Figure 3.

Linoleic acid (LA) and eicosapentaenoic acid (EPA) inhibit hypoxia-induced Zeb1 and SK3 expression. (A,B) FA effects on hypoxia-induced Zeb1 and SK3 expression. PC3 cells were treated with FAs for 48 h and the last 24 h cultured under hypoxia (1% O₂). qPCR results are expressed in 2^−ΔΔCt, (N = 3; n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; Kruskal–Wallis, post-test: Dunn’s test). (C) Zeb1 expression in human prostate cancer slices (Scale = 100 µm). Organotypic cultures were treated with FAs (60 µM) and cultured under normoxia for 24 h and then were cultured (with FAs) under hypoxia (1% O₂) for another 24 h; Zeb1 nuclear expression increased under hypoxia conditions. After palmitic acid (PA) treatment, most of the cancer cells (present in the upper right area) were still positive for Zeb1, whereas after treatment with either LA or EPA, only rare Zeb1 positive cancer cells were observed. Five slices were obtained for each patient (N = 4 patients) to test the different conditions. (D) Proposed schematic model for a positive feedback loop leading to prostate cancer (PCa) aggressiveness and inhibited by LA and EPA based on our results. In fact, hypoxia induces the transcription factor Zeb1 known to be regulated by calcium. Zeb1 targets the KCNN3 gene encoding for SK3. At the plasma membrane, the SK3 channel allows an increase in calcium entry by hyperpolarization of the plasma membrane. By incorporating Ohmline, LA, and EPA into the membrane, we observed that they inhibit this signaling pathway induced by hypoxia.