journal homepage: www.elsevier.com/locate/yexcr Available online at www.sciencedirect.com Research Article Histopathological and in vivo evidence of regucalcin as a protective molecule in mammary gland carcinogenesis Ricardo Marques a , Cátia V. Vaz a , Cláudio J. Maia a , Madalena Gomes b , Adelina Gama c , Gilberto Alves a , Cecília R. Santos a , Fernando Schmitt b,d,e,f , Sílvia Socorro a,n a CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal b IPATIMUP, Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal c Department of Veterinary Sciences, Animal and Veterinary Science Research Center (CECAV), University of Trás-os-Montes and Alto Douro (UTAD), Portugal d Medical Faculty, University of Porto, Porto, Portugal e Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Canada f Department of Pathology, University Health Network, Toronto, Canada articleinformation Article Chronology: Received 20 June 2014 Received in revised form 30 July 2014 Accepted 2 August 2014 Keywords: Apoptosis Breast cancer Carcinogen DMBA Mammary gland Regucalcin abstract Regucalcin (RGN) is a calcium-binding protein, which has been shown to be underexpressed in cancer cases. This study aimed to determine the association of RGN expression with clinico- pathological parameters of human breast cancer. In addition, the role of RGN in malignancy of mammary gland using transgenic rats overexpressing the protein (Tg-RGN) was investigated. Wild-type (Wt) and Tg-RGN rats were treated with 7,12-dimethylbenz[α]anthracene (DMBA). Carcinogen-induced tumors were histologically classified and the Ki67 proliferation index was estimated. Immunohistochemistry analysis showed that RGN immunoreactivity was negatively correlated with the histological grade of breast infiltrating ductal carcinoma suggesting that progression of breast cancer is associated with loss of RGN. Tg-RGN rats displayed lower incidence of carcinogen-induced mammary gland tumors, as well as lower incidence of invasive forms. Moreover, higher proliferation was observed in non-invasive tumors of Wt animals comparatively with Tg-RGN. Overexpression of RGN was associated with diminished expression of cell-cycle inhibitors and increased expression of apoptosis inducers. Augmented activity of apoptosis effector caspase-3 was found in the mammary gland of Tg-RGN. RGN overexpression protected from carcinogen-induced mammary gland tumor development and was linked with reduced proliferation and increased apoptosis. These findings indicated the protective role of RGN in the carcinogenesis of mammary gland. & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2014.08.007 0014-4827/& 2014 Elsevier Inc. All rights reserved. n Correspondence to: CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Fax: þ351 275 329 099. E-mail address: ssocorro@fcsaude.ubi.pt (S. Socorro). EXPERIMENTAL CELL RESEARCH ] ( ]]]] ) ]]] – ]]] Please cite this article as: R. Marques, et al., Histopathological and in vivo evidence of regucalcin as a protective molecule in mammary gland carcinogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.08.007