Figure 5.

Gal and VNPT55 inhibit migration and invasion of PC cells in vitro. (a) To evaluate whether PC-3 and DU145 cells used in migration and invasion assays at indicated compound concentrations did not significantly compromise cell numbers and viability, we performed a 24 h MTT cell viability assay with gal/VNPT55 (1–10 μM. (b) PC-3 (top panel) and DU145 (bottom panel) cultured in 24-well plates to a confluent monolayer were scratched with a 200 μl pipette tip and subsequently treated with indicated compounds for 12 h. (c) Wounds were measured before and after the 12 h time point. Distance migrated were quantified by measuring the difference at time 0 and 12 h and normalized to control. (Distance migrated = Distance at time 0 h - distance at 12 h/Distance migrated by control). Experiments were repeated at least 3 times and represented as mean ± S.E.M, shows significant inhibition of cell migration (**p<0.001) (d) PC-3, DU145 and CWR22Rv1 were seeded in BME pre-coated inserts. Cells treated with gal, VNPT55 and CGP at 5 μM in the upper chamber in serum free RPMI media. The bottom chamber was filled with 1ml RPMI media supplemented with 10% FBS. Set-up was placed in 37 °C incubator for 36 h. Cells were fixed in 3.7% paraformaldehyde for 10 minutes and stained with 0.05% crystal violet; cells in upper chamber were wiped off with cotton swabs and invaded cells at the bottom of inserts analyzed by counting. (e) Schematic illustration of invasion assay set-up, with upper and lower chambers. (f) Quantified invaded cells shows a significant inhibition of PC cell invasion (*p < 0.05, **p<0.001). (g and h) PC-3 cells grown to a monolayer and scratched with 200 μl were treated with EGF (10ng/ml) alone and in combination with gal (5 μM), CGP (5 μM) and U0126 (5 μM). Wound healing was analyzed as in (b and c), gal and CGP significantly inhibited migration in the presence of EGF ligand (*p<0.05). All experiments were repeated at least three times and represented as mean ± S.E.M. (i) To evaluate whether activated MMP-9 could be inhibited by galeterone as in Figure 4g, we treated cells with gal, VNPT55 with or without EGF at (gal-5 μM + EGF-10ng/ml) and incubated for 72 conditioned media was concentrated and separated on zymogram gels. It was observed, just as in Figure 5g, that galeterone even in the presence of EGF, was able to inhibit collagenase activity. Experiments were repeated three times (*p<0.05, **p<0.001)