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Cytotoxic activity of epidermal growth factor-genistein against breast cancer cells
1Department of Oncology, Wayne Hughes Institute, St. Paul, Minnesota 55103, USA. Fatih_Uckun@mercury.IH.org
Abstract:
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.
Insights
A novel EGF-Gen conjugate targets epidermal growth factor receptors (EGF-R) on breast cancer cells, inhibiting protein tyrosine kinases (PTKs) and inducing apoptosis. This targeted therapy is over 100-fold more potent than genistein alone.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Epidermal growth factor receptor (EGF-R) is highly expressed on human breast cancer cells.
- EGF-R associates with protein tyrosine kinases (PTKs) to form signaling complexes crucial for cell function.
- Targeting these complexes offers a potential therapeutic strategy for breast cancer.
Purpose of the Study:
- To develop and evaluate a novel cytotoxic agent targeting EGF-R.
- To conjugate the PTK inhibitor genistein (Gen) to epidermal growth factor (EGF).
- To assess the efficacy of the EGF-Gen conjugate in inhibiting EGF-R and inducing apoptosis in breast cancer cells.
Main Methods:
- Recombinant human EGF was conjugated to genistein (Gen).
- The EGF-Gen conjugate's binding and internalization into EGF-R-positive breast cancer cells were assessed.
- EGF-R tyrosine kinase inhibition and induction of apoptosis by EGF-Gen were measured.
- Clonogenic assays were performed to compare cytotoxicity of EGF-Gen and unconjugated Gen.
Main Results:
- EGF-Gen specifically bound and entered EGF-R-positive breast cancer cells (MDA-MB-231, BT-20).
- EGF-Gen potently inhibited EGF-R tyrosine kinase activity at nanomolar concentrations.
- EGF-Gen induced rapid, EGF-R-specific apoptosis in breast cancer cells.
- EGF-Gen demonstrated over 100-fold greater potency as a cytotoxic agent compared to unconjugated Gen.
Conclusions:
- The EGF-Gen conjugate is a highly potent and specific therapeutic agent for EGF-R-positive breast cancer.
- EGF-R-associated PTK complexes play critical anti-apoptotic roles in breast cancer.
- These complexes represent promising therapeutic targets for breast cancer treatment.