title: AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2.
abstract: Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal sequencing, MALDI-TOF/MS-MS analysis and enzyme kinetics studies. AkP could cleave the growth promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 75±1.18nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration and surface proteoglycans cleavage.
authors.affiliation: Drug Development Diagnostics & Biotechnology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032 West Bengal, India. Electronic address: rajib.iicb@gmail.com.
authors.forename: Rajib
authors.initials: R
authors.lastname: Majumder
authors.affiliation: Maulana Azad College, Department of Microbiology, University of Calcutta, 8, Rafi Ahmed Kidwai Road, Kolkata 700013, India. Electronic address: samudrapb@gmail.com.
authors.forename: Samudra Prosad
authors.initials: SP
authors.lastname: Banik
authors.affiliation: Drug Development Diagnostics & Biotechnology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata, 700032 West Bengal, India. Electronic address: sumankhowala@iicb.res.in.
authors.forename: Suman
authors.initials: S
authors.lastname: Khowala
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism; D003603:Cytotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D013379:Substrate Specificity; D055434:Termitomyces / Q000201:enzymology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494826:BAX protein, human; D003603:Cytotoxins; D005656:Fungal Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D010447:Peptide Hydrolases
keywords: Alkaline serine protease; HepG2 cell apoptosis; Termitomyces clypeatus