Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

蒙古马不同组织器官TLR1、TLR2、TLR4和TLR6 mRNA转录水平研究

根据GenBank中马Toll样受体基因序列设计特异性引物,建立检测马Toll样受体（TLRs）mRNA转录水平的SYBR GreenⅠ实时荧光定量PCR方法,检测TLR4、TLR2、TLR1和TLR6在蒙古马不同组织器官中的转录水平。4种TLRs在心脏、肝脏、脾脏、肺脏、肾脏、胃、十二指肠、空肠、盲肠和骨髓中均有转录。其中,TLR4mRNA除在空肠和肝脏外,在其他组织器官中的表达水平均高于TLR2、TLR1、TLR6。免疫器官中,TLR4、TLR1mRNA在骨髓中表达量高于脾脏,而TLR2、TLR6mRNA在脾脏中表达量高于骨髓。各肠段,TLR4、TLR2、TLR1、TLR6mRNA表达水平之间在空肠的差异不是很大,而在十二指肠和盲肠中差异很大。结果表明,TLRs mRNA在马各组织器官转录水平差异较大,可能与其对病原体的识别和抵抗能力有关。     

Effect of cyclooxygenase inhibitors in a xenograft model of canine mammary tumours

Inhibition of cyclooxygenase-2 (COX-2) represents a possible avenue for the prevention and/or treatment of some cancers. Our goal was to compare the effect of a selective inhibitor of COX-2, deracoxib, and a COX-1 and -2 inhibitor, piroxicam, on the growth of canine mammary tumours in a murine model. CMT-9 was used to induce xenografts in nude mice. Mice were treated with piroxicam (0.6 mg kg(-1)), deracoxib (6 mg kg(-1)) or a control solution. Tumour volumes between 0 and 24 days post-treatment showed no significant difference between all groups. A second series of experiments was performed with a higher dose of piroxicam (0.9 mg kg(-1)). Tumour volumes between 14 and 21 days post-treatment were significantly smaller in piroxicam-treated mice compared with controls. These results demonstrate that COX inhibition reduced the growth of canine mammary cancer xenografts in mice, suggesting that COX inhibitors could have a positive effect in dogs.     

Expression and significance of PTEN and VEGF in canine mammary gland tumours

To investigate the relationship between the expression of the PTEN (phosphatase and tensin homolog deleted on chromosometen) and VEGF (vascular endothelial growth factor) and the clinicopathological features in canine mammary gland tumours, the expression levels of PTEN and VEGF protein were assessed in 50 cases of canine mammary gland tumours tissues and 4 cases of normal mammary gland tissues with using immunohistochemical method. The over-expression rate of PTEN protein was 100% in normal and well-differentiated mammary gland tissues and 67% in breast cancer cases respectively with a significant difference between the two groups (P<0.01). Expression of PTEN was not related to age and tumour size, but closely correlated to lymph node metastasis (P<0.01). The over-expression rate of VEGF protein was 33.3% in normal mammary gland tissues, and 78% in canine mammary gland tumours with a significant difference between the two groups (P<0.01).Expression of VEGF was not related to age or tumour size, but closely correlated with lymph node metastasis and clinical stage (P<0.05).Therefore the combination detection of PTEN and VEGF could serve as an important index to estimate the biological behavior and prognosis of canine mammary gland tumours. Reduced expression of PTEN might be involved in carcinogenesis and progression of canine breast cancer by up-regulating the VEGF expression to enhance angiogenesis.     

VEGFR‐2 expression in malignant tumours of the canine mammary gland: a prospective survival study

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is the main receptor activated by vascular endothelial growth factor ‐A (VEGF‐A) to promote tumour angiogenesis. Its clinical prognostic value has not been studied in canine mammary tumours (CMTs). Dogs with mammary cancer were enrolled in a survival study and the immunohistochemical expressions of VEGFR‐2 and VEGF‐A were analysed and associated with clinicopathological features. VEGFR‐2 expression was associated with VEGF immunoreactivity in cancer cells, supporting the presence of an autocrine loop that may be involved in CMTs growth and survival. VEGFR‐2 was also expressed by endothelial cells from tumour vasculature and positively associated with stromal matrix metalloproteinase‐9 (MMP‐9), suggesting the existence of a link between endothelial cells activation and up‐regulation of matrix degrading proteins. Carcinosarcomas exhibited high VEGFR‐2 expression suggesting that it may be one of the activated molecular pathways in this aggressive histological type and that VEGFR‐2 inhibitors may constitute a potential treatment to improve the prognosis of these patients. Both VEGF and VEGFR‐2 immunoreactivities were independent of patients' overall survival (OS) and disease‐free survival (DFS).     

小鼠细胞因子实时定量PCR检测方法的建立及应用

采用荧光SYBR Green I建立小鼠细胞因子mRNA实时定量PCR的检测方法;并利用建立的实时定量PCR的检测方法时感染H5N1禽流感病毒的BALB/c小鼠不同时间采集的肺脏组织中几种重要致炎细胞因子及趋化因子mRNA表达水平进行了检测.对HSN1禽流感病毒感染的BALB/c小鼠肺脏细胞因子的检测具有高度的特异性,检测的灵敏度为10~1～10~2拷贝数.H5N1禽流感病毒感染BALB/c小鼠后,小鼠肺脏中IL-1β、IL-6、TNFα、MIG、IP-10、RANTES、MIF和HMGB1细胞因子mRNA表达水平与时照组小鼠相比均有明显差异.建立的实时定量PCR能在基因转录水平敏感和特异地反应细胞因子的表达水平,该技术在基础和临床免疫研究中,具有良好的应用价值.     

Metallothionein expression in benign and malignant canine mammary gland tumours

The presence of metallothioneins (MTs) were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in canine mammary tumours. In a semiquantitative analysis MT expression in the tumour cells was observed in 54/54 cases of benign and 32/40 malignant mammary neoplasms. A statistically significant difference at the level of P<0.01 was observed for MT expression between benign and malign mammary tumours in terms of immunoreactivity score. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with benign canine mammary tumours.     

Polymorphism of glutathione S-transferase P1 of dogs with mammary tumours

Mammary tumours constitute more than half of neoplasms in female dogs from different countries. Genome sequences are associated with cancer susceptibility but there is little information available about genetic polymorphisms of glutathione S-transferase P1 (GSTP1) in canine cancers. The aim of this study was to find single nucleotide polymorphisms (SNPs) in GSTP1 of dogs (Canis lupus familiaris) with mammary tumours compared to healthy dogs and to determine the association between GSTP1 polymorphisms and the occurrence of these tumours. The study population included 36 client-owned female dogs with mammary tumours and 12 healthy female dogs, with no previous diagnosis of cancer. DNA was extracted from blood and amplified by PCR assay. PCR-products were sequenced by Sanger method and analysed manually. The 33 polymorphisms were found in GSTP1: 1 coding SNP (exon 4), 24 non-coding SNPs (9 in exon 1), 7 deletions and 1 insertion. The 17 polymorphisms have been found in introns 1, 4, 5 and 6. The dogs with mammary tumours have significant difference from healthy in SNPs I4 c.1018 + 123 T > C (OR 13.412, 95%CI 1.574–114.267, P = .001), I5 c.1487 + 27 T > C (OR 10.737, 95%CI 1.260–91.477, P = .004), I5 c.1487 + 842 G > C (OR 4.714, 95% CI 1.086–20.472, P = .046) and I6 c.2481 + 50 A > G (OR 12.000, 95% CI 1.409–102.207, P = .002). SNP E5 c.1487 T > C and I5 c.1487 + 829 delG also differed significantly (P = .03) but not to the confidence interval. The study, for the first time, showed a positive association of SNPs in GSTP1 with mammary tumours of dogs, that can possibly be used to predict the occurrence of this pathology.     

Immunohistochemical study of the expression of E-cadherin in canine mammary tumours

To investigate the possible role of E-cadherin in canine mammary tumours 20 benign and 40 malignant tumours were evaluated by immunohistochemistry in formalin-fixed paraffin wax-embedded samples. In all the benign tumours, E-cadherin was strongly expressed at the intercellular borders of epithelial cells, but it was less strongly expressed in 17 (43 per cent) of the malignant tumours. Furthermore, poorly differentiated carcinomas were less immunoreactive for E-cadherin than moderately and well differentiated carcinomas.     

Xenobiotic-metabolizing enzymes in canine mammary tumours

Mammary tumours are the most common neoplasms in female dogs. The present study was designed to evaluate the relationship between different clinical stages with activities of phase I and phase II carcinogen-metabolizing enzymes in canine mammary tumours. The levels of cytochrome P450 and cytochrome b5 and the activities of glutathione S-transferase (GST), gamma-glutamyl transpeptidase (GGT), DT-diaphorase (DTD) and NADPH diaphorase in tumour tissues of 25 bitches was estimated. Enhanced levels of cytochrome P450 and b5 and phase II enzyme activities were observed in tumour tissues compared to the corresponding uninvolved adjacent tissues. The magnitude of the changes in phase I and phase II enzyme status was, however, more pronounced in stages I and II compared to stages III and IV. The results suggest that the balance between phase I carcinogen activation and phase II detoxification systems may play an important role in canine mammary tumour development.     

The metastatic potential of canine mammary tumours can be assessed by mRNA expression analysis of connective tissue modulators

Metastases are the crucial factor for the prognosis of canine mammary tumours (CMTs). In women, the peptide hormone relaxin is linked with metastatic breast cancer. Therefore, the impact of relaxin and its receptors on matrix metalloproteinase (MMP) expression, metastatic disease and survival was analysed using qRT‐PCR and immunohistochemistry of CMT samples from 59 bitches. The expression of relaxin and its receptor RXFP1 (relaxin family peptide receptor 1) was discovered on gene and protein levels. Intratumoural relaxin mRNA expression and relaxin plasma levels had no prognostic value. High mRNA levels RXFP1 were an independent marker of metastatic potential, with a more than 15‐fold risk increase, and a predictor for shorter survival. Also, MMP‐2 expression was associated with early death because of CMT. The mRNA expressions of relaxin, RXFP1 and MMP‐2 were positively correlated indicating a common pathogenetic linkage. Thus, RXFP1 is proposed as a new early marker of metastatic potential in CMT and a possible therapeutic target.     

Estrogen receptors in canine mammary gland tumours

The aim of the present study was to establish the relationship between estrogen receptor expression in neoplastic parenchymal cells and the type of canine mammary gland tumour. The research material included mammary gland tumours obtained from 66 dogs. Paraffin sections were stained with hematoxylin-eosin and immunohistochemically with monoclonal antibodies and LSAB/Peroxidase/Universal Kit. The estrogen receptor expression was observed in nuclei of neoplastic cells in about 59% of the cases and in the cytoplasm in about 89% of the cases. In about 20% of the cases the expression in the nuclei and in the cytoplasm was extremely weak. No correlation was found between the expression of estrogen receptors and the value of mitotic indexes in the neoplasms investigated.     

VEGF-A、VEGF-C及PLGF基因在奶牛乳腺发育各时期的表达变化

试验旨在研究奶牛VEGF-A、VEGF-C及PLGF基因在乳腺发育各个时期的mRNA表达情况,进而为下一步研究奶牛乳腺发育功能基因奠定基础。采用Real-time PCR SYBR GreenⅠ染料法,所得结果与Affymetrix公司的基因芯片差异筛选结果作比较分析。结果表明,3个基因在乳腺发育11个时期均有表达。VEGF-A基因在妊娠后期及泌乳期表达量较高且与其它时期差异显著(P<0.05);VEGF-C基因在泌乳期表达量较高;PLGF基因的表达量从青春期开始到妊娠期呈递增趋势,到妊娠期4个月达到最高表达量,其后表达量逐渐递减。从以上结果可以得出,VEGF-A及VEGF-C基因可能在奶牛泌乳中发挥作用,而PLGF基因对奶牛乳腺发育起到重要作用,因此通过PCR扩增出PLGF基因的全部CDS序列,为下一步研究PLGF基因功能奠定基础。     

Microtomographic characterization of calcifications in canine mammary tumours

The present work describes the microtomographic characterization of macro‐ and microcalcifications present in excised canine mammary glands. In human breast cancer, microcalcifications are highly relevant for diagnosis and prognosis, often being the sole element determining biopsy. Canine mammary tumours are considered a model for human breast cancer, but the morphological features of calcifications had still to be studied in this species. The objective of this research is to contribute to the characterization of the mineralization features of the canine mammary gland. In the present study, the excised mammary glands of 33 bitches underwent fluoroscopic examination. In 30 of the samples, the presence of calcification was suspected, and multiple biopsies were taken of these areas. Biopsy fragments underwent microtomographic scanning. Microcalcifications were found in non‐neoplastic glandular tissue, benign and malign lesions, as it is known to happen in humans. Qualitative evaluation regarding morphology of the imaged calcifications showed similarities to breast cancer findings, based on the BI‐RADS 2013 classification, such as pleomorphism and shape. No differences in the quantitative morphological parameters of volume, surface, surface/volume, SMI and structure thickness were found when macrocalcifications were considered. However, although significant differences existed in these parameters between microcalcifications from malignant canine mammary tumours and the two other groups, none were found between non‐neoplastic and benign tumours. Findings further support the use of this spontaneous animal model for the study of human breast cancer, considering how clinically relevant microcalcifications are in humans.     

Distribution of nuclear DNA-content in canine mammary tumours

It is well established that nuclear DNA-content contributes valuable information concerning tumour behaviour in a vast number of human neoplasms (compare Auer and Zetterberg, 1984 and Zetterberg and Auer 1984 for review). The authors have attempted for the first time to apply this concept to clinical veterinary medicine and in the present investigation 17 dogs with mammary tumour were studied retrospectively. Microspectro-photometric DNA-measurements were performed and correlated to the clinical course defined as distant recurrence free interval. The results suggest that a relationship exists between nuclear DNA-content of the breast cancer cells and prognosis. Tumours exhibiting DNA-values within the limits of normal tissue correlate with a favourable prognosis in the sense that all dogs with diploid tumour cell DNA-content under study had survived for more than one year without recurrence of the tumour. In contrast 50 per cent of the tumours with increased and scattered DNA-values developed metastasis within one year whereas the other 50 per cent were disease free after this period. It is therefore proposed that cytophotometric analysis of DNA may be used in the assessment of prognosis in dogs with malignant breast tumours.     

p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer‐related signalling pathways. In this context, the deregulated expression of p62 – Sequestosome1 (p62/SQSTM1) – a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62‐based medicine. In veterinary oncology the role of p62 in cancer‐related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non‐neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62‐negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour‐rejection antigen for an anti‐cancer immunotherapy.     

MUC1 expression in canine malignant mammary tumours and relationship to clinicopathological features

Mucin-1 (MUC1) is over-expressed in human breast carcinomas and is linked to a poorer prognosis. In this study, MUC1 expression in 32 spontaneous canine malignant mammary tumours was characterised in relation to histological type, mode of growth, grade, lymph node metastases and distant metastases. All tumours exhibited immunostaining for MUC1. In the normal canine mammary gland, MUC1 was expressed mainly in the apical cellular membrane, while in carcinomas MUC1 was detected in the cytoplasm only (56.3%) or in the cytoplasm with membrane accentuation (43.7%). There was a significant association between development of distant metastases and MUC1 over-expression (P=0.03), but no association with histological type, histological grade, mode of growth or lymph node metastasis.