Chemopreventive effect of pomegranate and cocoa extracts on ultraviolet radiation-induced photocarcinogenesis in SKH-1 mice.

Non-melanoma skin cancer (NMSC) has a high and increasing incidence all over the world. Solar radiation is the main aetiology for humans. Although most research into photocarcinogenesis uses UVB as a source of radiation, UVA is also carcinogenic in long term. Pomegranate (PGE) and cocoa (CE) extracts have been used for medicinal purposes for time immemorial. Recently, it has been claimed that some of their properties may be an effective preventative measure against photocarcinogenesis and photoaging, but to date in vivo models have not been tested using RUVA, the objective of the present work. A lower incidence of lesions was observed in SKH-1 mice treated with PGE (p<0.001), and lower incidence of invasive squamous carcinoma in both treatment groups (p<0.001 for PGE and p<0.05 for CE); the PGE group also showed a lower level of cell proliferation than the control group (p<0.001). Significantly greater p53 alteration was observed in the control group than the treatment groups (p<0.001 for PGE and p = 0.05 for CE). No significant differences were found in relation to TIMP-1 and MMP-9. Taken together, the results suggest that oral feeding of PGE and CE to SKH-1 mice affords substantial protection against the adverse effects of RUVA, especially PGE.

Introduction

The incidence of skin cancers exceeds all other malignant neoplasias together. Among these, the most aggressive is melanoma. Although it only represents 5% of all skin cancers, melanoma causes 80% of all deaths from skin cancer (Weinstock 2008) [1]. Non-melanoma skin cancer (NMSC) has a high incidence all over the world, with the number of cases increasing annually, see [2]. Although most are basal cell carcinomas (80%), which have an excellent prognosis, between 15 and 25% are squamous cell carcinomas (SCC), with worse prognosis, whereby they are associated with not inconsiderable rates of morbidity and mortality (Eisemann et al. 2014) [3]. In the U.S.A. incidence varies between 5 and 499 cases per 100.000 inhabitants depending on latitude. Its annual increase has been estimated at between 50 and 200% over the last three decades, and these figures continue to increase due to aging populations in the developed world (Waldman and Schmults, 2019) [4]. Although the rate of hospitalization due to melanoma is higher, the incidence of NMSC makes health care costs higher for this type of skin cancer in absolute terms (Duarte et al. 2018) [5]. Exposure to sunlight, pale skin, and immunosupression are risk factors for NMSC. Solar radiation is the main cause of this type of cancer in humans, particularly those with white skin. Ultraviolet A (UVA) radiation (320-400 nm) is the main component of solar radiation arriving on the Earth’s surface (90-99%) and is associated with both benign and malignant skin cancers. While UVB provokes more direct damage to cellular DNA, the effects of UVA radiation are indirect, derived from oxidative damage caused by singlet oxygen release, which provokes diverse genetic alterations which vary from point mutations to crude chromosomal dislocations (Bachelor and Bowden, 2004) [6].

Punica granatum L. (pomegranate) is a deciduous shrub which has been extensively used in traditional medicine in many regions of the world, including the Mediterranean area, Middle East, and central Asia (Shaygannia et al., 2015) [7]. Pomegranate fruit has biological effects including anti-inflammatory and antibacterial activities. It also contains flavonoids (kaempferol, luteolin and naringenin) and alkaloids (caffeic, gallic and ferulic acids, punicalagin, quercetin or catechin), which can prevent carcinogenesis both in vitro (Singh et al., 2002) [8] and in vivo (Justin et al., 2003) [9]. The seeds of the plant Theobroma cacao L. (Sterculiaceae) are the raw material from which one of the most widely consumed functional foods in the world is made–cocoa. The Mayas and the Aztecs used cocoa for both nutritional and curative purposes (Scapagnini et al., 2014) [10]. Cocoa extract is rich in polyphenols, mainly catechins and proanthocyanidin flavanols, but also gallocatechin and epicagallocatechin or methylxanthine compounds (theobromine and caffeine) among other components with reactive oxygen species (ROS) scavenging properties (Bosch et al., 2015) [11]. The quantity and proportion of these components is highly variable due to differences in processing and manufacturing. Although an increasing number of published papers have presented evidence that cocoa extract contributes to endogenous photoprotection and has anti-aging and anti-wrinkle effects, little research has investigated its role in photocarcinogenesis (Kim et al., 2016) [12].

SKH1/CRL mice are an excellent animal model for use in photodamage studies. Their skin is very sensitive to ultraviolet radiation; they are euthymic, immunocompetent, and easy to look after. Oral feeding of both pomegranate fruit and cocoa extracts to SKH-1/CRL mice has demonstrated substantial protection from the adverse effects of UVB radiation derived from modulation of early biomarkers of photocarcinogenesis (Afaq et al., 2010) [13], but this has not been proved using UVA radiation in vivo. In this context, the main objective of the present study was to evaluate the protective effects of pomegranate and cocoa extracts during UVA-induced skin cancer in SKH-1 hairless mice. See for other approaches [14, 15] or [16].

Materials and methods

Chemicals

The extracts were supplied by Naturex S.A. (Avignon, France). Pomegranate extract (reference EA140914, batch no D047/002/A11) was obtained from Punica granatum L. rinds (plant extract ratio between 5:1 and 7:1). The extraction solvent contains 70% ethanol, 30% water. The nutritional composition of the extract was as follows: energy 367 kcal/100 g; carbohydrates 84% (w/v); fat 1.4% (w/v); fiber 3.8% (w/v); proteins 2.4% (w/v), and total specific phenolic 12.62% (w/v)with, respectively, 2.77%; 7.92%, and 1.93% (w/v) ofpunicalagin A, punicaligin B, and ellagic acid. Cocoa extract (batch no TCP 19013) was obtained from Theobroma cacao L. complete seeds with the method patented by the company (EP2071961A1). The resulting product is a powdered semi-purified extract with a total polyphenol content of 20–45% and an organic fraction, from which, after concentration and drying thereof, a powdered purified extract is obtained with a total polyphenol content of 60–90%. The content of flavonols flavanol monomers catechin (24.2) and epicatechin (115) and 226.6 of total oligomeric procyanidins, flavonols (quercetin-3-O-glucoside 1.0; quercetin-3-O-arabinoside 1.0), anthocyanins and alkaloids (10.3 theobromine and 1.1 caffeine).

The extracts were supplied by Naturex S.A. (Avignon, France).

Animals and treatment

The study used 27 SKH-1/CRL female mice, four weeks old at the beginning of the experiment. The animals (euthymic and immunocompetent) were obtained from Charles River Laboratories (Wilmington, MA, U.S.A.) and acclimatized for 10 days at the Animal Laboratory Service of the University of Murcia (Spain) before the start of the experiment. They were randomly distributed into three groups. Group I (n = 9) were exposed to UVA radiation. Group II (n = 9) animals were also treated with PG, which was orally administered in the diet. Group III (n = 9) was treated with UVR and cocoa extract with ad libitum access. Complete experimental groups (9 animals) were housed in methacrylate cages transparent of 40 × 30 cm, covered with metal right and with space for food and water.

The standard chow (PanLab, Barcelona) had 14.3% protein, 4.0% fat, 48.0% carbohydrate, 4.1% crude fiber, 18.0% neutral detergent fiber and 4.7% ash; energy density, 2.9 kcal/g). The doses corresponded to 520 and 228 mg of extract per kg body weight per day of pomegranate and cocoa extract respectively, approximately 41.9 and 18.37 mg per kg body weight HED. The doses of the agents were selected in order to provide equivalent concentrations with other anticancer researches where has been previously tested the positive effect of these compounds (Vlachojannis et al., 2015) [17]) and Urpi-Sarda et al., 2009) [18].Dietary intervention lasted for 29 weeks (including 2 weeks of pretreatment). Body weight, food and water intake were recorded throughout the study. The animals were housed in climate controlled rooms (24°C at 50% humidity) with 12-hour light/dark cycles and free access to food and water, and treated in accordance to the rules of the European Union for the protection of animals used for experimentation (2010/63/EU). All experimental protocols comply with the ARRIVE guidelines and were approved by the Bioethics Committee of the University of Murcia.

UV irradiation

The mice were subjected to UV irradiation using a Philips lamp (Type HB 554/01/A) with 8 Philips Performance S 100W tubes. The lamp has an emission spectrum of 220-425 nm with a maximum peak of 364 nm (98.6% UVA and 1.4% UVB). The animals were exposed to radiation three times a week for a total of 80 sessions, each lasting 60 minutes (always at the same hour in the morning). For this purpose, the animals were placed in PVC cages, with individual separators and a metallic lattice ceiling, which were placed under the ultraviolet lamp with a distance of 20 cm between the light source and the skin. The energy received in each session was 21.1 J/cm2, so that at the end of the experiment the total energy received by each mouse was 1688 J/cm2.

Macroscopic study

A detailed macroscopic study of the dorsal skin was made after each session and digital photographs were taken on millimeter paper to measure the lesional areas at the end of the experiment (80 sessions). An application of the image processing and analysis software platform Leica Qwin was used to obtain a binary mask of the lesional areas, which were measured individually.

Microscopic study

At the end of the experiment, animals were sacrificed by CO2 overdose. A necropsy was performed on the dorsal skin and viscera. The samples collected were embedded in paraffin and stained with haematoxylin and eosin for microscopic analysis. The presence of normal skin, actinic keratoses, dysplasia, carcinoma in situ and invasive carcinoma was examined under light microscopy (Leica DM 6000B). Immunohistochemical study of tumors was conducted by the Pathology Department at the Reina Sofía University Hospital (Murcia, Spain) using the following antibodies: proliferating cellular nuclear antigen (PCNA); p53; anti-metaloproteinase-9 (MMP-9); and anti-metallopeptidase inhibitor 1 (TIMP-1) (Dako, S.A, Barcelona, Spain). Immunohistochemical staining was performed using the labeled streptavidin-biotin method. The sections were incubated overnight at 4°C with antibodies to PCNA (PC-10; Dako Corporation, Glostrup, Denmark); MMP; and TIMP-1 at a 1:200 dilution. Negative controls were treated with all reagents except the primary antibody. PCNA expression was quantified by means of image analysis. Firstly, the prepared specimens were digitalized by high resolution microscopy scanning with the Leica SCN400F. Then using an image analysis module from the Slidepath Digital Image Hub, three 100 μm2 areas were traced on each image. The areas selected were the tumor invasion front (in the case of tumors) and the epidermis basal layer when no carcinoma was present. The software produced a count of the number of positive nuclei and the total number of cells, using the Nuclear Algorythm 3.0, which detects PCNA marking by means of a previously defined color pattern. Inmunohistochemical quantification (PCNA and P53) was expressed as the mean percentage of PCNA and P53 immunohistochemical positive cells, in relation to the total number of cells (Ibrahim and Elwan, 2017) [19]. In semi-quantitative immunohistochemical analysis (TIMP, and MMP-9), scores were calculated by multiplying the graded intensity of stained stroma (grade 0-3) by the percentage of stained stroma surface (grade 0: no stained stroma, grade 1: less than 10% stained stroma, grade 2: 10-50% stained stroma, and grade 3: 51-100% stained stroma) (Preidl et al., 2019) [20]. All microscopic analyses were carried out by two pathologists blinded to the results of the study.

Statistical analysis

For the statistical analysis, we have used the SPSS software, version 20.0 (SPSS ®Inc., Chicago, IL, USA). A descriptive study of each involved variable was performed. The associations between qualitative variables were determined by Pearson’s Chi-square test. For the quantitative variables, the ANOVA and Tuckey tests were performed, determining in each case if the variances were homogeneous. Differences were regarded as significant if p≤0.05 and highly significant if p≤0.01.

Results and discussion

Skin cancer is more common among the white races and has undergone a dramatic increase during the last 40 years, due to social changes with regard to exposure to sunlight and UV radiation (UVR) (Weinstock, 2008) [1]. As a result, UVR has come to be regarded as the most important environmental carcinogen, and has driven the health-care sector on a quest for better information and management regarding the effects of solar radiation (Duarte et al., 2018) [5]. The UV spectrum can be divided into three wavelength ranges, UVA (320–400 nm), UVB (280–320) and UVC (200–280 nm). Of the solar radiation that reaches the surface of the earth, more than 90% is comprised of UVA and 1-10% is comprised of UVB. A large portion of UVB and all of the UVC wavelengths are absorbed by the ozone layer. So the biological effects of solar radiation are attributed to UV A and B radiations. For many years, it has been known that UVB is a complete carcinogen that can generate squamous-type carcinomas in animals. Its main biological target is DNA, where it produces CC to TT or C to T point mutations (Bosch et al., 2015) [11]. So, as far as most authors have been concerned, the spectrum in which skin carcinomas are produced has been the UVB region, attributing less carcinogenic activity to the UVA region (Forbes, 1981) [21]. Forbes considered that while UVB played a key role in initiating tumors, UVA energy intervened in promoting it. The current understanding is that although UVB is approximately 1,000-10,000 times more carcinogenic per J/m2 than UVA, chronic UVA radiation alone is also carcinogenic as shown in diverse in vivo experiments since the nineteen nineties (De Laat et al., 1997) [22]; (de Gruijl, 2004) [23]. Nevertheless, as tumor genesis is faster with UVB, most research into UV-related pathogenesis and particularly those works investigating preventative or palliative treatment have used UVB radiation sources (Kim et al., 2016) [12]; (Rigby et al., 2017) [24]; (Afaq et al., 2010) [13]; (Park et al., 2010) [25]. The equipment employed in the present study was a cosmetic tanning lamp that emitted 98.6% UVA and 1.45% UVB radiation. UVA radiation acts directly by generating ROS, which act on light-sensitive molecules such as porphyrins and nicotinamide adenine dinucleotide hydrate (NADH). One important aspect is that, while UVB radiation is almost completely absorbed by the epidermis (Seebode et al., 2016 [26]), UVA radiation is able to reach the dermis layers and even affects circulating blood cells (Möller et al., 2002) [27]. Among other effects, UVA radiation can inhibit DNA repair and induce metalloproteinase synthesis of the cell matrix (MMP), which can boost the skin cancer’s biological aggression (Fisher et al., 2001) [28]. The present study saw a 100% incidence of spinocellular carcinomas in the Control Group (Fig 1), which highlights the fact that UVA radiation is capable of provoking the entire spectrum of photoaging lesions. After their first exposure to UVA, all animals in the Control Group presented temporary diffuse erythema on their back, which disappeared or diminished 2 or 3 hours after exposure to radiation. After the tenth to twelfth session, the erythema became permanent accompanied by a markedly geometric skin pattern characterized by a reticular appearance, which became more evident over time. The appearance of prominent longitudinal wrinkles along the whole surface of the back was remarkable, presenting skin laxity and loss of elasticity, which progressively increased in length and thickness alternating with more local areas that were slightly raised and of irregular aspect, generally accompanied by a squamous surface. These areas were irregularly located between the wrinkles and from that time until the end of the UVA sessions the skin presented increasingly extensive areas of telangiectatic appearance. After the fortieth session, the skin took on a diffuse granular appearance, and around the fiftieth session, suffered extensive irregular surface ulceration. In control specimens, neoplastic lesions started to appear after the fiftieth session, while in the treatment groups, macroscopic lesions were not detected until after the sixtieth session. These lesions were of nodular type and attached to deep tissue planes. Some of them had irregular edges, were raised, with congestive borders and a verrucous appearance, while others were depressed in the center, of squamous appearance, with surface ulceration. These were dirty-based ulcers, which in some cases presented spontaneous bleeding. By the end of the 75 sessions of the study, all the control group animals developed lesions of neoplastic appearance, with similar lesions appearing in the group treated with cocoa extract (Fig 2). Numerous studies have backed the hypothesis that the use of plant-derived antioxidant substances has a favorable effect in the treatment or prevention of photoaging and photocarcinogenesis. Diverse studies have demonstrated reductions in inflammatory response, oxidative stress, DNA damage, the early appearance of erythema, premature wrinkling, or skin cancer after prolonged exposure to UVR as a result of treatment with natural compounds (Bosch et al., 2015) [11]. In the present study, we used purified extracts of cocoa and pomegranate, observing significant reductions in the incidence of skin carcinoma in both groups, but especially in the PGE group. In the latter group, hardly any of the animals presented lesions, their skins being classified as normal in histopathological analysis (Fig 1). No previous in vivo trials of cocoa extract have been conducted, although (Kim et al. 2016) [12] published an article in which it was shown that oral cocoa supplements protected against the formation of wrinkles provoked by UVB radiation by regulating the genes that intervene in the formation and degradation of the extracellular collagen matrix (cathepsin G and serpin B6c) (Kim et al., 2016) [12]. The pomegranate has been studied more often, although mainly in relation to UVB radiation. Pomegranate extract is a high source of ellagitannins, anthocyanins, and tannins and possesses a powerful antioxidant action (Bassiri-Jahromi, 2018) [29]; (Afaq et al., 2010) [13] carried out a trial using SKH-1 hairless mice treated with PGE in drinking water (0.2%, wt/vol) for 14 days before irradiation and then exposed to UVB radiation only once (180 mJ cm2). The results showed inhibition of: skin edema, hyperplasia, infiltration of leukocytes, lipid peroxidation, hydrogen peroxide generation, ornithine decarboxylase and cyclooxygenase-2 activity (Afaq et al., 2010) [13]. In particular, following UVA radiation, PGE inhibits UVA-mediated phosphorylation of STAT3, AKT, ERK1/2, mTOR and p70S6K, decreases up-regulation of PCNA and Ki-67 expression, and up-regulates Bax and Bad expression in human keratinocytes (Syed et al., 2005) [30], inhibiting the effects of photoaging (Silva et al., 2019) [31]. But its effects against UVA radiation have not been investigated in vivo. In the present study, we observed a clear decrease in macroscopic lesions, particularly in the PGE group, with statistically significant differences in comparison with the control and cocoa groups, which did not show significant differences between them (Fig 3). In microscopy analysis with hematoxylin and eosin staining, the incidence of squamous cell carcinoma was 100% in the control group, while animals treated with PGE only developed one carcinoma (11.11%) and those treated with CE developed four (22.22%). Statistically significant differences were found between both the treatment groups and the control. The very slight skin affection in seven of the nine animals treated with PGE was remarkable, their skin remaining normal (Fig 1).

Fig 1

Table of comparison of incidence of skin lesions between study groups (Pearson’s χ2 test).

Fig 2

Skins of animals at the end of the irradiation period (80 sessions).

Control, PE (pomegranate extract) and CE (cocoa extract).

Fig 3

Comparison of macroscopic morphometric analysis of skin area with lesions (mm2) between study groups (Tukey test).

Significant difference (*p≤0.050; **p<0.010).

The protein p53 has diverse and complex functions, including cell cycle regulation. Its alteration is directly related to cancer development, as it mutates in 50% of human cancers, including NMSC (Benjamin and Ananthaswamy, 2007) [32]. In SKH-1 mice chronically exposed to UVB radiation, inactivation of p53 mutations has been reported to occur in 50-70% of tumors, this being an early event, detected as early as 1 week post-UVB exposure (Rigby et al. 2017) [24]. The present study found lower mutated p53 expression in the treatment groups than in the control, with statistically significant differences, but without any significant difference between PGE and CE (Figs 4 and 5). After UVB exposure, oral treatment with PGE, has also shown a positive effect on the expression of tumor suppressor p53 and cyclin kinase inhibitor p21(Afaq et al., 2010) [13].

Fig 4

Comparison of percentage of P53 immunohistochemical positive cells in relation to the total number of cells between study groups (Tukey test).

Significant difference (*p ≤ 0.050; **p<0.010).

Fig 5

Spinocellular carcinomas in the different study groups (Haematoxilin-eosin) (a,b,c); MMP and TIMP-1 expression in surrounding tumour stroma (d, e, f and g, h, i respectively); p53 (j, k, l) and PCNA (m, n, o) immunostaining.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein necessary for DNA synthesis and repair. PCNA quantification is used as a marker of cell proliferation in tissues to assess the efficacy of chemopreventative drugs in cancer research (Smolarek et al., 2014) [33], and is considered an independent factor in the prognosis of survival, as its expression is seen to increase in lesions with high malignancy in both NMSC and melanoma (Ruksha et al., 2007) [34]. In the present study, PCNA expression was similar in the control and CE groups but lower cell proliferation was observed in the PGE group with significant difference in comparison with the other two groups (Figs 5 and 6). These results concur with other studies of human keratinocytes irradiated with UVA (Syed et al., 2005) [30], and of SKH-1 mice irradiated with UVB (Afaq et al., 2010) [13].

Fig 6

Comparison of percentage of proliferating cell nuclear antigen (PCNA) immunohistochemical positive cells in relation to the total number of cells between study groups (Tukey test).

Significant difference (*p≤0.050; **p<0.010).

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes capable of degrading different components of the extracellular matrix (ECM). Their role in healthy tissue is their physiological remodeling through both formation and repair. In cancer, they are related to important processes such as angiogenesis, cell proliferation, cellular invasion, and metastasis. MMP-9 belongs to a subgroup of gelatinases, and its function is the degradation of basement membrane components such as type IV collagen and laminin, as well as other ECM components (Andisheh-Tadbir et al., 2016) [35]. This process is regulated by tissue inhibitors of MMPs (TIMPS). Although there are four types, one of the most employed in NMSC is TIMP-1, which is usually inhibited in patients with skin cancer (Fu et al., 2012) [36]. In the present study, MMP-9 expression was higher in the treatment groups than the control group, even though tumors were more frequent in the latter group. But TIMP-1 showed bigger value in the treatment groups, and so it would appear that inhibition of ECM degradation tended to be more active in these groups, although without reaching statistical significance differences (Figs 5 and 7).

Fig 7

Table of comparison of degree of intensity (0–3) of stained stroma with TIMP and MMP-9 between study groups (Tukey test).

Although in many studies, MMP-9 expression is higher in precancerous tumors and lesions than healthy tissue (Gupta et al., 2014) [37] (Fu et al., 2012) [36] (Andisheh-Tadbir et al., 2016) [35], others such as (Poswar et al., 2013) [38] have found greater expression in microinvasive carcinomas than in those that invade in depth, concluding that MMP-9’s proteolytic activity takes place during the initial stages of carcinogenesis and decreases afterwards (Poswar et al. 2013) [38]. This could explain the present study’s observations, whereby control group tumors were more evolved than in the treatment groups.

While much research has focused upon the effects of UVB radiation, little is known about UVA-induced photocarcinogenesis in vivo. This lack was the motivation for the present study, which also set out to treat animals with extracts whose efficacy in UVB-induced skin cancer chemoprevention appears to have been demonstrated. Taken together, our results suggest that oral feeding of PGE and CE to SKH-1 mice affords substantial protection from the adverse effects of UVA radiation, especially PGE. Obviously further research is needed to investigate in detail the mechanisms of action of these substances, which could play an important role in NMSC prevention in the future.

(PDF)

Click here for additional data file.

29 Jan 2020

PONE-D-19-31596

Chemopreventive effect of pomegranate and cocoa extracts on ultraviolet radiation-induced photocarcinogenesis in SKH-1 mice

PLOS ONE

Dear Dra. Guerrero-Sánchez,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Mar 14 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

J. Alberto Conejero

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. As part of your revision, please complete and submit a copy of the ARRIVE Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://www.nc3rs.org.uk/arrive-guidelines. Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

3. We noticed you have some minor occurrence(s) of overlapping text with the following previous publication(s), which needs to be addressed:

https://doi.org/10.1111/jop.12435

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the Methods section. Further consideration is dependent on these concerns being addressed.

4. We note that you have not given a rationale for the chosen treatment doses. Please elaborate this aspect in the Methods section. Please also justify why group 3 received 'UVR' radiation, if this is a misspelling please correct it.

5. Please include additional information regarding pomegranate and cocoa extracts, such as: product name and number, lot/batch number, full ingredient list, method of chemical characterization, purity and yield. Please also include a description of the look and feel of the substance, and the species name of the cocoa plant used.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

7. Please ensure that you refer to Figures 6 and 7 in your text as, if accepted, production will need this reference to link the reader to the figure.

8. Please include a separate title for each table in your manuscript.

Additional Editor Comments:

We have received 3 reports of your work. In one of them, the reviewer points out some concerns respect to the model. Please, try to clarify these points when you submit the revision of your work.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study, the authors have examined the effect of pomegranate and cocoa extracts on UVA-induced skin cancer in SKH-1 hairless mice. Overall, whereas the studies are conducted properly, the model itself has serious concerns. Specifically, UVA radiation also does not induce skin cancers, and indeed the authors did not find any skin cancer in control group; except some kind of invasion which is not that common for non-melanoma skin cancer. Because of the same issue of using UVA, the effects of the test agents are also not evident, though they have shown efficacy against UVB-induced skin cancer; therefore, the studies lack both the novelty and appropriate model. Lastly, the molecules analyzed at the the study end are also not properly justified, and in most cases there is hardly any effect.

Reviewer #2: General Comments

This is an excellent article that investigates the protective effects of pomegranate and cocoa extracts during UVA-induced skin cancer in SKH-1 hairless mice. In general, it is very well written. The research question is clear. Robust, well-explored and well-described methods were used.

Authors demonstrated that 100% of control animals presented spinocellular carcinomas which highlights the fact that UVA radiation can provoke the entire spectrum of photoaging lesions. Animals that received purified extracts of cocoa and pomegranate showed significant reductions in the incidence of skin carcinoma in both groups. In addition, lower mutated p53 expression in the treatment groups than in the control as well as, control group demonstrated higher PCNA and MMP-9 labelling. The present study is a step toward better understanding the UVA photocarcinogenesis and new chemopreventive strategies. However, some changes in the article are needed.

I have the following suggestion and criticism.

- Please add the reference of GLOBOCAN report. Check the incidence of skin cancer reported on it.

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A.Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2018 Nov;68(6):394-424. doi: 10.3322/caac.21492.

- Separate the introduction into three paragraphs to make it easier to follow.

2nd- Punica granatum…

3rd- SKH1/CRL…

- Materials and methods

UV irradiation- Please inform the reference for the protocol used.

- At the end of the experiment, animals were sacrificed by CO2 overdose. Why did you use CO2 chamber? Nowadays, it is recommended euthanasia using an isoflurane anesthetic overdose .

-Results- Please change this title for Results and Discussion

Please separate the following information in new paragaraph-

“ The protein p53 has diverse and complex 227 functions, including cell cycle regulation. Its alteration is directly related to cancer 228 development, as it mutates in 50% of human cancers, including NMSC (Benjamin and 229 Ananthaswamy, 2007) [5]. In SKH-1 mice chronically exposed to UVB radiation, 230 inactivation of p53 mutations has been reported to occur in 50-70% of tumors, this 231 being an early event, detected as early as 1 week post-UVB exposure (Rigby et al. 232 2017) [25]. The present study found lower mutated p53 expression in the treatment 233 groups than in the control, with statistically significant differences, but without any 234 significant difference between PGE and CE (Figures 3 and 5). After UVB exposure, 235 oral treatment with PGE, has also shown a positive effect on the expression of tumor 236 suppressor p53 and cyclin kinase inhibitor p21(Afaq et al., 2010) [1].”

-Please change the following sentence.

“But TIMP-1 showed more expression in the treatment groups, and so it would appear that inhibition of ECM degradation was more 260 active in these groups, although differences did not reach statistical significance (Table 261 2, Figure 5).” If you did not have statistical differences the groups are equals. Rewrite the sentence!

Reviewer #3: The paper with the title Chemopreventive effect of pomegranate and cocoa extracts on ultraviolet radiation-induced photocarcinogenesis in SKH-1 mice is well write and the study design is conducted correctly.

For these reasons the paper is accepted for the publication on PLOS ONE.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

18 Mar 2020

Please see the attached file where we have answered in detail to all referee's remarks.

Submitted filename: Answers_to_the_reviewer.pdf

Click here for additional data file.

7 Apr 2020

Chemopreventive effect of pomegranate and cocoa extracts on ultraviolet radiation-induced photocarcinogenesis in SKH-1 mice

PONE-D-19-31596R1

Dear Dr. Guerrero-Sánchez,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

J. Alberto Conejero

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all the concerns related to the use and carcinogenic effects of UVA and UVB, as acceptable responses.

Reviewer #2: The authors performed all the modification suggested.I reccomend the aproval of the mansucript.Best Regards

Reviewer #3: The Authors presented all the revisions requested. For this reason the paper could be accepted for the publication

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

15 Apr 2020

PONE-D-19-31596R1

Chemopreventive effect of pomegranate and cocoa extracts on ultraviolet radiation-induced photocarcinogenesis in SKH-1 mice

Dear Dr. Guerrero-Sánchez:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr J. Alberto Conejero

Academic Editor

PLOS ONE