Purification and characterization of a novel antitumor protein with antioxidant and deoxyribonuclease activity from edible mushroom Pholiota nameko

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.