Apo-form of recombinant human lactoferrin changes the genome-wide DNA methylation level and the chromatin compaction degree in neuroblastoma cell line IMR-32

Abstract

BACKGROUND: Neuroblastoma is one of the most common extracranial solid tumors in childhood. At present, epigenetic disorders play a significant role in neoplasms development. Since epigenetic changes in the cell are quite dynamic and reversible, epigenome-modulating exogenous agents can be used in epigenetic targeted therapy for various types of tumors. Therefore, the identification of these agents is still significant. Lactoferrin is one such potential molecule from the transferrin family. Currently, the anti-tumor properties of lactoferrin have been identified, but its effect on the epigenome of cells of various tumors types, particularly on neuroblastomas, is practically unknown.
 AIM: To study the effect of the exogenous recombinant human apolactoferrin on the viability and epigenomic status of IMR-32 neuroblastoma cells.
 MATERIALS AND METHODS: We studied human IMR-32 neuroblastoma cells after 72 hours of exposure to 8 doses of recombinant human apolactoferrin: 0.1, 0.5, 1, 5, 10, 50, 100 and 500 g/ml. The level of genome-wide DNA methylation and the degree of chromatin compaction in IMR-32 cells were quantified using commercial kits 5-mC DNA ELISA Kit, Global DNA Methylation LINE-1 Kit, as well as enzymatic hydrolysis of MspI / HpaII and DNaseI.
 RESULTS: The recombinant apolactoferrin reduces the viability of IMR-32 and, depending on the dose, differentially affects the level of genome-wide DNA methylation (СpG dinucleotides, CCGG sites, LINE-1 repeats) and the degree of chromatin compaction. At the same time, a complex picture of the epigenomic cellular response to the effect of apo-lactoferrin was observed (nonlinear nonmonotonic dose-effect relationship).
 CONCLUSIONS: We assumed that apolactoferrin modulates gene activity through epigenetic mechanisms, in particular, by changing the DNA methylation pattern and affecting the chromatin structure, which may be one of the molecular mechanisms of its anti-tumor effect.