*2.9. Effect of the OOPs' Stability in the Calculation of EC50 Values*

A very important observation made in this study pertains to the instability of oleocanthal (**1**) in the cell culture medium. Representative results from the treatment of SK-BR-3 cells for 48 h with two different concentrations of oleocanthal (**1**) added to the cells directly or after a 15 min pre-incubation in a culture medium are shown in Figure 5. Oleocanthal (**1**) was partially deactivated when its addition was delayed by the 15 min pre-incubation step and this was time- and dose-dependent (Figure S3A). The other OOPs tested were not affected similarly (Figure S3B). The low stability of oleocanthal (**1**) in the culture medium resulted in a strong local antiproliferative/cytotoxic effect (i.e., in the vicinity of the positions where it was added) while cells located in the tissue culture wells distant from the site of oleocanthal (**1**) addition were less affected. These results raised questions about the chemical form of the active oleocanthal (**1**) and the ways of handling it in our assays. It was therefore decided for all the experiments presented in this manuscript and the assays performed to calculate the EC50 values to add all the OOPs tested directly to the culture medium. A recent study showed that oleocanthal (**1**) spontaneously reacts with amino acids, with high preferential reactivity to glycine, which is found in abundance in culture media. A glycine derivative with a tetrahydropyridinium skeleton was identified as the product of this reaction and was called oleoglycine [66]. This type of reaction with amino acids or generally peptides and proteins may be one of the reasons for the variations observed in the EC50 values reported for oleocanthal (**1**) by different laboratories for the same cancer cell lines.

**Figure 5.** Partial deactivation of oleocanthal (**1**) in culture medium. SK-BR-3 cells were treated with two different concentrations of oleocanthal (**1**) at two different concentrations (20 and 40 μM) for 48 h. Viable cell numbers were determined using the ATP-based luminescence assay after 48 h of treatment. The compound was added to the cells either directly or after incubation with the culture medium for 15 min. The results presented are from four independent experiments performed in triplicate. Bar graphs represent the mean cell count ± SE in each treatment group normalized to the control group (i.e., cells treated only with 0.2% (*v*/*v*) DMSO). \* *p* < 0.05; \*\* *p* ≤ 0.01 (*t*-test) comparing the two different treatment methods.
