Ferdowsi University of Mashhad

Document Type : Research Articles


1 Recombinant Proteins Research Group, The Research Institute of Biotechnology Group, Ferdowsi University of Mashhad, Mashhad, Iran

2 School of Biotechnology and Biomolecular Science, University of New South Wales, Sydney, Australia

3 Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

4 School of Medicine and Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast, 4222, Australia

5 Department of Animal Science, Faculty of Agriculture, University of Guilan, Rasht, Iran

6 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran


p53 is a tumor suppressor protein that plays an essential role in controlling the cell and vascular endothelial growth factor (VEGF) is one of the most strong and specific angiogenic factors. The main objective of this study was to evaluate the impact of p53 and VEGF-C gene expression in the neoplastic and normal mammary gland of canine as an animal model. Elleven benign and malignant specimens and 5 normal specimens were collected. After RNA extraction and cDNA synthesis, relative quantification of p53 and VEGF-C genes were accomplished by Real-time quantitative PCR (RT-qPCR) based on use of β-actin as a reference gene. The relative mRNA expression of the p53 and VEGF-C genes were analyzed by GLM procedure of SAS software v9.2. The results indicated that the VEGF-C and p53 mRNA expression in neoplastic specimens was over-and down-expressed respectively as compared with normal specimens and p53 mRNA expression was significantly negatively associated with VEGF-C (~4 fold) in neoplastic specimens (P <0.01). The findings emphasized that simultaneous evaluation of p53 and VEGF-C expression can be used as tumor biomarker for early diagnosis of malignancy in canine. Furthermore, RT-qPCR is a rapid and sensitive method to for monitoring and investigating of suspicious canine at the beginning stage of malignancy and may provide an alternative explanation for deregulated p53 signalling in breast cancer.


Abdelmegeed S.M. and Mohammed S. (2018) Canine mammary tumors as a model for human disease. Oncology Letter 15:6, 8195-8205.
Anadol E., Yar Saglam A.S., Gultiken N., Karakas K., Alcigir E., Alkan H. et al. (2017) Expression of iNOS, COX-2 and VEGF in canine mammary tumours and non-neoplastic mammary glands: Association with clinicopathological features and tumour grade. Acta Veterinaria Hungarica 65:3, 382-393.
Bae S.Y., Nam S.J., Jung Y., Lee S.B., Park B.W., Lim W. et al. (2018) Differences in prognosis and efficacy of chemotherapy by p53 expression in triple-negative breast cancer. Breast Cancer Research and Treatment 172, 437-444.
Bell R., Barraclough R. and Vasieva O. (2017) Gene expression meta-analysis of potential metastatic breast cancer markers. Current Molecular Medicine 17:3, 200-210.
Chen Y., Liu Y., Wang Y., Li W., Wang X., Liu X. et al. (2017) Quantification of STAT3 and VEGF expression for molecular diagnosis of lymph node metastasis in breast cancer. Medicine (Baltimore) 96, e8488.
Dolka I., Krol M. and Sapierzynski R. (2016) Evaluation of apoptosis-associated protein (Bcl-2, Bax, cleaved caspase-3 and p53) expression in canine mammary tumors: An immunohistochemical and prognostic study. Research in Veterinary Science 105, 124-133.
Garcia A.P.V., Reis L.A., Nunes F.C., Longford F.G.J., Frey J.G., de Paula A.M. and Cassali G.D. (2021) Canine mammary cancer tumour behaviour and patient survival time are associated with collagen fibre characteristics. Scientific Reports 11: 5668.
Gasco M., Shami S. and Crook T. (2002) The p53 pathway in breast cancer. Breast Cancer Research 4, 70-76.
Ghoncheh M., Pournamdar Z. and Salehiniya H. (2016) Incidence and mortality and epidemiology of breast cancer in the world. The Asian Pacific Journal of Cancer Prevention 17, 43-46.
Goldschmidt M., Pena L., Rasotto R. and Zappulli V. (2011) Classification and grading of canine mammary tumors. Veterinary Pathology 48:1, 117-131.
Howard E.M., Lau S.K., Lyles R.H., Birdsong G.G., Tadros T.S., Umbreit J.N. et al. (2004) Correlation and expression of p53, HER-2, vascular endothelial growth factor (VEGF), and e-cadherin in a high-risk breast-cancer population. International Journal of Clinical Oncology 9, 154-160.
Iovino F., Ferraraccio F., Orditura M., Antoniol G., Morgillo F., Cascone T. et al. (2008) Serum vascular endothelial growth factor (VEGF) levels correlate with tumor VEGF and p53 overexpression in endocrine positive primary breast cancer. Cancer Investigation 26:3, 250-255.
Karpanen T., Egeblad M., Karkkainen M.J., Kubo H., Yla-Herttuala S., Jaattela M. et al. (2001) Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Research 61:5, 1786-1790.
Kaszak I., Ruszczak A., Kanafa S., Kacprzak K., Krol M. and Jurka P. (2018) Current biomarkers of canine mammary tumors. Acta Veterinaria Scandinavica 60: 66.
Kato Y., Asano K., Mogi T., Kutara K., Teshima K., Edamura K. et al. (2007) Clinical significance of circulating vascular endothelial growth factor in dogs with mammary gland tumors. Journal of Veterinary Medicine Science 69:1, 77-80.
Klopfleisch R. and Gruber, A.D. (2009) Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands. Research in Veterinary Science 87:1, 91-96.
Lee C.H., Kim W.H., Lim J.H., Kang M.S., Kim D.Y. and Kweon O.K. (2004) Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors. Journal of Veterinary Science 5:1, 63-69.
Lee C.H. and Kweon, O.K. (2002) Mutations of p53 tumor suppressor gene in spontaneous canine mammary tumors. Journal of Veterinary Science 3: 4, 321-325.
Levine A.J. (2019) The many faces of p53: something for everyone. Journal of Molecular Cell Biology 11:7, 524-530.
Li X., Dang X. and Sun X. (2012) Expression of survivin and VEGF-C in breast cancer tissue and its relation to lymphatic metastasis. European Journal of Gynaecological Oncology 33: 2, 178-182.
Liang B. and Li Y. (2014) Prognostic significance of VEGF-C expression in patients with breast cancer: A meta-analysis. Iranian Journal of Public Health 43: 2, 128-135.
Linderholm B., Lindh B., Tavelin B., Grankvist K. and Henriksson R. (2000) p53 and vascular-endothelial-growth-factor (VEGF) expression predicts outcome in 833 patients with primary breast carcinoma. International Journal of Cancer 89: 51-62.
Linderholm B.K., Lindahl T., Holmberg L., Klaar S., Lennerstrand J., Henriksson R. et al. (2001) The expression of vascular endothelial growth factor correlates with mutant p53 and poor prognosis in human breast cancer. Cancer Research 61:5, 2256-2260.
Lu X., Gu Y., Ding Y., Song W., Mao J., Tan J. et al. (2008) Correlation of ER, PgR, HER-2/neu, p53, and VEGF with clinical characteristics and prognosis in Chinese women with invasive breast cancer. The Breast Journal 14: 308-310.
Lüder Ripoli F., Conradine Hammer S., Mohr A., Willenbrock S., Hewicker-Trautwein M., Brenig B. et al. (2016) Multiplex gene expression profiling of 16 target genes in neoplastic and non-Neoplastic canine mammary tissues using branched-DNA assay. International Journal of Molecular Sciences 17, 9.
Ma H., Wang Y., Sullivan-Halley J., Weiss L., Marchbanks P.A., Spirtas R. et al. (2010) Use of four biomarkers to evaluate the risk of breast cancer subtypes in the women's contraceptive and reproductive experiences study. Cancer Research 70:2, 575-587.
Millanta F., Caneschi V., Ressel L., Citi S. and Poli A. (2010) Expression of vascular endothelial growth factor in canine inflammatory and non-inflammatory mammary carcinoma. The Journal of Comparative Pathology 142: 36-42.
Mohammed R.A., Green A., El-Shikh S., Paish E.C., Ellis I.O. and Martin S.G. (2007) Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio- and lymphangiogenesis. British Journal of Cancer 96: 1092-1100.
Muto T., Wakui S., Takahashi H., Maekawa S., Masaoka T., Ushigome S. et al. (2000) p53 gene mutations occurring in spontaneous benign and malignant mammary tumors of the dog. Veterinary Pathology 37:3, 248-253.
Noranizah W., Siti-Aishah M.A., Munirah M.A., Norazlin M.H., Rohaizak M., Naqiyah I., Sharifah N.A. et al. (2010) Immunohistochemical expression of vascular endothelial growth factor (VEGF) and p53 in breast lesions. Clinical Therapeutics 161:2, 129-137.
Pena L., Gama A., Goldschmidt M.H., Abadie J., Benazzi C., Castagnaro M. et al. (2014) Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry. Veterinary Pathology 51: 127-145.
Pfaffl M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29: 9, e45.
Qiu C., Lin D., Wang J. and Wang L. (2008a) Expression and significance of PTEN in canine mammary gland tumours. Research Veterinary Science 85:2, 383-388.
Qiu C., Lin D.D., Wang H.H., Qiao C.H., Wang J., Zhang T. (2008b) Quantification of VEGF-C expression in canine mammary tumours. Australian Veterinary Journal 86:7, 279-282.
Qiu C.W., Lin D.G., Wang J.Q., Li C.Y., Deng G.Z. (2008c) Expression and significance of PTEN and VEGF in canine mammary gland tumours. Veterinary Research Communications 32:6, 463-472.
Queiroga F.L., Pires I., Parente M., Gregorio H., Lopes C.S. (2011) COX-2 over-expression correlates with VEGF and tumour angiogenesis in canine mammary cancer. Veterinary Journal 189:1, 77-82.
Raposo T.P., Arias-Pulido H., Chaher N., Fiering S.N., Argyle D.J., Prada J. et al. (2017) Comparative aspects of canine and human inflammatory breast cancer. Seminars in Oncology 44: 4, 288-300.
Saba C.F., Rogers K.S., Newman S.J., Mauldin G.E. and Vail D.M. (2007) Mammary gland tumors in male dogs. Journal of Veterinary Internal Medicine 21: 5, 1056-1059.
Santos A.A., Oliveira J.T., Lopes C.C., Amorim I.F., Vicente C.M., Gartner F.R. et al. (2010) Immunohistochemical expression of vascular endothelial growth factor in canine mammary tumours. The Journal of Comparative Pathology 143: 4, 268-275.
Sorenmo K.U., Rasotto R., Zappulli V. and Goldschmidt M.H. (2011) Development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms. Veterinary Pathology 48: 85-97.
Thammineni K.L., Thakur G.K., Kaur N. and Banerjee B.D. (2019) Significance of MMP-9 and VEGF-C expression in North Indian women with breast cancer diagnosis. Molecular and Cellular Biochemistry 457:1-2, 93-103.
Veldhoen N., Watterson J., Brash M., Milner J. (1999) Identification of tumour-associated and germ line p53 mutations in canine mammary cancer. British Journal of Cancer 81: 3, 409-415.
Visan S., Balacescu O., Berindan-Neagoe I. and Catoi C. (2016) In vitro comparative models for canine and human breast cancers. Clujul Medical 89: 38-49.
Wang L. (2017) Early diagnosis of breast cancer. Sensors17:7, 1572.
Wijnhoven S.W., Zwart E., Speksnijder E.N., Beems R.B., Olive K.P., Tuveson D.A. et al. (2005) Mice expressing a mammary gland-specific R270H mutation in the p53 tumor suppressor gene mimic human breast cancer development. Cancer Research 65: 8166-8173.
Yang P., Du C.W., Kwan M., Liang S.X. and Zhang, G.J. (2013) The impact of p53 in predicting clinical outcome of breast cancer patients with visceral metastasis. Scientific Reports 3, 2246.
Zajkowska M., Glazewska E.K., Bedkowska G.E., Chorazy P., Szmitkowski M. and Lawicki S. (2016) Diagnostic power of vascular endothelial growth factor and macrophage colony-stimulating factor in breast cancer patients based on ROC analysis. Mediators of Inflammation 2016: 5962946.
Zhang J., Chen X., Kent M.S., Rodriguez C.O. and Chen X. (2009) Establishment of a dog model for the p53 family pathway and identification of a novel isoform of p21 cyclin-dependent kinase inhibitor. Molecular Cancer Research 7: 67-78.