Abstract
To glean insights into the mechanism of their action, we assessed the effects of two flavonoids, quercetin (Qu) and luteolin (Lu), on the growth and epidermal growth factor receptor (EGFR) tyrosine kinase activity of MiaPaCa-2 cancer cells. Exposure of these EGFR-expressing cells to 20 microM Qu or Lu resulted in concomitant decreases in cellular protein phosphorylation and growth. On the cellular level, Qu and Lu sensitivity correlated with EGFR levels and rapid cell proliferation, indicating the possibility of targeting those cells most prone to neoplastic progression. Cell treatment with the flavonoids markedly diminished the extent of cellular protein phosphorylation, by effectively modulating protein tyrosine kinase (PTK) activities, including that of EGFR. Immunocomplex kinase assay revealed that both Qu and Lu inhibited the PTK activities responsible for the autophosphorylation of EGFR as well as for the transphosphorylation of enolase. Treatment of the cells with Qu or Lu also reduced the phosphotyrosyl levels of 170-, 125-, 110-, 65-, 60-, 44-, 30- and 25-kDa proteins. We identified the 170-kDa phosphotyrosylprotein as EGFR. Qu and Lu exhibited a specific action in hampering the levels of phosphorylation of this and the aforementioned proteins, while having no discernible effect on their synthesis. A time-dependent attenuation of the phosphorylation of the above proteins was demonstrable. Treatment of the cells with Qu or Lu for 6 hours showed little inhibition, but prolonging the cell treatment for 24 hours caused the suppression of phosphorylation. Further continuation of the cell treatment culminated in the induction of apoptosis, characteristically exhibiting shrinkage of the cell morphology, DNA fragmentation and poly(ADP-ribose)polymerase (PARP) degradation. The onset of apoptosis and associated events occurred in a time-dependent fashion. The data clearly demonstrate that MiaPaCa-2 cells respond to Qu and Lu by a parallel reduction in cellular protein phosphorylation and cellular proliferation. The flavonoid-evoked attenuation of the phosphorylation of EFGR and of other proteins appeared to be transient, since removal of the flavonoid from the cell growth medium after 24 hours of incubation followed by exposure to 10 nm EGF, restored protein phosphorylation and cellular proliferation. Such an addition of EGF was also able to reverse Qu- or Lu-induced cell growth inhibition and diminish nuclear digestion evoked by 20 microM Qu or Lu. Both Qu and Lu were able to reverse the effect of EGF biochemically as well as functionally. Based on the evidence accrued, the above proteins could be implicated in growth signal transduction and the subtle changes in their phosphorylation, as effected by flavonoids, utilized as a reliable guide to predict growth response. The antiproliferative effect of flavonoids might result, at least in part, from the modulation of the EGF-mediated signaling pathway. The results indicate that the blockade of the EGFR-signaling pathway by the PTK inhibitors Qu and Lu significantly inhibits the growth of MiaPaCa-2 cells and induces apoptosis. The modulation of EGFR kinase appears to be a critically important, intrinsic component of Qu- and Lu-induced growth suppression, even though other mechanisms could also have contributed to the net effect.
