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Published on: October 27, 2020
Effects of peptidic antagonists of Grb2-SH2 on human breast cancer cells
Chiu-Heng Chen1, Meng-Kai Chen, Kee-Ching G Jeng
1Department of Chemistry, Tunghai University, Taichung, Taiwan, ROC.
Abstract:
The growth factor receptor-bound protein Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, the antagonist of Grb2-SH2 has become a potential target for developing anticancer agents. Recently, we discovered a peptide 1 (Fmoc-Glu-Tyr-Aib-Asn-NH(2)) with high affinity for the Grb2-SH2 domain by using surface plasmon resonance (SPR)-biosensor technology. Herein, we report the further design of the lead peptide 1 by addition of an Arg-Gly-Asp sequence to 1 to enhance binding to Grb2-SH2 and inducing apoptosis in cancer cells. Both the linear and cyclic analogs of the newly designed compound were prepared along with an analog in which the N(alpha)-Fmoc group was removed. These peptide analogs were assayed for their affinity for the Grb2-SH2, their antiproliferative effect on human breast cancer cells, their specificity for cancer cells, and their effects on cytotoxicity and the cell cycle. MCF-7 and MDA-MB-453 breast cancer cells were treated with various concentrations of each peptide. The cell viability and cytotoxicity of peptide-treated cells were determined by using the cell proliferation kit (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-tetrazolium bromide, MTT) and cytotoxicity kit (lactate dehydrogenase, LDH), respectively. Effects of peptides on the cell cycle progression of cancer cells and apoptosis were analyzed by using flow cytometry. Results demonstrated that the peptide analog 2 (H-Arg-Gly-Asp-Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH(2)) had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with an IC(50) of 45.7 microM and 47.4 microM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells when cells were treated with higher concentration of the Arg-Gly-Asp-containing peptide 2. These results provide important information for the development of anti-cancer agents.
Insights
Researchers developed a new peptide analog that inhibits cancer cell growth by targeting the Grb2-SH2 domain, a key player in cell proliferation pathways. This peptide shows promise as a potential anticancer agent by inducing apoptosis and affecting the cell cycle in breast cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The Grb2-SH2 domain is crucial in the Ras signaling pathway, regulating cell proliferation and differentiation.
- Dysregulation of this pathway is implicated in cancer development, making Grb2-SH2 a potential therapeutic target.
- Previous research identified a peptide with high affinity for Grb2-SH2, serving as a basis for further development.
Purpose of the Study:
- To design and synthesize novel peptide analogs by incorporating an Arg-Gly-Asp sequence into a lead peptide.
- To evaluate the binding affinity, antiproliferative effects, cytotoxicity, and cell cycle impact of these new peptide analogs on human breast cancer cells.
- To assess the potential of these modified peptides as anticancer agents.
Main Methods:
- Peptide synthesis of linear and cyclic analogs, including modifications like N(alpha)-Fmoc removal.
- Surface Plasmon Resonance (SPR) biosensor technology for assessing binding affinity to Grb2-SH2.
- In vitro assays using MCF-7 and MDA-MB-453 breast cancer cells to determine antiproliferative (MTT assay) and cytotoxic (LDH assay) effects.
- Flow cytometry to analyze cell cycle progression and apoptosis induction.
Main Results:
- Peptide analog 2, containing the Arg-Gly-Asp sequence, demonstrated significant anti-proliferative effects against MCF-7 and MDA-MB-453 cells, with IC(50) values of 45.7 microM and 47.4 microM, respectively.
- Increased cytotoxicity and a higher percentage of cells in the sub-G1 phase of the cell cycle were observed with higher concentrations of peptide analog 2.
- The study successfully generated and characterized novel peptide analogs with enhanced Grb2-SH2 binding and anticancer activity.
Conclusions:
- The Arg-Gly-Asp-containing peptide analog 2 exhibits potent anticancer properties by inhibiting proliferation and inducing apoptosis in breast cancer cells.
- These findings highlight the therapeutic potential of targeting the Grb2-SH2 domain with rationally designed peptide-based agents.
- The developed peptide analogs represent promising leads for the future development of novel anticancer therapeutics.
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