For these reasons molecules of the ET family

For these reasons, molecules of the ET family and in particular ET-1 are considered useful factors in the evaluation of malignancy and in consequence, in the prognosis for several forms of neoplasia, such as ovarian (Bagnato et al., 2005), prostate (Nelson et al, 1995, Nelson et al, 1996), colorectal (Asham et al., 2001) and mammary tumours (Alanen et al, 2000, Kojima, Nihei, 1995). To the best of our knowledge, little is known about the role ETs play in canine neoplasms (Borzacchiello et al., 2010). The aim of the present study was, on one hand, to evaluate the expression of ET-1 and ETAR in normal and in neoplastic canine mammary tissue by immunohistochemistry, double immunofluorescence staining and western blot analysis, and, on the other, to correlate results with the histological grade of those tumours.
Materials and methods
Results
Discussion The “angiogenic switch”, by which neoplastic sphingosine 1 phosphate receptor acquire the ability to provide for their own oxygen and their nutrient requirements, is a key event in malignant transformation and in tumour growth (Hanahan and Folkman, 1996). This vasculogenic potential has already been studied in several spontaneous tumours in animals (Maiolino et al, 2001, Queiroga et al, 2011, Restucci et al, 2000, Restucci et al, 2002, Sleeckx et al, 2014, Wolfesberger et al, 2008). In this study we found a significant increase of ET-1 and its receptor (ETAR) expression in G1 and G2 mammary carcinomas both by immunohistochemical staining and western blot analysis; this demonstrates an involvement of this protein and its receptor in the early stages of malignant cell transformation and tumour progression when the intensity of cell proliferation and metabolic demands strongly increase. It has been shown previously that ET-1 is involved in neoplastic proliferation, activating a network of signals that results in the transfer of a mitogen signal to the nucleus, promoting endothelial cell growth (Nelson et al., 2003) which is the first step of the angiogenetic process. It has also been demonstrated in some tumours, such as in prostate, ovarian and cervical cancer (Bagnato, Rosanò, 2008, Bagnato et al, 1997, Bagnato et al, 1999, Nelson et al, 1995, Nelson et al, 1996, Venuti et al, 2000), that the spontaneous growth of malignant cells was inhibited in the presence of an ETAR receptor antagonists, which is in further support of ET-1 stimulating cell proliferation, by acting through an autocrine mechanism (Bhalla et al., 2009). Therefore, the ETAR receptor expression in mammary gland tumours and its progressive intensification in respect to malignant progression, as observed in our set of canine mammary tumours, further confirm this mechanism. In addition, the interaction ET-1/ETAR increases the expression of matrix metalloproteinases (MMPs), modulating the invasive capacity of some tumour cells (Rosanò et al., 2001) and can phosphorylate connexin 43, thus altering the gap junction intercellular connections (GJIC) (Spinella et al., 2003). Destabilization of GJIC has been associated with decrease in intercellular communication and the acquisition of a local invasive and metastatic phenotype in human (Carystinos et al., 2002) and animal tumours (Torres et al., 2005). An increased ET-1/ETAR expression is positively correlated with vascular endothelial growth factor expression (VEGF) and with angiogenesis in different malignancies including those of human (Wulfing et al., 2003) and canine mammary gland tumours (Martano et al., 2008). In particular ET-1 can stimulate endothelial cell proliferation and can induce the production of VEGF (Salani et al., 2000), which in turn stimulates angiogenesis and tumour growth, respectively, through paracrine and autocrine mechanisms (Restucci et al., 2004). In human ovarian neoplasia a correlation between the increase of ET-1 and cyclooxygenase-2 (COX-2) was also shown (Spinella et al., 2004). COX-2 induces the synthesis of PGE2 and VEGF, confirming both, the link between ET-1 and VEGF, and the involvement of ET-1 in neoplastic angiogenesis.