RESUMEN
The aim of this study were characterize acellular collagen matrices derived from porcine pericardium (PP) and to evaluate their properties after sterilization by ethylene oxide and gamma ray. PP matrices were subjected to alkaline hydrolysis (AH), and samples were characterized for biological stability, membrane thickness measurements, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Subsequently, the matrices were frozen, lyophilized and sterilized by ethylene oxide or gamma radiation. For in vitro assays, CHO-K1 cell culture was used and evaluated for cytotoxicity, clonogenic survival assay, genotoxicity and mutagenicity. Analysis of variance (ANOVA) was used, followed by Dunnett's post-test, with a significance level of 5%. After AH, there was no significant change in matrix thickness. The relative biodegradability of the material after implantation was observed. Morphology and dimensions had small changes after AH. As for cell viability, none of the tested matrices showed a statistically significant difference (p > 0.05; Dunnett) regardless of the sterilization method. Furthermore, it was found that PP matrices did not interfere with the proliferation capacity of CHO-K1 cells (p > 0.05; Dunnett). As for genotoxicity, when sterilized with ethylene oxide (NP, P12 and P24), it showed genotoxic potential, but it was not genotoxic when sterilized by gamma radiation. No mutagenic effects were observed in either group. PP-derived collagen matrices hydrolyzed at different times were not cytotoxic. It is concluded that the best method of sterilization is through gamma radiation, since no significant changes were observed in the properties of the PP matrices.
RESUMEN
BACKGROUND: Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. METHODS: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. RESULTS: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. CONCLUSION: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.
RESUMEN
Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)
