous versus normalcells. An encouraging fact is that, in the last few decades, nearly 70%of all anticancer drugs originated from natural sources or are deriva-tives of natural products [12].

The most extensively studied group of plant secondary metabo-lites are polyphenols, characterized by their structure of multiple phe-nol (benzene) rings [13]. Polyphenols, ranging in their structure fromsmall molecules to highly complex compounds, are widely distrib-uted in various vegetables, fruits, legumes, coffee, wine, beer, spicesand nuts [4,10]. Polyphenols are further divided into flavonoids, phe-nolic acids, stilbenes, lignans and other polyphenols. Their structuralvariance is the reason why polyphenols possess many different bio

https://doi.org/10.1016/j.canlet.2017.10.0410304-3835/© 2017.

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logical functions; most importantly anticancer activity. This activity isdependent on their structure, concentration and the type of cancer.

Flavonoids, a group representing 60% of all natural polyphenols,are present in all parts of plants, especially in flowers and leaves.Based on their structure, flavonoids can be classified into distinctsub-groups including anthoxanthins (flavones and flavonols), fla-vanones, flavanols, isoflavonoids and anthocyanidins. They exert theiranticancer activity through the induction of apoptosis in cancer cells.Studies have shown that high dietary intake of flavonoids is associatedwith reduced occurrence of many types of cancer [12,13], but thereis still insufficient data on the precise mechanisms of flavonoid anti-cancer effects. Apart from the anticancer activity, flavonoid-mediatedhealth benefits include anti-oxidant activity through the removal offree radicals, which are capable of damaging lipids, proteins and DNA[14], anti-inflammatory, neuroprotective and antiproliferative activity,as well as an ability to modulate signalling pathways involved in cen-tral cell processes.

One of the most abundant and most studied flavonoid is 4′,5,7-tri-hydroxyflavone, commonly referred to as apigenin, with molecularstructure C15H10O5 and molecular mass 270.24 g mol−1 (Fig. 1). Api-genin is found in significant quantities in a variety of vegetables andfruits such as parsley, celery, chamomile, oranges, thyme, onions,honey and spices, as well as beverages derived from plants; tea, beerand wine [4,15]. The name “apigenin” comes from Apium genus inApiaceae family, a group of mostly aromatic flowering plants includ-ing celery, carrot and parsley [13]. It is a secondary plant metabolite,usually found in nature in glycosylated form, more soluble than itspure form which is unstable and quite insoluble in water and organicsolvents [9,15]. The first reference of apigenin in science literaturecomes from the 1950s at what time Spicak and Subrt [16] analysedits effect on histamine release. It was not until the 1980s that api-genin was associated with the process of carcinogenesis when Birt etal. [17] reported its effective anti-mutagenic and anti-promotion prop-erties. Since then, apigenin has been investigated in many studies asa potential cancer chemopreventive agent against a wide selection ofdifferent cancer types.

Interest in apigenin, as a beneficial and health promoting agent, hasgrown in recent years because of its low intrinsic toxicity and remark-able effects on normal vs cancerous cells [18,19]. There is also verylittle evidence that suggests that apigenin promotes adverse metabolicreactions in vivo when consumed as part of a normal diet. Moreover,apigenin has been increasingly recognized as a cancer chemopreven-tive agent in a series of studies done both in vitro and in vivo. Thisinterest could be largely attributed to its potent antioxidant and anti-in-flammatory activities [19]. Indirect support to this hypothesis is pro-vided by a study where consumption of flavonoid free diets by healthyhuman volunteers has reportedly led to a decrease in the oxidativestress markers such as antioxidant vitamins and superoxide dismutase(SOD) activities, which are commonly associated with enhanced dis-ease risk and progression [20].

A variety of biological effects of apigenin in a number of mam-malian systems in vitro as well as in vivo are mainly related to its an

Fig. 1. . Chemical structure of dietary flavonoid apigenin.

tioxidant effects and its role in free radical scavenging. Besides, api-genin exhibits anti-mutagenic, anti-inflammatory and antiviral effects[21]. The actions of apigenin in inhibiting the cell cycle, diminishingoxidative stress, improving the efficacy of detoxification enzymes, in-ducing apoptosis, and stimulating the immune system are also known[21–23]. One human study demonstrated that apigenin was absorbedsystemically by a subject fed a diet rich in parsley. Results showedthat this subject had elevated levels of the antioxidant enzymes glu-tathione reductase and SOD [24], while the activities of catalase andglutathione peroxidase were found to be unchanged. Other biologicaleffects induced by flavonoids include the reduction of plasma levelsof low-density lipoproteins, inhibition of platelet aggregation, and re-duction of cell proliferation [21–23,25]. This is also apparent from an-other cross-sectional study conducted in Japan in which the total in-take of flavonoids among women was found to be inversely correlatedwith plasma total cholesterol and low-density lipoprotein concentra-tion, after adjustment for age, body mass index, and total energy in-take [26]. Moreover, the effects of flavonoids on the hematologic sys-tems were also performed in a 7-day study of 18 healthy men andwomen examining the effects of a daily dietary supplement, providingapigenin from parsley, on platelet aggregation and other haemostaticvariables. The authors of that study observed no significant changesin collagen- or ADP-induced platelet number, factor VII, plasmino-gen, and PAI-1 activity or fibrinogen concentrations [27]. These spe-cific properties categorise apigenin as part of a class of beneficialcompounds which possess health-promoting and disease-preventingdietary effects.

In the following chapters, including Tables and Figures, we pre-sent a summary of apigenin anticancer activities including dose rangesused in both in vitro and in vivo studies that exerted beneficial effectsagainst cancer growth and development.

2. Apigenin effects in cancers

2.1. Head and neck cancer

Several studies have evaluated the effect of apigenin on head andneck cancers. Apigenin was shown to inhibit proliferation in humanhead and neck squamous carcinoma cells which was followed withG2/M cell cycle arrest and increase in intracellular reactive oxygenspecies (ROS) levels. Growth inhibition was accompanied by apop-tosis through the up-regulation of both tumour necrosis factor re-ceptor (TNF-R) and TNF-related apoptosis-inducing ligand recep-tor (TRAIL-R) signalling pathways [28]. A study by Chakrabarti etal. [29] investigated the synergistic effect of human telomerase re-verse transcriptase (hTERT) knockdown and apigenin treatment in hu-man malignant neuroblastoma cells. This combination therapy led tocell proliferation with the inhibition of invasion and induced apop-tosis characterized by down-regulation of MMP-2 and -9, N-Myc,PCNA, CDK-2, CDK-4 and cyclin D1. In the next study, same au-thors showed that combination of N-Myc knockdown and apigeninexposure induced differentiation and apoptosis in SK-N-DZ andSK-N-BE2 neuroblastoma cell lines which are known for N-Myc am-plification. These effects were followed by a decrease in cell survival,invasive potential and angiogenesis [30]. Additional study by the samegroup reported that the overexpression of tumour suppressor miR-138enhanced the pro-apoptotic effects of apigenin in human neuroblas-toma cells, through both intrinsic and extrinsic pathways of apopto-sis [31]. Furthermore, apigenin-mediated induction of apoptosis waspotentiated by the overexpression of Krüppel-like factor 4 (KLF4), azinc-finger transcription factor, in neuroblastoma cells [32].

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Similarly, induction of apoptosis was also observed in api-genin-treated FRO anaplastic thyroid carcinoma (ATC) cells. Treat-ment with apigenin prompted apoptosis through the increase in c-Mycexpression followed by the phosphorylation of p53 and p38 [33]. Fur-ther research revealed that the treatment with PLX4032, an inhibitorof mutated B-Raf kinase, increased the apigenin cytotoxic effect inATC cells which was further potentiated with subsequent Akt inhibi-tion [34]. Recently, same authors evaluated the effect of apigenin incombination with TRAIL on survival of ATC cells [35]. Combinationtherapy reduced cell viability via modulation of the Bcl2/Bax ratio andthe suppression of Akt augmented the observed synergistic anticancereffect of apigenin in ATC cells. Apoptosis in human papillary thy-roid carcinoma (PTC) cells exposed to apigenin was associated withROS production, induction of DNA damage and G2/M cell cycle ar-rest, which led to autophagy as witnessed by LC3-II accumulation intreated cells [36].

Similar anticancer effects were observed in oral squamous cell car-cinoma cells. Treatment with apigenin inhibited cell growth and in-duced apoptosis accompanied by cell cycle arrest at both G0/G1 andG2/M checkpoints. Therefore, authors suggested apigenin could beused as an effective cell cycle regulating agent in cells with deregu-lated, but still active cell cycle [37]. In other study, administration ofapigenin and another flavonoid, hydroxygenkwanin (HGK), has beenshown to exert antiproliferative effects in C6 rat glioma cells. Api-genin strongly sensitized glioma cells to apoptotic activity of HGKthrough processes associated with Akt activation and suppression ofSOD activity [38]. A study using the same model cells confirmedthe apigenin's anticancer potential, which was mediated via growth-and migration inhibition through both apoptosis and autophagy. In-duction of differentiation and a decrease in the expression of immuneresponse factors was observed in rat glioma cells after apigenin treat-ment [39]. Another study, which aimed to investigate the effect of api-genin on glioma cells, demonstrated that apigenin inhibited cell via-bility through apoptosis induction in U87 human glioma cells [40].Apoptosis was followed by an increased miR-16 expression and sup-pression of Bcl2 and NF-κB pathway.

Various studies have established that glucose transporter-1(GLUT-1) is associated with chemoresistance in cancers and is con-sidered to be an important marker of hypoxia in malignant tumours.Study by Xu et al. [41] reported that apigenin treatment inhibitsGLUT-1 expression and by doing so sensitizes laryngeal carcinomaHep-2 cells to cisplatin chemotherapy. An in vivo study by the samegroup showed that apigenin enhanced radiosensitivity of laryngealcarcinoma and suppressed xenograft tumour growth through GLUT-1and PI3K/Akt inhibition [42]. Another recent study demonstrated api-genin's cytotoxic effects in human oesophageal cancer cells. These ef-fects were related to enhanced membrane permeability and inducedleakage of lactate dehydrogenase (LDH), which caused membranetoxicity and led to apoptosis in apigenin-treated cells [43].

Moreover, Kim et al. [44] investigated possible therapeutic effectsof apigenin in treating CSC-like phenotypes in human glioblastomacells and found that apigenin markedly decreased cell viability, neu-rosphere formation and invasiveness in glioblastoma stem-like cells.Furthermore, apigenin inhibited the c-Met receptor tyrosine kinasesignalling pathway and suppressed the expression of stem-like mark-ers CD133, Nanog, and Sox2. Similarly, it was also shown recentlythat apigenin exposure significantly suppressed hypoxia-induced ex-pression of CSC markers CD105, CD44, Nanog and VEGF in headand neck squamous cell carcinoma cells. This effect was accompaniedby the apigenin-induced decrease of CSC marker-expressing cells inoverall population [45].

2.2. Breast cancer

Apigenin has been shown to block the progression and devel-opment of progestin-dependent BT-474 breast cancer cell (BCC)xenograft tumours in nude mice through apoptosis induction and de-clined HER2/neu expression [46]. This was accompanied by the api-genin-mediated down-regulation of VEGF expression. A later studyevaluated the effect of apigenin exposure in HER2-transfected MCF-7(ER-positive) BCC. Results have shown that apigenin inhibits cellgrowth by promoting extrinsic apoptotic pathway, induces p53 andp21 expression and suppresses STAT3 and NFκB signalling [47].The same group demonstrated that apigenin exposure led to p53-de-pendent apoptosis in MDA MB-453 [48], SKBR3 [49] and BT-474[50] BCC lines. The observed cell growth inhibition was associatedwith caspase-dependent extrinsic pathway of apoptosis and suppres-sion of STAT3/VEGF signalling pathway in these HER2-overexpress-ing BCC.

A study by Cao et al. [51] evaluated the crosstalk of autophagyand apoptosis in apigenin-treated BCC. Autophagy was induced si-multaneously with apoptosis in T47D and MDA MB-231 BCC af-ter apigenin treatment, which was confirmed by increased levels ofLC3-II. Furthermore, subsequent inhibition of autophagy potentiatedthe apigenin-induced apoptosis. Unlike most other studies, Harrisonet al. [52] concentrated their research on a low-dose apigenin effectin BCC. Their results showed that sub-cytotoxic concentration of api-genin blocked DNA synthesis, elevated ROS levels and repressed pro-liferation in a panel of BCC lines, which authors attributed to Aktinhibition. Furthermore, apigenin has exhibited cytotoxic effects inMCF-7 BCC, which was accompanied by morphology changes, re-duced motility and decreased intracellular communication in treatedcells due to a disturbed structure and decreased amount of intracellularα-tubulin [53].

A study conducted in order to elucidate the effect of apigenin onER + MCF-7 and ER− SKBR3 BCC revealed that there were no sig-nificant differences between the response of these two BCC lines toapigenin treatment and concluded that the apigenin's antiproliferativeeffect did not depend on the steroid hormone receptor status in BCC[54].

It was shown recently that apigenin treatment blocked the ex-pression of cytokine-activated programmed death-ligand 1 (PD-L1),which enabled a T cell-mediated anticancer activity in BCC [55]. Thedetected immune-modulating effect of apigenin was followed by G2/M cell cycle arrest and apoptosis. Interestingly, studies have shownthat apigenin is capable of antiproliferative activity in BCC throughepigenetic regulation. The findings by Tseng et al. [56] demonstratedthat apigenin increased p21 expression via H3 acetylation inductionand histone deacetylase (HDAC) inhibition, which led to cell cycle ar-rest in G2/M phase in MDA MB-231 BCC.

2.3. Prostate cancer

Prostate cancer is one of the most prevalent cancers diagnosed inmen and the second leading cause of male cancer-related death af-ter lung cancer. The abnormal changes in the insulin-like growth fac-tor (IGF) axis are one of the markers of prostate cancer development,progression and metastasis. In a review by Babcook and Gupta [57],the authors reported that apigenin was capable of modulating the IGFaxis and its signalling in prostate cancer cells (PCC). Oral consump-tion of apigenin decreased prostate cancer levels of IGF-1 and inhib-ited downstream signalling followed by cell cycle arrest, growth inhi-bition and apoptosis in PCC. Furthermore, the same group indicated

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that apigenin administration to TRAMP (transgenic adenocarcinomaof the mouse prostate) mice (strain which spontaneously developsprostatic adenocarcinoma) supressed prostate carcinogenesis bydown-regulating IGF-I/IGFBP-3 signalling. This was accompaniedwith reduced angiogenesis and metastasis due to the inhibition ofVEGF, uPA, MMP-2, and MMP-9 expression. This study was de-signed to be comparable with consumption in humans so each mousewas treated with 20 and 50 μg/day apigenin over a period of 20 weeks[58].

Another study described the apigenin-mediated growth inhibitoryactivity in PCC, caused by the inhibition of class I histone deacety-lases (HDACs) [59]. Apigenin treatment induced cell cycle arrest fol-lowed by apoptosis in PC-3 and 22Rv1 prostate cancer cell lines,which was further confirmed when the authors used an in vivo modelof athymic nude mice with prostate cancer xenografts. Furthermore,apigenin was shown to bind and inhibit adenine nucleotide translo-case-2, an important transporter in the inner mitochondrial membrane,which up-regulated the death receptor 5 (DR5) and consequently in-duced tumour necrosis factor-related apoptosis in LNCaP and DU145human prostate cancer cell lines [60].

A study by Shukla et al. [61] showed that apigenin supressed theactivity of the inhibitor of apoptosis (IAP) proteins, which was ac-companied by a decrease in Bcl-xL and Bcl- 2 and an increase inthe active form of Bax protein. This triggered apoptosis in PC-3 andDU145 PCC. The same research group also reported that apigeninsuppressed prostate cancer progression by targeting the PI3K/Akt/FoxO-signalling pathway [62]. Apigenin treatment activated the Fox-O3a transcription factor and induced the expression of its downstreamtargets; BIM and p27, which led to cell cycle arrest and reduced via-bility in prostate tumours. Further studies by the same authors demon-strated apigenin's ability to bind and suppress IKKα thereby inhibit-ing NF-ĸB activation. This was followed by a decreased cell prolifera-tion and invasiveness of PCC [63]. Apigenin treatment also down-reg-ulated the expression of NF-κB-regulated genes involved in prolifer-ation (cyclin D1 and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL) andangiogenesis (VEGF) [64].

Development of metastases is the main reason behind prostate can-cer-associated mortality because primary site disease is organ-con-fined and rather treatable nowadays. An important factor in prostatetissue angiogenesis is the vascular endothelial growth factor (VEGF).Apigenin was shown to inhibit the TGF-β-induced VEGF productionin human PCC and consequently was able to supress prostate carcino-genesis by modulating the Smad2/3 and Src/Fak/Akt pathways [65].Similarly, a study by Zhu et al. [66] also recognized the importanceof decreasing the rate of metastasis in prostate cancer treatment. Theydemonstrated the apigenin-mediated inhibition of migration and inva-sion of DU145 PCC through the reversal of epithelial to mesenchymaltransition (EMT) followed by G2/M cell cycle arrest and apoptosis.

Another study revealed that apigenin selectively inhibited protea-somal degradation of estrogen receptor-β (ER-β), an important tu-mour suppressor in prostate cancer, through precise inhibition of chy-motrypsin-like activity of proteasome. This resulted in PCC apoptosis[67]. One of the mechanisms behind apigenin's chemopreventive ac-tivity in prostate cancers could be the inhibition of androgen hormoneproduction as was suggested by Wang et al. [68]. Apigenin was ableto inhibit steroidogenic enzymes by competing with the binding sitesand acting as steroid substrate.

Cancer stem cells (CSCs) are involved in metastasis, relapse ofcancers and drug resistance in various cancer types. Treatment withapigenin was shown to inhibit the survival of prostate CSC throughan extrinsic apoptotic pathway [69]. Apigenin-treated prostate CSC

sustained cell cycle arrest and a decrease in migration caused by par-tial down-regulation of PI3K/Akt and NF-κB signalling. The same re-search group analysed the effect of combined therapy by cisplatin andapigenin on prostate CSC growth and migration and found that api-genin significantly increased cisplatin cytotoxic and anti-migration ac-tivity in prostate CSC [70].

2.4. Colorectal cancer

Colorectal cancer (CRC) is a type of cancer which affects men andwomen equally. Even though most colorectal tumours can be surgi-cally treated, recurring metastases are the reason for high CRC-asso-ciated mortality. This clearly emphasizes the need for effective treat-ment of this disease. A study by Chunhua et al. [71] showed thatapigenin was capable of suppressing proliferation and migration inseveral colorectal adenocarcinoma cell lines through the up-regula-tion of an actin-binding protein and potential CRC suppressor, trans-gelin, with a subsequent down-regulation of MMP-9. Additionally,apigenin was observed to exert similar antiproliferative and anticancereffects in CRC cell lines via inhibition of the Wnt/β-catenin signallingpathway. Apigenin supressed β-catenin/TCF/LEF signalling activa-tion and prevented the expression of Wnt target genes by blocking thetransport of β-catenin into the nucleus [72]. Furthermore, it was shownthat apigenin improved the apoptotic activity of ABT-263, a BH3mimetic inhibitor in colon cancer cells. Apigenin down-regulated theexpression of prosurvival protein Mcl-1 and inhibited Akt and ERKsignalling pathways, which synergistically enhanced ABT-263-in-duced anticancer effects, both in cell and in vivo models [73]. A recentstudy by Wang and Zhao [74] found that apigenin induced apoptosisin colon carcinoma cells equally through intrinsic mitochondrial andextrinsic pathways, which was caused by the apigenin-mediated pro-duction of ROS and induction of endoplasmic reticulum (ER) stress.

There are data in the literature describing apigenin being able toinduce both apoptosis and autophagy in colon cancer cells. Treatmentwith apigenin inhibited growth of HCT116 cells through G2/M cellcycle arrest and induced cell death, both by apoptosis and autophagy,which authors attributed to apigenin's effect on PI3K/Akt/mTOR path-way [75]. Interestingly, Banerjee and Mandal [76] recently reportedthat while high concentrations of apigenin in shorter treatment causedapoptosis, lower concentrations over longer period of treatment led tooxidative stress and premature senescence in CRC cell lines HT-29and HCT-15.

Recently, it was confirmed that apigenin acted as an inhibitor ofNEDD9, a scaffolding protein strongly associated with cancer metas-tasis and development. Apigenin treatment decreased NEDD9 expres-sion resulting in the suppression of CRC cell migration, invasion andmetastasis [77]. Similar antiproliferative effects were observed in gas-tric cancer cells where apigenin induced apoptosis through the in-crease of Bax/Bcl-2 expression ratio, this was followed by a decreasein the mitochondrial membrane potential and activation of caspase-3cascade. At the same time, apigenin showed no substantial effects onproliferation and apoptosis of normal gastric cells [78].

Using an azoxymethane (AOM)-induced intestinal adenocarci-noma in Wistar rat model, Tatsuta et al. [79] found that apigenindecreased the incidence of lymphatic vessel invasion of adenocarci-nomas and together with in vitro results showed that apigenin su-pressed cancer metastasis by inhibiting phosphorylation of MAPK.The formation of colon aberrant crypt foci (ACF) and high activityof ornithine decarboxylase (ODC) are considered to be the prognosticmarkers of colon cancer. A study by Au et al. [80] reported that di-etary apigenin reduced the formation of ACF and decreased ODC ac

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tivity in AOM-induced CF-1 mice model. Furthermore, ODC activ-ity was significantly inhibited by 10 and 30 μM apigenin in Caco-2CRC cells. The authors also evaluated colon tumorigenesis using Minmice, a strain with mutant APC gene, predisposed to intestinal ade-noma formation. Contrary to the two AOM-injected mice studies,dietary apigenin did not supress adenoma formation in Min mice.Similarly, it has been shown that dietary apigenin reduced the inci-dence of high multiplicity aberrant crypt foci (HMACF) in the colonof Sprague-Dawley rats. Because HMACF are one of the earliestpre-cancer changes seen in the colon, these results suggest that api-genin may have a protective role against colon carcinogenesis. Fur-thermore, expression of proinflammatory enzymes COX-2 and iNOSwas analyzed but the authors found that apigenin treatment had no ef-fect on their expression in the aforementioned in vivo cancer model[81].

An in vitro study by Zhong et al. [82] revealed that apigenin in-duced apoptosis and significantly reduced proliferation of human CRCcells HCT-116 through PKCδ and ATM kinase pathways, affectingthe expression of NAG-1, p53, and p21. Apigenin modulated the ex-pression of p53 and p21 at the translational level whereas NAG-1 wasaffected at the transcriptional level. As opposed to previous studies inMin mice [80], the results of this study showed that apigenin inhibitedintestinal tumorigenesis with evident reduction in polyp number andload. The differences between these two in vivo studies could be dueto dissimilar treatment parameters such as way of feeding, timing oftreatment, and dosage of apigenin. A recent study by Banerjee et al.[83] described apigenin as a potential chemotherapeutic agent againstCRC, both in vitro and in vivo, and aimed to develop a lipid nanocar-rier for more effective delivery of apigenin. Using athymic nude mice,the authors demonstrated that administration of apigenin: 1) decreasedtumour volume in both apigenin and liposomal-apigenin treated an-imals, being more significant with the carrier; 2) attenuated tumourvasculature; and 3) inhibited cellular proliferation as witnessed by re-duction of Ki-67. These in vivo results are of great importance becausethey indicate future clinical potential of apigenin-based vesicles.

2.5. Pancreatic cancer

A study conducted in order to evaluate the effect of apigenin inpancreatic cancer cells (PaCC) discovered that apigenin was able torestore the activity of mutated p53 in MiaPaCa-2 and BxPC-3 PaCClines and by doing so, decrease proliferation and induce apoptosis inthose cells [84]. Apigenin treatment also promoted the binding of p53to DNA and up-regulated the expression of p21 and pro-apoptoticPUMA. Using the same model cells, the authors showed that apigeninsupressed mutagen-induced β-adrenergic receptor (β-AR) signallingand subsequent activation of its downstream targets; focal adhesionkinase (FAK) and ERK kinase. As a result, apigenin exposure re-duced the mutagen-enhanced proliferation and migration of β-AR-ex-pressing PaCC [85]. Furthermore, apigenin has been shown to effec-tively potentiate the anticancer effect of chemotherapeutic drugs inBxPC-3 PaCC. Such combination therapy was able to inhibit cell pro-liferation where timing and concentration were shown to play a cru-cial role; pretreatment with apigenin was more effective than the si-multaneous application of apigenin and chemotherapeutic drug [86].A later study by the same group demonstrated that apigenin treatmentin BxPC-3 and PANC-1 PaCC lines inhibited the GSK-3β/NF-κBsignalling pathway, which led to G2/M cell cycle arrest and acti-vated the intrinsic pathway of apoptosis [87]. Analysis of gene ex-pression showed that growth inhibition could be related to the api-genin-enhanced expression of inflammatory genes. Similarly, Wu etal. [88] observed that apigenin exposure in PaCC led to suppres

sion of the IKK-β-mediated NF-κB activation. Apigenin blocked theIKK-β activity, NF-κB DNA binding, induced apoptosis and reducedthe growth of pancreatic cancer xenografts in nude mice.

A recent study reported of an interesting activity of apigenin inpancreatic cancer. Ikaros, a transcription factor important in lympho-cyte development and anticancer immune response, is negatively reg-ulated by casein kinase 2 (CK2). The findings of Nelson et al. [89]showed that apigenin treatment caused specific CK2 inhibition, whichin turn stabilized Ikaros expression in pancreatic cancer. This was fur-ther confirmed using an in vivo Panc02 mice model where apigeninexposure facilitated survival, reduced tumour size and, overall, im-proved the anticancer immune response in pancreatic cancer.

2.6. Skin cancer

Previous studies have evaluated the effect of apigenin in melanomacancer cells. Apigenin treatment elevated ROS production, depletedglutathione (GSH) and SOD levels, which triggered apoptosis throughits intrinsic pathway in human A375 melanoma cells. Cells also under-went apigenin-mediated alterations in mitochondrial functions char-acterized mainly by oxidative phosphorylation impairment [90]. Re-cently, it was shown that A2058 and A375 melanoma cells exposedto apigenin exhibited reduced cell migration and diminished FAK andERK 1/2 activity, which sensitized cells to anoikis; detachment-in-duced apoptosis [91]. A study by Chao et al. [92] demonstrated thatnon-toxic doses of apigenin supressed VEGF expression in uvealmelanoma SP6.5 and C918 cell lines in a dose- and time-dependentmanner by inhibiting PI3K/Akt and ERK1/2 signalling.

Further studies have also evaluated the role of STAT3 signallingon the apigenin anticancer activity in melanoma cells. Exposure ofmurine melanoma B16F10 to apigenin resulted in metastasis, migra-tion and invasion inhibition via down-regulated STAT3 signalling.This was accompanied by apigenin-influenced suppression of STAT3downstream targets: MMP-2, -9, VEGF and Twist1 [93]. Interest-ingly, a recent study using same model cells showed that natural de-rivative of apigenin; apigenin-7-glucoside exerts antiproliferative anddifferential activity in B16F10 melanoma cells. Treatment inducedapoptosis and significantly promoted melanin synthesis, as well as theactivity of tyrosinase, melanogenesis-related enzyme [94].

Study designed to assess the effect of apigenin on A375 and C8161melanoma cell lines (different in their BRAF mutation status), re-vealed that apigenin inhibited cell growth through G2/M cell cycle ar-rest and induced apoptosis which was attributed to apigenin-mediatedinhibition of Akt/mTOR pathway [95]. Moreover, it was observed thatapigenin exposure triggered changes in the dendrite morphology inboth cell lines which authors associated with glutamate signalling in-hibition.

An in vivo study by Kiraly et al. [96] suggested that apigenin mayinhibit skin cancer development by down-regulating COX-2. Api-genin exposure of non-tumour epidermis in tumour bearing SKH-1mice decreased the expression of COX-2, prostaglandin PGE2, recep-tors EP1 and EP2, and increased terminal differentiation. Even thoughapigenin failed to inhibit the COX-2 pathway or promote differen-tiation in tumours, the apigenin-treated mice exhibited diminishedCOX-2 in their epidermis and developed fewer tumours.

2.7. Liver cancer

As other studies have demonstrated, apigenin treatment in com-bination with cytokine TRAIL is able to induce apoptosis with bet-ter efficacy that either compound alone [35]. A similar effect was ob

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served in Huh-7 hepatocellular carcinoma cells (HCC), which weresimultaneously exposed to apigenin and TRAIL. Huh-7 cells, other-wise resistant to TRAIL therapy, were sensitized by apigenin/TRAILtreatment, which prompted the upregulation of Bax/Bcl-2 ratio and ac-tivated caspase-dependent apoptosis [97]. A later study by the samegroup reported of the similar apigenin-mediated priming of HepG2HCC to TRAIL-induced apoptosis. The authors showed that com-bination therapy induced apoptosis by stimulating DR5 expressionthrough the ERK signalling pathway [98]. Similarly, a study by Huet al. [99] revealed that apigenin potentiated the cytotoxic activity of5-flurouracil (5-FU) in SK-Hep-1 and BEL-7402 HCC. Co-treatmentactivated the mitochondrial apoptotic pathway via ROS accumula-tion and mitochondrial membrane depolarization. The synergistic anti-cancer effect of apigenin/5-FU was also observed in HCC xenografts.

Apigenin has been associated with epithelial-to-mesenchymal tran-sition in HCC. Recent findings demonstrated that exposure ofBel-7402 and PLC HCC to apigenin resulted in change of EMTmarker expression accompanied by the inhibition of NF-κB/Snail sig-nalling. Apigenin also inhibited proliferation, invasion and migrationof HCC cells and reduced HCC xenografts growth, which suggeststhat apigenin inhibits EMT through NF-κB/Snail suppression in HCC[100]. The use of apigenin as an efficient therapeutic drug in HCCtreatment was also confirmed in a recent study where selective toxi-city of apigenin on cancerous versus non-cancerous hepatocytes wasevaluated. The results showed that apigenin enhanced ROS generationand facilitated cytochrome c release, which led to apoptosis only incancerous hepatocytes [101].

There are still few in vivo studies of apigenin as a potential HCCchemopreventive agent. Wistar albino rats treated with apigenin dis-played a decreased level of lipid peroxidation (LPO) and increasedantioxidant status in induced hepatocellular carcinoma [102]. More-over, a recent study by Li et al. [103] using bioluminescence and flu

orescence molecular imaging observed a significant inhibition of livertumour formation in apigenin-treated Balb/c nude mice (injected withHepG2 HCC) in comparison with the control group.

2.8. Cervical and ovary cancer

Oral administration of apigenin has been shown to inhibit themetastasis of ovarian cancer cell xenograft tumours in mice while invitro treatment supressed invasion and migration of ovarian cancercells. These effects were attributed to down-regulation of MMP-9 byapigenin in exposed cells and tissues [104].

A study was designed to examine whether apigenin could be usedin the treatment of cervical cancer by targeting cervical CSC. Its re-sults revealed that apigenin treatment inhibited the proliferation ofHeLa-derived sphere-forming cells (SFCs) and caused a loss of theirself-renewal capacity. The authors ascribed these antiproliferative ef-fects of apigenin to the suppression of CK2, kinase associated withmaintenance of CSC properties [105]. In a similar study, Tang et al.[106] confirmed that apigenin was able to target cervical CSC usingSFCs derived from a SKOV3 cervical cancer cell line. Apigenin ex-posure interfered with the self-renewal ability of SFCs through the de-crease of CK2 expression and inactivation of Gli1, an oncogene in-volved in the Hedgehog signalling pathway. Fig. 2.

The potential antiproliferative effects of apigenin have been re-cently evaluated in chemoresistant ovarian cancer cells. Apigenin de-creased the viability of both parental and chemoresistant SKOV3 cellsthrough the downregulation of TAM receptor tyrosine kinases ex-pression. It also downregulated their downstream targets; Akt andBcl-xL [107]. Furthermore, apigenin cytotoxic activity was recentlytested in a panel of cervical cancer cell lines. The findings indi-cated that apigenin possessed a selective cytotoxic effect, which wasachieved through the elevation of ROS and LPO, and mitochondrial

Fig. 2. Schematic summary of apigenin targets.

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membrane potential's decrease. Apigenin exposure also inhibited mi-gration and invasion in cervical cancer cells [108]. Table 1, Table 2.

3. Role of apigenin in chemotherapy

As was already mentioned in previous chapters, apigenin is ableto interact with compounds used in conventional drug chemother-apy. Earlier studies have reported of the apigenin's ability to blockcytochrome P450 (CYP) enzymes, proteins involved in the metabo-lism of many chemotherapeutics, which can overcome chemoresis-tance in many cancer types [109]. By doing so, apigenin augmentsthe anticancer activity of these drugs and decreases their negative ef-fects [9,64]. A study by Johnson and de Mejia [86] described the in-teractions between apigenin and various chemotherapeutics in PaCC.Their results showed that apigenin pretreatment sensitized PaCC tochemotherapeutics 5-FU and gemcitabine and enhanced their antipro-liferative effect. A similar synergistic activity of apigenin and 5-FUwas observed in hepatocellular carcinoma where apigenin exposureincreased the cytotoxic activity of 5-FU through ROS elevation anddepolarization of mitochondrial membrane [99].

Furthermore, simultaneous administration of apigenin and pacli-taxel, a widely used antimicrotubule chemotherapeutic, increasedhalf-life of paclitaxel, and elevated its oral bioavailability and in-testinal absorption in rats [110]. This was accomplished through api-genin-mediated inhibition of CYP3A4 and P-glycoprotein (P-gp), atransporter involved in the extrusion of paclitaxel from the intestines.An earlier study by Choi and Choi [111], reported of apigenin affect-ing the metabolism of oral paclitaxel, while the intravenously-admin-istrated drug was not affected, proving that the improved bioavail-ability was primarily due to apigenin's inhibition of P-gp and thesubsequent increased intestinal absorption. In another study, apigeninmodulated the pharmacokinetic characteristics of orally-administeredetoposide, topoisomerase II inhibitor, by suppressing P-gp andCYP3A4 activity, which enhanced oral exposure and increasedplasma half-life and bioavailability of etoposide. At the same time,body clearance of intravenous etoposide was partially down-regu-lated by concurrent apigenin administration [112]. A study designed toanalyse the interaction between raloxifene, a breast cancer chemother-apeutic, and apigenin, demonstrated that co-administration of apigeninincreased the bioavailability of raloxifene in rats by competitively in-hibiting its phase II conjugation (glucuronidation and sulfation) [113].

The ATP-binding cassette (ABC) transporter family are proteinsinvolved in the efflux of many chemotherapeutics and are often over-expressed in multidrug-resistant (MDR) tumour cells. P-gp is a mem-ber of the ABC family and is encoded by the ABCB1 gene. A studyconducted on docetaxel-resistant prostate cancer cells confirmed thatthe overexpression of ABCB1 facilitated docetaxel resistance in thosecells. Exposure of resistant cells to apigenin down-regulated ABCB1expression and re-established docetaxel sensitivity [114]. Saeed et al.[115] recently investigated the interaction between apigenin and ABCtransporters BCRP (breast cancer resistance protein), ABCB5 andP-gp in chemotherapeutic-sensitive and -resistant cancer cell lines.The findings of their in vitro study showed apigenin acting as amulti-specific inhibitor of all three ABC transporters, which aug-mented the intracellular concentration of doxorubicin and docetaxeland improved drug cytotoxicity in chemoresistant cells. Moreover, insilico molecular docking studies have indicated that apigenin binds totransporters and interferes with ATP binding and cleavage, thus deny-ing the energy necessary for the outward transport of chemotherapeu-tics.

Although most studies report that apigenin possesses favourabledrug-drug interaction profile, it may also, in certain circumstances,interfere with the activity of chemotherapeutics. The results of Ru-ela-de-Sousa et al. [116] revealed that apigenin weakened the cy-totoxic effect of the chemotherapeutic vincristine in leukemia cellsvia the induction of autophagy and G0/G1 cell cycle arrest. As vin-cristine acts by disrupting microtubules, the apigenin-mediated cellcycle arrest disabled this mechanism and protected the cells fromvincristine-induced cell death. Furthermore, it has been shown thatlong-term administration of apigenin accelerated the metabolism ofimatinib and consequently lowered its circulating concentration inrats. Contrastingly, single-dose exposure to apigenin had a negativeeffect on imatinib's metabolism which resulted in its increasedbioavailability and higher plasma concentrations [117].

4. Conclusions, knowledge gaps and future perspectives

In this review, we presented a summary of a number of articlesand studies which evaluated natural flavonoid apigenin as a compoundwith great potential in chemotherapy of various types of cancer. Mostchemotherapeutics target and selectively kill cells that are dividingrapidly. Unfortunately, normal fast-dividing cells such as hair folliclesand digestive epithelial cells are also affected during this approach.For this reason, the focus of the research in the last few years hasbeen directed to the discovery and development of new generation ofchemotherapeutics that would target and exclusively kill only cancercells without any adverse effects in normal cells. Dietary flavonoidssuch as apigenin represent an interesting link between diet and treat-ment of chronic diseases including cancer.

Previous studies have discovered a presence of cells with stem-likeproperties in hematopoietic and some solid tumours. It has been es-tablished that those cancer stem cells (CSC) possess a self-renewingcapacity and can regenerate tumour, but to a lesser extent, becausethey represent only a small fraction in tumour tissue. They share someof the properties with normal stem cells such as resistance to apopto-sis and drugs, which makes them an interesting target for anticancerstrategies. Evaluation of the currently available articles indicates thatapigenin can be used as an agent that targets CSC in various types ofcancer and in this way exerts its anticancer activity. Furthermore, api-genin is able to synergistically augment the effect of chemotherapeu-tics by overcoming the acquired resistance in cancer cells. It should benoted that further in vivo studies are needed to confirm the apigeninanticancer stem cell effect. Additionally, one of the present knowledgegaps is apigenin's activity in normal stem cells, which should also beevaluated in the future.

One of the promising future anticancer approaches is the induc-tion of cellular senescence, reported here to be easily achieved by ex-tended low-dose treatments of apigenin. This kind of chemotherapywould selectively kill only those cells which are unable to respondproperly to induced stress, such as cancer cells, due to their genomicinstability. Another important issue which should be further exploredin the future studies is the ability of apigenin to act as a small-mole-cule inhibitor. The studies reviewed so far show that apigenin down-regulates several important proteins involved in the onset and pro-gression of cancer, such as casein kinase 2 and histone deacetylases,which can be exploited in the development of new anticancer thera-pies. In this context, it is important to note the synergistic activity api-genin displays when combined with the inhibitors of signalling path-ways involved in carcinogenesis. Additionally, as mentioned in theprevious chapters, apigenin shows an interesting interaction with ep-ithelial to mesenchymal transition, a process crucial in the invasionand metastasis of cancer cells. Therefore, future efforts should focus

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Table 1Targets and mechanisms of apigenin activity in cancers.

Mechanism Effect Targets References

Apoptosisdecreased expression and/or activity Bcl-2 [28,30–33,35,40,51,59,61,64,70,76,78,87,90,97,99]

Bcl-xL [35,38,51,61,64,107]Mcl-1 [32,73]XIAP, cIAP-1 and -2 [61,87]N-Myc [29,30]Survivin [31,61,70]Sharpin [70]HDAC-1 and -3 [56,59,61]hTERT [29,30]GLUT-1 [41,42]Her2/neu [46,47,54]Axl, Tyro-3 [107]GSH, SOD [90]ΔΨM [38,74,76,78,90,99,108]

increased expression and/or activity Bax, Bim, Bid [28–33,51,59,61,62,64,69,73,74,76,78,90,97]Noxa, Puma [32,84]Caspase-3, -6, -7, -8, -9 [28–34,38,47–51,61,74,75,78,87,88,90,91,95,97–99]PARP-1 [33–35,47–51,54,61,75,88,90,91,95,97–99]Cytochrome c [61,69,74,84,87,90]miR-16 and -138 [31,40]c-Myc [33,34]Fas [33]DR5 [60,74,98]TNF-α [28,38,69]TRAIL [28]Calpain [30,31]CHOP [74]Smac [31]Apaf-1 [70]ROS [28,36,52,53,74,76,90,99,108]catalase [108]

Autophagydecreased expression and/or activity p62 [36]increased expression and/or activity LC3-II [36,51,75]

Beclin-1 [36]Nrf-2 [36]HO-1 [36]

Cell cycledecreased expression and/or activity pRb [76]

Cyclin A, B1, D1 and E [29,37,56,64,72,75,76,87]CDK-1, -2, and -4 [29,37,56,75]Cdc25C [36]PCNA [29,30]CK2α [89,105,106]

increased expression and/or activity p16, p21 and p27 [29,47,48,56,59,62,69,70,75,76,84]p53 [32,70,75]

altered phosphorylation p53 [33,47,48,50]MDM2 [47]

altered cellular location p53 [84]Angiogenesis, metastasis and invasiondecreased expression and/or activity VEGF [30,45,46,48–50,64,65,92,93]

COX-2 [64]MMP-2 and -9 [29,30,32,40,50,69,71,93,104]HIF-1α [48–50]b-FGF [30]Snail, Slug [66,69,70,100]Oct-3 and -4 [69]Sox-2 [44]Nanog [44,45]Twist-1 [93]Vimentin [66,100]N-cadherin [93,100]NEDD9 [77]CD44, 105 and 133 [44,45]fibronectin [93]Integrin subunits [91]

increased expression and/or activity E-cadherin [30,66,93,100]transgelin [71]Claudin-3 [100]Occludin [100]keratin-8 [93]

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Table 1 (Continued)

Mechanism Effect Targets References

altered phosphorylation Smad-2 and -3 [65]altered cellular location Smad-2 and -3 [65]

Snail [100]Signalling pathways modulated by apigenin activity

PI3K/Akt/FoxO [62]Src/FAK/Akt [65]NEDD9/Src/Akt [77]β-AR/Src/FAK/ERK [85]integrin/FAK/ERK [91]PI3K/Akt [30,35,41,42,52,69–71,73,92,107]Akt/P70S6K1/MMP [104]NF-κB [30,63,64,69,70,88]GSK-3β/NF-κB [87]NF-κB/MMP [40]NF-κB/Snail [100]ERK/Mcl-1 [73]Wnt/β-catenin/TCF/LEF [72]c-Myc/p38/p53 [33]MAPK/ERK [34,35,92,95]c-Met/STAT3/Akt/ERK [44]Her2/neu [46]p53 [47]JAK/STAT [47–50,55,93]Akt/mTOR [95]

Table 2Apigenin concentration ranges used in reviewed cancer studies.

Study type Dose References Study type Dose References

in vitro 1-10 μM [92,97] in vivo 20 or 50 μg/mouse/d [58,59,61–64]1-20 μM [60,62,63,78,89,104] 50 or 100 μg/mouse/d [42]1-30 μM [73,98] 20 mg/kg/w [77]1-40 μM [47,56,59,61,105–107] 5 or 25 mg/kg/d [56]3-50 μM [28,32,38,52,54,65,75,91] 25 mg/kg/t.i.w. [89]10-60 μM [77] 15 or 30 mg/kg/d [88]2-80 μM [33,35,51,66,67,72,88] 25 mg/kg/d [73]1-100 μM [34,36,37,39,43–45,48–50,53,76,84,85,87,99,108] 50 mg/kg/d [46,71]10-160 μM [41,71,74,90] 75, 150 mg/kg/d [104]10-200 μM [100] 150 mg/kg/d [93]40-280 μM [95] 200 or 300 mg/kg/d [100]

/d: once daily; /w: once per week; t.i.w.: three times per week.

on identifying the precise mechanisms behind this interaction. An-other relevant subject for future research will be apigenin's role in theanticancer immune response, an activity which was observed in sev-eral different cancer types. Many studies reviewed here reveal that api-genin treatment induces both apoptosis and autophagy in cancer cells.Consequently, a better clarification of the interplay between these twocell death modes in apigenin-treated cancer cells is needed to betterunderstand apigenin's role in cell death induction.

In conclusion, a few unanswered questions remain before we canstart using apigenin as a chemopreventive and chemotherapeuticagent, specifically; its limited bioavailability and poor absorption inhumans, limited data on pharmacokinetics and accumulation in or-gan sites, lack of specific cellular targets, toxicological safety and in-consistency between in vivo and in vitro therapeutic dosage. Evenwith these uncertainties and aforementioned knowledge gaps, the as-sessment of the existing literature indicates that this natural flavonoidcould be used as an effective chemopreventive and possible anticanceragent. The beneficial effects of apigenin are indeed promising but theyrequire further in vitro and in vivo studies to enable its translation frombench to bedside.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

Supported by the University of Zagreb, Faculty of Science and theInstitute for Medical Research and Occupational Health.

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