Fig. 1. Expression of LOX enzymes in colorectal tumour cell lines. Total RNA was isolated from sub confluent cultures of colorectal tumour cell lines and LOX expression determined by RT-PCR. PCR products were analysed on acrylamide gels and semi-quantified relative to GAPDH. The results shown represent the average of 3 experiments ± SEM.

Fig. 2. HETE production by 12(S)-LOX overexpressing cells SW480 cells transfected with a 12(S)-LOX overexpressing construct or a control vector were incubated with 10 μM AA to permit 12(S)-HETE production. Control groups were incubated without AA. The eicosanoid was determined from the culture supernatant by indirect ELISA. (a) 12(S)-LOX dependent 12(S)-HETE production in transiently expressing cells after 24 h of incubation with AA. (b) Media were collected for several days after transfection. (c) 12(S)-LOX dependent 12(S)-HETE production in stably expressing cells after 24 h of incubation with AA. (d) AA incubation was terminated at the times indicated in the figure. (e) CDC was added together with the AA to inhibit 12(S)-LOX and incubation performed for 24 h. The results shown represent the average of 3 independent experiments ± SEM. # increased above control at p < 0.05; * decreased compared to control at p < 0.05.

Fig. 3. Growth inhibition by bioactive plant constituents. Quercetin, kaempferol, baicalein, NDGA, and resveratrol were added to the culture medium of SW480 cells and (a) cell numbers determined by neutral red uptake 48 h after addition. The results shown represent the average of 3 independent experiments ± SEM. (b) Cultures were stained with Hoechst 33285 to visualise nuclear morphology indicative of apoptosis and incidence of apoptotic nuclei counted from 1000 cells each in triplicate cultures.

Fig. 4. 12(S)-LOX-inhibitory activity of bioactive plant constituents 12(S)-LOX overexpressing SW480 cells and control cells were plated at 5 × 10⁴ cells/ well in 24-well plates and 12(S)-HETE production determined after 24 h of incubation with 10 μM AA and increasing concentrations of the inhibitors. Inhibition was calculated relative to the transgene dependent 12(S)-HETE production as described in Materials and Methods. The results shown represent the average of 3 independent experiments ± SEM. * decreased compared to control at p < 0.05.

PCR conditions (supplied by P. Krieg, German Cancer Research Centre, Heidelberg)

Relationship between inhibition of cell growth and 12(S)-LOX activity