A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro

Yin,Jikai; Cai,Zhongliang; Zhang,Li; Zhang,Jian; He,Xianli; Du,Xilin; Wang,Qing; Lu,Jianguo

doi:10.3892/ijo.2013.1768

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro

Authors:
- Jikai Yin
- Zhongliang Cai
- Li Zhang
- Jian Zhang
- Xianli He
- Xilin Du
- Qing Wang
- Jianguo Lu
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Affiliations: Department of General Surgery, Tangdu Hospital of The Fourth Military Medical University, Ba'qiao, Xi'an, Shaanxi 710038, P.R. China, Research Institute of General Surgery, Nanjing General Hospital of Nanjing Military Command, Xuan'wu, Nanjing, Jiangsu 210002, P.R. China, Department of Ultrasound Diagnosis, Tangdu Hospital of The Fourth Military Medical University, Ba'qiao, Xi'an, Shaanxi 710038, P.R. China, State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Ba'qiao, Xi'an, Shaanxi 710032, P.R. China
Published online on: January 10, 2013 https://doi.org/10.3892/ijo.2013.1768
Pages: 1061-1069

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Abstract

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

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