br Materials and methods br Results br

Materials and methods
Results
Discussion In the absence of vascularization phenomena, endothelial cells (ECs) are among the most quiescent cells of the body [27]. ECs proliferation is significant during embryogenesis, as the vasculature is being formed, while in the adult life takes place only during physiological of pathological neovascularization processes [28]. Detailed knowledge of regulation and control of the proliferation process of ECs may significantly affect the efficiency of anticancer therapy treatment by limiting the pathological cancer-associated neovascularization [29]. In particular, many potential angiostatic agents targeting enzymes involved in epigenetic processes, such as histone deacetylases, HMTs and DNA methyltransferases have been described [30], suggesting a novel approach to inhibit tumor neovascularization. In the presented study, we focused on the role of euchromatic histone methyltransferase G9a – an enzyme found overexpressed in multiples cancer types – in the regulation of human microvascular endothelial cell proliferation. Using experimental models of (i) pharmacological and (ii) transcriptional inhibition of the enzyme, we analyzed the consequences of G9a HMT inhibition on chromatin methylation, cell proliferation, and cellular redox processes. Firstly, the effectiveness of G9a HMT inhibitors BIX-01294 and chaetocin on HMEC-1 was analyzed by assessing cell viability(Fig. 1A, B). Next, changes in histone methylation and chromatin remodeling upon BIX-01294 and chaetocin administration were evaluated using inhibitor concentrations not affecting cell viability. 10μM BIX-01294 – a concentration of the compound used for inhibition of G9a HMT in other cell types [31], [32] – inhibited both mono- and di- methylation of H3K9 (Fig. 1C, middle panel), whereas 20nM of chaetocin was insufficient to affect methylation of lysine 9 (Fig. 1C, bottom panel). Immunofluorescent analysis of HP-1α and γ localization, two proteins interacting with methylated histone H3, revealed that BIX-01294 decreased HP-1α and γ nuclear localization, while the effect of 20nM chaetocin were less pronounced (Fig. 2). HP-1 proteins are essential structural components of mitotic chromatin, protecting the integrity of chromosomes during cell division [33], and gene knockdown of HP-1 by RNAi in Drosophila melanogaster Kc cells have been shown to alter FH1 synthesis progression, with loss of S and G2/M cell populations and accumulation of aopototic cells [34]. In a comparable manner, upon BIX-01294 treatment of HMEC-1, we observed an increased fraction of cell in the G0/G1 phase, paralleled by a loss of cells in the S and G2/M phases (Fig. 3). In the search for a rationale to the decrease in the S and G2/M cell populations in HMEC-1 treated with G9a HMT inhibitors, we quantified the gene expression of the checkpoint genes Rb and cyclin-dependent kinase (CDK) inhibitor p21 [35]. We found that gene expression levels of both Rb and p21 were upregulated by BIX-01294 in a dose-dependent fashion, accounting – at least in part – for the accumulation of HMEC-1 cells in the G0/G1 phase (Fig. 3C). Furthermore, G9a HMT pharmacological inhibition likely induced a feed-forward inhibitory loop, as BIX-01294 induced gene repression of G9a HMT and its heterodimerizing partner GLP (Fig. 3C). Another key checkpoint protein is Chk1 kinase, which acts downstream of the ATM/ATR kinases and plays an important role in cell cycle progression [34]. As the activation of Chk1 occurs by phosphorylation at Ser317 by the ATM/ATR protein kinases [35], we monitored phosphorylated Chk1 (on serine 317) in response to BIX-01294 and chaetocin treatment (Fig. 3E), observing a significant nuclear accumulation in BIX-01294-treated cells. Previous investigations, mainly performed on tumor cell lines treated with G9a HTM inhibitor concentrations affecting cell viability, are concordant is asserting that BIX-01294 and chaetocin induce an increase in intracellular ROS, leading to apoptosis [36], [37], autophagic cell death [30], or NF-κB – dependent activation of autophagy [38]. At odds with these previous findings, we observed a dose-dependent decrease of reactive oxygen species upon administration of BIX-01294 or chaetocin to HMEC-1 cells (Fig. 4A, B), and, accordingly, the gene expression levels of the anti-oxidant enzymes catalase and SOD1 were significantly increased in BIX-01294 treated cells (Fig. 4C), as well as the total antioxidant capacity (Fig. 4E). We hypothesize that the antioxidant effects of BIX-01294 or chaetocin is due to the fact that we exposed HMEC-1 to low concentrations of the inhibitors, not affecting cell viability. Registered changes in the redox homeostasis of HMEC-1 due to decreased activity of G9a HMT, are in line with obtained cell cycle results, and with the number of studies showing that slight overproduction of reactive oxygen species, to the level that enhances their signaling messengers function, stimulates cell proliferation [39], [40]. These findings, in relation to our data, may suggest multilevel control of cell proliferation process by the H3K9 methyltransferase.