The melatonin hormone act in the regulation of immune system, proliferation, differentiation and cellular apoptosis; besides that, it has been shown effective in tumor growth inhibition. The interleukin (IL)-25, secreted by non-malignant mammary epithelial cells, induces apoptosis in neoplastic cells, and has been implicated as a new immunotherapeutic agent for cancer.

The aims were to evaluate the treatment with melatonin and IL-25 in canine mammary tumor cell lines CF-41 and CMT-U229 through the control of cell proliferation and induction of apoptosis by western blotting and immunofluorescence in three-dimensional culture cell. For both tumor cells, there was reduction in cell viability in response to treatment with 1 mM of melatonin. It was observed significantly increased of cleaved caspase-3 in both treatments in CF-41 cells and were evaluated jointly to confirm the capability of the combined action of these agents. There was increase of apoptosis in CMT-U229 cells treated with melatonin. To evaluate apoptosis in spheroid cells, the antibodies cleaved caspase-3 and 9 were labeled and it was found a qualitative increase in both cells; furthermore, an enhanced effect was observed when joining the treatments. The melatonin has proven to be effective in inhibiting tumor growth and there was activation of apoptosis in cancer cells by enhanced caspases. In addition, it is directly related to the modulation of immune activity on the production of cytokines. The IL-25 has shown satisfactory results, indicating its role as breast cancer apoptosis inducer; therefore, our attention is verify the joint action such as cancer therapeutic agents.