Triflorcas interferes with Met phosphorylation, its cellular localization, and activation of the PI3K/Akt pathway.

<p>(A) Met phosphorylation was analyzed by immuno-cytochemistry on H1437 cells untreated, treated with HGF, with Triflorcas (TFC; 10 µM for 24 hours) or with Triflorcas (10 µM for 24 hours) plus chlorpromazine (Chlor; 10 µg/ml for 2 hours) followed by HGF stimulation (20 ng/ml for 30 minutes). Triflorcas reduced the levels of Met phosphorylation induced by HGF. Note also that HGF-induced phospho-Met is localized at the plasma membrane of control cells, whereas it appears internalized in cells exposed to Triflorcas. Endocytosis inhibition with the chlorpromazine drug restored phospho-Met localization at the cellular membrane. Arrows indicate cluster of phosphorylated Met at the plasma membrane (40X magnification). (B) HGF-induced (20 ng/ml) phosphorylation levels of Met, Gab1, Akt, and p70^S6K were reduced in H1437 cells exposed to Triflorcas. In contrast, no changes were observed on ERKs phosphorylation levels. Similar expression levels of total Gab1, Akt, and p70^S6K were also found, indicating that Triflorcas interfered with their phosphorylation rather than with their expression levels. Western blot analyses were performed on total protein lysates. (C) Phosphorylation levels of Akt, p70^S6K, and S6 ribosomal protein, but not ERKs, were reduced in GTL-16 cells exposed to Triflorcas. Actin or Tubulin protein levels were used as loading controls in all experiments (lower panels in B and C). (D and E) Anchorage-independent growth of H1437 (D) and of GTL-16 (E) cells was impaired in the presence of Triflorcas (TFC), LY294002 (PI3K inhibitor), or A443654 (Akt inhibitor). Values are expressed as means ± s.e.m. **P<0.01; ***P<0.001; Student-<i>t</i> test.