Antioxidants in cervical cancer: Chemopreventive and chemotherapeutic effects of polyphenols

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Review

Antioxidants in cervical cancer: Chemopreventive and chemotherapeutic effects

of polyphenols

F. Di Domenico

a

, C. Foppoli

b

, R. Coccia

a

, M. Perluigi

a,

a

Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro, 5-00185 Rome, Italy bCNR Institute of Molecular Biology and Pathology, Sapienza University of Rome, Rome, Italy

a b s t r a c t

a r t i c l e i n f o

Article history: Received 27 August 2011

Received in revised form 5 October 2011 Accepted 6 October 2011

Available online 12 October 2011 Keywords: Cervical cancer HPV Polyphenol Apoptosis Oxidative stress

Cervical cancer lesions are a major threat to the health of women, representing the second most common cancer worldwide. The unanimously recognized etiological factor in the causation of cervical cancer is the fection with human papilloma virus (HPV). HPV infection, although necessary, is not per se sufficient to in-duce cancer. Other factors have to be involved in the progression of infected cells to the full neoplastic phenotype. Oxidative stress represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. Oxidative stress is known to perturb the cellular redox status thus leading to alteration of gene expression responses through the activation of several redox-sensitive transcription fac-tors. This signaling cascade affects both cell growth and cell death. The ability of naturally occurring antiox-idants to modulate cellular signal transduction pathways, through the activation/repression of multiple redox-sensitive transcription factors, has been claimed for their potential therapeutic use as chemopreven-tive agents. Among these compounds, polyphenols have been found to be promising agents toward cervical can-cer. In addition to acting as antioxidants, polyphenols display a wide variety of biological function including induction of apoptosis, growth arrest, inhibition of DNA synthesis and modulation of signal transduction path-ways. They can interfere with each stage of carcinogenesis initiation, promotion and progression to prevent can-cer development. The present review discusses current knowledge of the major molecular pathways, which are involved in HPV-driven cancerogenesis, and the ability of polyphenols to modulate these pathways. By acting at specific steps of viral transformation cascade, polyphenols have been demonstrated to selectively inhibit tumor cell growth and may be a promising therapeutic tool for treatment of cervical cancer. In addition, recent results obtained in clinical trials using polyphenols are also discussed. This article is part of a Special Issue entitled: An-tioxidants and Antioxidant Treatment in Disease.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction: cervical cancer

Cervical cancer lesions are a major threat to the health of the women, representing the second most common cancer worldwide. Human papillomaviruses (HPVs) have been identified as the major etiological factor in cervical carcinogenesis. The hypothesis of a corre-lation between HPV infection and cervical neoplasia was formulated first time at the beginning of the 70's by Harold zur Hausen[1]. HPVs can be clinically classified as “low-risk” (LR-HPV) and “high-risk” (HR-HPV) depending on the relative propensity of the HPV-associated lesions to undergo malignant progression. HPV 16 is the most prevalent HR-HPV type, followed by HPV 18 and others. LR-HPVs generate moderate dysplasia and genital warts, while high-risk HPVs are associated with cervical dysplasia or cervical intraepithelial neoplasia (CIN), currently indicated as squamous intraepithelial lesions

(SIL), graded as low (LSIL) or high (HSIL). Cervical cancers are thought to arise from these lesions after long persistent infection. The majority of HPV infections are subclinical and transitory (80% disappears) and in most of the cases they are resolved spontaneously by an effective im-mune response that eliminates virus or suppresses it down to unde-tected levels[2]. In few subjects, when the virus is not suppressed, the infection progress to CIN 1 and evolve to CIN 2/3 but the development of cervical carcinoma is a multi-step carcinogenesis, however the steps in the progression from LSIL to tumor remain unknown. It has been estimated that approximately 10% of LSIL and about 20–30% of HSIL actually progress to cancer[3, 4].

2. HPV: structure, infection and transformation activity

HPVs are small, non-enveloped DNA viruses with a circular genome of around 8 kb. The viral genome can be divided into three regions. The up-stream regulatory region is largely responsible in determining the host range and tissue tropism of each HPV type and regulates viral gene expression after infection. This region contains the viral origin of

☆ This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

⁎ Corresponding author. Tel.: +39 0649910900. E-mail address:marzia.perluigi@uniroma1.it(M. Perluigi). 0925-4439/$– see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2011.10.005

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replication and numerous binding sites for cellular transcription factors. The rest of the genome comprises eight open reading frames divided between the early region, that encodes the E1, E2, E4, E5, E6 and E7 pro-teins and the late region, that encodes the L1 and L2 propro-teins[5]. E6, E7 and in minor grade E5 are well-recognized oncoproteins responsible for cell transformation events.

HPVs have a unique life cycle, which is tightly linked to the differ-entiation program of the infected host epithelium. HPV infection starts in the basal epithelium (basal cells or stem cells) in prolifera-tion, where HPVs are in the phase of latent infection and the viral ge-nome is maintained at a low copy number without production of virions. In the upper layer, as the daughter cells move towards the surface and undergo differentiation, vegetative replication of the viral genome coordinates with expression of capsid proteins to form viral particles. Finally, the progeny virions are released from the up-permost layer within terminally differentiated epithelial squamae, to search for new host cells. HPV infection thus results in enhanced proliferation of the infected cells and their lateral expansion[2, 6, 7]. The failure of the immune system, in persistent HPV infection clear-ance, can lead, after about 10–20 years, to the development of cervical cancer. In LSIL, most HPV genomes are in episomal state whereas, in many HSIL, genomes are integrated into the host cell. Over one-half of HPV 16-positive cancers and most HPV 18-positive malignancies con-tain integrated HPV genomes, suggesting that integration may, in some cases, contribute to malignant progression. In lesions containing episomal HPV, the viral E2 protein directly represses early gene expres-sion regulating copy numbers. Integration of viral DNA usually disrupts E2 expression, leading to the deregulated expression of early viral genes, including E6 and E7, as well as increased proliferative capacity, a crucial step in progression to cancer[6].

In cervical carcinoma cells, the E6 and E7 proteins are continuous-ly expressed and this is required for transformation. Prior to viral in-tegration these proteins are believed to cooperate in order to overcome normal cell cycle controls. Expression of the E6 and E7 pro-teins from HR- but not from LR-HPV types lead to cell immortaliza-tion necessary for malignant progression.

The most characterized role of HR-HPV E6 is its ability to bind to the p53 tumor suppressor protein in conjunction with the cellular ubiquitin ligase E6AP and target p53 for degradation through the ubi-quitin–proteasome pathway[8, 9]. This impairs the normal cellular response to cell stress and DNA damage and allows infected cells to proliferate. Moreover since p53 plays a key role in preserving genome integrity, HPV infected cells accumulate chromosomal abnormalities thus increasing their intrinsic susceptibility towards malignancy. E6 can also interfere with the apoptotic pathway via its association with proteins belonging to the bcl-2 family, including Bax and Bak [10, 11]. Another way by which E6 confers resistance to apoptosis is the up-regulation of inhibitors of apoptosis proteins, as c-IAP2 and survivin, through a mechanism involving NF-κB[12, 13].

E7 is a small nuclear phosphoprotein known to bind to the retino-blastoma tumor suppressor gene product, pRb, and its family members, p107 and p130. HR-HPV E7 protein, by binding to pRb leads to the re-lease of E2F proteins and prematurely induces cells to enter the S phase prevailing the normal cell cycle control. E7 from HR-HPV can also induce the degradation of pRb via a proteasome-dependent path-way[14]. p16INK4a, a cyclin-dependent kinase (CDK) inhibitor, is over-expressed when pRb is inactivated by E7[15]in additional E7 interact with other CDK inhibitors such as p27 and p21 confirming its involve-ment in the abrogation of cell-cycle inhibition[2]. Finally, in targeting pRb, E7 causes deregulated E2F activity and E2F-1 can induce apoptosis in the presence of p53[5]. E2F-1 activates expression of p19ARF, which inhibits Mdm2 and thereby stabilizes p53[5]. A number of studies dem-onstrated that HR-HPV oncogenes activate MAPKs and the AP-1 family of transcription factors[16]. Moreover, increased COX-2 levels were shown in cervical cancer cells, via the EGFR→Ras→MAPK→AP-1 path-way and in correlation to E6-mediated p53 degradation[16–19].

3. Oxidative stress and cervical cancer

Persistent infection of the cervix with a HR-HPV is necessary to develop cervical cancer, other associated co-factors are required for the malignancy progression. Oxidative stress, among others, is receiv-ing great interest for its role durreceiv-ing the progression of neoplasias. Several risk factors of cervical cancer development, such as exposure to cigarette smoke and chronic inflammation, are well documented to increase oxidative stress, which could account for higher risk of cervical cancer in these two conditions[20, 21].

Oxidative stress is a condition arising from an increased production of reactive oxygen species (ROS) associated with a decreased antioxi-dant capability of the cell. ROS are constantly generated in aerobic cells by the incomplete reduction of molecular O2to H2O during

mito-chondrial oxidative phosphorylation, as well as during a number of pro-cesses such as inflammation, infections, mechanical and chemical stresses, exposure to UV and to ionizing irradiation[22–24]. The effect of ROS on cell depends on the level at which they are present. Low levels provide a beneficial effect, support cell proliferation and survival path-ways. However, at high levels, ROS can oxidatively damage biological macromolecules and cause detrimental oxidative stress that can lead to cell death. ROS can induce genotoxic damage, including single- and double-strand breaks, DNA–protein cross-links, basic sites and modified bases[25–27].

Cells withstand and counteract oxidative stress by the use of several and different defense mechanisms, ranging from free radical scavengers and antioxidant molecules/enzymes to sophisticated and elaborate DNA repair mechanisms to reduce excessive levels of ROS and prevent irreversible cellular damage[28, 29].

An increased rate of ROS production occurs in highly proliferative cancer cells, owing to the presence of oncogenic mutations that pro-mote aberrant metabolism. Increased oxidative stress is well documen-ted in transformed cells[30–33]and growing evidence suggests that ROS act as second messengers in intracellular signaling cascades which induce and maintain the oncogenic phenotype of cancer cells [34]. During cervical carcinogenesis an increase of oxidative DNA dam-age, as shown by progressive increase in the levels of 8-OHdG from nor-mal to SIL to invasive carcinomas, has been reported[35]. In our recent work we demonstrated that UVB-induced oxidative stress provoked an increased and selective protein oxidation in keratinocyte cells trans-fected with the whole HPV-16 genome[36]. Such oxidative modi fica-tions, combined with UVB-induced modulation of protein expression, likely lead to protein dysfunctions that might contribute to the malig-nant progression of transformed cells.

However, ROS regulation is crucial for transformed cells that coun-teract ROS accumulation by up regulating antioxidant systems, seem-ingly creating a paradox of high ROS production in the presence of high antioxidant levels[37]. We further demonstrated that UVB irra-diation caused, in HPV-transformed cells, a general suppression of viral transcription, accompanied by a moderate growth arrest, an ap-propriated response of cellular antioxidant enzymes, the activation of cell repair mechanisms and a mild induction of apoptosis[38]. This response was absent in transformed cells devoid of HPV sequences (HaCaT) that appeared extremely susceptible to apoptosis. We postu-lated that the suppression of viral oncogenes, restoring the cell con-trol on growth and repair mechanisms, allows the damage repair, ultimately resulting in a surviving response. Thefine mechanisms ac-tivated by cancer cells to counteract oxidative attack, allowing them to avoid ROS-induced cytotoxicity, also make them more exposed to additional ROS-mediated mutagenic events and accumulation of DNA damage.

Studies on serum of patients with CIN and carcinoma of the cervix evi-denced changes in lipid peroxidation and impairment of antioxidant sys-tems, either enzymatic[21, 39]or not[40]. Other blood-related studies showed that LSIL, HSIL or cervical cancer can be associated with changes in three indicators of oxidative stress: increase in erythrocyte TBARS,

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ALA-D reactivation index and a decrease in vitamin C content[41]. Nitro-sative stress was also evidenced in patients with cervical squamous cell carcinoma, as shown by increased plasmatic NO levels[21].

These data support the hypothesis about the involvement of both oxidative and nitrosative stress in the pathogenesis of cervical cancer. 4. Antioxidants, chemoprevention and chemotherapy

In recent years, many studies have been carried out to investigate the potential cancer chemopreventive activities of natural antioxi-dants, in particular dietary polyphenols[42]. Representative exam-ples of polyphenols include epigallocatechin-3-gallate (EGCG) from green tea, curcumin from turmeric, and resveratrol from grapes (Fig. 2). These compounds have been proved to block carcinogenesis and to inhibit tumor growth both in vitro and in vivo models (animal or in cell culture). These effects can be attributed to non-scavenging role of antioxidants including induction of apoptosis, growth arrest, inhibition of DNA synthesis and modulation of signal transduction pathways[43]. Intracellular signaling cascade involve several molec-ular events, which are initiated by the specific interaction of an extra-cellular ligand with its receptor at the cell membrane[44]. After this first event, second messengers participate to amplify signal transduc-tion pathways, which ultimately lead to modulatransduc-tion of several cellu-lar functions. Within this frame, ROS/RNS have been proposed as second messengers in the activation of several signaling pathways leading to mitogenesis or apoptosis. The effective transmission of sig-nals occurs through the recruitment of highly specific intermediates, and how ROS signaling may be activated with specificity and without oxidative damage is still poorly understood[45]. In addition, gene ex-pression is significantly regulated in response to oxidative stress and is necessary to ensure cell survival involving specific redox-sensitive transcription factors such as Nrf2, NF-κB, AP-1, and MAPKs[46]. Mod-ulation of their activity leads to changes in the cellular redox status and alteration of gene expression responses to oxidative stresses, pre-sumably via sulfhydryl modification of critical cysteine residues found on these proteins and/or other upstream redox-sensitive molecular targets[47]. For example, AP-1 is activated by low levels of oxidants resulting in AP-1/DNA binding and an increase in gene expression. In turn, AP-1 activation leads to the induction of JNK activity resulting in the phosphorylation of the c-Jun transactivation domain[48]. On the contrary, high concentrations of ROS inhibited AP-1 and AP-1 in-duced gene expression. The inhibition of AP-1/DNA interactions is caused by the oxidation of specific cysteine residues in c-Jun's DNA binding region.

Similarly, NF-κB contains a redox-sensitive critical cysteine resi-due that is involved in DNA binding[47, 49]. NF-κB is normally se-questered in the cytoplasm by IκB, but under oxidative conditions, IκB is phosphorylated by IκB kinase (IKK), ubiquitinated, and subse-quently degraded. ROS production appears to be necessary to initiate the events leading to the dissociation of the NF-κB/IκB complex[50], but excessive ROS production results in the oxidation of cysteine res-idue which does not affect its translocation to the nucleus, but rather, interferes with DNA binding and decreases gene expression[45].

The ability of naturally occurring antioxidants to modulate cellular signal transduction pathways, through the activation/repression of multiple redox-sensitive transcription factors has been claimed for their potential therapeutic use as chemopreventive agents. They can interfere with each stage of carcinogenesis—initiation, promotion and progression—to prevent cancer development[51].

Decreased mutagenic risk could result from the induction of several phase 2 detoxifying and antioxidant enzymes, such as glutathione-S-transferase, NAD(P)H quinone reductase and heme oxygenase among others[46]. This process is known to involve the antioxidant response element (ARE), which is present in the promoter region of all the afore-mentioned genes[52–56]. Nuclear factor-E2-related factor-2 (Nrf2) is a basic leucine zipper, redox-sensitive transcription factor that has been

widely studied to gain an insight into its role in chemoprevention. It is known to be involved in the regulation of ARE-mediated gene transcrip-tion. Polyphenols, such as curcumin and resveratrol, are known to in-duce ARE-mediated gene expression by increasing the levels of Nrf2 protein or suppressing its turnover by ubiquitination[53]. They activate Nrf2-mediated gene expression either by directly modifying the cysteine residues on Keap1 to disrupt the Nrf2–Keap1 complex or by activating kinase signaling pathways such as MAPKs, protein kinase C (PKC), and PI3K to phosphorylate Nrf2/Keap1 complex and/or facilitate the release of Nrf2 or to increase the nuclear translocation of Nrf2 and regulate the transcriptional activity of Nrf2 nuclear co-activators.

One of the major research focus is to solve the“redox paradigm”, that is to understand how these compounds can differentiate be-tween“normal” versus “abnormal tumor” cells in terms of signaling, gene expression, and pharmacological effects. Oxidative stress and perturbation of intracellular redox status can have a profound in flu-ence on the direction of signaling between cell survival and cell death. The comprehension of the mechanisms which lead to cell death of cancerous cells and cell survival of normal cells will need to be better investigated.

5. Polyphenols and cervical cancer

Polyphenols, from natural and herbal extract are raising great in-terest as powerful and safe anticancer strategy for their broad range targeting capability and low side effects. Many herbal extract are both able to target against viral oncogenes and to inhibit the cooper-ative signaling and deregulated gene expression of the host cells[3]. Recently an inverse association between antioxidant nutrients and HPV persistence, SIL, and cervical cancer was shown, suggesting pro-tective effect of natural extract against HPV persistence and cervical dysplasia[57, 58]. A new approach in the use of natural extract con-cern the sensitization of cancer cells to radio- and chemo-therapy by a pretreatment before the prolonged cure[59, 60].

In the following sections are described the most recent research about the anti-cancerogenic activities of curcumin, resveratrol and epigallocatechin-3-gallate (EGCG), three of the most commonly studied compounds from herbal extract, for the treatment of cervical cancer (Figs. 1 and 2). Moreover few preliminary data on the therapeutic use of these antioxidant compounds are reported.

5.1. Curcumin and ferulic acid

Curcumin (1,7-bis (4-hydroxy 3-methoxy phenyl)-1,6-heptadiene-3,5-dione) is a natural polyphenolic compound extracted from the rhi-zome of the medicinal plant Curcuma longa Linn (also known as turmeric) found in South and Southeast tropical Asia. Curcumin has been used for centuries throughout Asia as a food additive, cosmetic, and as a tradition-al herbtradition-al medicine for its various biologictradition-al activities.

Over the past decade, several studies have indicated the potential therapeutic value of curcumin and have clearly demonstrated that curcu-min possesses anti-inflammatory, antioxidant, anticarcinogenic, throm-bosuppressive, cardioprotective, antiarthritic, and anti-infectious properties[61–68]. Several studies reported that curcumin suppresses all 3 stages of carcinogenesis: initiation, promotion, and progression. In-hibition of nuclear factor kappa B (NF-κB) and subsequent downregula-tion of various NF-κB-related proinflammatory pathways is very likely the primary target accounting for its efficacy[59]. Moreover curcumin suppresses a number of key elements in cellular signal transduction pathways, including c-Jun/AP-1 activation[69, 70]and phosphorylation reactions catalyzed by protein kinases[71]. Further, curcumin suppresses angiogenesis in vivo, abrogates FGF-2-induced angiogenic response, ma-trix metalloprotease expression and cyclooxygenase-2[72–75](Table 1). The varied biologic properties of curcumin and lack of toxicity, even when administered at doses as high as 8 mg/d[76], make curcumin an ideal and very attractive compound in the attempt of therapeutic agents

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against various tumors. Curcumin has been shown to prevent cancer in the colon, skin, stomach, duodenum, soft palate, and breasts of rodents after oral administration[77, 78]. Recent data indicate that in addition to its chemopreventive role, curcumin has great potential as a chemo-and radio-sensitizer[59]. Considering the toxic side effects of radio and chemotherapy, achieving radio and chemosensitization with mini-mal toxicity represents a goal for the treatment of cancer patients. In the other hand, one concern regarding curcumin therapeutic use is its low bioavailability and poor pharmacokinetics[76].

In regard of curcumin antitumoral properties, several studies were conducted on cervical cancer. Cervical cancer cell lines were often used as a preferred carcinogenic model to understand molecular targets and mechanism of curcumin action. Here are reported all the in vitro and in vivo advances for the use of curcumin in the treatment of cervical cancer. Initial studies about the effect of curcumin on NF-κB activity in cervi-cal cancer cells were conducted by Venkatraman et al. in 2005[79]. They demonstrated in SiHa cell line, normally resistant to cisplatin, that inhi-bition of NF-κB activity induced by curcumin determined increased sen-sitization to cisplatin treatment evaluated as increased cell-death. According with this study Bava et al.[80]showed that curcumin sensi-tizes cervical cancer cells to the therapeutic effect of taxol, acting in the down-regulation of both NF-κB and serine/threonine kinase AKT path-way, a survival signal related to NF-κB.

A further step in the investigation of curcumin molecular target was made by the study of Prusty et al.[81], which showed that curcu-min can downregulate HPV-18 transcription, selectively inhibit AP-1

binding activity and reverse the expression dynamics of c-fos and fra-1 in HeLa cells. Subsequently, Divya et al.[82]indicated that cur-cumin is cytotoxic to cervical cancer cells in a concentration-dependent and time-concentration-dependent manner and the cytotoxicity was higher in HPV infected cells. The authors confirmed the inhibitory ac-tion of curcumin on NF-κB activation, through the degradation of IκB, and on AP-1 binding that results in the downregulation of COX-2 and increased tumor cell apoptosis. Moreover, it was shown that curcu-min downregulates the expression of HPV-16 and -18 E6 and E7 resulting in loss of the transforming phenotype and the cessation of cellular growth. Current studies by Singh and Singh[83, 84]explored other molecular mechanisms exerted by curcumin in cervical cancer cells, showing that it inhibits telomerase activity, Ras and ERK signal-ing pathway, cyclin D1, COX-2 and iNOS activity. Moreover, curcumin decreases c-Myc transcriptional factor and Hsp 70 chaperone protein, activates AIF, releases cytochrome c and enhances apoptosis via the mitochondrial pathway with the upregulation of BAX and the down-regulation of Bcl-2 and Bcl-XL. The authors further demonstrated that the activity of curcumin on these multiple targets was able to revert the proliferative action due to a pretreatment with estradiol on cervi-cal cancer cell. A different approach was used by Madden et al.[85] that with a proteomics approach analyzed the effect of curcumin on HeLa cells, reporting significant changes in tumor-related proteins linked to cell metabolism, cell cycle, and carcinogenic process.

Other recent mechanistic studies confirmed the suppression of cancer cell growth by curcumin also in a three-dimensional raft

Fig. 1. Molecular mechanisms of human papillomavirus-induced carcinogenesis. Oncogenic action of high-risk HPV E6 and E7 oncoproteins is exerted through the inactivation of tumor suppressors p53 and pRb, respectively. Abrogation of p53 by E6 ensures cell survival by preventing apoptosis inactivation while pRb inactivation by E7 forces infected cells to remain in a proliferative state. The dark red box represent the target of action of the polyphenols considered: C = curcumin; E = EGCG; R = resveratrol.

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culture system[86]. This effect was exerted through the induction of apoptosis, the inhibition of cell motility, the decrease in expression of HPV oncoproteins, and the restoration of expression of tumor sup-pressor proteins. Interestingly they also showed that curcumin was able to inhibit benzo[a]-pyrene (BaP) induced increase in HPV oncoproteins.

Several studies proposed curcumin as a powerful radio- and chemo-sensitizer. Javvadi et al.[87]showed that curcumin, with ion-izing radiation (IR), produced a significant increase of ROS, which fur-ther led to sustained ERK 1/2 activation, favoring the radiofur-therapic effect on cervical cancer cells. Moreover Javvadi et al. showed later the involvement thioredoxin reductase enzyme in the radiosensitiz-ing action of curcumin in cervical cancer cells[88]. Very recently the group of Anto R.J. showed that curcumin given during paclitaxel treatment act as a down-regulator of paclitaxel pro-survival path-ways linked to NF-κB and Akt activation in cervical cancer cells and mouse models of cervical cancer [89, 90]. These studies, both in vitro and in vivo, indicate that NF-κB is one of the central players in the synergism of paclitaxel and curcumin and act as a regulator of Cox-2, Cyclin D1, XIAP and cIAP1. AKT instead is the regulator of NF-κB, which through the phosphorylation of MAPKs regulates a set of survival signals.

The degradation of curcumin brings the formation of two products: vanillic acid and ferulic acid (FA) and FA represent the major compo-nent of curcumin.

FA (3-methoxy-4-hydroxycinnamic acid) is a dietary phytochemical that occurs primarily in the seeds and leaves of most plants such as rice, wheat, barley, oat, roasted coffee, tomatoes, and citrus fruits. FA ethyl ester, FAEE, has been largely investigated in the protection against OS during neurodegeneration, driven by Alzheimer disease, for its ability to cross the brain blood barrier and exert its activity directly on OS chal-lenged brain cells[91–94]. Moreover FA and FAEE are well-known anti-oxidant agents used to prevent damage to cells caused by ultraviolet light[95]. For this property FA is used in a wide range of cosmetics, such as skin lighteners, sunscreens, anti-aging creams and moisturizers. However several studies show that FA exhibits chemotherapic proper-ty. It was described as a specific inhibitor of the anti-apoptotic proteins Bcl-XL and Bcl-2, thereby inducing apoptosis [96]. However, a

prooxidant activity of FA was observed in different experimental models that might help to enhance radiation effects in cancer cells, as was previously proven for other phenolic compounds[97].

A recent study from Karthikeyan et al.[98]investigated the radio-sensitizing effect of FA on two human cervical carcinoma cell lines. The authors showed that FA enhances radiation effects by decreasing cancer cell viability through the influence on mitochondrial activity, cell survival, and antioxidant status. Further was showed that FA and IR increase lipid peroxidative markers, ROS levels, oxidative DNA damage and apoptotic morphological changes, corroborating that treatment of cancer cells with FA may help in augmenting the an-ticancer effect of radiation.

From the studies aforementioned in this review, it is clear that curcumin and its product FA holds great potential in the prevention and therapy of cervical cancer. Researchers suggest that curcumin represents an ideal compound for cancer patient treatment, consider-ing the encouragconsider-ing data as a safe and effective antitumoral and chemo- and radio-sensitizer agent, and considering also its ability to prevent depression, fatigue, lack of appetite and most of the symp-toms induced by cancer.

5.2. Epigallocatechin-3-gallate

Green tea is one of the most widely consumed beverages. It has been proven that it possesses beneficial anticarcinogenic and antipro-liferative properties attributed to the biological properties of green tea polyphenolic compounds. Consumption of green tea has been reported to lower the risk of developing gastric, pancreatic, and colo-rectal cancers in human populations[99, 100]. The polyphenols ac-count for up to 30% of the dry weight of green tea and include flavanols, flavandiols, flavonoids, and phenolic acids[101]. In particu-lar,flavanols, known as catechins, are a major component of most green tea. Generally, a typical cup of green tea contains 100 to 150 mg catechins, including 50% of epigallocatechin-3-gallate, 15% of epigallocatechin, 15% of epicatechin-3-gallate, and 8% of epicate-chin[102]. Epigallocatechin-3-gallate (EGCG) is the major bioactive polyphenol present in green tea and displays antiproliferative, anti-angiogenic, antimetastatic, proapoptotic, and cell cycle perturbation

Fig. 2. Polyphenolic antioxidants. Chemical structure, properties and sources of curcumin (1,7-bis (4-hydroxy 3-methoxy phenyl)-1,6-heptadiene-3,5-dione), ferulic acid (3-methoxy-4-hydroxycinnamic acid), EGCG (epigallocatechin-3-gallate) and resveratrol (trans-3,5,4′-trihydroxy-trans-stilbene).

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activities in various in vitro and in vivo tumor models [103–109] (Fig. 2). It has also been reported that there exists a threshold level of EGCG that induces apoptosis and growth inhibition in cancer cell lines, but not in normal cells[110, 111]. EGCG acts as a potent antiox-idant and can scavenge ROS, such as lipid free radicals, superoxide radicals, hydroxyl radicals, hydrogen peroxide and singlet oxygen [112–115].

EGCG has been shown to inhibit several critical signal transduc-tion pathways as well the activatransduc-tion of the redox-sensitive transcrip-tion factors, NF-κB and AP-1 in cultured cells[116–118].

One of thefirst study about the effect of EGCG on the HPV-16 as-sociated cervical cancer cell line CaSki, by Ahn et al.[119] demon-strated that an EGCG antiproliferative effect depends by the arrest of cell cycle in G1 phase that precedes in time the consequent pro-grammed cell death by apoptosis. Furthermore, antitumor effects of EGCG were also observed in nude mice challenged with CaSki cells and then treated orally with EGCG. Siddiqui et al.[120], in a recent study on cervical cancer cells extracted from fresh tissue, observed apoptosis induction by EGCG, confirming results previously obtained. Afterwards, Khun et al.[121]showed that EGCG potently inhibits the proteasomal activity in HeLa cells, as evident by accumulation of

ubiquitinated proteins and three natural proteasome targets (p27, IκB-α and Bax). Sah et al.[116]investigated the effect of EGCG on EGFR signaling in several cervical cells lines, demonstrating that EGCG inhibits EGFR, the initial kinase in the EGF signaling cascade, while activating the ERK1/2 and AKT activity. This inhibition is associ-ated with reduced phosphorylation, levels and activity of ERK1/2 and AKT downstream substrates, which lead again to G1 arrest and in-creased apoptosis. Yokoyama et al. and Noguchi et al.[122, 123] indi-cated that EGCG, beyond cell growth and apoptosis, target also telomerase, inhibiting its activity required for the initiation and pro-gression of cervical lesions. It is well recognize that angiogenesis is a key step in the progression cervical cancer. Zhang et al.[124]reported that EGCG exhibit anti-angiogenic activities for the suppression of HIF-1a protein accumulation and the decrease of VEGF expression at both mRNA and protein levels, through the enhancement of protea-some system activity and the blocking of PI3K/AKT and ERK1/2 sig-naling pathways. The anti-angiogenic effect of EGCG was shown recently also by Tudoran et al.[125]through the analysis on the alter-ations of the expression pattern of 84 genes known to be involved in the angiogenesis process. Li et al.[126]showed that EGCG abrogates anchorage-independent growth and inhibits cell proliferation and

Table 1

Biochemical effects and molecular targets of chemopreventive action of polyphenols (curcumin, ferulic acid, EGCG, and resveratrol) in cervical cancer models. Effects in cervical

cancer models

Compound Molecular targets References

Cell growth inhibition Curcumin F.A. WAF-1/p21; Cdc2; p16; PCNA; cyclin D1

Venkatraman et al. (2005); Prusty et al. (2005); Bava et al. (2005); Divya et al. (2006); Javvadi et al. (2008); Singh et al. (2009); Maher et al. (2011); Singh et al. (2011); Sreekanth et al. (2011); Bava et al. (2011); Karthikeyan et al. (2011)

EGCG G1 phase arrest; WAF-1/ p21; KIP-1/p27; cyclin; CDK

Ahn et al. (2003); Sah et al. (2003); Yokoyama et al. (2004); Noguchi et al. (2006); Yokoyama et al. (2008); Qiao et al. (2009); Zou et al. (2010)

Resveratrol S phase arrest Zoberi et al. (2002); Roy et al. (2002); Kramer et al. (2009) Apoptosis induction Curcumin

F.A.

Caspases 3, 8, 9; DNA damage; p53; PARP; cytocrome c; Bax, AIF; Bcl-2; Bcl-XL; annexin V

Venkatraman et al. (2005); Prusty et al. (2005); Bava et al. (2005); Divya et al. (2006); Singh et al. (2009); Bava et al. (2011); Maher et al. (2011); Karthikeyan et al. (2011)

EGCG p53; BAD; caspase 3 Ahn et al. (2003); Sah et al. (2003); Yokoyama et al. (2004); Yokoyama et al. (2008); Qiao et al. (2009); Siddiqui et al. (2010); Zou et al. (2010)

Resveratrol DNA damage; caspase 3; cathepsin L

Roy et al. (2002); Hsu et al. (2009) Angiogenesis

inhibition

Curcumin F.A.

VEGF Sreekanth et al. (2011) EGCG VEGF; HIF-1α Zhang et al. (2007) Resveratrol VEGF; HIF-1α Tang et al. (2007) Transcriptional

activity inhibition

Curcumin F.A.

AKT; NF-κB; AP-1; telomerase; c-fos; fra-1

Venkatraman et al. (2005); Bava et al. (2005); Prusty et al. (2005); Divya et al. (2006); Javvadi et al. (2008); Singh et al. (2009); Singh et al. (2011); Sreekanth et al. (2011);

Bava et al. (2011); Maher et al. (2011) EGCG AKT; RNA pol. III;

telomerase

Sah et al. (2003); Yokoyama et al. (2004); Noguchi et al. (2006); Li et al. (2007); Zhang et al. (2007); Jacob et al. (2007); Yokoyama et al. (2008)

Resveratrol AKT Sexton et al. (2006) Signal transduction

pathways regulation

Curcumin F.A.

ERK 1/2; JNK; p38; c-Jun Javvadi et al. (2008); Singh et al. (2009); Sreekanth et al. (2011); Bava et al. (2011) EGCG ERK 1/2; p90RSK Sah et al. (2003); Li et al. (2007); Zhang et al. (2007)

Resveratrol ERK; JNK; PKC-δ Woo et al. (2004) HPV suppression Curcumin

F.A.

E6/E7; HPV-18 mRNA Prysty et al. (2005); Divya et al. (2006); Maher et al. (2011) EGCG E6/E7 Qiao et al. (2009); Zou et al. (2010)

Resveratrol Cyclooxygenase

inhibition

Curcumin F.A.

COX-2; Divya et al. (2006); Singh et al. (2009); Madden et al. (2009); Sreekanth et al. (2011); Bava et al. (2011)

EGCG

Resveratrol COX-1; COX-2 Zoberi et al. (2002); Sexton et al. (2006) Cell redox status/cell

stress response

Curcumin F.A.

iNOS; HSP7; TRX-R; ROS production

Javvadi et al. (2008); Singh et al. (2009); Javvadi et al. (2010); Karthikeyan et al. (2011) EGCG

Resveratrol Other effects Curcumin

F.A.

MMP-9, -2; ICAM-1; survivin

Sreekanth et al. (2011); Bava et al. (2011) EGCG IGF-IR; EGFR; aromatase;

ERα, β; proteasome inhibition

Sah et al. (2003); Li et al. (2007); Qiao et al. (2009); Bonfili et al. (2011) Resveratrol MMP-9; autophagy Woo et al. (2004); Hsu et al. (2009)

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transformation induced by IGF-IR overexpression by competing with ATP, that result in a prevention of IGF-IR downstream signaling and expression of cervical cancer cell phenotype. Another target of EGCG anti-proliferative activity in cervical cancer cells is RNA pol III tran-scription, as reported by Jacob et al.[127]. The authors found that EGCG treatment inhibits transcription by RNA pol III, from both gene internal (tRNA) and gene external (U6 snRNA) promoters. Moreover EGCG inhibits expression of the TFIIIB subunits Brf1 and Brf2, and inhibits the activity of the human Brf2 promoter. An essen-tial effect of EGCG on cervical cancer cell lines is represented by the inhibition of HPV oncogenes E6/E7 expression. Quiao et al.[128] ob-served that EGCG, in cervical cancer cells, could suppress mRNA and protein expression of ERα and aromatase limiting in this way the ex-pression of E6/E7, and indirectly inhibiting cervical cancer cell growth and inducing apoptosis. Zou et al. [129] achieved a similar result showing decreased expression of E7 oncogene, increased apoptosis coupled with increased expression levels and increased cell growth inhibition during treatment of cervical cancer cells with EGCG. Re-cently, Bonfili et al. [130]sustained that the observed EGCG effects on proteasome functionality and on pro-apoptotic protein intracellu-lar concentrations in cervical cancer cells may be the result of a com-bined action of EGCG and its oxidation product. In fact the authors observed that EGCG, under a common experimental condition un-dergoes modification, involving the hydrolysis of the gallate ester, and the disruption of the catechin A ring, with the formation of a bi-ologically active product with inhibitory activity towards cellular proteasomes.

Green tea represents, for its common use, one of the most studied antioxidant compounds. From the aforementioned studies, it is clear that EGCG, a green tea major constituent, holds great potential in the prevention and therapy of cervical cancer. Taken together, these data support the idea that EGCG provides an additional option for a new and potential drug approach for cervical cancer patients. 5.3. Resveratrol

Resveratrol (trans-3,5,4′-trihydroxy-trans-stilbene) is a natural polyphenolic phytoalexin most commonly found in nuts, berries, grapes, and red wine. In several plants, including grapevine, resvera-trol is synthesized in response to stress, injury, ultraviolet irradiation, and fungal infection.

After the discovery of resveratrol pronounced pharmacological ef-fects, it became a topic of intense investigations. It has been proposed to prevent or counteract cardiovascular diseases (result from a mod-erate wine drinking known as“The French Paradox”)[131, 132]and inhibit cellular events associated with tumor initiation, promotion and progression as a result of anti-inflammatory, proapoptotic, anti-angiogenic, chemo-, and radio-sensitizing properties [132–139] (Fig. 2). Numerous reports also indicate that resveratrol has antiviral effects against HIV-1 and the herpes simplex virus[140, 141]. The high concentration of resveratrol in grape skin (50–100 mg/g) and thereby the high amounts in red wine (from 0.2 mg/l to 7.7 mg/l) suggests that wine consumption might be protective against several pathologies. However, the amount of resveratrol consumed daily by a person drinking one glass of red wine is considerably lower than the dose needed to benefit of resveratrol activity as antitumoral agent[131, 142]. Studies focused on resveratrol anti-tumor activity showed that this multi-hit compound exerts its action interacting with numerous molecular targets involved in tumor progression, such as Fas pathway, Rb-E2F/DP pathway, NF-κB and AP-1 transcrip-tional factors, MAPK and many others[142]. However just few of them were investigated in cervical cancer (Table 1). Recently a num-ber of studies were conducted to investigate the effects of resveratrol intake during cervical cancer development and the results are reviewed below. Zoberi et al.[60]showed that resveratrol pretreat-ment, in cervical cancer cell lines, has the ability to inhibit cell

division inducing an early S-phase arrest, suggesting a role for resver-atrol in the regulation of cell cycle progression. Kramer et al.[131] using a double dose of resveratrol confirmed this result adding that resveratrol-induced S-phase block was transient and reversible. Cy-clooxygenase (COX) is well recognized as one of the main targets of the resveratrol mechanism of action[142] [143]; in their study Zoberi et al. showed that COX-1 is overexpressed in cervical cancer cells while resveratrol treatment inhibits COX-1 activity. PGs are implicat-ed in the response of tumor cells to ionizing radiation and other cyto-toxic insults; COX-1 inhibition suppresses PGs biosynthesis, suggesting that COX-1 inhibition by resveratrol might alter how tumor cells respond to the cytotoxicity of ionizing radiation. Zoberi et al. supported this hypothesis demonstrating that HeLa and SiHa cell line cytotoxicity was enhanced by pretreatment with resveratrol prior to ionizing radiation exposure. Sexton et al.[144]showed, in six different human uterine cancer cell lines, that treatment with res-veratrol decrease endogenous COX-2 protein levels, concomitant with a decrease in production of COX metabolites PGE2 and PGF2α. They suggest in their study that resveratrol regulates COX expression not directly but possibly thru other enzymes involved in prostaglan-din synthesis that act downstream of the COXs. Moreover they also showed that resveratrol reduced cellular levels of the phosphorylat-ed/active form of anti-apoptotic kinase AKT. Lin et al. obtained similar results, about COX-2 suppression by resveratrol, in ovarian and breast cancer cells supporting the role of resveratrol on cervical cancer cells [145–147]. Another recognized mechanism of resveratrol chemopre-vention is represented by the inhibition of metalloproteinases (MMPs). A positive correlation between the expression of MMP-9 and tumor metastasis for several types of cancer was recently shown[148, 149]. Woo et al.[150]demonstrated, in a CaSki cervical cancer cell line, a decreased expression of MMP-9 during treatment with resveratrol that exerts its actions through a tight regulation of MMP-9 transcription. The authors also show that reduced MMP-9 levels depend by reduced PKC-δ activity, JNK activation as well as

diminished NF-κB and AP-1 activity during treatment with

resveratrol.

The progression of cervical cancer relies on tumor angiogenic ac-tivity and several studies showed that HPV-16 oncoproteins, E5, E6, and E7, induce VEGF expression in several established cervical cancer cells lines[151–153], suggesting that high-risk HPV-16 oncoproteins can promote tumor angiogenesis possibly via HIF-1α. Tang et al. [154]revealed that overexpression of HPV-16 E6 and E7 oncoproteins significantly promoted HIF-1α protein accumulation and VEGF ex-pression in human cervical cancer cells. However activation of HIF-1α and VEGF can be reverted by resveratrol through the inhibition of AKT and ERK1/2 in cervical cancer cells, conceivably via the PI3K/ AKT and ERK1/2 signaling pathways. These results suggest that res-veratrol can suppress tumor angiogenic activity in vitro and thereby tumor progression and proliferation. Recently Hsu et al.[155] indicat-ed that resveratrol induces autophagy, inhibits proliferation and in-duces apoptotic death in HeLa and SiHa cervical cancer cell lines. The authors showed that resveratrol destabilize lysosomes, which cause lysosomal leakage, increased cytosol translocation, and enzymatic activity of cathepsin L (cat L). The increased cat L activity in cytosol in-duces cytochrome c release from mitochondria, and subsequently re-sults in apoptotic cell death. An essential event in the pathway is the lysosomal membrane permeabilization led by resveratrol induced autophagy and the release of lysosomal proteases to the cytosol. The re-leased cathepsins may trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues, and induce cyto-chrome c release.

Studies by Rezk et al.[156]on the chemotherapic use of resvera-trol showed that this compound used with cisplatin or doxorubicin demonstrated an additive growth-inhibitory anticancer effect on uterine cancer cells supporting its possible use in the treatment of cervical cancer.

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In conclusion, all these studies demonstrated that resveratrol is ca-pable of: regulating cell cycle, progression, suppressing angiogenesis, inhibiting MMP activities, inducing autophagy with consequent pro-grammed cell death and increasing IR-induced cytotoxicity (Table 1). These properties strongly support the beneficial effects of resveratrol as a chemopreventive, a chemotherapeutic and a radiosensitizing com-pound in the treatment of high-risk HPV-associated human cervical cancers as well as other tumors[157]. However supplementary studies to elucidate all the molecular mechanisms activated by resveratrol treatment during cervical cancer are needed. Moreover, the lack of spe-cific clinical trials to prove the efficiency of resveratrol for the treatment of cervical cancer still raises the question about its real suitability. 6. Clinical trial in cervical cancer

The effects and molecular mechanisms of action of curcumin, EGCG and resveratrol clearly sustain the chemopreventive and che-motherapic properties of these phenolic compounds, as well as their chemosensitizer and radiosensitizer abilities in cervical cancer models. However, despite the confirmed beneficial effect of these an-tioxidant compounds on cervical cancer progression, only few clinical studies have been conducted until now.

Cheng et al. in 2001[76]piloted a prospective phase-I study to evaluate anticancer properties and also toxicology, pharmacokinetics and biologically effective dose of curcumin in patients with uterine CIN. The study demonstrated that curcumin is not toxic to humans up to 8 mg/d when taken by mouth for 3 months and suggested a bi-ologic effect of curcumin in the chemoprevention of cancer lesions at least in 25% of the cases under treatment. A curcumin testing started in 2008 for the investigation of its safety and efficacy against HPV in-fection, in a Phase II multicentric clinical trial on 280 women enrolled on the basis of clinical examination, cytology, histo-pathology and presence of HPV[158].

Ahn et al. in 2003[159]investigated clinical efficacy of EGCG and an-other green tea component, poly E, delivered vaginally, orally, or both, in patients with HPV-infected cervical lesions. Fifty-one patients with such lesions, ranging from chronic cervicitis through mild and moderate dysplasia to severe dysplasia, were divided into four groups and com-pared with 39 controls. For oral delivery, a 200 mg of EGCG or Poly E capsules was taken every day for eight to 12 weeks while Poly E oint-ment was applied locally to 27 patients twice a week. The 60% of sub-jects under EGCG capsule therapy, the 50% under poly E capsule therapy, the 74% under poly E ointment therapy and the 75% under poly E ointment plus poly E capsule therapy showed a response. Overall, a 69% response rate was noted for treatment with green tea extracts, as compared with a 10% response rate in untreated controls. These results indicate that green tea extracts used orally and/or vaginally are effective for treating HPV-related cervical lesions suggesting that green tea ex-tracts can be a potential therapy routine for patients with HPV infected cervical lesions

As previously stated, despite the promising results in preclinical models, clinical trials, focusing on the health promoting effect of res-veratrol, in subjects with cervical neoplasia are currently missing [160]. To date most of the available clinical studies in humans are fo-cused on resveratrol bioavailability, pharmacokinetics and metabolism [161]and so far only two recently reports showed that resveratrol could represent a promising agent in the treatment of cancerogenesis [162, 163].

7. Conclusions

Cervical cancer is the second most common cancer among women worldwide. Thus, it still represents a major concern for public health although current screening methods are already available to allow early diagnosis and treatment. Several therapeutic strategies are being explored which can effectively target various phases of viral

infection and its oncogenic expression. However, apart from the cur-rent pharmacological intervention it becomes essential to explore al-ternative approach. In recent years there has been increasing interest in the potential cancer chemopreventive properties of diet-derived agents. Among these, polyphenolic compounds, such as curcumin, resveratrol and ECGC, received much attention for prevention and treatment of cervical cancer.

Polyphenols were demonstrated to inhibit the proliferation of HPV-immortalized and HPV-positive cancer cells, through induction of apoptosis, growth arrest, inhibition of DNA synthesis and modula-tion of signal transducmodula-tion pathways. They are able to act at several steps in the cascade of cell transformation promoted by HPV infec-tion. However, further studies will likely provide additional insights into the mechanism of redox control of cancerogenesis events.

From the studies reviewed in this paper, it is clear that dietary polyphenols hold a great potential in the prevention and therapy of cervical cancer. Encouraging data as a safe and effective antitumoral and chemo- and radiosensitizer agent have been demonstrated in clinical trials. Further, considering that curcumins, EGCG and resvera-trol present peculiar targets of action the synergistic use of these polyphenols might favor their anticarcinogenic effect, however no such studies has been conducted yet on cervical cancer models. In ad-dition, they also prevent depression, fatigue, lack of appetite and most of the symptoms induced by cancer.

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