BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

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.

Triptolide Inhibits the Biological Processes of HUVECs and HepG2 Cells via the Serine Palmitoyltransferase Long Chain Base Subunit 2/Sphingosine-1-Phosphate Signaling Pathway

“…Bernhardt et al [39] studied the interaction between primary human osteoclasts and mature human osteoclasts in a coculture model. Machado et al [40] investigated the effect of HepG2 cells on endothelial cells through a coculture model. Few studies have investigated the effect of endothelial cells on tumor cells.…”

Antimicrobial properties of L-amino acid oxidase: biochemical features and biomedical applications

“…Inhibit by Zn 2+ Bactericidal activity Leu X2L4E2 Lazo et al ( 2017 ) H1 : Bothrops jararacussu BjussuLAAO-I (BjsuLAAO) MW = 54 kDa, pI5.7, flavoprotein (venom) Antiprotozoal effects Met > Leu > Phe > Ile > Tyr > Trp > Cys > others Q6TGQ9, 4E0V França et al ( 2007 ), Ullah et al ( 2012 ) França et al ( 2007 ), Ullah et al ( 2012 ) H2 : Bothrops jararacussu BjussuLAAO-II MW = 60 kDa, pI3.9, glycosylated flavoprotein (venom) pH 7.0, 65 °C, inhibited by Zn 2+ , Al 2+ , Cu 2+ , Ni 2+ Antiprotozoal effects, tumor resistance, cytotoxicity effects, cytotoxicity and apoptosis in MCF-7, but not MCF10A. DNA methylation of cell-cycle genes in HepG2, upregulates the inflammatory genes, downregulates the apoptotic-related genes Met > Leu > Ile > Phe > Tyr > others Costa et al ( 2018 ), Carone et al ( 2017 ), Machado et al ( 2018 ), Machado et al ( 2019a , 2019b ) I : Bothrops moojeni BmooLAAO-I MW = 65 kDa, homodimeric glycoslyated flavoprotein (venom) pH 7.0–8.0, 60 °C, inhibited by Zn 2+ , Al 2+ , Cu 2+ , Ni 2+ Bactericidal activity, trypanocidal, antitumor effects Met > Leu > Ile > Phe > Tyr > others Q6TGQ8 Stábeli et al ( 2007 ), Costa et al ( 2018 ) J : Bungarus multicinctus BM-Apotxin MW = 65 kDa, homodimeric glycoslyated flavoprotein (venom) pH 8.4, 45 °C, deglycoslyation can significantly reduce the oxidation and antitumor activity Antitumor effects, selectively kill tumor, with less cytotoxicity to the normal cells Met > Leu > Phe > Ile > Tyr > Pro > others A8QL51, ABN72539.1 Lu et al ( 2018 ) K1 : Cerastes cerastes CC-LAAO MW = 58 kDa, homodimeric (115 kDa), g...…”

“…1 illustrates the molecular phylogenetic tree of LAAOs from various organisms. LAAOs are involved in innate immunity with selective and specific antibacterial properties (Kasai et al 2015a (Table 1 V1), Kasai et al 2015b (Table 1 V1), Teixeira et al 2016 (Table 1 M2), Lazo et al 2017 (Table 1 G), Rey-Suárez et al 2018 (Table 1 N), Salama et al 2018 (Table 1 L), Elsheimer-Matulova et al 2020 (Table 1 D), Soares et al 2020 (Table 1 E)), antiparasites (Carone et al 2017 (Table 1 H2), Wiezel et al 2019 (Table 1 M1), Soares et al 2020 (Table 1 E), Barbosa et al 2021 ) and induce DNA fragmentation (Mukherjee et al 2015 , Burin et al 2016a , 2016b , Machado et al 2018 (Table 1 H2), Machado et al 2019a , 2019b (Table 1 H2)), apoptosis (Mukherjee et al 2015 , Fung et al 2015 (Table 1 O), Burin et al 2016a , 2016b , Ribeiro et al 2016 , Tan et al 2017 , Carone et al 2017 (Table 1 H2), Machado et al 2018 (Table 1 H2), Mukherjee et al 2018 , Rey-Suárez et al 2018 (Table 1 N), Bedoya-Medina et al 2019 (Table 1 N)), regulation of immune responses (Cousin et al 2015 , Scarlata et al 2015 , Romagnani 2016 , Bod et al 2018 , Ramspott et al 2018 (Table 1 A), Aubatin et al 2018 (Table 1 A), Elsheimer-Matulova et al 2020 (Table 1 D), Sadik et al 20...…”