The receptor (R) for
epidermal growth factor (
EGF) is expressed at high levels on human
breast cancer cells and associates with ErbB2, ErbB3, and Src proto-oncogene family
protein tyrosine kinases (PTKs) to form membrane-associated PTK complexes with pivotal signaling functions. Recombinant human
EGF was conjugated to the soybean-derived PTK inhibitor
genistein (Gen) to construct an
EGF-R-directed
cytotoxic agent with PTK inhibitory activity. The
EGF-Gen conjugate was capable of binding to and entering
EGF-R-positive MDA-MB-231 and BT-20
breast cancer cells (but not
EGF-R-negative NALM-6 or HL-60
leukemia cells) via its
EGF moiety, and it effectively competed with unconjugated
EGF for target
EGF-R molecules in
ligand binding assays.
EGF-Gen inhibited the
EGF-R
tyrosine kinase in
breast cancer cells at nanomolar concentrations, whereas the IC50 for unconjugated Gen was >10 microM. Notably,
EGF-Gen triggered a rapid apoptotic cell death in MDA-MB-231 as well as BT-20
breast cancer cells at nanomolar concentrations. The
EGF-Gen-induced apoptosis was
EGF-R-specific because cells treated with the control
granulocyte-colony stimulating factor-Gen conjugate did not become apoptotic. Apoptosis was dependent both on the PTK inhibitory function of Gen and the targeting function of
EGF, because cells treated with unconjugated Gen plus unconjugated
EGF did not undergo apoptosis. The IC50s of
EGF-Gen versus unconjugated Gen against MDA-MB-231 and BT-20 cells in clonogenic assays were 30 +/- 3 nM versus 120 +/- 18 microM (P < 0.001) and 30 +/- 10 nM versus 112 +/- 17 microM (P < 0.001), respectively. Thus, the
EGF-Gen conjugate is a >100-fold more potent inhibitor of
EGF-R
tyrosine kinase activity in intact
breast cancer cells than unconjugated Gen and a >100-fold more potent
cytotoxic agent against
EGF-R+ human
breast cancer cells than unconjugated Gen. Taken together, these results indicate that the
EGF-R-associated PTK complexes have vital antiapoptotic functions in human
breast cancer cells and may therefore be used as therapeutic targets.