As done in Fig. 2, we investigated the effects of PI3K and Src inhibition on MMP-2 activity and cell invasion. Gelatin zymography unveiled that either LY294002 or PP2 alone, reduced both EGF-stimulated MMP-2 secretion (from 2.1 ± 0.4 fold-increase in pro-MMP-2 for untreated
GSK-923295 to values of 1.5 ± 0.3 and 1.7 ± 0.3 fold-increase, respectively; Fig. 3C and D) and activation (from 3.7 ± 0.6 fold-increase in active-MMP-2 for untreated cells to values of 2.2 ± 0.4 and 2.7 ± 0.4 fold-increase, respectively; Fig. 3C and D), after 24 h incubation in serum free media. Similar results as those observed for NSC23766-treated cells were obtained by treatment with both LY294002 and PP2 together. Incubation of unstimulated and EGF-stimulated
cells with the combination of these inhibitors resulted in decreased pro-MMP-2 secretion (0.6 ± 0.1 and 0.7 ± 0.1 fold-increase, respectively) and lack of MMP-2 activation (Fig. 3C and D). In the invasion assays, we found that either PI3K or Src inhibition alone, only mildly abated EGF-stimulated invasion by PANC-1 cells (from 249 ± 30% for untreated cells to values of 159 ± 22% and 189 ± 26%, respectively; Fig. 3E). However, both unstimulated and EGF-stimulated cells treated with LY294002 and PP2 together showed diminished invasion (71 ± 10% and 74 ± 8%, respectively; Fig. 3E). These results indicate that PI3K and Src synergistically activated Rac1 upon EGF stimulation in PANC-1 cells.
