Possible Mechanism Underlying The Cytotoxic Effect Of Gentisic Acid And Protocatechuic Acid Triphenylphosphonium Derivatives In Human Breast Cancer Cells

Mitochondrion has been recently acknowledged as a potential molecular target in the treatment of cancer, especially considering that such organelle exhibits a significantly higher transmembrane potential (MTP) in cancer cells, and is thus more susceptible to be targeted with lipophilic delocalized cations such as triphenylphosphonium (TPP+) derivatives. Previous work conducted by our laboratory has shown that TPP+-derivatives of gallic acid were highly cytotoxic towards TA3/Ha mouse breast cancer cells, inducing an uncoupling of the oxidative phosphorylationsystem (OXPHOS) (1). Therefore, aiming to further expand our knowledge on the possible mechanisms underlying the cytotoxic effect of TPP+ derivatives, gentisic acid (GA) and protocatechuic acid (PCA), both linked to the TPP+moiety by a ten-atom carbon chain, were evaluated as potential cytotoxic agents in several human breast cancer cell lines (thatdiffer primarily in their expression of estrogen (ER) and epidermal growth factor (HER2) receptors and in their metabolic profile). Indeed, MDA-MB-361 cells (ER+/HER2+), MCF7 cells (ER+/HER2-), AU565 cells (ER-/HER2+) and MDA-MB-231 cells (ER-/HER2-) were all sensitive to the cytotoxic action of the mentioned compounds. Although the three former cell lines showed similar IC50 values (about 3 µM after 48 hours of incubation), MDA-MB-231 cells appeared to be slightly more resistant, showing an IC50 value of near 9 µM after 48 hours of incubation. The apparent difference could be based in the almost completely glycolytic metabolism exhibited by MDA-MB-231 cells, in contrast to a more balanced metabolism (approximately 50% glycolytic/50% oxidative) shown by the other three cell lines. The latter concept is supported by the fact that the mechanism underlying the cytotoxicity of the TPP+-linked compounds seems to be defined by their mitochondrial uncoupling effect (evidenced by an increase in mitochondrial oxygen consumption and a loss of MTP comparable to those induced by 0.5 µM CCCP). Interestingly, while the uncoupling effect of our compounds could result in a drop in cellular ATP levels, we have found that in MCF7 and MDA-MB-231 cells, the incubation with both TPP+-derivatives for 4 hours led to almost negligible losses of ATP. However, when the compounds were added in the presence of either dorsomorphin (AMPK inhibitor) or diadenylate-pentaphosphate (adenylate kinase inhibitor), the levels of cellular ATP fell greatly (down to almost 40% after 4 hours of incubation with a 20 µM concentration). Thus we have hypothesized that the absence of effect of our compounds in cellular ATP levels would be due to a compensatory effect, induced by both AMPK and adenylate kinase, two well-known metabolic-stress sensors. Furthermore, we have determined that our compounds were also able to decrease the migratory capacity of highly metastatic MDA-MB-231 cells (by almost 80% after 72 hours of incubation with 5 µM in a wound healing assay), a process that is widely relying on high ATP levels. Based on our results, we postulate that the GA-TPP+ and PCA-TPP+derivatives uncoupledOXPHOS and decreased MTP, and that such effects might underlie their cytotoxicity towards breast cancer cells, observed regardless of the expression of receptors for growth signals or the metabolic profile of the cell lines.

(1) Jara J et al. (2014). J Med Chem 57: 2440−2454