Immunology and mammary cancer development: addressing the role of mast cells

Background: Mammary cancer is one of the most frequent cancers worldwide. Mast cells are among the cells of tumor microenvironment and have been associated with increased angiogenesis and poor prognosis. Despite this, the role of mast cells on mammary cancer is not fully elucidated. In this way, this work studied the role of mast cells in a rat model of mammary cancer chemically-induced. Materials and Methods: All experiments were performed in accordance with the Portuguese and European legislation on the protection of animals used for scientific purposes. The experiments were approved by the Portuguese (no.008961) and University (CE_12-2013) Ethics Committees. Thirty- four female Sprague-Dawley rats were randomly divided into five experimental groups. At seven weeks of age, mammary tumors’ development was induced in animals from groups I, II, III (n = 10+10+10) by a single intraperitoneal injection of the carcinogen N-methyl-N-nitrosourea (MNU). Groups II and IV (n = 2) were treated with ketotifen in drinking water (1 mg/kg/day, 7 days/week) immediately after the MNU ad- ministration for 18 weeks, while the group III received the ketotifen after the development of the first mammary tumor. Groups I and V (n = 2) received only water. Animals were sacrificed at 25 weeks of age by an overdose of ketamine and xylazine, followed by an exsanguination by cardiac puncture. Mammary tumors were collected and immersed in formalin for posterior analysis. Tumors’ vascularization, proliferation and apoptosis were also assessed by immunohistochemistry (Vascular Endothelial Growth Factor (VEGF)-A, Ki-67, and caspase-3 and caspase-9). Results: Animals from groups IV and V did not develop any mammary tumor. Twenty-one animals (six animals from group I, eight animals from group II and seven animals from group III) developed a total of 58 mammary tumors, mainly classified as papillary non-invasive carcinomas. Tumors’ vascularization was similar among groups (P > 0.05). Mammary tumors from group II exhibited the lowest prolif- eration (P < 0.05) and apoptotic indexes. Conclusions: The mainly positive effect of the ketotifen administration seems to be the reduction of tumor prolifera- tion when the drug was administered before mammary tumor development

The influence of physical exercise on oestrogen and androgen receptor expression in a chemically and hormonally-induced rat model of prostate cancer

Background: Oestrogen (ER) and androgen (AR) recep- tors play an important role in normal prostate development and are also implied in prostate cancer (PCa) development. Several studies suggested that physical activity may decrease the risk of PCa development and also changes sexual hor- mones and their receptors. This study aimed to evaluate the effects of physical exercise on ERα and AR expression in a rat model of chemically and hormonally-induced PCa. Materials and Methods: Fifty-five male Wistar Unilever rats of 12 weeks of age were randomly divided into four groups: control sedentary (n = 10), control exercised (n = 10), induced sedentary (n = 15) and induced exercised (n = 20). Animals from exercised groups started the exercise training in a treadmill (Treadmill Control LE 8710, Harvard Apparatus, USA), at the age of 8 weeks, for 35 weeks (5 days/week). The protocol for PCa induction started at 12 weeks of age and consisted of sequential administration of flutamide (50 mg/kg, TCI Chemicals), testosterone propion- ate (100 mg/kg, TCI Chemicals) and N-methyl-N-nitrosourea (30 mg/kg, Isopac®, Sigma Chemical Co.), followed by sub- cutaneous implants of crystalline testosterone. Animals were sacrificed at 61 weeks of age and a complete necropsy was performed. All experiments were approved by DGAV (no. 021326). Antibodies for Erα (1:500, clone 6F11, Novocastra) and AR (clone PG21, Merck Millipore) were used for the immunohistochemical study. The staining extension was evaluated in normal prostate tissue and in dorsolateral pros- tate lesions (hyperplasia, dysplasia, prostatic intraepithelial neoplasia (PIN) and microinvasive carcinoma) and assessed to five levels (0%, 75%), con- sidering the extension of immunopositive tissue. Data was analysed with SPSS 25.Results: The normal prostate tissue and dorsolateral prostate lesions of animals from all groups were immunopositive for Erα and AR. However, the groups showed high immunoposi- tivity for AR and low positivity for Erα ( 0.05). The malignant lesions (PIN and microinvasive carcinoma) showed lower AR expression when compared with normal prostate tissue in all groups. Conclusions: As expected, the AR expression was lower in malignant lesions. Inversely to that reported in other studies, the exercise training did not modify the ERα and AR expres- sion, which may be related to the duration and type of exer- cise performed

Rat prostate: practical tips for ultrasonographic monitoring

Background: Prostate is the largest accessory gland of the male reproductive tract. The prostate of men over 40 years- old is frequently affected by several pathologies, like benign prostate hyperplasia and cancer. Rats have been used as model to study prostate cancer. This study intended to address the usefulness of ultrasonography for rat prostate monitoring. Materials and Methods: Eight male Wistar Unilever rats were acquired from Charles River Laboratories and main- tained under controlled conditions of temperature, humidity, air system filtration and light/dark cycle. The prostate was evaluated by ultrasonography in awake animals. The animals were restrained by a researcher and placed in supine position. The skin of the inguinal region was shaved using a machine clipper (AESCULAP® GT420 Isis, USA). A real-time scan- ner (Logic P6®, GE, USA) and a 12 MHz linear transducer were used. Acoustic gel (Parker Laboratories Inc., USA) was applied. A complete transverse scan using B mode was per- formed from the cranial to the caudal region of the prostate, and a sagittal scan was performed moving the probe from the right to the left side. Procedures were approved by the Portuguese Ethics Committee (no.021326). Results: Prostate was easily evaluated by ultrasonography in all animals. In the transverse scan, the urinary bladder presents as a round to oval shape filled with urine (anechoic structure) and the prostate lobes were visible around it. The ventral prostate lobes appear as hypoechoic elongated struc- tures (one right and one left) with a hyperechoic capsule, placed ventrally to the urinary bladder. In this scan, the dorsal prostate was observed close to the urinary bladder neck, as a round hypoechoic structure with a hyperechoic capsule, dor- sally to the urinary bladder. In the sagittal scan, the urinary bladder was observed as an elongated structure filled with urine (anechoic content). The ventral prostate lobes were occasionally observed ventrally to the neck of the urinary bladder, as previously described. The dorsal prostate was ob- served dorsally to the neck of the urinary bladder, presenting as a round to elongated shape, with a hypoechoic appearance and a hyperechoic capsule.Conclusions: The ultrasonography is a non-invasive and ac- cessible tool for prostate monitoring in the rat model. Acknowledgments: This 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 RUNawayPCa (POCI-01-0145-FEDER-016728 and PTDC/DTP-DES/6077/2014), UIDB/04033/2020, UIDB/ CVT/00772/2020 and UIDB/50006/2020 (LAQV)

Effects of physical exercise in biochemical parameters and dorsolateral prostate lesions: data from a rat model of prostate cancer

Background: Prostate cancer (PCa) is among the most prevalent cancers worldwide. Physical exercise is widely recognized due to its beneficial effects. This study aimed to evaluate the effects of physical exercise on biochemical pa- rameters and in dorsolateral prostate lesions in a rat model of PCa. Materials and Methods: Ninety-five male Wistar Unilever rats were randomly divided into eight groups sacrificed at 35 (groups I) or 61 weeks of age (groups II): control sedentary groups (Cont+Sed I (n = 10); Cont+Sed II (n = 10)); induced sedentary group (PCa+Sed I (n = 10); PCa+Sed II (n = 15)); control exercised groups (Cont+EX I (n = 10); Cont+EX II (n = 10)) and induced exercised groups (PCa+EX I (n = 10); PCa+EX II (n = 20)). All procedures were approved (DGAV, no. 021326). Animals from exercised groups started the exer- cise program in a treadmill at 8 weeks of age, for 28 weeks or 53 weeks. The animals were trained 5 days/week, 60 min per day. Prostate lesions were induced at 12 weeks of age, with sequential administration of flutamide, testosterone propion- ate and N-methyl-N-nitrosourea, and subcutaneous implants of crystalline testosterone. Animals were sacrificed at 35 or 61 weeks of age. Peripheral blood of all animals was col- lected by intracardiac puncture. A complete necropsy was performed. The dorsolateral prostate tissues sections were processed for histological analysis. Data were analysed using SPSS 25. p 0.05). Dorsolateral prostate lesions were classified as dysplasia, prostatic intraep- ithelial neoplasia (PIN) and microinvasive carcinoma. The number of prostate lesions was higher in animals from groups II than in those from groups I, mainly in PCa+Sed II animals when compared with PCa+Sed I (p 0.05). Conclusions: Overall, the animals sacrificed at 61 weeks of age developed more dorsolateral prostate lesions than ani- mals sacrificed at 35 weeks of age, which may be related to a longer testosterone exposure

SETDB2 and RIOX2 are differentially expressed among renal cell tumor subtypes, associating with prognosis and metastization

Increasing detection of small renal masses by imaging techniques entails the need for accurate discrimination between benign and malignant renal cell tumors (RCTs) as well as among malignant RCTs, owing to differential risk of progression through metastization. Although histone methylation has been implicated in renal tumorigenesis, its potential as biomarker for renal cell carcinoma (RCC) progression remains largely unexplored. Thus, we aimed to characterize the differential expression of histone methyltransferases (HMTs) and histone demethylases (HDMs) in RCTs to assess their potential as metastasis biomarkers. We found that SETDB2 and RIOX2 (encoding for an HMT and an HDM, respectively) expression levels was significantly altered in RCTs; these genes were further selected for validation by quantitative RT-PCR in 160 RCTs. Moreover, SETDB2, RIOX2, and three genes encoding for enzymes involved in histone methylation (NO66, SETD3, and SMYD2), previously reported by our group, were quantified (RT-PCR) in an independent series of 62 clear cell renal cell carcinoma (ccRCC) to assess its potential role in ccRCC metastasis development. Additional validation was performed using TCGA dataset. SETDB2 and RIOX2 transcripts were overexpressed in RCTs compared to renal normal tissues (RNTs) and in oncocytomas vs. RCCs, with ccRCC and papillary renal cell carcinoma (pRCC) displaying the lowest levels. Low SETDB2 expression levels and higher stage independently predicted shorter disease-free survival. In our 62 ccRCC cohort, significantly higher RIOX2, but not SETDB2, expression levels were depicted in cases that developed metastasis during follow-up. These findings were not apparent in TCGA dataset. We concluded that SETDB2 and RIOX2 might be involved in renal tumorigenesis and RCC progression, especially in metastatic spread. Moreover, SETDB2 expression levels might independently discriminate among RCC subgroups with distinct outcome, whereas higher RIOX2 transcript levels might identify ccRCC cases with more propensity to endure metastatic dissemination.This study was funded by research grants from Research Center of Portuguese Oncology Institute - Porto (CI-IPOP 4-2012 and CI-IPOP 27) and from Associacao Portuguesa de Urologia (APU-2010). ASP-L was supported by FCT-Fundacao para a Ciencia e a Tecnologia fellowship (SFRH/SINTD/94217/2013). CSG is supported by FCT- Fundacao para a Ciencia e Tecnologia PhD fellowships (SFRH/BD/92786/2013) and BMC is funded by FCT-Fundacao para a Ciencia e a Tecnologia (IF/00601/2012).info:eu-repo/semantics/publishedVersio

Effects of testosterone and exercise training on bone microstructure of rats

Background and Aim: Male hypogonadism results from failure to produce physiological levels of testosterone. Testosterone in men is essential in masculine development, sperm production, and adult man’s health. Osteoporosis is one of the consequences of hypogonadism. Regular physical exercise and exogenous testosterone administration are frequently used to prevent or treat this condition. This study aimed to understand the effects of lifelong exercise training and testosterone levels (isolated and together) in the main bone structure parameters. Materials and Methods: A total of 24 rats were used and randomly divided into four groups: Control group (CG; n=6), exercised group (EG, n=6), testosterone group (TG, n=6), and testosterone EG (TEG, n=6). A micro-computed tomography equipment was used to evaluate 15 bone parameters. Results: Both factors (exercise training and testosterone) seem to improve the bone resistance and microstructure, although in different bone characteristics. Testosterone influenced trabecular structure parameters, namely, connectivity density, trabecular number, and trabecular space. The exercise promoted alterations in bone structure as well, although, in most cases, in different bone structure parameters as bone mineral density and medullar mineral density. Conclusion: Overall, exercise and testosterone therapy seems to have a synergistic contribution to the general bone structure and resistance. Further studies are warranted, comparing different individual factors, as gender, lifestyle, or testosterone protocols, to constantly improve the medical management of hypogonadism (and osteoporosis)

The red seaweed Grateloupia turuturu prevents epidermal dysplasia in HPV16-transgenic mice

Abstract: The role of dietary profiles in promoting or reducing the risk of multiple types of cancer is increasingly clear, driving the search for balanced foods and nutraceuticals. The red seaweed Grateloupia turuturu has been used as human food showing a balanced nutritional profile. This study aims to test in vivo chemopreventive effects of G. turuturu against cutaneous pre-malignant lesions in transgenic mice for the human papillomavirus type 16 (HPV16). Forty-four female HPV+/− or HPV−/− mice received a standard diet or were supplemented with 10% G. turuturu for 22 consecutive days. Cutaneous lesions (ear and chest skin) were identified histologically. Complementarily, the weights and histology of internal organs as well as blood biochemical and DNA integrity parameters were also assessed. G. turuturu consistently reduced the incidence of epidermal dysplasia induced by HPV16 on both cutaneous sites. Moreover, biochemical, DNA integrity and histological analyses confirmed G. turuturu edibility as no signs of toxicity were found. Dietary supplementation with G. turuturu is an effective and safe chemopreventive strategy in this model

Antioxidant activities of sulfated polysaccharides from brown and red seaweeds

The in vitro antioxidant activities of the following six sulfated polysaccharides were investigated: iota, kappa and lambda carrageenans, which are widely used in the food industry, fucoidan (homofucan) from the edible seaweed Fucus vesiculosus and fucans (heterofucans) F0.5 and F1.1 from the seaweed Padina gymnospora. With respect to the inhibition of superoxide radical formation, fucoidan had an IC50 (the half maximal inhibitory concentration) of 0.058 mg·mL−1, while the IC50 for the kappa, iota and lambda carrageenans were 0.112, 0.332 and 0.046 mg·mL−1, respectively. All of the samples had an inhibitory effect on the formation of hydroxyl radicals. The results of peroxidation tests showed that fucoidan had an IC50 of 1.250 mg·mL−1 and that the kappa, iota and lambda carrageenans had an IC50 of 2.753 and 2.338 and 0.323 mg·mL−1, respectively. Fucan fractions showed low antioxidant activity relative to fucoidan. These results clearly indicate the beneficial effect of algal polysaccharides as antioxidants