Fig. 2.

n-3 PUFAs inhibit growth by inducing apoptosis and inhibiting cell proliferation.

Annexin-V assays showing the effect of (a) DHA and (b) EPA on apoptosis 48 h after adding doses of 5 and 10 µM to normal (HEK27), premalignant (SVHFK) and malignant (SCC-13 and SCC-25) keratinocytes. Early apoptotic cells are indicated by the black part of the bar, and the late apoptotic and necrotic cells (Annexin-V +ve/DAPI +ve) by the white part of the bar.

The results in (a) and (b) are the means of three experiments ± SEM.

Significantly different from the mean value of normal keratinocytes [*P < 0.05; **P < 0.01; ***P < 0.001 as measured by one-way ANOVA followed by post hoc Bonferroni test].

(c) Western blot analysis of caspases 3, 8 and 9 in SCC-25 cells treated for 5 h with DHA and 17 h with EPA 10 µM compared with the ethanol vehicle control. C = cleaved caspase; T = total caspase; GAPDH is the loading control. The positive control was an extract from normal keratinocytes NHEK treated with cisplatin for 24 h. The blots are representative of three independent experiments.

(d) ³H-thymidine incorporation assay performed on SCC-25 cells treated with a dose of 3 µM DHA (white bars) and EPA (hatched bars) for 48 h compared with the vehicle control (black bars). The results are the means of six independent experiments ± SEM. SCC-25 cells were incubated in 3 µM DHA and EPA for 48 h in keratinocyte growth medium serum-free medium and ³H-thymidine was added 18h prior to the incorporated thymidine measurement. The values were normalized to a percentage of the untreated control, which was taken as 100%. Significantly different from the mean value of the untreated control (***P < 0.001 as measured by one-way ANOVA followed by post hoc Bonferroni test).