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 many cell types, induces apoptosis in some cell lines that overexpress the EGF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined by microscopy, flow cytometry, immunoblotting, enzyme assays, and affinity labeling after treatment with EGF, paclitaxel, or 5-fluoro-2'-deoxyuridine (5FUdR). Apoptosis induced by all three agents was accompanied by activation of caspases-3, -6, and -7, as indicated 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 labeling. Further analysis indicated involvement of the cytochrome c/Apaf-1/caspase-9 pathway of caspase activation, but not the Fas/Fas ligand pathway. Interestingly, caspase activation was consistently lower after EGF treatment than after paclitaxel or 5FUdR treatment. Additional experiments revealed that the majority of cells detaching from the substratum after EGF (but not paclitaxel or 5FUdR) were morphologically normal and retained the capacity to readhere, suggesting that EGF-induced apoptosis involves cell detachment followed by anoikis. These observations not only indicate that EGF- and chemotherapy-induced apoptosis in this cell line involve the same downstream pathways but also suggest that detachment-induced apoptosis is responsible for the paradoxical antiproliferative effects of EGF.
Kottke
T J
TJ
Division of Oncology Research, Mayo Clinic, Mayo Graduate School, Rochester, Minnesota 55905, USA.
Blajeski
A L
AL
Martins
L M
LM
Mesner
P W
PW
Davidson
N E
NE
Earnshaw
W C
WC
Armstrong
D K
DK
Kaufmann
S H
SH
D000345:Affinity Labels / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1; D001943:Breast Neoplasms / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D005467:Floxuridine / Q000494:pharmacology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017239:Paclitaxel / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014407:Tumor Cells, Cultured
C506592:APAF1 protein, human; D000345:Affinity Labels; D000970:Antineoplastic Agents; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D005467:Floxuridine; D004815:Epidermal Growth Factor; D020169:Caspases; D017239:Paclitaxel