Department of Clinical Analysis, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo - USP, Ribeirão Preto, SP, Brazil.
Department of Genetics, Ribeirão Preto Medical School, University of São Paulo - USP, Ribeirão Preto, SP, Brazil.
Int J Biol Macromol. 2019 Apr 15;127:425-432. doi: 10.1016/j.ijbiomac.2019.01.059. Epub 2019 Jan 15.
Hepatocellular carcinoma incidence rates have increased worldwide, which encouraged the development of new chemotherapeutic drugs. l-Amino acid oxidases from snake venoms are cytotoxic towards human tumor cells in in vitro monoculture systems, which do not simulate the tumor microenvironment. We examined the antitumor potential of BjussuLAAO-II, an l-amino acid oxidase from Bothrops jararacussu venom, in hepatocarcinoma cells (HepG2) in monoculture and co-culture with human umbilical vein endothelial cells (HUVEC) in vitro. All the concentrations tested (0.25-5.00 μg/mL) were cytotoxic (MTT and clonogenic survival assays) towards HepG2 and HUVEC cells in monoculture, and increased oxidative stress by 2',7'-dichlorofluorescin diacetate fluorescence assay. Only 1.00 and 5.00 μg/mL exerted these effects in HepG2 cells co-cultured with HUVEC cells, and were genotoxic (comet assay) to HUVEC cells in monoculture. BjussuLAAO-II at 5.00 μg/mL induced DNA, but not chromosomal damage (micronucleus assay) in HepG2 cells in mono- and co-culture. The cytotoxicity and genotoxicity was more pronounced in monoculture, indicating that the tumor microenvironment influences the cellular response. BjussuLAAO-II caused cell death and DNA damage in HepG2 cells in vitro by inducing oxidative stress. Therefore, BjussuLAAO-II is a promising molecule for the development of new antitumor drugs.
肝细胞癌的发病率在全球范围内有所增加，这促使人们开发新的化疗药物。蛇毒液中的 l-氨基酸氧化酶在体外单细胞培养系统中对人类肿瘤细胞具有细胞毒性，但这种系统并不能模拟肿瘤微环境。我们研究了来自巴西矛头蝮蛇毒液的 l-氨基酸氧化酶 BjussuLAAO-II 在体外与人脐静脉内皮细胞（HUVEC）共培养的肝癌细胞（HepG2）中的抗肿瘤潜力。所有测试浓度（0.25-5.00μg/mL）对 HepG2 和 HUVEC 细胞在单细胞培养中的细胞毒性（MTT 和集落形成存活测定），并通过 2'，7'-二氯荧光素二乙酸荧光测定法增加氧化应激。只有 1.00 和 5.00μg/mL 在 HepG2 与 HUVEC 共培养的细胞中发挥这些作用，并且对 HUVEC 细胞在单细胞培养中的遗传毒性（彗星试验）。BjussuLAAO-II 在 5.00μg/mL 时诱导 HepG2 细胞的 DNA 损伤，但不诱导染色体损伤（微核试验），在单和共培养中。在单细胞培养中，细胞毒性和遗传毒性更为明显，表明肿瘤微环境影响细胞反应。BjussuLAAO-II 通过诱导氧化应激在 HepG2 细胞中引起细胞死亡和 DNA 损伤。因此，BjussuLAAO-II 是开发新型抗肿瘤药物的有前途的分子。
