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Research article - Vol. 25, 2019 |
BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells
1 Department of Clinical Analysis, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo - USP, Ribeirão Preto, SP, Brazil.
2 Department of Genetics, Ribeirão Preto Medical School, University of São Paulo - USP, Ribeirão Preto, SP, Brazil.
3 Center for the Study of Venoms and Venomous Animals (CEVAP), São Paulo State University - UNESP, Botucatu, SP, Brazil.
ABSTRACT
Background:
The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells.
Methods:
BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array.
Results:
BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05).
Conclusion:
Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.
Keywords: snake venom; epigenetics; GADD45A; CCND1; CDKN1A
Received: June 29, 2018.
Accepted: October 22, 2018.
Correspondence: lusania@fcfrp.usp.br
Competing interests The authors declare that they have no competing interests.
Authors’ contributions ARTM was responsible for project development, designed the experimental approaches, interpreted the data, performed the experiments and drafted the manuscript. DLR and AFA participated in the designed experiment, interpreted data and drafted the manuscript. RSFJR and SSV provided the toxin and drafted the manuscript. LMGA coordinated and designed all the experiments, analyzed and interpreted the data, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.