Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies

Highlights

• Acra3 has highly selective effect on cell lines.

• It effects on cellular proliferation of BC3H1 cell lines determined as apoptotic and necrotic.

• Acra3 has antigenic property and polyclonal antibodies were capable of neutralize crude venom of Androctonus crassicauda.

• Monoclonal antibody specific for Acra3 antigen (5B9) was developed by hybridoma technology and antibody was found to be IgM.

Abstract

Constitutes of the venom scorpion are a rich source of low molecular mass peptides which are toxic to various organisms, including man. Androctonus crassicauda is one of the scorpions from the Southeastern Anatolia of Turkey with public health importance. This work is focused on the investigation of biological effects of Acra3 peptide from Androctonus crassicauda. For this purpose, Acra3 isolated from crude venoms was tested for its cytotoxicity on BC3H1 mouse brain tumor cells using tetrazolium salt cleavage and lactate dehydrogenase activity assays. To determine whether the cytotoxic effects of Acra3 was related to the induction of apoptosis, the morphology of the cells and the nuclear fragmentation was examined by using Acridin Orange staining and DNA fragmentation assay, respectively. Caspase 3 and caspase 9 activities were measured spectrophotometrically and flow cytometric assay was performed using Annexin-V FITC and Propidium Iodide staining. Furthermore toxic peptide Acra3 was used as an antigen for immunological studies.

Results showed that Acra3 exerted very strong cytotoxic effect on BC3H1 cells with an IC₅₀ value of 5 μg/ml. Exposure of the cells to 0.1 and 0.5 μg/ml was resulted in very strong appearance of the apoptotic morphology in a dose dependent manner. On the other side, not any DNA fragmentation was observed after treatment of the cells. Caspase 3 and 9 activities were slightly decreased with Acra3. Results from flow cytometry and lactate dehydrogenase activity assays indicate that Acra3 exerts its effects by inducing a stronger necrosis than apoptosis in BC3H1 cells. To evaluate its immunogenicity, monoclonal antibody (MAb) specific for Acra3 antigen (5B9) was developed by hybridoma technology using spleen and lymph nodes of mice and immunoglobulin type of antibody was found to be IgM.

We suggest that Acra3 may exert its effects by inducing both necrotic and apoptotic pathway in some way on mouse brain tumor cells. These findings will be useful for understanding the mechanism of cell death caused by venom in vitro. Anti-Acra3 monoclonal antibody can be further used as a bioactive tools for exploring the structure/function relationship and the pharmacological mechanism of scorpion peptide neurotoxins.

Introduction

Scorpion venoms are known highly complex mixtures of components such as enzymes, peptides, nucleotides, lipids, mucoproteins, biogenic amines and other unknown substances. Among them peptides plays a fundamental role (Bosmans and Tytgat, 2007, Possani and Rodriguez de la Vega, 2006). Four different groups of toxic peptides have been described, which specifically interact with ion channels for sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺) and chloride (Cl⁻). Based on the length of the sequences, generally two groups of peptides were identified: The first group is formed by long chain peptides containing 60–76 amino acids residues which mainly affect sodium channels. The second group is represented by short chain peptides containing 21–40 amino acids residues which are active on potassium, chloride and calcium channels (Possani et al., 2000). While most long chain scorpion toxins are known to be specific for Na⁺ channels, there are small number of peptides are known specific to K⁺ channels and also the peptides without disulphide bridges have been identified and characterized from scorpion venom (Bergeron and Bingham, 2012, Zeng et al., 2005). The venom alters the activity of the enzymes, receptors, or ion channels, thus disarranging the autonomic central and peripheral nervous system, the cardiovascular and neuromuscular systems, blood coagulation and homeostasis. In addition, peptides from scorpion venoms have a great potential as pharmacological tools (Calvete et al., 2009).

In Turkey, twenty-two different species of scorpions distributed into four families are reported (Koç and Yağmur, 2007). Among these species Androctonus crassicauda belongs to the Buthidae family play an important role in deadly accidents in humans (Altinkurt and Altan, 1980). The accidents mostly can cause severe pain, hyperemia, autonomic central nervous system and muscle function disturbances through a mechanism not yet fully understood (Dehghani and Khamehchian, 2008, Ozkan et al., 2006, Radmanesh, 1990). Application of specific anti-venom immunotherapy is one of the specific medical treatments for scorpion sting (De Roodt et al., 2010). In Turkey, horse anti-venom against to A. crassicauda crude venom has been prepared by Ministry of Health since 1942 (Adiguzel et al., 2007).

The biochemical composition of A. crassicauda venom is poorly known, despite the fact that it is most dangerous scorpion and many serious envenomation cases of humans have been reported. Although the crude venom consist of at least 80 different peptide components of molecular masses vary from 267 Da to 44,541 Da, up to date a few number of peptide toxins were described by our group (Caliskan et al., 2006). Recently, another new lethal peptide named as Acra3 composed of 66 amino acids residues with a molecular mass 7620 Da has been discovered from the A. crassicauda crude venom (Caliskan et al., 2012a). On the other hand, recently apoptotic and the anti-proliferative effects of A. crassicauda crude venom on SH-SY5Y and MCF-7 cells were reported (Zargan et al., 2011a). Cell death through apoptosis or necrosis is known to play an important role on normal tissue homeostasis (Jang et al., 2003). Apoptosis is a programmed cell death characterized by biochemical (mitochondrial fragmentation, activation of protease caspases) and morphological alterations (nuclear condensation and fragmentation, cytoplasmic shrinkage and membrane blebbing). Beside, necrosis is characterized by mitochondrial changes, uncontrolled osmotic pressure and finally swells and ruptures of cells. Up to date, several groups have declared principal toxicity mechanism of peptide toxins from venomous animals using in vitro tests with various cell lines, indicating their apoptotic or necrotic effects (D'Suze et al., 2010, Heinen and da Veiga, 2011).

Our previous results showed that Acra3 is highly toxic to mice when injected at low concentration whereas it was non-toxic to insects and crustaceans. Amino acid sequence of Acra3 was similar to Na⁺-channel specific scorpion toxins with between 60 and 76 amino acid residues cross-linked by 4 disulfide bridges. Although the presence of these structural similarities, Acra3 was not found effective on 6 different sub-types of Na⁺-channels in vitro (Nav1.1–Nav1.6) (Caliskan et al., 2012a). In the present study, cytotoxic effects and antigenic properties of Acra3 and crude venom were investigated both in vitro and in vivo methods, respectively. First of all, the crude venom was obtained by the electrostimulation of animals and then several fractions were fractioned by means of high performance liquid chromatography (HPLC) separations. Cytotoxic effects of the crude venom were determined on six different cell types in vitro by MTT assay. The cell lines were F2408 (embryonic rat fibroblasts), CO25 (mouse myoblasts) 5RP7 (H-ras active embryonic rat fibroblasts), A549 (human lung adenocarcinoma cells), WM115 (human malignant melanoma cells), NIH3T3 (mouse embryo fibroblasts), and BC3H1 (mouse brain tumor cells). BC3H1 cells were used for further investigations by several assays as described in material methods section. Furthermore, purified Acra3 peptide was used as an antigen for the development of polyclonal and monoclonal antibody (MAb 5B9). Cross-reactivity of monoclonal antibody was tested with enzyme-linked immunosorbent assay (ELISA). 5B9 MAb was produced in a large scale and purified with S300 size exclusion chromatography. As a result of the study, biological and immunological effects were described for Acra3 peptide from Turkish species of A. crassicauda.