Elsevier

Toxicon

Volume 119, 1 September 2016, Pages 203-217
Toxicon

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

https://doi.org/10.1016/j.toxicon.2016.06.009Get rights and content

Highlights

  • 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.

Abstract

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.

Introduction

Crotalus durissus terrificus (Family: Viperidae; Subfamily: Crotalinae; Genus: Crotalus; Species: Crotalus durissus) is a snake species of public health importance in South America because it is responsible for most of the lethal snakebite events in Brazil. Crotalus durissus is a rattlesnake species found in all South American countries, with the exception Ecuador and Chile (Calvete, 2009). The species can be found in five geographic areas in Brazil; three of them are widely distributed. Crotalus durissus terrificus is the most common rattlesnake in Brazil, and it may also be found in the western part of the country, including some areas of Mato Grosso, Rondônia, Amazonas and Pará States (Pardal et al., 2007). Its venom is formed by components responsible for necrosis, neurotoxicity, renal failure, myotoxicity, cardiotoxicity and coagulopathy. The most well-studied components are proteins and enzymes, including crotoxin, crotalin, gyroxin, thrombin-like enzymes and convulxin (Pinho and Pereira, 2001, Azevedo-Marques et al., 1985, de Sousa-e-Silva et al., 2003, Fernandes et al., 2008, Boldrini-França et al., 2010).

L-amino acid-oxidases (LAAOs, EC 1.4.3.2) are homodimeric flavoenzymes that utilize FAD, similar to that of their prosthetic group. They catalyze the oxidative and stereo-specific deamination of L-amino acids by converting them into α-keto acids and producing ammonia and H2O2 (Zeller, 1977). LAAOs exhibit preferential catalytic specificity for hydrophobic and aromatic L-amino acids, and they are active at a wide range of pHs and temperatures. These classes of enzymes are acidic or basic (pI = 4.4 to 8.5); they contain glycosylation sites (approximately 4%) and have a relative molecular mass of 120–150 kDa (native form) or 55–66 kDa (reduced form) (Curti et al., 1992, Du and Clemetson, 2002).

LAAOs are present in bacteria, fungi, green algae, and in some plant and animal species. In addition to their role in supporting the amino acid metabolism pathways, their presence in different organisms suggests that they play an active role in cell growth and the defense metabolism. Two coupled oxidative reactions are observed during the catalytic cycle of LAAOs and produce ammonia and hydrogen peroxide (Curti et al., 1992, Du and Clemetson, 2002).

Snake venom L-amino acid-oxidases (SV-LAAO) have been described to induce a series of toxic effects, such as platelet aggregation, hemorrhaging, edema, cytotoxicity and apoptosis, among others. In addition, they exhibit a wide spectrum of bactericidal and anti-parasitic activities (Du and Clemetson, 2002, Souza et al., 1999, Ali et al., 2000, Torii et al., 1997, Costa Torres et al., 2010, Ehara et al., 2002, Stábeli et al., 2004, Butzke et al., 2005, Toyama et al., 2006, Zuliani et al., 2009). The hydrogen peroxide generated during the enzymatic reaction was described as the main factor responsible for the cytotoxic effects of SV-LAAO (Torii et al., 1997, Tempone et al., 2001, Li et al., 2006, Suhr and Kim, 1999, Zhang et al., 2004). However, according to several studies, the role played by hydrogen peroxide in the biological activities of SV-LAAO is uncertain. It is likely that there are other mechanisms involved in the pharmacological and toxicological effects of L-amino acid-oxidase (Suhr and Kim, 1999, Zhang et al., 2004, Izidoro et al., 2014).

It is known that these enzymes may significantly differ in their structure, pharmacological properties and biotechnological potential. According to several researchers, both the three-dimensional structure and the amino acid composition generally show that SV-LAAOs are quantitatively similar because they contain a significant amount of asparagine, glutamic acid, aspartic acid, methionine and tryptophan residues. Despite the fact that most SV-LAAOs show high identity, a number of dissimilar regions are observed in both the C- and the N-terminal regions of the molecule; therefore, these regions reveal the intra- and interspecies differences in the composition of the venom (Izidoro et al., 2014).

Crotalus venom is considered to be one of the most harmful venoms due to its previously reported systemic effects. In addition to the complex chemical nature of this venom, its pharmacological activities show that there is significant interest in its different components. Currently, little is known about the LAAOs from this genus or their primary and tertiary structures. Casca-LAO and Apoxin 1 were isolated from Crotalus durissus cascavella and Crotalus cumanensis, respectively, and LAAO was isolated from Crotalus durissus adamanteus. The first two were the only identified enzymes, based on their biochemical and functional properties (Toyama et al., 2006, Torii et al., 1997, Raibekas and Massey, 1998). Thus, the current study aims to report the isolation and biochemical and functional characterization of a new 113 kDa L-amino acid oxidase (LAAOcdt) by considering the different biological functions of LAAOs.

Section snippets

Reagents and venom

Crotalus durissus terrificus venom, which is yellowish in color, was purchased from Koemitã Me (Mococa, SP, Brazil). Sephadex G-75 and Phenyl Sepharose FF were purchased from Amersham Life Science, Inc. The Müeller-Hinton agar was purchased from Himedia (India), Sabouraud-dextrose agar was purchased from Accumedia (USA) and the platelets were purchased from Bio/Data (USA). All of the other reagents used in this study were of analytical grade and purchased from Sigma Chem. Co, Merck and/or

Purification and biochemical characterization

A new L-amino acid oxidase with a relative molecular mass of 113 kDa was purified from Crotalus durissus terrificus venom after two chromatographic steps (Fig. 1a). The active fraction (F1 G-75) was identified through an enzymatic assay. The F1 G-75 was subsequently separated with hydrophobic interaction chromatography using Phenyl-Sepharose FF (Fig. 1b), which resulted in twelve additional fractions, F1 to F12. The LAAO activity was detected in the F4 fraction and named LAAOcdt, as shown in

Discussion

We isolated the first 113 kDa L-amino acid-oxidase (LAAOcdt) from Crotalus durissus terrificus venom, which has a yellowish coloration. As LAAOs have several biological functions, the aim of the present study was to explore the antitumor effect of the LAAOcdt isolated from C. durissus terrificus using different cancer cell lines from 4 distinct tumor types. In addition to the pharmacological properties and very attractive biotechnological potential of these enzymes, it is known that they may

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical statement

The experiments follow the methodology recommended by the international ethical standards of the scientific committee of our university (process nº 048/2011).

Human cancer cell lines

(Kyse 30 and Kyse 270, Esophageal cancer); (GAMG, U373 and HCB151, Glioma primary culture); (PSN-1 and PANC-1, Pancreatic cancer); (HeLa and SiHa, Ovarian cancer).

Molecular markers

(Parp total, 116 kDa nuclear poly (ADP-ribose) polymerase; P21, cyclin-dependent kinase inhibitor; p-H2AX, phospho-Histone; p-AKT(pan), phosphor-protein kinase; p42/44, Mitogen-activated protein kinases; SOD, Superoxide Dismutase).

Acknowledgments

The current study was supported by grants from FAPESP (grant number: 2011/12267-6) and partially supported by FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT) and the Hermínio Ometto University Center.

We acknowledge the Mass Spectrometry Laboratory of the Brazilian Biosciences National Laboratory, CNPEM, Campinas County, Brazil, for providing support for the mass spectrometry analysis.

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