Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines

• Published in: Toxicon (Oxford) 2016-09, Vol.119, p.203-217
• Main Authors: Teixeira, Tuila Leveghim, Oliveira Silva, Viviane Aline, da Cunha, Daniel Batista, Polettini, Flávia Lino, Thomaz, Camila Daniele, Pianca, Ariana Aparecida, Zambom, Fabiana Letícia, da Silva Leitão Mazzi, Denise Pimenta, Reis, Rui Manuel, Mazzi, Maurício Ventura
• Format: Article
• Language: English
• Subjects: Activation; Animals; Anti-Infective Agents - pharmacology; Antitumor activity; Biotechnology; Cancer; Cell Line, Tumor; Chromatography, Ion Exchange; Coagulants - pharmacology; Crotalid Venoms - enzymology; Crotalus; Crotalus durissus terrificus; Cytotoxicity; Drug Screening Assays, Antitumor; Electrophoresis, Polyacrylamide Gel; Enzymes; Flow cytometry; Homogeneity; Humans; L-Amino Acid Oxidase - chemistry; L-Amino Acid Oxidase - isolation & purification; L-Amino Acid Oxidase - pharmacology; L-amino acid-oxidase; Oxidase; Phenyls; Platelet Aggregation - drug effects; Staphylococcus aureus
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2016.06.009

An L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom was purified to homogeneity in a two-step procedure using molecular exclusion on Sephadex G-75, followed by Phenyl Sepharose FF chromatography. The molecular mass of the purified enzyme was 113 kDa, as determined by SDS-PAGE under reducing conditions. LAAOcdt showed amino acid homology to other L-amino acid oxidases isolated from different snake venoms. The comparative analysis of the internal peptide sequences of the NNPGILEYPVKPSEEGK fragments by LC-MS/MS spectrometry revealed 100% identity with C. durissus cumanensis LAAO. The purified protein catalyzed the oxidative deamination of L-amino acids, and the most specific substrates were L-Tyr and L-Phe. The enzyme presented optimum activity at pH 7.4 and at 44 °C. LAAOcdt also showed hemolytic activity (0.6–20 μg/μL) and induced both the formation plasma clots (5–100 μg/μL) and platelet aggregation (2.5 × 10−3, 5.0 × 10−3 and 10 × 10−3 μg/mL), as well as bactericidal activity (2.5–10 μg/μL) against Staphylococcus aureus. Moreover, LAAOcdt exhibited cytotoxicity in distinct cancer cell lines, which presented a heterogeneous response profile. The mean IC50 value was 10.5 μg/mL. Glioma and pancreatic carcinoma cells were the most sensitive cell lines; they showed mean IC50 values of 7.2 μg/mL and 7.4 μg/mL, respectively. The exposure of the drug-sensitive cells to LAAOcdt for 24 h upregulated activated p-H2AX and efficiently decreased P42/P44 (ERK) activation in glioma cells (HCB151), which suggested an anti-proliferative effect. In addition, increased p21 expression was observed in SiHa cells, which showed a resistant phenotype. On the other hand, the flow cytometry and immunoblotting analyses showed that the enzyme did not induce cancer cell apoptosis. These results suggest that another cell death mechanism might contribute to the LAAOcdt-induced cytotoxicity. Taken together, this work may help to elucidate the function and structure of LAAOcdt by providing the basis for further investigations on its efficacy in cancer treatment. •A new 113 kDa L-amino acid oxidase was purified from Crotalus durissus terrificus venom.•The enzyme presented bactericidal activity against Staphylococcus aureus.•LAAOcdt showed antitumor effect in different cancer cell lines.•Apoptosis does not seem to be the main mechanism involved in cell death process.•Glioma cells and pancreatic carcinomas presented the most sensitive profile.