title: A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation             of breast cancer cells in vitro.
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.
authors: Department of General Surgery, Tangdu Hospital of The Fourth                     Military Medical University, Ba'qiao, Xi'an, Shaanxi 710038, P.R. China.
authors: Jikai
authors: J
authors: Yin
authors: Zhongliang
authors: Z
authors: Cai
authors: Li
authors: L
authors: Zhang
authors: Jian
authors: J
authors: Zhang
authors: Xianli
authors: X
authors: He
authors: Xilin
authors: X
authors: Du
authors: Qing
authors: Q
authors: Wang
authors: Jianguo
authors: J
authors: Lu
mesh_terms: D017209:Apoptosis; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D005260:Female; D051380:GRB2 Adaptor Protein / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D018909:src Homology Domains / Q000235:genetics*
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011993:Recombinant Fusion Proteins