Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway

Abstract

This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS-PAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (Δψ(m)), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of Δψ(m) besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.