L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes

Abstract

Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC50) of 5.1 μg/mL. Cytotoxicity caused by LAAO was mediated by H2O2 and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms.

Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and l-amino acid oxidase

“…In addition to apoptosis, the venom LAAO exhibits a dose-dependent transition of apoptosis to necrosis when its concentration increases [22,33,80,83]. This is presumably related to the levels of H 2 O 2 produced by the enzyme, as the treatment with catalase significantly reduced the number of necrotic cells [85].…”

“…Although apoptosis remains as the predominant cell death mode in LAAO-induced cytotoxicity in cancer cells, the venom LAAO is able to cause autophagy in normal human keratinocyte [33]. Autophagy refers to a self-degenerative cell death process in which cellular components are degraded in autophagic vacuoles of dying cells [86].…”

“…Autophagy refers to a self-degenerative cell death process in which cellular components are degraded in autophagic vacuoles of dying cells [86]. The LAAO-induced cytotoxic effects are dose dependent and follow a sequential manner of cells undergoing autophagy, apoptosis to necrosis within 24 h [33,35]. On the other hand, preclinical trials of LAAO from Ophiophagus hannah revealed that LAAO suppresses PC-3 Solid Tumour Growth in a tumour xenograft mouse model [87].…”

“…The membrane perturbation by PLA 2 is a secondary process to its catalytic actions on membrane phospholipids [32], indicating that venom PLA 2 exhibits remarkable cytotoxicity. On the other hand, venom LAAO has also been demonstrated to induce cell death due to the generated H 2 O 2 [33][34][35]. Cancer is characterised by an uncontrolled cells proliferation, the ability to escape apoptosis and evading growth suppressors with active metastasis.…”

Structure–Function Studies and Mechanism of Action of Snake Venom L-Amino Acid Oxidases

“…Snake venom LAAO (LAAOs, EC 1.4.3.2) is an FAD-containing dimeric enzyme that stereospecifically deaminates an L-amino acid to an a-keto acid with the concomitant production of hydrogen peroxide and ammonia (Pawelek et al, 2000;Du and Clemetson, 2002;Moustafa et al, 2006;Ullah et al, 2012b;Ullah et al, 2014;Costal-Oliveira et al, 2019). These enzymes are widely distributed in the snake venom and have been found to be toxic (Li et al, 1994;Torii et al, 1997).…”