Department of Virology and Immunology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19 Street, 20-033 Lublin, Poland.
Department of Cell Biology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19 Street, 20-033 Lublin, Poland.
Molecules. 2022 Dec 22;28(1):97. doi: 10.3390/molecules28010097.
Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon.
由于天然物质被广泛探索为基因表达的表观遗传调节剂，并且表观遗传异常是癌症发生的重要原因，因此受表观遗传控制的具有促肿瘤活性的因素，例如中性内肽酶（NEP， Neprilysin），是通过基因表达的表观遗传调控作用的有前途的抗癌靶标。α-酮戊二酸（AKG）是参与组蛋白和 DNA 去甲基化的酶的天然共底物，具有潜在的抗癌活性。因此，我们研究了 AKG 对源自各种癌症（宫颈癌、结肠癌、骨肉瘤）和正常上皮细胞和成骨细胞的细胞中 NEP 表达的潜在影响。此外，还探索了组蛋白 H3 的整体甲基化状态，以确定 AKG 活性的分子靶标。此外，还研究了 AKG 与硫普罗宁（NEP 特异性抑制剂）联合使用是否表现出增强的抗癌活性。结果表明，仅在高度侵袭性骨肉瘤 HOS 细胞中，AKG 在蛋白质水平下调 NEP 的表达（流式细胞术和荧光测定法），并且发现该蛋白酶参与 AKG 诱导的骨肉瘤细胞生长抑制（siRNA NEP 沉默，BrdU 测定法，流式细胞术）。出乎意料的是，AKG 诱导 HOS 细胞中 H3K27 的超甲基化，这部分依赖于 EZH2 活性。然而，这种作用与 AKG 诱导的 NEP 下调无关（流式细胞术）。最后，结果表明 AKG 与硫普罗宁联合治疗可显著增强每种药物对 HOS 细胞的生长抑制潜力（BrdU 测定法）。这些初步研究首次表明，下调 NEP 表达是治疗涉及 NEP 的 HOS 细胞的有前途的靶标。此外，这种治疗目标可以通过 AKG 诱导的 NEP 下调和 AKG 与硫普罗宁的协同作用来实现，即 NEP 特异性抑制剂。此外，本研究首次报道外源性 AKG 可以影响组蛋白甲基转移酶 EZH2 的活性。然而，需要进一步研究来阐明这一现象的机制。
