title: Purification and characterization of a novel antitumor protein with antioxidant and deoxyribonuclease activity from edible mushroom Pholiota nameko.
abstract: A novel antitumor protein from the edible mushroom Pholiota nameko (PNAP) was purified through a two-step chromatographic procedure including SP cation exchange chromatogram and Superdex gel filtration. The approximate molecular weight was demonstrated to be 18.5 kDa by SDS-PAGE and ultracentrifugation analysis and N-terminal sequence was detected as AGRTFIGYNG by Edman degradation. Biochemical characterization showed that it exhibited significant antioxidant activity by effectively scavenging hydroxyl and 1,1-diphenyl-2-picrylhydrazyl radicals compared to standard antioxidant butylated hydroxy anisole. PNAP had deoxyribonuclease activity with the optimum pH and temperature were 5.0 and 60 °C respectively, as well as it can act on both double-stranded and single-stranded DNA, but preferentially on double-stranded DNA. PNAP displayed antitumor activity against cancer cell lines such as MCF7 and Hela cells. Human breast cancer MCF7 cells treated with PNAP produced typical apoptotic morphological changes including chromatin condensation, accumulation of sub-G1 cells and alternation of mitochondrial permeability. The PNAP induced apoptosis of MCF7 cells entailed loss of mitochondrial membrane potential resulting in release of cytochrome c into cytosol, activation of caspase-9 and caspase-3, which are responsible for the execution of apoptosis, implying intrinsic signal pathway is involved in PNAP induced apoptosis.
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Yeni
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Zhaokun
authors.initials: Z
authors.lastname: Liu
authors.affiliation: School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Electronic address: b021770@mailserv.cuhk.edu.hk.
authors.forename: Tzi Bun
authors.initials: TB
authors.lastname: Ng
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Chen
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin 300071, China.
authors.forename: Wentao
authors.initials: W
authors.lastname: Qiao
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin 300071, China. Electronic address: liufang312@nankai.edu.cn.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004354:Drug Screening Assays, Antitumor; D016166:Free Radical Scavengers / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D048690:Fruiting Bodies, Fungal / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D061986:MCF-7 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D055368:Pholiota / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D014461:Ultracentrifugation
chemical_list: D000970:Antineoplastic Agents; D016166:Free Radical Scavengers; D005656:Fungal Proteins
keywords: Antioxidant; Antitumor; Apoptosis; Deoxyribonuclease; Pholiota nameko; Purification