• Corpus ID: 207789341

protein anti-HER 2 antibody and caspase-3 fusion HER 2-directed induction of apoptosis with Potent inhibition of human gastric cancer

@inproceedings{ZhaoproteinA2,
  title={protein anti-HER 2 antibody and caspase-3 fusion HER 2-directed induction of apoptosis with Potent inhibition of human gastric cancer},
  author={Peng Zhao and Lin Xia and Li-Li Liu and Jie Liang and Hui-hong Zhai and Hong-bo Zhang and Xue-Gang Guo and Kai-chun Wu and Yanfen Xu and Lintao Jia and An-gang Yang and Si-Yi Chen and Dai-ming Fan}
}

• P. Zhao, L. Xia, +10 authors D. Fan
• Biology, Medicine

Background and aims HER2, an oncogene, has been found to be over-expressed in 10e40% of human gastric carcinomas. The aims of this study were to investigate if a fusion protein consisting of anti-HER2 sFv and constitutively active caspase-3 was capable of inducing apoptosis in HER2-expressing human gastric cancer cells and blocking the growth of human gastric cancer xenografts in nude mice. Methods NIH3T3 cells stably transduced with the pcDNA3.1-HER-PE-CP3 recombinant plasmid containing a secretion signal, a single-chain anti-HER2 monoclonal antibody fragment, a Pseudomonas exotoxin A translocation domain and a constitutively active caspase3 molecule were used to induce apoptosis in human gastric cancer cells both in vitro and in vivo. Immunofluorescence staining and western blotting were used to examine the expression of the recombinant protein HER-PE-CP3. Apoptosis was determined by flow cytometry and TUNEL assay. Results Co-cultivation of HER-PE-CP3/ NIH3T3 with human gastric cancer cells led to internalisation of HERPE-CP3 and apoptosis in HER2-expressing human gastric cancer cells but not in HER2-negative cancer cells. Inoculation of HER-PE-CP3/NIH3T3 in nude mice resulted in potent inhibition of human gastric cancer xenografts and much prolonged survival time of the tumour-bearing mice compared with the control. Significantly more apoptotic cells were detected in xenografts in mice receiving HER-PE-CP3/NIH3T3 than in control mice. Conclusions The HER-PE-CP3 chimeric molecule could induce selective apoptosis and potent growth inhibition of HER2-positive human gastric cancer cells and might represent a novel HER2-directed treatment option for human gastric cancer. INTRODUCTION Gastric cancer is one of the most common types of human malignancy and the second leading cause of cancer death in the world. In most countries the majority of gastric cancer cases are diagnosed at the advanced stage, when only around 50% of patients with gastric cancer could receive surgery as treatment. The efficacy of chemotherapy for gastric cancer is limited due to the relative insensitivity of gastric cancer to chemotherapeutic agents and the development of multidrug resistance. Other treatment options are even less optimal. The 5-year survival rates of gastric cancer patients are around 25%, which represents a serious health problem. Multiple genetic and epigenetic alterations have been found in gastric cancer and some have been shown to be associated with the development and progression of the cancer. Human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, is a cell membrane receptor-associated tyrosine kinase and is believed to be an oncogene. Amplification of the HER2 gene or over-expression of HER2 protein has been found in a variety of human tumours, including breast, ovary and stomach cancers. According to previous reports, over-expression of HER2 could be detected in around 10e40% of gastric cancer patients. Activation of HER2 has been suggested to play an important role in progression and metastasis of advanced gastric cancer. Caspases are a family of cysteine proteases that cleave targetproteins at specific aspartate residues. 10 Caspase-3 is a key effecter molecule in the caspase-dependent cell apoptosis pathway that cleaves a number of cellular proteins, leading to the apoptotic changes. Wild-type caspase-3 exists as a zymozyme in the cell and consists of an Nterminal prodomain, a large subunit and a small subunit at the C-terminus. Upon activation, caspase-3 is cleaved at the caspase processing sites (CPSs) between these domains, which results in the release of the prodomain and the subsequent reassociation of the large and small subunit as a heterodimer. The reunification of the large and small subunits produces an active form of caspase-3, in which the small subunit is located at the Nterminus, opposite to the order in which the large and small subunits are positioned in the precursor molecule. In the present study, we constructed an artificial chimeric molecule which consists of a secretion signal, a single-chain anti-HER2 monoclonal antibody fragment (e23sFv), a Pseudomonas exotoxin A-derived transportation domain (PEII) and a constitutively active caspase-3 component and tested its efficacy in inducing selective apoptosis in HER2-expressing gastric cancer cells and in suppressing the growth of human gastric cancer xenografts in a nude mouse model. MATERIALS AND METHODS Cell culture Human gastric cancer cell lines MKN28 and MKN45 were obtained from RIKEN Cell Bank State Key Laboratory of Cancer Biology & Xijing Hospital of Digestive Disease, Fourth Military Medical University, Xi’an, P.R. China Department of Pathology and Pathophysiology, Fourth Military Medical University, Xi’an, P.R. China State Key Laboratory of Cancer Biology & Department of Immunology, Fourth Military Medical University, Xi’an, P.R. China Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA