Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line

Abstract

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 μM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.

In Vitro Insights Regarding the Role of Melanin in Melanoma Cells’ Response to Betulinic Acid Treatment

“…The current results indicate the following: (i) BA at low concentrations (< 50 µM) exerts a selective cytotoxicity (HDFa cells' viability was not affected, while in melanoma cells was significantly decreased -Figures 2, 4 and 6), still by increasing the concentration the selectivity is lost; and (ii) BA treatment proved to be highly cytotoxic on both pigmented (B16-F10) and non-pigmented (SK-MEL-5) cells (with a higher susceptibility recorded in the case of SK-MEL-5) by decreasing cells' viability percentages and by inducing morphological changes in melanoma cells shape, changes that indicate an apoptosis-like effect. The selectivity of BA towards cancer cells and a lack/a very low toxicity against healthy cells was also described in previous studies [21,31].…”

“…In another study developed by Farcas et al, it has been revealed that BA decreases the viability of B164A5 and A375 MM cells at the concentrations of 5 and 25 µM following 24 and 48 h [34]. In a recent study by Coricovac et al, BA is shown to induce a dose-dependent cytotoxicity in A375 MM cells after 24 h, accompanied by apoptotic nuclear features (shrinkage, chromatin condensation), up-regulation of pro-apoptotic markers (Bax, Bad, and Bak), inhibition of mitochondrial respiration and glycolysis, and decrease in the mitochondrial membrane potential [21]. A very recent study showed that BA proved to be very active against equine melanoma cells [35].…”

“…BA, the pentacyclic triterpene also known as "mitocan" due to its capacity to target mitochondria in tumour cells, was highly investigated as an antitumour agent [21]. This natural compound was initially described by Pisha et al as an antimelanoma agent and further studies proved its antitumour properties against multiple types of cancer [22].…”

“…Previous in vitro studies directed the utilization of BA towards the treatment of MM [23]. Our previous work established several new potential antimelanoma mechanisms of action for BA as antimetastatic agent by interfering with the epithelialto-mesenchymal transition [30] and by impairing mitochondrial metabolism of melanoma cells [21].…”

“…In order to verify the impact of BA solution on both healthy (HDFa) and melanoma cells' (SK-MEL-5 and B16-F10) viability it was applied the MTT technique, a standardized assay to determine the percentage of viability cells following exposure to a specific treatment. The protocol applied was previously described [21,32] and was adapted to our laboratory facilities. In brief, the following steps were performed: (i) seeding of 10 4 cells/well/200 µL culture medium in 96-well plates;…”