Effect of serum starvation and down-regulation of cd146 protein-expression on the cellular life cycle of oral canine melanoma cells.

Abstract

The oral cavity is place of 6% of all tumors in dogs, being the fifth most important localization of neoplasms in this specie. Melanoma is a type of cancer that originates from melanocytes and its establishment involves several stages, until the formation of an invasive and metastatic tumor. The exact molecular mechanisms of the dysregulation in melanoma are yet unknown. Many studies have advanced our knowledge towards understanding the multiple roles of CD146 in biology and pathology and have pointed to the significance of CD146 as a potential diagnostic marker for certain lesions including melanomas. In the present study we examine the impact of different serum starved conditions and reduction of CD146 expression on the cellular life cycle of canine melanoma cell in order to obtain more information about the functional relationship of CD146 and nutritional undersupply on tumor cell proliferation and metastatic behavior. After cell culture, melanoma cells were serum starved and then proliferation assay was performed through ELISA technique. Then, FACS staining could analyze the CD146 down-regulation so the cell cycle could be done. Statistics was performed between the different cell densities, media times and concentrations, using the data obtained from proliferation assay, through procedures of SAS® University Edition. The 5,000 cells per well seeding density had a R2 = 0.70, being the best density to perform the proliferation assay; specific siRNA number 34 proved a reduction in the percentage of cells expressing CD146; and compared to the siRNA scrambled control CD146 silenced cells showed an accumulation of cells within the G0/G1 phase demonstrating an inhibition in cell proliferation and cellular senescence. Further studies are needed to provide additional molecular information about the functional relationship of CD146 and the cell cycle analysis using melanoma cells.