Comparison of paclitaxel-, 5-fluoro-2 '-deoxyuridine-, and epidermal growth factor (EGF)-induced apoptosis - Evidence for EGF-induced anoikis

Epidermal growth factor (EGF), a hormone that stimulates proliferation of m any cell types, induces apoptosis in some cell lines that overexpress the E GF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined by microscopy, flow cytometry, immunoblo tting, enzyme assays, and affinity labeling after treatment with EGF, pacli taxel, or 5-fluoro-2'-deoxyuridine (5FUdR). Apoptosis induced by all three agents was accompanied by activation of caspases-3, -6, and -7, as indicate d by disappearance of the corresponding zymogens from immunoblots, cleavage of substrate polypeptides in situ, and detection of active forms of these caspases in cytosol and nuclei using fluorogenic assays and affinity labeli ng. Further analysis indicated involvement of the cytochrome c/Apaf-1/caspa se-9 pathway of caspase activation, but not the Fas/Fas ligand pathway. Int erestingly, caspase activation was consistently lower after EGF treatment t han after paclitaxel or 5FUdR treatment. Additional experiments revealed th at the majority of cells detaching from the substratum after EGF (but not p aclitaxel or 5FUdR) were morphologically normal and retained the capacity t o readhere, suggesting that EGF-induced apoptosis involves cell detachment followed by anoikis, These observations not only indicate that EGF- and che motherapy-induced apoptosis in this cell line involve the same downstream p athways but also suggest that detachment-induced apoptosis is responsible f or the paradoxical antiproliferative effects of EGF.