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

Abstract

Previously, we showed that desethylamiodarone (DEA), a major metabolite of the widely used antiarrhythmic drug amiodarone, has direct mitochondrial effects. We hypothesized that these effects account for its observed cytotoxic properties and ability to limit in vivo metastasis. Accordingly, we examined DEA’s rapid (3–12 h) cytotoxicity and its early (3–6 h) effects on various mitochondrial processes in B16F10 melanoma cells. DEA did not affect cellular oxygen radical formation, as determined using two fluorescent dyes. However, it did decrease the mitochondrial transmembrane potential, as assessed by JC-1 dye and fluorescence microscopy. It also induced mitochondrial fragmentation, as visualized by confocal fluorescence microscopy. DEA decreased maximal respiration, ATP production, coupling efficiency, glycolysis, and non-mitochondrial oxygen consumption measured by a Seahorse cellular energy metabolism analyzer. In addition, it induced a cyclosporine A–independent mitochondrial permeability transition, as determined by Co2+-mediated calcein fluorescence quenching measured using a high-content imaging system. DEA also caused outer mitochondrial membrane permeabilization, as assessed by the immunoblot analysis of cytochrome C, apoptosis inducing factor, Akt, phospho-Akt, Bad, and phospho-Bad. All of these data supported our initial hypothesis.

Involvement of Mitochondrial Mechanisms and Cyclooxygenase-2 Activation in the Effect of Desethylamiodarone on 4T1 Triple-Negative Breast Cancer Line

“…The MCF-7 and 4T1 cells were treated with 0, 5, or 10 μM of DEA for 3 h before the assessment of mitochondrial fragmentation. Similarly, as it did in melanoma cells previously [34], DEA treatment caused mitochondrial fragmentation in both BC cell lines in a concentration-dependent manner (Figure 6A). Figure 6.…”

“…Previously, we reported DEA's cytotoxicity in the bladder, cervix, and melanoma cell lines and its metastasis-limiting properties in a rodent lung metastasis model [31][32][33]. Furthermore, by using the B16F10 melanoma cell line, we demonstrated that DEA affected mitochondrial processes in live cells [34] similarly as it did previously in isolated liver and heart mitochondria [35]. A uniform anti-neoplastic effect on three very different, highly proliferative cell lines indicated that DEA interferes with fundamental survival-promoting processes likely present in most cancer cell lines of different tissue and species of origin.…”

“…DEA induced fragmentation of the mitochondria in both BC cell lines (Figure 6) that could contribute to its anti-neoplastic properties. Since DEA depolarized the ΔΨm (Figure 5), it seems logical that the DEA-induced mitochondrial fragmentation was caused by inhibiting fusion that was supported by the release of OPA1 in the case of B16F10 melanoma previously [34]. In contrast, in the BC cell lines, mitochondrial fragmentation seemed to be caused by promoting fission, as it was supported by the increased Drp1 expression that was accompanied by its decreased inhibitory phosphorylation (Figure 6).…”

“…They have to fine-tune their metabolism to meet this challenge [52,56], which makes them vulnerable against drugs that interfere with their metabolism [52]. DEA may represent such a drug candidate, since it decreased the ΔΨm in a concentrationdependent manner in both BC cell lines (Figure 5) similarly as it did in isolated liver and rat mitochondria [35] and in the B16F10 melanoma line [34].…”

“…Accordingly, drugs that significantly interfere with mitochondrial energy production may have therapeutic value in these tumors [50]. Based on its mitochondrial effects in B16F10 melanoma cells [34], DEA fulfills this criterion. Furthermore, and in agreement with our previous results on T24 bladder and HeLa cervix carcinomas and B16F10 melanoma [31][32][33], DEA at low μM concentrations reduced the viability of the BC cell lines MCF-7 and 4T1 in a concentration-and time-dependent manner (Figure 8A).…”