Cytotoxic Effect of Calcineurin B Against Melanoma and Adenocarcinoma Cells In Vitro.

Chagas disease caused by the obligate intracellular flagellate protozoan infects about 6 million people. From the 1930s to the present, the antitumor capacity of has been studied; however, the identification of the responsible molecules for this effect remains undiscovered. Calcineurin, a calcium/calmodulin-dependent serine/threonine phosphatase, is a heterodimer consisting of a catalytic subunit (CaNA) and a regulatory subunit (CaNB). It has been described that CaN is involved in the cell invasion and proliferation of the parasite. Recently, extracellular human CaNB has been demonstrated to be capable of inhibiting tumor growth cells, conferring an antitumor effect; however, the extracellular role of CaNB (CaNB) is still unknown. The objective of this work was to investigate the antitumor potential of CaNB by interacting with membrane proteins and evaluating its effects on the viability, proliferation, and morphology of tumor cells in vitro. Additionally, the possible mechanism of action of CaNB was explored. Murine melanoma (B16-F10), human cervical adenocarcinoma (HeLa), and African green monkey kidney epithelial (Vero) cell lines were employed for in vitro assays. Far Western blot and immunofluorescence were performed to assess the interaction of CaNB with membrane proteins, and the effect of CaNB on cell viability and proliferation was evaluated using the MTS assay and the CyQUANT NF assay, respectively. The effect of the caspase inhibitor Z-VAD-FMK on CaNB-stimulated tumor cells was investigated to determine if CaNB-induced cell death was associated with apoptosis. To assess cell cycle progression, CaNB-treated cells were analyzed by flow cytometry. In this study, the results showed an interaction of CaNB with cell membrane proteins in B16-F10 and HeLa tumor lines, indicating that CaNB is capable of decreasing viability and proliferation of B16-F10 and HeLa cells, with no significant effect observed in Vero cells. Furthermore, morphological changes were observed in tumor cells treated with TcCaNB. DNA fragmentations and inhibition of caspases with Z-VAD-FMK partially counteracted the cytotoxic effects of CaNB on tumor cells, suggesting that CaNB-induced cell death might be associated with apoptosis. Additionally, CaNB caused S phase cell cycle arrest in HeLa cells, with an increase in the sub-G1 population indicative of apoptosis, while no significant effects were observed in Vero cells.