FIGURE 5.

Migration of LNCaP hepsin-overexpressing prostate cancer cells is enhanced on Ln-332. A, to determine the potential biological significance of hepsin cleavage of the Ln-332 β3 chain, we examined the migratory behavior of hepsin-overexpressing LNCaP-34 prostate cancer cells on Ln-332 versus low hepsin-expressing LNCaP-17 cells. Using a modified Boyden chamber assay, we applied either Ln-332 (10 μg/ml) or PBS to transwells, and allowed cells (5 × 10⁴) to migrate at 37 °C for 24 h, as described under “Experimental Procedures.” LNCaP-34 cells exhibited a significant increase in migration on Ln-332 (∼2.1-fold) compared with LNCaP-17 cells on Ln-332 (n = 3, in duplicate; p < 0.01). To confirm that degradation of Ln-332 by cells influenced migration, we also measured both cell lines on PBS-treated inserts. As expected, both clones weakly migrated on PBS-treated transwells (n = 3, in duplicate; p > 0.05). These results suggest that cleavage of Ln-332 by hepsin may physiologically enhance migration. B, Western blot analysis of LNCaP-17 and LNCaP-34 cells, each in the presence or absence of Ln-332, revealed that hepsin-overexpressing cells (LNCaP-34) created an additional band at ∼100 kDa. The bands in lanes 3 and 4 (cells alone) are background, possibly due to endogenous expression of β3 by these LNCaP cells.