Figure 6

Etomoxir, an inhibitor of β-oxidation, suppresses hGX-induced LD biogenesis and cell survival. (A, B) Quiescent MDA-MB-231 cells were treated with hGX (10 nM) in serum-free medium containing 0.02% FAF BSA for 96 h in the presence or absence of etomoxir (Eto; 20 μM) and bezafibrate (Bez; 500 μM). Etomoxir, but not bezafibrate, prevented both hGX-induced LD formation (A) and cell survival (B). (C) MDA-MB-231 cells were grown in complete medium for 24 h and then treated with hGX (1 nM) in complete medium for 48 h in the presence or absence of either etomoxir (20 μM) or bezafibrate (500 μM). Etomoxir significantly reduced the level of hGX-induced LDs in proliferating cells, while bezafibrate stimulated control LD formation (#, P < 0.05) and supported hGX-induced LD formation. (D, E) MDA-MB-231 cells were pre-treated with hGX (10 nM) for 48 h in complete medium to form LDs; the cells were then washed (0 h) and incubated in serum-free and hGX-free medium for an additional 48 h in the presence or absence of either etomoxir (250 μM) or bezafibrate (500 μM). The levels of LDs remaining after serum deprivation (48 h) were compared with those at the beginning of starvation (0 h). Both agents significantly prevented LD breakdown during starvation (D). (E, F) MDA-MB-231 cells were treated as described in (D) and the percentage of apoptotic cells determined following 48 h (E) or 96 h (F) serum deprivation. Etomoxir (250 μM) alone significantly increased the percentage of apoptotic cells in samples with preformed LDs, and also in hGX-untreated cells (E). Values on the graphs are means ± SD of at least two experiments performed in duplicate. Results that are statistically significant are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni adjustment).