Amiodarone’s major metabolite, desethylamiodarone inhibits proliferation of B16-F10 melanoma cells and limits lung metastasis formation in an in vivo experimental model

Abstract

Previously, we demonstrated the in vitro anti-tumor effects of desethylamiodarone (DEA) in bladder and cervix cancer cell lines. In the present study, we intended to establish its potentiality in B16-F10 metastatic melanoma cells in vitro and in vivo. We assessed cell proliferation, apoptosis and cell cycle by using sulforhodamine B assay, Muse™ Annexin V & Dead Cell and Muse® Cell Cycle assays, respectively. We determined colony formation after crystal violet staining. For studying mechanistic aspects, immunoblotting analysis was performed. We used a C57BL/6 experimental lung metastasis model for demonstrating in vivo anti-metastatic potential of DEA. DEA inhibited in vitro proliferation and colony formation, and in vivo lung metastasizing properties of B16-F10 cells. It arrested the cells in G0/G1 phase of their cycle likely via p21 in a p53-dependent fashion, and induced caspase mediated apoptosis likely via inversely regulating Bcl-2 and Bax levels, and reducing Akt and ERK1/2 activation. In this study, we provided in vitro and in vivo experimental evidences for DEA's potentiality in the therapy of metastatic melanomas. Since DEA is the major metabolite of amiodarone, a worldwide used antiarrhythmic drug, safety concerns could be resolved more easily for it than for a novel pharmacological agent.

Involvement of Mitochondrial Mechanisms in the Cytostatic Effect of Desethylamiodarone in B16F10 Melanoma Cells

“…Future studies should seek to identify missing mechanisms. On the other hand, all the aforementioned data are in line with our previous findings on DEA [15][16][17][18], and they account for the time-and concentration-dependent cytostatic effect of DEA treatment (Figure 7). Based on the data presented in this study, it is difficult to propose a mechanism that can explain the said multiple effects on several mitochondrial processes.…”

“…This assay is considered the most suitable for assessing the toxicity of substances in cultured cells, especially when the toxicity affects the mitochondria [31]. In complete agreement with our previous results [17], DEA decreased the viability of B16F10 melanoma cells in a time-and concentration-dependent manner, although at 5 µM and for up to 6 h of incubation, this effect did not reach statistical significance (Figure 7).…”

“…Decreases in the mitochondrial copy number compromise normal mitochondrial function and promote cellular migration, thus contributing to tumor progression [53]. Accordingly, the fragmentation-inducing effect of DEA (Figure 3) may contribute to its cytotoxic properties and ability to limit in vivo metastasis, which we observed previously in cultured bladder and cervix carcinoma and melanoma cells [15][16][17]. Increased mitochondrial fragmentation results from increased fission or decreased fusion.…”

“…In complete agreement with our previous results obtained in isolated liver and heart mitochondria [18], 10 µM of DEA induced a CsA-independent mPT (Figure 5). This likely contributes to DEA's cytotoxicity in cultured bladder carcinoma, cervical carcinoma, and melanoma cells [15][16][17]. Furthermore, DEA definitely compromised IMM integrity, as calcein fluorescence was not quenched in the absence of DEA or Ca 2+ stimulation (data not shown).…”

“…The accumulation of DEA in the myocardium, as well as in lung, liver, thyroid gland, pancreas, and skin, but not in adipose tissue, exceeds that of the parent molecule [9,12,13], and its mean elimination half-life is about 40 days [14]. Based on the tissue accumulation properties and toxic effects of DEA, we previously proposed that the compound has a potential use in cancer therapy and provided experimental evidence for its cytostatic and metastasis-limiting properties in the bladder, cervix, and melanoma cell lines [15][16][17].…”