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Hepatocellular carcinoma (HCC) is a neoplasia representing the fifth most common malignancy worldwide and the third cause of death from cancer. Diets with high content in fruits and vegetables are widely recommended for their health-promoting properties, among them, the protection against diabetes, cancer, and cardiovascular diseases. Hesperidin is the most important phenol in the orange fruit with well-known health benefits. Diet components have been used as possible modulator agents of DNA methylation in cancer cells and epigenetic therapy against their harmful effects could be a potential tool in chemotherapy. The purpose of the present study was to evaluate the methylation patterns induced by hesperidin in HL60 cell line as an in vitro model in order to analyze its chemopreventive effects in epigenetic cancer therapies. A parallel in vivo pilot experience using a rat diethyl nitrosamine hepatocarcinogenesis-induced model was carried out to validate the therapeutic efficacy of this orange flavonol. Results showed that: (i) Hesperidin is cytotoxic in a dose-dependent manner and the IC50 was 12.5 mM; (ii) Hesperidin exerts a significant hypomethylating effect on the LINE-1 sequence (up to 47% hypomethylation at 12.5 mM) and on the ALU-M2 repetitive sequences (up to 32% at 6 mM) in HL60 tumor cells. (iii) Hesperidin does not affect the rat body and liver weight and it is able to reduce the diethyl nitrosamine-induced nodules at 1,000, 500, and 250 ppm. In conclusion, hesperidin could be proposed as a candidate molecule in chemoprevention in epigenetic therapy purposes.

Mammary tumours are the most common neoplasms of female dogs. Compared to mammary tumours of humans and cats, myoepithelial (ME) cell involvement is common in canine mammary tumours (CMT) of any subtype. Since ME cell involvement in CMT influences both histogenetic tumour classification and prognosis, correct identification of ME cells is important. This review describes immunohistochemical methods for identification of canine mammary ME cells used in vivo. In addition, phenotypic and genotypic methods to isolate ME cells for in vitro studies to analyse tumour-suppressor protein production and gene expression are discussed. The contribution of ME cells to both histogenetic classifications and the prognosis of CMT is compared with other species and the potential use of ME cells as a method to identify carcinoma in situ is discussed.

Background The use of plasma rich in growth factors (PRGF) has been proposed to improve the healing of Achilles tendon injuries, but there is debate about the effectiveness of this therapy. The objective of the present study was to evaluate the histological effects of PRGF, which is a type of leukocyte-poor platelet-rich plasma, on tendon healing. Methods The Achilles tendons of twenty-eight sheep were divided surgically. The animals were randomly divided into four groups of seven animals each. The repaired tendons in two groups received an infiltration of PRGF intraoperatively and every week for the following three weeks under ultrasound guidance. The tendons in the other two groups received injections with saline solution. The animals in one PRGF group and one saline solution group were killed at four weeks, and the animals in the remaining two groups were killed at eight weeks. The Achilles tendons were examined histologically, and the morphometry of fibroblast nuclei was calculated. Results The fibroblast nuclei of the PRGF-treated tendons were more elongated and more parallel to the tendon axis than the fibroblast nuclei of the tendons in the saline solution group at eight weeks. PRGF-treated tendons showed more packed and better oriented collagen bundles at both four and eight weeks. In addition to increased maturation of the collagen structure, fibroblast density was significantly lower in PRGF-infiltrated tendons. PRGF-treated tendons exhibited faster vascular regression than tendons in the control groups, as demonstrated by a lower vascular density at eight weeks. Conclusions PRGF was associated with histological changes consistent with an accelerated early healing process in repaired Achilles tendons in sheep after experimental surgical disruption. PRGF-treated tendons showed improvements in the morphometric features of fibroblast nuclei, suggesting a more advanced stage of healing. At eight weeks, histological examination revealed more mature organization of collagen bundles, lower vascular densities, and decreased fibroblast densities in PRGF-treated tendons than in tendons infiltrated with saline solution. These findings were consistent with a more advanced stage of the healing process. Clinical implications Based on the findings in this animal model, PRGF infiltration may improve the early healing process of surgically repaired Achilles tendons.

The PI3K/AKT/PTEN pathway is involved in the pathogenesis of several human cancers. This study investigated the biological and prognostic value of PI3K/AKT/PTEN pathway dysregulation in feline mammary tumours. Expression of p-AKT, HER2, PTEN and steroid receptors was assessed by immunohistochemistry (IHC) in 27 malignant and 12 benign mammary tumours from 39 female cats followed up over a 24-month period. Feline mammary carcinoma (FMC) cell lines were analyzed by Western blot and the feline AKT gene sequence was characterized. p-AKT expression statistically correlated with tumour malignancy, histological dedifferentiation and clinical recurrence. The animals with tumours expressing p-AKT had a shorter disease-free period than those with p-AKT-negative tumours. AKT activation was associated with HER2 expression and PTEN down-regulation, as occurs in human breast cancer, and feline AKT sequencing showed high homology with the human AKT gene. No AKT activation was observed in relation to either oestrogen receptor α (ERα) or progesterone receptor expression. Taken together, these data offer an explanation for AKT signalling and its role in FMC pathogenesis and prognosis, shedding new light on similarities between feline mammary tumours and hormone-independent breast cancer.

Summary The aim of this study was to determine whether the myoepithelial (ME) cell marker calponin could be used to analyze the integrity of the ME cell layer as a means of identifying canine mammary carcinoma in situ. Tissue from 74 canine mammary lesions was evaluated (two dysplasia, eight benign tumours and 64 carcinomas including one carcinoma in situ). The 63 carcinomas included examples of histological grade 1 (n = 32), grade 2 (n = 23) and grade 3 (n = 8). Expression of calponin was determined by immunohistochemistry. The percentage of proliferating cells surrounded by a single layer of calponin-positive cells formed the basis of classification as type I (≥90%), type II (70–90%) and type III (≤70%). Expression of Ki67 was used to determine the proliferation index (PI). The malignant tumours comprised of an approximately equal mixture of type I, II and III lesions. The two examples of dysplasia, the carcinoma in situ and two thirds of the benign tumours were classified as type I lesions. Some overlap in the level of calponin expression was observed between benign and malignant tumours. Positive correlations between the degree of calponin expression and the type of lesion (i.e. benign versus malignant; R = +0.3, P = 0.08) and the histological grade of malignancy (R = +0.54, P = 0.000001) were found. A negative correlation between the degree of calponin expression and PI (R = +0.027, P = 0.016) was found. The ME cell marker calponin may be used as an aid in the identification of canine carcinoma in situ, but the study of the ME cell layer integrity is not definitive for the diagnosis of malignancy in canine mammary tumours.

Background: Progesterone receptor (PR) antagonist aglepristone (RU534) has been used successfully for pregnancy termination and therapy of pyometra, vaginal tumors, and mammary hyperplasia in bitches and queens. All of these conditions share with canine mammary carcinomas the expression of PR. Objectives: To study the effect of RU534 on proliferation and apoptosis in canine mammary carcinomas in relation to PR expression. Animals: Twenty-seven nonspayed bitches with mammary carcinomas were treated with either 2 doses of 20 mg/kg RU534 (n = 22, RU534-treated group) or oil placebo (n = 5, control group) on days 1 and 8. Methods: Tumor samples were collected before (day 1) and after (day 15) treatment for immunohistochemistry. PR expression, proliferation index (PI), and apoptotic index (AI) were determined using antibodies against PR, Ki67, and cleaved lamin A/C antigens, respectively. The effect of treatment on these parameters was analyzed. Results: Differential expression of PR between day 1 (59.1% PR-positive tumors) and day 15 (36.4% PR-positive tumors) was observed in RU534-treated tumors exclusively. After RU534 treatment, mean PI was significantly decreased in PR-positive but unchanged in PR-negative RU534-treated tumors. A reduction of ≥20% in PI was found in 61.5% of RU534-treated tumors with PR expression. Conversely, no effect on AI was observed after RU534 treatment. Conclusions and Clinical Importance: Neoadjuvant RU534 treatment had PR expression-related inhibiting effects on proliferation of canine mammary carcinoma cells.

Scope Wine has shown anticarcinogenic benefits in hepatocarcinoma and polyphenols seem to be responsible for these effects. Wine lees are the sediments produced during fermentation and they endow wine with organoleptic and physicochemical properties. However, the anticarcinogenic role of these compounds is still unknown. Thus, the purpose of this work is to determine the phytochemical profiles of wine lees and then to analyze their anticarcinogenic effect and DNA methylation on a model of hepatocarcinogenesis. Methods and results The phytochemical composition of lees is determined by the Folin-Ciocalteu method and high-performance liquid chromatography. An in vivo study using a diethyl nitrosamine-hepatocarcinogenesis-induced model is performed to investigate the hepatoprotective properties of different doses of wine lees. For the DNA methylation analysis, a bisulfite-based method is used. Both types of lees mostly contain pyrogallol, gallic, and syringic acid with a high content of catechins in red lees. The carcinogen hypermethylates the Alu-M2 repetitive sequence and white lees decreases the hypermethylation at all tested concentrations. Low concentration of red and white lees and high concentration of white lees significantly improve the hepatocellular architecture and decrease the mitotic index in the murine model. Conclusion These findings suggest that wine lees are promising agents for chemoprevention of hepatocarcinoma.

Passive immunization against inhibin with an anti-inhibin serum (AIS) during the diestrous phase in cycling rats increased follicle-stimulating hormone secretion, stimulated the ovaries and reduced the magnitude of the luteinizing hormone (LH) surge in the afternoon of proestrus. The involvement of gonadotrope progesterone receptor (PR) expression/action in the inhibitory effects of the follicle-stimulating hormone-dependent putative ovarian factor gonadotropin surge-attenuating factor on preovulatory LH secretion was studied in the absence of circulating free inhibin. Proestrous pituitaries from rats injected with AIS or a non-immune serum (NIS) were studied for determination of PR-AB and PR-B mRNAs by RT-PCR and PR-B and PR-A isoform proteins by Western blot. In addition, pituitaries from AIS- and NIS-injected rats were incubated and studied for PR-dependent LH secretion parameters: LH-releasing hormone (LHRH)-stimulated LH secretion, progesterone-potentiated LHRH-stimulated LH secretion and LHRH self-priming. Also, the effects of the antiprogestagen RU486 on these LH secretion parameters were evaluated and compared with those of AIS. Finally, gonadotrope PR phosphorylation was evaluated by immunohistochemistry. Results showed that the hyperstimulated ovaries of AIS-injected rats produce a factor, different from inhibin, that blocked LHRH self-priming and P-potentiation of LHRH-stimulated LH secretion. These effects were not due to decreased pituitary PR mRNAs, PR protein expression or PR protein B/A ratio. The inhibitory effect of AIS on PR-dependent LH secretion seemed to be due to gonadotrope PR dephosphorylation. Taken together, the findings indicated that the putative gonadotropin surge-attenuating factor affected LH surge through an inhibition of PR phosphorylation/action but not PR expression.

Thickening of the uterine cervix and bilateral ovarian cystic change was identified in a 6-month-old pig during routine abattoir inspection. Microscopically, the cervical lesion comprised a non-encapsulated mass of densely packed, large and monomorphic spindle cells within the myometrium. Immunohistochemically, the majority of these neoplastic cells expressed the cytoplasmic terminal smooth muscle differentiation marker calponin, the nuclear oestrogen receptor alpha and the progesterone receptor. The ovarian cysts were classified as follicular cysts. A diagnosis of leiomyoma of the uterine cervix with bilateral ovarian follicular cysts was made. The expression of calponin as a marker of smooth muscle differentiation in tumours of the genital tract of the pig has not previously been reported. The expression of steroid hormone receptors suggests a role for steroid hormones derived from the ovarian follicular cysts in tumourigenesis.

The expression of oestrogen receptor-alpha (ERalpha) and progesterone receptor (PR) was examined in 32 canine genital tract tumours diagnosed as smooth muscle tumours (benign or malignant, pure or mixed). The immunohistochemical expression of calponin was used to assess the smooth muscle differentiation of the tumours. Nineteen human uterine leiomyomas were also examined. Calponin expression was detected in 89.3% of canine and 100% of human genital tract tumours diagnosed as leiomyomas, as well as in the majority of other tumours examined (canine or human, genital or extragenital, benign or malignant) with the exception of canine negative control tumours (cutaneous fibroma and hepatoid gland adenoma). ERalpha was found in 56.3% of canine and 52.6% of human leiomyomas, while PR was found in 84.4% of canine and 94.7% of human tumours. These results indicate that calponin is a good marker for differentiating neoplasia of the canine genital system of uncertain origin, as in human patients. They also show that canine tumours with smooth muscle differentiation of the genital tract of the bitch express steroid hormone receptors, a finding that opens up the possibility of hormone therapy.

A spontaneous trichoepithelioma occurred in a Swiss OF1 outbred, four-month-old, intact, nulliparous female mouse from a breeding colony. At necropsy, the tumour was a single, well-delineated mass measuring 4.2 cm in major diameter, located in the thoracic region and had an intact haired surface. The regional lymph nodes were not enlarged and no other abnormalities were found. Microscopically, it was composed of a random admixture of budding epithelial islands and cystic structures variable in size. The epithelial islands were composed of basaloid cells. The cystic structures were lined by squamous epithelium with or without a granular cell layer and contained lamellar or amorphous keratin, as well as wide areas of matrical keratinization (ghost cells) with or without a peripheral layer of basaloid cells and calcified contents. Mitotic activity of basaloid cells was moderate to high, but nuclear or mitotic atypia were not observed. High and low molecular weight cytokeratins, profilaggrin and involucrin expression were observed in the tumour. The immunohistochemical profile of this rare type of tumour of the skin of mice, which includes a first-time description of involucrin expression, confirms the histological evidence of differentiation towards more than one segment of follicular epithelium.

Integrins are heterodimeric cell adhesion proteins with major roles in a variety of biological processes ranging from cell migration to tissue organization, immune and non-immune defense mechanisms and oncogenic transformation. Members of the beta(1) integrin subfamily are composed of a beta(1) subunit (CD29) non-covalently associated with different alpha subunits to constitute a group of transmembrane glycoproteins that participate in many physiologically important events. Here, we have studied the CD29 expression in porcine tissues and cells at two different levels: expression of the CD29 mRNA by RT-PCR and localization of the protein by immunohistochemistry. CD29 transcripts were detected in a variety of tissues and cells: platelets, PBMC, granulocytes, alveolar macrophages, smooth muscle, intestine, lung, liver, spleen, lymph node, skin, testis, heart, kidney and bone marrow. Our results suggest that CD29 gene transcription occurs in all organs examined, although with different intensities. The precise localization of CD29 protein in paraffin-embedded tissues was detected by using a specific polyclonal antibody indicating that its expression is limited to smooth muscle, epithelium cells, endothelium of blood vessels and myeloid cells and is no detectable in cells of the lymphoid lineage. The distribution of the CD29 in normal tissues provide insight into the physiological function of the porcine beta(1) integrins and should be of importance in understanding the role of this integrin family in pathological processes.

Immunohistochemical (IHC) HER-2/neu protein overexpression was found in 17.6% of canine mammary gland carcinomas, a percentage similar to that observed in human breast carcinoma, but there was no gene amplification by chromogenic in situ hybridization (CISH). Canine mammary carcinoma would be a suitable natural model of that subset of human breast carcinomas with HER-2 protein overexpression without gene amplification.