Characterization of the Eugenol Effects on the Bioenergetic Profile of SCC-4 Human Squamous Cell Carcinoma Cell Line

Abstract

Eugenol (EU), the active ingredient in clove oil, is commonly used as successful therapeutic compound in dentistry due to its antiseptic and anti-inflammatory effects. Recent research studies suggest that eugenol has also a potential anti-cancer effect. This study was thereby purported to assess the effects of EU on the bioenergetic profile of the SCC-4 human squamous cell carcinoma cell line. To this aim, SCC-4 cells were treated for 24 hours with free EU and EU incorporated in polyurethane structures (50 �M each). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF-24e extracellular flux analyzer (Agilent Technologies Inc.). Analysis of the SCC-4 bioenergetic profile was performed in the presence of the classic modulators of the electron transport chain: oligomycin, FCCP, and antimycin A+rotenone. Our data showed that cells stimulated with free EU induced a decrease of OCR linked parameters and an increase of ECAR, effects that were abolished by the incorporation of EU in polyurethane structures. In conclusion, free eugenol elicits inhibitory effects on mitochondrial respiration in the SCC-4 cell line, a result that might be suggestive for its anti-tumoral effects.

Pleiotropic Effects of Eugenol: The Good, the Bad, and the Unknown

“…In line with these observations, we aimed to investigate the effect of eugenol, free, or encapsulated in polyurethane nanoformulations [ 25 ], on both mitochondrial bioenergetics and glycolysis in SCC-4 human squamous cell carcinoma cells by means of the extracellular flux analyzer Seahorse XF24e (Agilent Technologies Inc.). This automatic platform provides a simultaneous measurement of oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, and the extracellular acidification rate (ECAR) as an indirect measurement of anaerobic glycolysis, according to a previously described method [ 116 ]. In brief, cellular metabolic activity was challenged with the classic modulators of the mitochondrial electron transport chain: the first automatic injection was performed using oligomycin (1 μ g/ml), the inhibitor of the mitochondrial ATP synthase; FCCP (3 μ M), a classic uncoupler, was further injected, followed by antimycin A (5 μ M), the inhibitor of mitochondrial complex III.…”

“…OCR was reported in units of pmoles/min and ECAR in mpH/min. SCC-4 human squamous carcinoma cells were incubated for 24 h with eugenol (free or incorporated in polyurethane structures), and we found that free eugenol (50 μ M) induced a decrease of OCR parameters (i.e., inhibition of mitochondrial respiration) coupled with an increase of ECAR (i.e., stimulation of glycolysis); surprisingly, opposite effects were recorded for eugenol nanoformulations, i.e., an increase in basal and maximal respiration (OCR) plus a decrease in glycolysis (ECAR) [ 116 ]. The effects of free eugenol are in line with the abovepresented literature data, yet the paradoxical effect of nanostructures requires further investigations.…”