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.
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.
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: tuila@ead.uniararas.br.
Tuila Leveghim
TL
Teixeira
Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP, Brazil. Electronic address: vivianeaos@gmail.com.
Viviane Aline
VA
Oliveira Silva
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: dbcunha@live.com.
Daniel Batista
DB
da Cunha
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: fla_poletini@hotmail.com.
Flávia Lino
FL
Polettini
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: camila.daniele@hotmail.com.
Camila Daniele
CD
Thomaz
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: arianapianca@hotmail.com.
Ariana Aparecida
AA
Pianca
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: fabbyleticia@hotmail.com.
Fabiana Letícia
FL
Zambom
Department of Cell and Molecular Biology and Pathogenic Bioagents, University of São Paulo-FMRP-USP-Ribeirão Preto, SP, Brazil. Electronic address: denise_psl@yahoo.com.br.
Denise Pimenta
DP
da Silva Leitão Mazzi
Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP, Brazil; Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga, Portugal. Electronic address: ruireis.hcb@gmail.com.
Rui Manuel
RM
Reis
Graduate Program in Biomedical Sciences Hermínio Ometto University Center, UNIARARAS, Av. Dr. Maximiliano Baruto, 500, CEP 13607-339, Araras, SP, Brazil. Electronic address: maumazzi@uniararas.br.
Maurício Ventura
MV
Mazzi
D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002852:Chromatography, Ion Exchange; D003029:Coagulants / Q000494:pharmacology; D003435:Crotalid Venoms / Q000201:enzymology*; D017839:Crotalus; D004354:Drug Screening Assays, Antitumor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D050830:L-Amino Acid Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects
D000890:Anti-Infective Agents; D003029:Coagulants; D003435:Crotalid Venoms; D050830:L-Amino Acid Oxidase
Antitumor activity; Crotalus durissus terrificus; Cytotoxicity; L-amino acid-oxidase