*2.11. Effect of O2 Concentration on the OOPs' Antiproliferative/Cytotoxic Activity*

The effects of drugs on cancer cell lines are usually tested under atmospheric conditions (20% (*v*/*v*) O2). However, the O2 content in tissues (normoxia) and solid tumors (hypoxia) is ≤ 5% (*v*/*v*). In some tumors (i.e., pancreatic tumors) the % O2 is even ≤ 1% (*v*/*v*) [54,86,87]. This affects cell metabolism and signaling pathways which depend on the hypoxia-induced Factor 1A (HIF1A) and it could affect the activity of certain drugs [59,63,88–90]. To examine if low O2 levels modify the activity of OOPs, oleocanthal (**1**), oleuropein aglycone (**3a,b**) and ligstroside aglycone (**4a,b**) were tested under low O2 concentration (i.e., 1% (*v*/*v*) (hypoxia)) in comparison to 20% (*v*/*v*) (atmospheric O2 levels in tissue culture) at three different concentrations close to the EC50 value for each OOP for the cell lines analyzed (Figure 6). The effects of these three OOPs were tested on the MDA-MB 231, SK-BR-3 and MCF-7 breast cancer and on the AGS stomach cancer cell lines.

O2 concentration did not affect in the same way the antiproliferative/cytotoxic bioactivity of the three OOPs on all four cancer cell lines tested (Figure 6). Treatment with oleuropein or ligstroside aglycones (**3a,b** and **4a,b**) at low oxygen O2 tension (i.e., 1% (*v*/*v*) (hypoxia)) rendered some cell lines more resistant. Ligstroside aglycone (**4a,b**) appeared to be less effective under hypoxic conditions on the AGS stomach cancer cells, while the bioactivity of oleocanthal (**1**) and oleuropein aglycone (**3a,b**) in the same cells was similar. Similarly to AGS cells, the MDA-MB 231 cells in hypoxia were more resistant to treatment with both aglycones ((**3a,b**), (**4a,b**)). Finally, the MCF-7 cells and SK-BR-3 breast cancer cells appeared to respond similarly to treatment with the three OOPs under hypoxic and atmospheric O2 conditions. These results indicated once more the variability observed in the bioactivity of each OOP in each cell line, as described in the previous paragraphs. Overall, oleocanthal (**1**), oleuropein aglycone and ligstroside aglycone (**3a,b** and **4a,b**) were equally or less effective at low oxygen O2 tension (i.e., 1% (*v*/*v*) (hypoxia)) as compared to atmospheric O2 levels. This is an important piece of information for the design of future in vivo experiments aiming at the evaluation of the OOPs' anti-tumor properties.

**Figure 6.** Effect of O2 concentration on the antiproliferative/cytotoxic effect of OOPs. The effect of three different concentrations close to the EC50 value of oleocanthal (**1**), oleuropein aglycone (**3a,b**) and ligstroside aglycone (**4a,b**) was evaluated after 72 h treatment under either 1% (*v*/*v*) O2 or 20% (*v*/*v*) O2. Cell viability was determined on (**A**) AGS, (**B**) MDA-MB 231, (**C**) MCF-7 and (**D**) SK-BR-3 cell lines using ATP-based luminescence assay. The results presented are from two 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; \*\*\* *p* ≤ 0.001 (*t*-test) comparing the two different conditions.
