The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected.
The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5' AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0. 15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.
Frontiers in plant science. 2015 Oct 07*** epublish ***
Shayan Sarkar, Sumeet Jain, Vineeta Rai, Dipak K Sahoo, Sumita Raha, Sujit Suklabaidya, Shantibhusan Senapati, Vivek M Rangnekar, Indu B Maiti, Nrisingha Dey
Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India Bhubaneswar, India. , Department of Translational Research and Technology Development, Institute of Life Sciences, Department of Biotechnology, Government of India Bhubaneswar, India ; Manipal University Manipal, India. , Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India Bhubaneswar, India. , Kentucky Tobacco Research & Development Center, Plant Genetic Engineering Research and Services, College of Agriculture, Food and Environment, University of Kentucky, Lexington KY, USA ; Department of Agronomy, Iowa State University, Ames IA, USA. , Department of Radiation Oncology, Feinberg School of Medicine, Northwestern University, Chicago IL, USA. , Department of Translational Research and Technology Development, Institute of Life Sciences, Department of Biotechnology, Government of India Bhubaneswar, India. , Department of Translational Research and Technology Development, Institute of Life Sciences, Department of Biotechnology, Government of India Bhubaneswar, India. , Department of Radiation Medicine, Markey Cancer Center, University of Kentucky, Lexington KY, USA. , Kentucky Tobacco Research & Development Center, Plant Genetic Engineering Research and Services, College of Agriculture, Food and Environment, University of Kentucky, Lexington KY, USA. , Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India Bhubaneswar, India.