Sirtuin 1 Expression in Canine Mammary Tumors: A Pilot Study

Abstract

Sirtuin 1 (SIRT1) is a protein involved in aging, cell protection, and energy metabolism in mammals. Recently, SIRT1 has been intensively studied in medical oncology, but the role of SIRT1 is still controversial, as it has been proposed as both an oncogene and a tumor suppressor. The aim of this study is to investigate the expression of SIRT1 by immunohistochemistry in canine mammary tissues, and by Western blot and immunofluorescence analysis in different canine mammary cell lines. Our results showed a decrease in SIRT1 expression from normal mammary gland tissue, and from benign and well-differentiated malignant tumors (G1) to less differentiated ones (G2-G3). Furthermore, a shift in the subcellular localization of SIRT1 from the nucleus to the cytoplasm was observed in less differentiated malignant tumors. However, further studies are needed to investigate the subcellular localization of SIRT1 in canine cancer cells and the role it may play in oncogenesis in animals.

Keywords: canine mammary tumors; culture cells; sirtuin family; veterinary oncology.

Figure 1

In normal canine mammary gland (NMG), case number 2, strong nuclear SIRT 1 protein expression was found in epithelial ductal cells (A). In benign tumor, case number 4, the expression was similar to NMG (B). In G1 carcinomas, case number 8, the intensity of immunostaining was strong and localized in the nuclei (C). A progressive decrease in IRS values and a loss of nuclear localization was observed in G2, case number 6, (D). A further decrease in IRS values and a loss of nuclear localization was observed in G3 case number 7 (E). Scale bar: 50 µm.

Figure 2

The graph shows the mean ± SEM of the immunoreactive (IR) score for the expression of SIRT1 in normal and tumor samples with different degrees of malignancy. * p < 0.05 G1 carcinomas vs NMG; * p < 0.05 G2 carcinomas vs. NMG; *** p < 0.001 G2 carcinomas vs. NMG; NMG, normal mammary gland; G1, grade 1 carcinoma; G2, grade 2 carcinoma; G3, grade 3 carcinoma.

Figure 3

Expression of SIRT1 in CMT cell lines. Western blot analysis of SIRT1 protein expression in different CMT cell lines. The levels of the ß-actin were evaluated to normalize the amount of protein loading. The graphs show the expression of SIRT1 in the CMT cells analyzed.

Figure 4

Absence of SIRT1 is evident in CMT-U309, CF33, and CMT-U131 cell lines. Weak SIRT1 expression, characterized by cytoplasmic small granules, is shown in CMT-U229 and P114. Strong SIRT1 expression is evident as larger cytoplasmic granules in CMT-U27.

Figure 5

CTCF values, calculated by ImageJ software from Figure 4, quantify the protein expression of SIRT1 in all CMT cell lines used.