HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols via NF-κB inhibition

    HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols via NF-κB inhibition Rui M. Gil da Costa, Sofia Arag˜ao, Magda Moutinho, Antonieta Alvarado, Diogo Carmo, F´atima Casaca, Sandra Silva, Joana Ribeiro, Hugo Sousa, Rita Ferreira, Rita Nogueira-Ferreira, Maria Jo˜ao Pires, Bruno Colac¸o, Carlos Venˆancio, Maria Manuel Oliveira, Margarida M. S. M. Bastos, Carlos Lopes, Paula A. Oliveira PII: DOI: Reference:

S0024-3205(16)30668-3 doi: 10.1016/j.lfs.2016.10.031 LFS 15082

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

28 June 2016 4 October 2016 17 October 2016

Please cite this article as: Gil da Costa Rui M., Arag˜ao Sofia, Moutinho Magda, Alvarado Antonieta, Carmo Diogo, Casaca F´atima, Silva Sandra, Ribeiro Joana, Sousa Hugo, Ferreira Rita, Nogueira-Ferreira Rita, Pires Maria Jo˜ao, Cola¸co Bruno, Venˆancio Carlos, Oliveira Maria Manuel, Bastos Margarida M. S. M., Lopes Carlos, Oliveira Paula A., HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols via NF-κB inhibition, Life Sciences (2016), doi: 10.1016/j.lfs.2016.10.031

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ACCEPTED MANUSCRIPT HPV16 induces a wasting syndrome in transgenic mice: amelioration by dietary

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polyphenols via NF-κB inhibition.

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Rui M. Gil da Costa1,2*, Sofia Aragão3, Magda Moutinho3, Antonieta Alvarado3,4,

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Diogo Carmo5, Fátima Casaca5, Sandra Silva5, Joana Ribeiro2,6, Hugo Sousa2,6, Rita Ferreira7, Rita Nogueira-Ferreira7, Maria João Pires3,8, Bruno Colaço3,8, Rui Medeiros2,6, Carlos Venâncio9, Maria Manuel Oliveira10, Margarida M. S. M. Bastos1, Carlos

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Lopes11, Paula A. Oliveira3,8

1. LEPABE, Faculty of Engineering - University of Porto (FEUP), Porto, Portugal 2. Molecular Oncology and Viral Pathology Group, CI-IPOP, Porto, Portugal

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3. University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal 4. Area de Patologia, Decanato de Ciencias Veterinarias, UCLA, Lara, Venezuela.

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5. BMAC - Botelho Moniz Análises Clínicas, Porto, Portugal. 6. Virology Service, IPO-Porto, Porto, Portugal

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7. QOPNA, Department of Chemistry, University of Aveiro, Aveiro, Portugal 8. CITAB, UTAD, Quinta de Prados, Vila Real, Portugal. 9 Animal Science Dept., School of Agrarian and Veterinary Sciences, UTAD, Vila Real, Portugal. 10. Centro de Química de Vila Real, UTAD, Vila Real, Portugal. 11. Experimental Pathology and Therapeutics Group, CI-IPOP, Porto, Portugal.

*Corresponding author: Rui M. Gil da Costa, [email protected], Phone: +351 225081687, Fax: +351 225081440

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ACCEPTED MANUSCRIPT Abstract Cancer patients often show a wasting syndrome for which there are little therapeutic

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options. Dietary polyphenols have been proposed for treating this syndrome, but their

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usefulness in cases associated with human papillomavirus (HPV)-induced cancers is

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unknown. We characterized HPV16-transgenic mice as a model of cancer cachexia and tested the efficacy of long-term oral supplementation with polyphenols curcumin and rutin.

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Both compounds were orally administered to six weeks-old HPV16-transgenic mice

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showing characteristic multi-step skin carcinogenesis, for 24 weeks. Skin lesions and blood, liver and spleen inflammatory changes were characterized histologically and hematologically. Hepatic oxidative stress, skeletal muscle mass and the levels of muscle

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pro-inflammatory transcription factor NF-κB were also assessed. Skin carcinogenesis was associated with progressive, severe, systemic inflammation

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(leukocytosis, hepatitis, splenitis), significant mortality and cachexia. Curcumin and rutin totally suppressed mortality while reducing white blood cells and the incidence of

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splenitis and hepatitis. Rutin prevented muscle wasting more effectively than curcumin. Preservation of muscle mass and reduced hepatic inflammation were associated with down-regulation of the NF-κB canonical pathway and with reduced oxidative stress, respectively. These results point out HPV16-transgenic mice as a useful model for studying the wasting syndrome associated with HPV-induced cancers. Dietary NF-κB inhibitors may be useful resources for treating this syndrome.

Keywords: rutin, curcumin, cancer, cachexia, HPV, NF-κB.

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ACCEPTED MANUSCRIPT Introduction Many cancer patients develop a severe systemic para-neoplastic syndrome, related to

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tumoral inflammation, metabolic imbalances and loss of appetite [1]. In approximately

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50% of cancer patients, these factors may join to cause cancer cachexia for which there

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are currently little therapeutic options [2]. This is often the case with patients presenting with malignancies induced by human papillomavirus (HPV), which include cervical cancer and some cases of head-and-neck, anal and skin cancers [3, 4]. Many animal

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species show similar lesions induced by species-specific papillomavirus [5]. HPV-

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induced pre-malignant and malignant lesions elicit a significant local inflammatory response, and frequently induce a systemic inflammatory state, leading to cancer cachexia [4]. Dietary polyphenols have been studied for their antitumor properties

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against different types of cancer, and some of them were also shown to inhibit cachexia induced by, e.g. colon and mammary cancers [6, 7], and melanoma [8]. An important

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part of the anti-cachectic potential of dietary polyphenols is believed to depend on their ability to inhibit nuclear factor κB (NF-κB), a transcription factor that underpins

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inflammatory processes [9]. The same ability is likely to explain part of these compound's activity against HPV-induced cell transformation, as NF-κB plays an important role in carcinogenesis induced by papillomavirus [10, 11]. In fact, several polyphenols have shown significant activity in preventing or treating HPV-induced lesions in vivo, by targeting multiple signaling pathways [12]. Among these compounds, curcumin and its derivatives occupy a prominent place. Curcumin inhibits NF-κB signaling, has a chemopreventive effect concerning HPV-induced carcinogenesis and shows some activity against HPV-related malignancies [13, 14]. Curcumin also shows potential to treat the wasting syndrome induced by several malignancies [7]. However, these effects are limited by curcumin's poor pharmacokinetics, a handicap that has been

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ACCEPTED MANUSCRIPT addressed by developing several derivatives and innovative formulations [15]. Other polyphenols such as quercetin [6, 16] and resveratrol [17] have been shown their anti-

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tumoral and anti-cachectic potential. Despite the extensive research on polyphenols, no

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studies addressed their efficacy against the wasting syndrome caused by HPV-induced

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cancers. This would require either an HPV-transgenic mouse strain or mice bearing xenografted HPV-positive tumor cells, but the use of xenograft models is poorly adequate for this purpose, as these models often lack a functional immune system in

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order to sustain transplanted cells. On the other hand, the systemic effects of

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carcinogenesis in the existing HPV-transgenic mouse strains are not yet reported. Using K14-HPV16 mice, our group recently reported that extensive cutaneous lesions are associated

with

significant

local

inflammation

and

are

influenced

by

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microenvironmental factors [18, 19]. This mouse strain recapitulates HPV16-induced carcinogenesis on the uterine cervix and the epidermis. The present work aims to

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characterize the systemic inflammatory changes and the wasting syndrome in K14HPV16 transgenic mice, and to study the efficacy of two well-known dietary

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polyphenols, curcumin and rutin, in preventing these symptoms.

Materials and methods Animals K14-HPV16 mice generation has been previously reported [20]. K14-HPV16 mice on a FVB/n background were generously donated by Drs. Jeffrey Arbeit and Douglas Hanahan, from the University of California, through the USA National Cancer Institute Mouse Repository. Animals were bred in-house and genotyping was performed as previously described [18, 19]. Hemizygotic (HPV16+/-) and wild-type (HPV16-/-) 6 weeks-old females were selected for use in the study. The study was authorized by the

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ACCEPTED MANUSCRIPT University de Trás-os-Montes e Alto Douro ethics committee (approval no. 10/2013)

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and the Portuguese Veterinary Directorate (approval no. 0421/000/000/2014).

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Chemicals

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Curcumin and rutin (in the form of rutin-3-rutinoside hydrate) were acquired from Sigma-Aldrich, Saint-Louis, Missouri. Both substances were dispersed in sunflower oil and coated unto diet pellets using an industrial mixer, to a final concentration of

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2.0mg/g. The expected animal's daily food intake was estimated at 5.0g/mouse,

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allowing for a daily intake of 10.0mg curcumin or rutin. The food intake was slightly lower than expected, and so the average daily doses of rutin and curcumin were 9.42mg/animal ± 0.62, or 413.0mg/Kg (rutin) and 422.4mg/kg (curcumin). The final

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sunflower oil concentration was 3.0% (w/w).

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Experimental procedures

The animals were maintained in accordance with Portuguese (Decreto-Lei 113/2013,

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dated August the 7th) and European (EU Directive 2010/63/EU) legislation, under controlled conditions of temperature (23±2 ºC), light-dark cycle (12 h light/12 h dark) and relative humidity (50±10%), using hardwood bedding. A standard diet (4RF21 GLP, Mucedola, Italy) and water were provided ad libitum, and health checks were performed daily. Fifty mice were divided into 4 groups: groups 1 (HPV16-/-, n=12), 2 (HPV16+/-, n=13), 3 (HPV16+/-, n=12), 4 (HPV16+/-, n=13). Groups 3 and 4 were administered the modified diets containing rutin and curcumin for 24 consecutive weeks, respectively, while groups 1 and 2 received a control diet, containing 3.0% sunflower oil. The animal's weights and their food and water consumption were recorded weekly. The ponderal weight gain was calculated as previously described [21].

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ACCEPTED MANUSCRIPT At 30-weeks-old, the experimental groups were humanely sacrificed, by intraperitoneal pentobarbitol overdose, followed by cardiac puncture and exsanguination, as

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recommended by FELASA.

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Hematology

Blood smears were immediately made, coloured with a Romanowsky stain, and used for a differential count of leukocytes. Total leukocytes were counted using a Neubauer

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chamber, and leukograms were obtained for all animals.

Histopathology

All animals were thoroughly necropsied. The whole digestive and reproductive systems,

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kidneys, adrenal glands, spleen, heart, lungs, mandibular lymph nodes were collected and examined. The spleen and liver were weighted. Relative organ weights were

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calculated as the ratio of the organ weight to the animal’s bodyweight [22]. Chest skin samples were used to classify cutaneous tumours and pre-neoplastic lesions. All

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samples were fixated in 10% neutral buffered formalin for 24h, dehydrated through graded alcohols and xylene and paraffin-embedded. Three μm-thick sections were stained with hematoxylin-eosin for histological examination on light microscopy. Skin samples were classified as normal skin, epidermal hyperplasia or epidermal dysplasia. Inflammatory hepatic lesions were classified as grade I (mild), II (moderate) or III (severe) hepatitis. Splenic changes were classified as white pulp hyperplasia or granulomatous splenitis.

Hepatic oxidative stress

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ACCEPTED MANUSCRIPT Frozen and stored -80ºC liver samples were washed, minced and homogenized with a motor-driven Teflon Potter-Elvjhem homogenizer in the presence of 10 w/v of 100 mM

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phosphate buffer. The homogenate were centrifuged at 1000 g for 10 min at 4ºC and

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then the supernatant were centrifuged at 15000 g for 15 min. The resultant supernatant

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were used for antioxidant enzymatic activities and the pellet for lipid peroxidation determination. Protein concentration was measured according to the biuret method [23].

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Antioxidant enzymatic activities

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Superoxide dismutase (SOD) activity was assayed according to [24] with minor modifications [25]. Catalase (CAT) activity was assayed polarographically as described previously [26]. Glutathione reductase (GR) activity was measured as described [25].

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The lipid peroxidation was evaluated in terms of thiobarbituric acid reactive substance (TBARS) formation as described [27], with minor modifications. Briefly, the liver

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samples were mixed with TCA (37.5%), TBA (0.38%) and BHT (0.015%) and the reaction mixture was incubated at 100ºC for 15 min on a water bath and then cooled on

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ice. The major oxidative product of phospholipids i.e. malondialdehyde (MDA) was estimated, after centrifugation at 3000 rpm during 10 min, by measuring the supernatant absorbance at 530 nm using a microplate spectrophotometer (Power Wave XS2, BioTek, Bad Friedrichshall, Germany). The data were expressed as nanomoles MDA equivalents formed per milligram protein using molar extinction coefficient of 1.56 × 10 M−1 cm−1.

Muscle weights and western blot analysis of NF-κB subunits The left hind leg gastrocnemius muscle was dissected and weighted. A portion (~10 mg) of gastrocnemius muscle was homogenized in 100 mM phosphate buffer, pH 7.4

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ACCEPTED MANUSCRIPT supplemented with the protease inhibitor PMSF (200 mM, 1:1000) using a Teflon pestle on a motor-driven Potter-Elvehjem glass homogenizer at 0-4 ºC. The protein content of

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the homogenate was assayed with the Bio-Rad DC method, following the instructions of

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the manufacturer, using bovine serum albumin (BSA) as a standard. Equivalent amounts

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of muscle protein of each experimental group were electrophoresed on a 12.5% SDSPAGE. Gels were blotted onto a nitrocellulose membrane (Whatman®, Protan®) in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3 and 20% methanol) during 2

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hours (200 mA). Then, nonspecific binding was blocked (5% (w/v) nonfat dry milk in

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TBS-T: 100 mM Tris, 1.5 mM NaCl, pH 8.0 and 0.5% Tween 20). The membrane was incubated with primary antibody solution diluted 1:1000 in 5% (w/v) nonfat dry milk in TBS-T (rabbit anti-NF-κB p105/p50, ab32360, abcam; rabbit anti-NF-κB p65, ab16502,

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abcam; mouse anti-NF-κB p100/p52, ab71108, abcam rabbit anti-Rel B, ab150305, abcam). After incubation at room temperature with agitation, the membrane was washed

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with TBS-T and incubated with anti-mouse or anti-rabbit IgG peroxidase secondary antibody (Sigma-Aldrich) diluted 1:1000 in 5% (w/v) nonfat dry milk in TBS-T.

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Immunoreactive bands were detected with enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech) according to the manufacturer's procedure. Images were recorded using X-ray films (Amersham Hyperfilm ECL, GE Healthcare), which were scanned in Molecular Imager Gel Doc XR+System (Bio-Rad) and analyzed with Image Lab software (v4.1, Bio-Rad). Protein loading was controlled by Ponceau S staining once the content of cytoskeletal proteins was found to be modulated by cancer cachexia.

Statistical analysis Kolmogorov-Smirnov test was performed to check the normality of the data. Since all variables were normal distributed, significant differences were assessed using analysis

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ACCEPTED MANUSCRIPT of variance (ANOVA) followed by Tukey post-hoc test for multiple comparisons. For categorical variables, Fisher’s exact test and Bonferroni’s correction was applied.

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Survival time was estimated using Kaplan-Meier estimates from first day of treatment

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until death. The four groups were compared using the Log-rank, Breslow and Tarone-

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Ware tests and, if there were an overall difference between the groups, pair-wise comparisons were made among all pairs.

The minimum requirement to reject the null hypothesis was p < 0.05. All statistical

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analyses were carried out using SPSS 17.0. Results were expressed as mean ± standard

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deviation and as percentages, when appropriate.

Results

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General findings

Five group 2 (HPV16+/- untreated mice) animals died during the experimental period,

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but no mortality was observed in negative controls and in HPV16+/- mice treated with rutin and curcumin (Figure 1). There was a statistical significant difference in

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cumulative survival between group 2 and other groups (p <0.05).

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Fig. 1 Kaplan-Meier survival analysis (a). Note significantly reduced survival for group 2 (HPV16+/- untreated mice, n=13), compared with HPV16-/- (group 1, n=12) controls

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and with HPV16+/- mice treated with rutin and curcumin (groups 3 and 4, n=12 and

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n=13 respectively). Chemical structures of rutin (b) and cucumin (c)

As depicted on Figure 2, all groups maintained a stable food intake throughout the experimental period (4.71g/animal/day ± 0.31). HPV16+/- animals showed increased

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water consumption compared with HPV16-/- animals, and their water intake increased linearly during the experimental period, while that of HPV16-/- animals remained stable.

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Fig. 2 The evolution of food (a) and water (b) consumption and bodyweight (c) of animals from groups 1 (n=12), 2 (n=13), 3 (n=12) and 4 (n=13) during the experimental period

During the last 3 experimental weeks, untreated HPV16+/- animals markedly reduced their water intake, compared with the quercetin and curcumin-treated HPV16+/- groups. HPV16-/- animals (groups 2, 3 and 4) showed slower and smaller weight gains along the

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ACCEPTED MANUSCRIPT 24 weeks experimental period, compared with HPV16-/- (group 1) animals. At the end of the experimental period, HPV16-/- animals were significantly heavier compared with

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any of the HPV16+/- groups (group 2, 3 and 4) (p ˂ 0.05), and no differences were found

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between treated and untreated HPV16+/- groups. The same was observed concerning

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ponderal weight gains, with HPV16-/- animals showing significantly (p ˂ 0.05) higher gains (7.79g ± 1.30), compared with groups 2, 3 and 4 (5.45g ± 1.81; 5.09g ± 2.00; and

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7.14g ± 3.63, respectively).

Cutaneous carcinogenesis

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Histologically, all control HPV16-/- mice (group 1) showed normal skin, whereas all treated and untreated HPV16+/- mice (group 2, 3 and 4) presented with extensive pre-

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malignant cutaneous lesions, ranging between epidermal hyperplasia and dysplasia (Figure 3). Simple to papillary, diffuse, variably severe epidermal hyperplasia was

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accompanied by papillomatosis and orthokeratotic hyperkeratosis. Hyperplasia was characterized by epidermal thickening with acanthosis and by retention of normal,

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orderly keratinocytic differentiation. Inflammation was mild, with a few macrophages, lymphocytes and mast cells present in the superficial dermis.

Fig. 3 Skin histology among surviving HPV16-/- and HPV16+/- mice in groups 1 (n=12), 2 (n=8), 3 (n=12) and 4 (n=13). a- group 1 (HPV16-/-) animal. Normal skin histology. H&E 200x, bar = 50µm. b- group 3 (rutin-treated HPV16+/-) animal. Simple epidermal 12

ACCEPTED MANUSCRIPT hyperplasia with orthokeratotic hyperkeratosis. Note sebaceous gland hyperplasia and light dermal inflammatory infiltrate. H&E 200x, bar = 50µm. c- group 4 (curcumin-

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treated HPV16+/-) animal. Epidermal dysplasia. Note parakeratotic hyperkeratosis, loss

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of cell polarity and enhanced dermal inflammation and angiogenesis. H&E 200x, bar =

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50µm.

Dysplastic lesions presented as small epidermal dysplastic foci in a hyperplastic

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background or as variably severe diffuse epidermal dysplasia. Dysplastic changes

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involved loss of normal keratinocytic differentiation and cell polarity, parakeratothic hyperkeratosis and moderate nuclear pleomorphism. Enhanced sub-epidermal angiogenesis and dermal inflammatory infiltrates became prominent, with numerous

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mixed mononuclear leukocytes (macrophages, lymphocytes, mast cells, plasma cells) and neutrophils. The inflammatory infiltrate often assumed a band-like distribution,

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obscuring the dermal-epidermal junction.

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Systemic inflammation

Untreated HPV16+/- (group 2) animals showed a severe leukocytosis compared with HPV16-/- (group 1) animals (Figure 4), exhibiting neutrophilia, lymphocytosis and monocytosis. Curcumin was able to reduce this leukocytosis: although group 4 is not statistically different from untreated HPV16+/- animals (group 2) (p = 0,284) it is also not different from healthy controls (group 1) (p = 0.065). Rutin reduced the circulating neutrophil and monocyte counts and increased circulating lymphocytes, resulting in only a moderate reduction in total circulating leukocytes. Mandibular lymph nodes were not conspicuous among HPV16-/- animals, but were consistently enlarged and macroscopically evident in 100% of HPV16+/- animals.

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Fig. 4 Hematological analysis in surviving HPV16-/- and HPV16+/- mice in groups 1 (n=12), 2 (n=8), 3 (n=12) and 4 (n=13). a- total leukocyte counts for each experimental group. b- differential leukocyte counts for each experimental group.* Statistically different from group 1 (p <0.05)

Lymphadenomegaly was also occasionally noted on mesenteric, as well as inguinal and axillary lymph nodes. HPV16+/- animals also showed a remarkable absence of subcutaneous or mesenteric adipose tissue. The livers and spleens of HPV16+/- animals

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ACCEPTED MANUSCRIPT were significantly enlarged (p ˂ 0.05), compared with HPV16-/- animals (Figure 5). The weight of each organ was compared with the animal's weight and expressed as a

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percentage in Table 1. Curcumin- and rutin-treated groups showed a slight reduction in

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hepatic mass compared with untreated HPV16+/- (group 2) animals, but this did not

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reach statistical significance. Histologically, the spleens and livers of transgenic animals (HPV16+/-, groups 2, 3 and 4) showed variably severe inflammatory changes (Figure 5 and Table 2). Control animals (HPV16-/-, group 1) showed normal hepatic and splenic

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histology.

Fig. 5 Liver and spleen findings among surviving HPV16-/- and HPV16+/- mice in groups 1 (n=12), 2 (n=8), 3 (n=12) and 4 (n=13). a- spleens from HPV16-/- (group 1) and HPV16+/- (group 2) mice. b- livers from HPV16-/- (group 1) and HPV16+/- (group 2) 15

ACCEPTED MANUSCRIPT mice. c to e- splenic histology. c- HPV16-/- (group 1) animal. Normal spleen histology. H&E 100x, bar = 100µm. d- rutin-treated HPV16+/- (group 3) animal. White pulp

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hyperplasia. H&E 100x, bar = 100µm. e- untreated HPV16+/- (group 2) animal.

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Fibrohystiocytic splenitis. H&E 100x, bar = 100µm. f to i- liver histology. f- HPV16-/-

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(group 1) animal. Normal liver histology. H&E 100x, bar = 100µm. g- curcumin-treated HPV16+/- (group 4) animal. Grade I hepatitis, displaying Kupffer cell hyperplasia. H&E 400x, bar = 20µm. h- rutin-treated HPV16+/- (group 3) animal. Grade II hepatitis. H&E

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400x, bar = 20µm. i- untreated HPV16+/- (group 6) animal. Grade III hepatitis. H&E

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100x, bar = 100µm.

Splenic lesions consisted of a diffuse white and red pulp hyperplasia or a granulomatous

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splenitis characterized by intense macrophagic infiltration and less numerous

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neutrophils, associated with granulation tissue leading to extensive fibrosis, which

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replaces normal splenic tissues. Hepatic lesions ranged between moderate to intense, diffuse Kupffer cell hyperplasia, accompanied by scattered microabscesses (grade I hepatitis) and severe periportal, centrilobular or panlobular infiltration of macrophages

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and neutrophils, accompanied by smaller numbers of lymphocytes and plasma cells.

Table 1 - Liver, spleen and gastrocnemius weights among wild-type and HPV-transgenic mice, as a percentage of total live weight Group

Spleen

Liver

Muscle

(mean % ± STD)

(mean % ± STD)

( mean % ± STD)

1 (HPV-/-, n=12)

0.50 ± 0.07

5.17 ± 0.40

0.35±0.07

2 (HPV+/-, n=8)

1.91 ± 0.691

9.07 ± 2.311

0.25±0.092

3 (HPV+/- rutin, n=12)

1.84 ± 0.821

8.68 ± 3.641

0.37±0.08

4 (HPV+/- curcumin, n=13)

1.97 ± 0.911

8.62 ± 3.171

0.31±0.03

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ACCEPTED MANUSCRIPT STD- standard deviation;1 Statistically different from group 1 (p <0.05);

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Statistically different from

group 3 (p <0.05).

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Grade II hepatitis showed periportal or centrilobular to midzonal inflammatory cell

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infiltration with absent or minimal necrosis, fibrosis and/or bile duct hyperplasia. Grade

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III hepatitis showed portal and centrilobular bridging inflammatory infiltrates, with

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severe necrosis, fibrosis and/or bile duct hyperplasia).

Table 2- Incidence of splenic and hepatic lesions in wild-type and HPV-transgenic mice.

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Spleen Incidence (%)

100%

2 (n=8)

-

3 (n=12)

-

4 (n=13)

-

-

Splenitis

Normal

Incidence (%) Grade I

Grade II

Grade III

hepatitis

hepatitis

hepatitis

-

100%

-

-

-

-

100%

-

13%

13%

74%

67%

33%

25%

25%

8%

42%

38%

23%

15%

15%

47%

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1 (n=12)

WPH

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Normal

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Group

Liver

62%

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WPH- white pulp hyperplasia.

Considering the pivotal role of oxidative stress in triggering and perpetuating hepatic damage and the anti-inflammatory and anti-oxidant properties of rutin and curcumin, we investigated the hepatic status of various oxidative stress markers (Figure 6).

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Fig. 6 Hepatic oxidative stress markers in mice from groups 1 (n=12), 2 (n=8), 3 (n=12) and 4 (n=13). A- Superoxide dismutase (SOD); B- Glutathione reductase (GR); CThiobarbituric acid reactive substances (TBARS). Values are given as mean ± standard deviation. * p <0.05, ** p <0.01, *** p <0.001

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ACCEPTED MANUSCRIPT Untreated HPV16+/- (group 2) animals exhibited increased oxidative stress compared with HPV16-/- (group 1) animals, as reflected by significantly increased GR activity and

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TBARS and a mild, near-significant, increase in SOD activity. There were no

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statistically significant differences between groups concerning CAT activity (data not

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shown). Both rutin and curcumin-treated groups (groups 3 and 4) showed reduced SOD and GR activity and also MDA levels, compared with untreated HPV16+/- animals

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(group 2).

Skeletal muscle wasting

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Untreated HPV16+/- (group 2) animals showed significantly reduced skeletal muscle mass compared with HPV16-/- (group 1) animals (p ˂ 0.05) (Table 1). Rutin-treated

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mice (group 3) showed control-level muscle mass while curcumin-treated (group 4) animals showed only a partial loss, with no statistical difference from group 2.

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Considering the pivotal role of NF-κB in driving protein breakdown in muscle fibers and rutin's anti-inflammatory and NF-κB inhibitory properties, we investigated the

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phosphorylation status of NF-κB subunits, involved in the canonical and non-canonical signaling pathways. Rutin-treated animals were selected for analysis, since they showed the most promising results concerning muscle mass preservation. Western blot analysis (Figure 7) showed a significant up-regulation of phosphorylated p65 and p50, the two subunits that usually form active heterodimers involved in the canonical pathway, in HPV16+/- (group 2) animals compared with HPV16-/- (group 1) animals (p ˂ 0.01). No differences were observed between groups concerning those subunits involved in the non-canonical pathway. In animals treated with rutin (group 3), the p65 and p50 levels were again reduced, which correlates with preservation of muscle mass. Taken together, these findings indicate that the canonical NF-κB signaling pathway is activated in

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ACCEPTED MANUSCRIPT muscle fibers leading to wasting, and that the current rutin dosage is able to abrogate it, preserving skeletal muscle mass in the context of severe carcinogenesis-associated

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inflammation.

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Fig. 7 Western blot analysis of NF-κB subunits p105/50, p65, Rel-B and p100/52 in gastrocnemius muscle samples from mice of groups 1 (n=5), 2 (n=5), 3 (n=5) and 4 (n=5). Representative immunoblots are shown above the correspondent graph. Values are given as mean ± standard deviation for all variables. * p <0.05, ** p <0.01, *** p <0.001

Discussion Human papillomavirus induce a range of benign and malignant lesions, as observed with other papillomavirus in animal species [9]. Malignant lesions, induced by high-risk

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ACCEPTED MANUSCRIPT HPV types such as HPV16 and HPV18, include cervical cancer and some head-andneck, anal and skin cancers [3].

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Carcinogenesis is often associated with systemic inflammatory phenomena, reducing

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life quality and worsening patient prognosis, through appetite loss, weight loss and

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multi-organic lesions and dysfunction leading to cancer cachexia [2, 28]. This is often the case with patients suffering from HPV-related malignancies [4], highlighting the need for effective strategies for treating this syndrome. K14-HPV16 transgenic mice are

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a particularly useful model for studying HPV-associated cancers and testing

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immunomodulatory strategies [20, 29]. Pre-neoplastic and neoplastic lesions develop on the uterine cervix and skin, through a multi-stage process, starting with hyperplasia that progresses to dysplasia, in situ carcinoma (CIS) and, eventually, invasive carcinoma,

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accompanied by an increasingly severe inflammatory process [30-32]). Progressive cutaneous lesions develop over the whole skin surface. Since they are more extensive

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than cervical lesions, they are also more likely to show systemic effects. Accordingly, we focused our attention on skin lesions, which consisted essentially of epidermal

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dysplasia, as expected for K14-HPV16 mice at this age [20]. The development of epidermal dysplasia is associated with enhanced sub-epidermal inflammation and vascularization, triggered by an "angiogenic switch" [18-19, 33]. This correlated with a strong systemic inflammatory response, as evidenced by the significant elevation of white blood cells and by severe spleen and liver enlargement with inflammation. The severity of this paraneoplastic syndrome is evidenced by the reduced bodyweight gains and significant mortality observed in transgenic animals. Depression and appetite loss were not a feature of this syndrome, as sometimes observed in cancer patients, and cage behavior and food intake were normal.

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ACCEPTED MANUSCRIPT HPV16+/- animals exhibited an increased water intake, suggesting that dehydration may also be involved. HPV-induced epithelial changes progressively impair the skin's barrier

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function [34] and may explain the progressive water consumption observed in HPV16+/-

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animals. The loss of skeletal muscle mass in HPV16+/- animals was associated with

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activation of the canonical NF-κB pathway, as observed in other models of cancer cachexia [8]. NF-κB activation in HPV-induced cancers has been recently reviewed [35]. Taken together, these data validate K14-HV16 as a model for studying the wasting

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syndrome observed in patients with HPV-induced malignancies. The multi-step process

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of carcinogenesis (as opposed to the inoculation of a cell line) and the presence of a fully functional immune system (as opposed to the immune depression of xenograft models) make this a particularly useful model for testing new therapies against cancer

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cachexia. Curcumin is the yellow pigment from Curcuma longa, while rutin is a quercetin glycoside and one of its most common presentations in plants (Erlund et al.,

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2000 [36]. Long-term oral administration of curcumin and rutin had a positive impact on animal health, improving survival and reducing inflammation. Rutin showed a strong

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immunomodulatory effect, restoring the lymphocyte:neutrophil ratio, through a significant increase in circulating lymphocytes and a minor decrease in neutrophils. However, curcumin was more effective in diminishing white blood cells. None of the compounds was able to significantly reduce splenic or hepatic mass, although both attenuated the incidence of splenitis and hepatitis. Hepatitis observed in HPV16+/animals was associated with up-regulation of oxidative stress markers. Curcumin and rutin reduced the expression of these markers, in some cases back to control level. These findings suggest that the antioxidant properties of both polyphenols may have contributed to minimize hepatocellular damage and pro-inflammatory stimuli, reducing hepatic inflammation. Curcumin and rutin also displayed a muscle-protective effect,

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ACCEPTED MANUSCRIPT which reached statistical significance in the case of rutin. In line with previous findings, this compound exerted its effect by inhibiting NF-κB activation [2, 9]. However, a

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recent study reports that an antioxidant mixture accelerated cachexia by promoting

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tumor growth in a xenograft mouse model of colon [37], highlighting the importance of

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employing adequate models for specific cancers. As a secondary finding in this study, neither compound was able to interfere with multistep skin carcinogenesis induced by the HPV16 oncogenes, although numerous studies report on the chemopreventive and

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therapeutic efficacy of polyphenols - and especially curcumin - against HPV-induced

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lesions [12-13]. This negative result should be interpreted with care, as in K14-HPV16 mice, all basal keratinocytes express HPV16 oncogenes under the control of the cytokeratin 14 gene promoter. This leads to continuous high-level oncogene expression,

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while leaving no normal keratinocytes to replace transformed ones, and limiting this model's applicability for testing chemopreventive anti-tumor compounds.

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Overall, these data indicate that K14-HPV16 mice are a useful model for studying the wasting syndrome frequently associated with HPV-induced malignancies. Dietary

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curcumin and rutin have potential for preventing this syndrome and rutin shows a particularly strong muscle-protective effect, mediated through NF-κB down-regulation.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgements The authors acknowledge Prof. Paulo Rema and his team from UTAD, for their help in preparing modified diets. This work was financially supported by: European Investment Funds

by

FEDER/COMPETE/POCI

–

Operational

Competitiveness

and

23

ACCEPTED MANUSCRIPT Internationalization Program, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, and POCI-01-0145-FEDER-006939

(Laboratoy

for

Process

Engineering,

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Project

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Environment, Biotechnology and Energy - LEPABE funded by FEDER funds through

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COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT. Rui Gil da Costa is supported by grant nº SFRH/BPD/85462/2012 from FCT, financed by the Portuguese Government and the

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Social European Fund.

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