MPX INDUCES NECROSIS IN MELANOMA, BUT NOT IN MELANOCYTES

Cationic peptides can play an important role in addressing the issue of anticancer therapies. MPX, a mastoparan cationic peptide, is composed of 14 amino acid residues, three of which are basic, assigning it a positive residual net electric charge. This structural feature may change the way this peptide interacts with different surfaces. Given the neutral and anionic nature, respectively, of healthy and cancerous cells’ plasma membranes, we investigated the cytotoxic, metabolic, and mechanistic effects of the MPX peptide on healthy (Melan-a) and cancerous (B16-F10) cells. A dose-response curve was obtained by MTT assays employed to test cell viability following treatment with a variety of concentrations for two hours. Although toxic for both cell lines, MPX is selective, achieving its half-maximum response at approximately 20 µM for B16-F10 and 40 µM for Melan-a. Fluorescence microscopy was employed to allow visualization of the process and further investigation of this selectivity. Metabolic aspects were monitored by the quantification of the mean fluorescence intensity of the fluorophores TMRE and Fluo-4. After treatment of both cell lines individually with 20 µM MPX, mitochondrial potential has decreased in both of them, but more pronouncedly in B16-F10. Intracellular calcium level, on the other hand, has increased in B16-F10 and decreased in Melan-a. The membrane modification, triggered by MPX action, was then visualized with the fluorophores Annexin V-FITC and Propidium Iodide (PI), which denote, respectively, phosphatidylserine exposure and pore formation. Both cell lines treated with 20 µM MPX were equally stained with Annexin V-FITC, indicating that there was a common phosphatidylserine exposure in their plasma membrane. However, this treatment resulted in almost complete staining of the B16-F10 cell line with PI, while only 25% staining of Melan-a. This data suggests that the main mechanism of cell death involved in MPX treatment is necrosis, but somehow this process is only fully developed in the cancerous cell lines. This may occur because of the stronger electrostatic attraction between MPX and the anionic membrane of B16-F10. These findings strengthen the antitumoral selective action of mastoparan peptides and provide insights into their mechanism of action. This work was supported by CAPES.