Sublineage A1 Drives Multi-Organ Carcinogenesis

by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/ 04462/2020); from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020—Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Pro- Int. J. Mol. Sci. 2022, 23, 12371 8 of 10 gramme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). This work was also supported by Fundos FEDER through the Programa Operacional Factores de Competitividade—COMPETE and by Fundos Nacionais through the Fundação para a Ciência e a Tecnologia within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health-ToxOmics); and from LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC); 2SMART (NORTE-01-0145- FEDER-000054), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).The study of human papillomavirus (HPV)-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.publishersversionpublishe

Dietary supplementation with chestnut (Castanea sativa) reduces abdominal adiposity in FVB/n mice: a preliminary study

The production of chestnut (Castanea sativa Miller) is mostly concentrated in Europe. Chestnut is recognized by its high content of antioxidants and phytosterols. This work aimed to evaluate the e ects of dietary chestnut consumption over physiological variables of FVB/n mice. Eighteen FVB/n male 7-month-old mice were randomly divided into three experimental groups (n = 6): 1 (control group) fed a standard diet; 2 fed a diet supplemented with 0.55% (w/w) chestnut; and 3 supplemented with 1.1% (w/w) chestnut. Body weight, water, and food intake were recorded weekly. Following 35 days of supplementation, the mice were sacrificed for the collection of biological samples. Chestnut supplementation at 1.1% reduced abdominal adipose tissue. Lower serum cholesterol was also observed in animals supplemented with chestnut. There were no significant di erences concerning the incidence of histological lesions nor in biochemical markers of hepatic damage and oxidative stress. These results suggest that chestnut supplementation may contribute to regulate adipose tissue deposition.This work is supported by National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020, CIMO (UIDB/00690/2020) and UIDB/CVT/00772/2020 Interreg Program for the financial support of the Project IBERPHENOL, Project Number 0377_IBERPHENOL_6_E; co-financed by European Regional Development Fund (ERDF) through POCTEP 2014-2020. This work was also supported by VALORIZEBYPRODUCTS Project, reference n.º 029152, funded by Portuguese Foundation for Science and Technology (FCT) and co-financed by the European Regional Development Fund (FEDER) through COMPETE 2020 - Operational Competitiveness and Internationalization Programme (POCI). This work was also financially supported by Project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC) and Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), by the Research Centre of the Portuguese Institute of Oncology of Porto (CI-IPOP 37-2016) and by the Interact R&D project, operation number NORTE-01-0145-FEDER-000017, in its ISAC research line, co-financed by the ERDF through NORTE 2020. This work was also supported by PhD fellowship SFRH/BD/136747/2018.info:eu-repo/semantics/publishedVersio

Laurus nobilis (laurel) aqueous leaf extract's toxicological and anti-tumor activities in HPV16-transgenic mice

Cancers induced by human papillomavirus (HPV) infection remain a significant public health threat, fueling the study of new therapies. Laurel (Laurus nobilis) compounds and extracts recently showed in vitro activity against HPV-transformed cell lines. This work aims to evaluate the in vivo efficacy and hepatic toxicity of a laurel extract in a transgenic mouse model of HPV16-induced cancer. The extract was administered in drinking water (20 mg per animal per day) for three consecutive weeks, using four experimental groups (n = 10) (group I: HPV16−/− without treatment, group II: treated HPV16−/−, group III: HPV16+/− without treatment and group IV: treated HPV16+/−). Following the treatment period, animals were sacrificed and skin samples were used to classify skin lesions histologically. Toxicological parameters included hematological and biochemical blood markers, splenic and hepatic histology and hepatic oxidative stress. The extract did not prevent the progression of HPV16-induced cutaneous lesions in this model. The treated wildtype animals showed mild hepatitis, while transgenic animals suffered weight loss. However, there were no changes concerning hematological, biochemical and hepatic oxidative stress markers.This work was supported by: Integrative Research in Environment, Agro-Chains and Technology no. NORTE-01- 0145-FEDER-000017, in its line of research entitled ISAC, cofinanced by the European Regional Development Fund (ERDF) through NORTE 2020 (North Regional Operational Program 2014/2020). European Investment Funds by FEDER/COMPETE/ POCI– Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013. This study was also funded by Liga Portuguesa Contra o Cancro, by the Research Center of the Portuguese Institute of Oncology of Porto (CI-IPOP 37-2016), by project POCI-01-0145- FEDER-006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE), project POCI-01-0145-FEDER-006958 and UID/AGR/04033/2013, funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT – Fundação para a Ciência e a Tecnologia; Rui M. Gil da Costa was funded by grant number SFRH/BPD/85462/2012 from FCT, funded by the Portuguese Government and the Social European Fund. The authors are also grateful to FCT, Portugal and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/ 00690/2013), and to the Interreg España-Portugal for financial support through the project 0377_Iberphenol_6_E.info:eu-repo/semantics/publishedVersio

The cyclooxigenase-2 inhibitor parecoxib prevents epidermal dysplasia in HPV16-transgenic mice: efficacy and safety observations

Carcinogenesis induced by high-risk human papillomavirus (HPV) involves inflammatory phenomena, partially mediated by cyclooxigenase-2. In pre-clinical models of HPV-induced cancer, cyclooxygenase-2 inhibitors have shown significant e cacy, but also considerable toxicity. This study addresses the chemopreventive e ect and hepatic toxicity of a specific cyclooxigensase-2 inhibitor, parecoxib, in HPV16-transgenic mice. Forty-three 20 weeks-old female mice were divided into four groups: I (HPV16+/-, n = 10, parecoxib-treated); II (HPV16+/- n = 11, untreated); III (HPV16+/-, n = 11, parecoxib-treated) and IV (HPV16+/- n = 11, untreated). Parecoxib (5.0 mg/kg once daily) or vehicle was administered intraperitoneally for 22 consecutive days. Skin lesions were classified histologically. Toxicological endpoints included genotoxic parameters, hepatic oxidative stress, transaminases and histology. Parecoxib completely prevented the onset of epidermal dysplasia in HPV16+/- treated animals (0% versus 64% in HPV16+/- untreated, p = 0.027). Parecoxib decreases lipid peroxidation (LPO) and superoxide dismutase (SOD) activity and increases the GSH:GSSG ratio in HPV16+/- treated animals meaning that oxidative stress is lower. Parecoxib increased genotoxic stress parameters in wild-type and HPV16-transgenic mice, but didn’t modify histological or biochemical hepatic parameters. These results indicate that parecoxib has chemopreventive e ects against HPV16-induced lesions while maintaining an acceptable toxicological profile in this model.This work is supported by National Funds by FCT—Portuguese Foundation for Science and Technology, under the projects UID/AGR/04033/2019, UID/CVT/00772/2019 and UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION”—NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund.info:eu-repo/semantics/publishedVersio

role of female sex hormone receptors

Funding Information: Funding: This study was supported by grant IECT-FAPEMA-05796/18 and FAPEMA IECT 30/2018-IECT Saúde, by the Research Center of the Portuguese Oncology Institute of Porto (project no. PI86-CI-IPOP-66-2017); by European Investment Funds by FEDER/COMPETE/POCI—Operational Competitiveness and Internationalization Program, and national funds by FCT—Portuguese Foundation for Science and Technology under projects UID/AGR/04033/2020, UIDB/CVT/00772/2020 and by Base Funding-UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology, and Energy—LEPABE—funded by national funds through the FCT/MCTES (PID-DAC); Project 2SMART-engineered Smart materials for Smart citizens, with reference NORTE-01-0145-FEDER-000054, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.A growing proportion of oropharyngeal squamous cell carcinomas (OPSCC) are associated with infection by high-risk human papillomavirus (HPV). For reasons that remain largely unknown, HPV+OPSCC is significantly more common in men than in women. This study aims to determine the incidence of OPSCC in male and female HPV16-transgenic mice and to explore the role of female sex hormone receptors in the sexual predisposition for HPV+ OPSCC. The tongues of 30-weeks-old HPV16-transgenic male (n = 80) and female (n = 90) and matched wild-type male (n = 10) and female (n = 10) FVB/n mice were screened histologically for intraepithelial and invasive lesions in 2017 at the Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Por-tugal. Expression of estrogen receptors alpha (ERα) and beta (ERβ), progesterone receptors (PR) and matrix metalloproteinase 2 (MMP2) was studied immunohistochemically. Collagen remodeling was studied using picrosirius red. Female mice showed robust ERα and ERβ expression in intraepithelial and invasive lesions, which was accompanied by strong MMP2 expression and marked collagen remodeling. Male mice showed minimal ERα, ERβ and MMP2 expression and unaltered collagen patterns. These results confirm the association of HPV16 with tongue base cancer in both sexes. The higher cancer incidence in female versus male mice contrasts with data from OPSCC patients and is associated with enhanced ER expression via MMP2 upregulation.publishersversionpublishe

Toxicological and anti-tumor effects of a linden extract (Tilia platyphyllos Scop.) in a HPV16-transgenic mouse model

Tilia platyphyllos Scop. is a popular broad-leaved tree, native to Central and Southern Europe. Hydroethanolic extracts rich in phenolic compounds obtained from T. platyphyllos Scop. have shown in vitro antioxidant, anti-inflammatory and antitumor properties. The aim of this work was to evaluate the therapeutic properties of a hydroethanolic extract obtained from T. platyphyllos in HPV16-transgenic mice. The animals were divided into eight groups according to their sex and phenotype. Four groups of female: HPV+ exposed to linden (HPV linden; n = 6), HPV+ (HPV water; n = 4), HPV- exposed to linden (WT linden; n = 5) and HPV- (WT water; n = 4) and four groups of male: HPV+ exposed to linden (HPV linden; n = 5), HPV+ (HPV water; n = 5), HPV- exposed to linden (WT linden; n = 5) and HPV- (WT water; n = 7). The linden (Tilia platyphyllos Scop.) extract was orally administered at a dose of 4.5 mg/10 mL per animal (dissolved in water) and changed daily for 33 days. The hydroethanolic extract of T. platyphyllos consisted of protocatechuic acid and (-)-epicatechin as the most abundant phenolic acid and flavonoid, respectively, and was found to be stable during the studied period. In two male groups a significant positive weight gain was observed but without association with the linden extract. Histological, biochemical, and oxidative stress analyses for the evaluation of kidney and liver damage support the hypothesis that the linden extract is safe and well-tolerated under the present experimental conditions. Skin histopathology does not demonstrate the chemopreventive effect of the linden extract against HPV16-induced lesions. The linden extract has revealed a favourable toxicological profile; however, additional studies are required to determine the chemopreventive potential of the linden extract. This journal isThis work was supported by the project IBERPHENOL, project number 0377_IBERPHENOL_6_E; Interact R&D project, operation number NORTE-01-0145-FEDER-000017, National Funds by FCT – Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 (CITAB), and project UIDB/ CVT/00772/2020 (CECAV) and the post-graduation grant SFRH/ BD/136747/2018 and 2020.04789.BD; the authors are also grateful to FCT, Portugal and FEDER under programme PT2020 for financial support to CIMO (UIDB/00690/2020) and L. Barros acknowledges the national funding by FCT, P. I., through the institutional scientific employment program-contract. The authors would like to thank Cantinho das Aromáticas organic farmers from Vila Nova de Gaia (Portugal) for providing the samples. This work was financially supported by: Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC).info:eu-repo/semantics/publishedVersio

Study on the antineoplastic and toxicological effects of pomegranate (Punica granatum L.) leaf infusion using the K14-HPV16 transgenic mouse model

Punica granatum L. (pomegranate) has been used in functional foods due to its various health benefits. However, the in vivo biological potential of its leaf remains little known. This study has aimed to characterize the antineoplastic and toxicological properties of using pomegranate leaf infusion (PLI) on transgenic mice carrying human papillomavirus (HPV) type 16 oncogenes. Thirty-eight mice were divided into 3 wild-type (WT) and 3 transgenic (HPV) groups, with exposure to 0.5% PLI, 1.0% PLI, and water. The animals' body weight, drink and food consumption were recorded. Internal organs, skin samples and intracardiac blood were collected to evaluate toxicological parameters, neoplastic lesions and oxidative stress. The results indicated that PLI was safe as no mortality, no behavioural disorders and no significant differences in the levels of microhematocrit, serum biochemical markers, internal organ histology, and oxidative stress was found among the WT groups. Histological analysis revealed that HPV animals that consumed PLI exhibited reduced hepatic, renal and cutaneous lesions compared with the HPV control group. Low-dose PLI consumption significantly diminished renal hydronephrosis lesions and relieved dysplasia and carcinoma lesions in the chest skin. Oxidative stress analysis showed that low-dose PLI consumption may have more benefits than high-dose PLI. These results suggest that oral administration of PLI has the potential to alleviate non-neoplastic and neoplastic lesions against HPV16-induced organ and skin injuries, though this requires further scientific research studies.info:eu-repo/semantics/publishedVersio

Effect of hidroethanolic extract of Lavandula pedunculata (mill.) Cav. On morphometric parameters in HPV-16 trangenic mice

Lavandula penduculata (Mill.) Cav., common English name French lavender, belongs to the Lamiaceae family and has been used as a medicinal plant in infusions for respiratory and digestive systems and as a therapeutic agent with antiseptic action for cleaning wounds [1,2]. The K14HPV16 mice is a skin squamous carcinoma model that can be used to test the antitumoral properties of several chemical and natural products [3]. The aim of this work was to evaluate the effect of the hydroethanolic French lavender extract (FLE) on body weight, relative organs weights, food and water consumption in an HPV-16-transgenic mice model. The extract was obtained from a maceration with ethanol/water (80:20, v/v), and the phenolic composition was determined through HPLC-DAD-ESI/MS. Twenty-eight male mice were randomly divided into four groups (n=7/group) according to their genotype: group I (HPV16- control); II (HPV16- FLE); III (HPV16+ control) and IV (HPV16+ FLE). The FLE was administrated orally in drinking water at 6.8 mg/10mL/animal to animals from groups II and IV and changed every 4 days. The animals were kept under controlled conditions such as temperature, light and humidity. Food and water were kept ad libitum body weight, food and water consumption were measured weekly as well as animal welfare. After twentynine days, all animals were sacrificed by anaesthetic overdose and blood was obtained from cardiac punction. The organs were collected and immediately weighted. Data was analysed using SPSS 25. The differences were considered statistically significant at p<0.05. A total of thirteen compounds were identified in the hydroethanolic extract, being salvianolic acid B and rosmarinic acid the main molecules present. Moreover, the compounds revealed to be stable in the drinking water during the 5 tested days. HPV animals exposed to FLE (group III) showed higher values of body weight variation than HPV animals not exposed to lavender in week 1, 2 and 3 (p<0.05), suggesting that the FLE was highly palatable. However, the values of food consumption were identical between groups and water intake was higher in transgenic animals as expected. The relative organ weight of heart, lung, kidneys, adrenals and liver did not demonstrate differences between groups (p<0.05). According to our results the consumption of French lavender demonstrated a favourable and safe toxicological profile using these experimental conditionsThis work was supported by European Investment Funds by FEDER/ COMPETE/POCI - Operational Competitiveness and Internationalization Program and National Funds by FCT - Portuguese Foundation for Science and Technology, under the projects Project UIDB/04033/2020 (CITAB), and PhD fellowship SFRH/BD/136747/2018 and 2020.04789.BD. The authors are also grateful to FCT for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020). L. Barros thanks FCT, P.I for her contract through the institutional scientific employment program. This work was also funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Norte-01-0145-FEDER-000042. The authors would like to thank BPGV for the samples provided.info:eu-repo/semantics/publishedVersio

The antioxidant effect of spearmint (Mentha spicata) in a HPV-16-transgenic mouse model

The Human Papillomavirus (HPV) is the prime cause of cervix cancer in womeninfo:eu-repo/semantics/publishedVersio

Histological lesions in hpv16-transgenic model: the effect of hidroethanolic extract of Lavandula pedunculata (mill.) Cav.

The K14HPV16 mice is a skin squamous carcinoma model that can be used to test antitumoral properties of several chemical and natural compounds1. Lavandula penduculata (Mill.) Cav., known as lavender, belongs to the Laminaceae family and has been used in traditional medicine as infusions to treat several conditions2. This work aimed to evaluate the effects of the hydroethanolic French lavender extract (FLE) in an HPV16-transgenic mice model lesions. The extract was obtained through a maceration with ethanol/water (80:20, v/v) and its phenolic composition was determined by HPLC-DADESI/ MS. The FLE was dissolved in drinking water at 6.8 mg/10mL/animal and the animals were supplemented during 29 consecutive days. Twentyeight male mice were randomly divided into four groups: (n=7/group): group I (HPV16- control); II (HPV16- FLE); III (HPV16+ control) and IV (HPV16+ FLE). After 29 days all animals were sacrificed by xylazineketamine overdose following cardiac puncture to obtain blood samples. Skin samples (chest and ear), kidney, liver and spleen were processed for histological analysis.A total of thirteen compounds were identified in the hydroethanolic extract, being salvianolic acid B and rosmarinic acid the main molecules present. Moreover, the compounds revealed to be stable in the drinking water for 5 days. Histological analyses of skin samples from wild-type mice exposed (group II) and not exposed (group I) to FLE showed normal skin histology. Group III showed skin chest epidermal hyperplasia in 100% of the mice while group IV showed less epidermal hyperplasia frequency (66.6%) (p>0.05). Concerning to liver, kidney, and spleen lesions there are no differences between groups (p>0.05). The lavender extract did not prevent the progression of HPV-16 induced cutaneous lesions in this model. These data deserve more investigation to clarify the effect of lavender extract on HPV-16 lesions.info:eu-repo/semantics/publishedVersio