Bückreiß Nico, Schulz-Fincke Marie, König Philipp, Maccarana Marco, van Kuppevelt Toin H, Li Jin-Ping, Götte Martin, Bendas Gerd
Pharmaceutical Institute, Pharmaceutical and Cell Biological Chemistry, University of Bonn, 53121 Bonn, Germany.
Department of Medical Biochemistry and Microbiology, SciLifeLab Uppsala, The Biomedical Center, University of Uppsala, 75123 Uppsala, Sweden.
ACS Pharmacol Transl Sci. 2024 Jul 30;7(8):2484-2495. doi: 10.1021/acsptsci.4c00295. eCollection 2024 Aug 9.
The deregulation of cell surface heparan sulfate proteoglycans (HSPGs) is a main issue of cancer cells for increasing their malignancy. In these terms, the sulfation pattern of HS, created by an orchestrated activity of enzymes balancing a site-specific sulfation, is of key importance. These enzymes are often deregulated by epigenetic processes in cancer, e.g., being silenced by DNA hypermethylation. Here, we address this issue in human breast cancer cell lines aiming to target epigenetic processes to reactivate HS sulfation, shifting HS into an antithrombotic phenotype for which 3--sulfation is particularly important. Treatment of MCF-7 and MDA-MB-231 cells with nontoxic concentrations of 5-azacytidine (azacytidine) and 5-fluoro-2'-deoxycytidine (FdCyd) as DNMT inhibitors or vorinostat for targeting HDAC increased HS3--sulfation remarkably, as confirmed by fluorescence microscopy, by upregulating HS3--sulfotransferases, detected by quantitative real-time polymerase chain reaction and Western blot. Flow cytometry and microscopic approaches confirm that upon inhibitor treatment, increased HS3--sulfation improves cell binding to antithrombin, leading to an antithrombotic activity. Nevertheless, only azacytidine- and vorinostat-treated cells display anticoagulative properties, represented by attenuated thrombin formation, a lower activation of human platelet aggregation, or ATP release. In contrast, FdCyd additionally upregulated tissue factor expression in both cell lines, overshadowing the anticoagulant effects of HS, leading to an overall prothrombotic phenotype. Our data provide evidence for the first time that targeting epigenetic processes in HS sulfation is a valuable means to foster anticoagulative cell properties for decreasing malignancy and metastatic potency. These data warrant further investigations to fine-tune epigenetic targeting and to search for potential biomarkers attributed to these activities.
细胞表面硫酸乙酰肝素蛋白聚糖（HSPGs）的失调是癌细胞增加其恶性程度的一个主要问题。就此而言，由平衡位点特异性硫酸化的酶的协同作用所产生的HS硫酸化模式至关重要。这些酶在癌症中常常因表观遗传过程而失调，例如因DNA高甲基化而沉默。在此，我们在人乳腺癌细胞系中探讨这个问题，旨在针对表观遗传过程来重新激活HS硫酸化，使HS转变为抗血栓形成表型，其中3 - 硫酸化尤为重要。用无毒浓度的5 - 氮杂胞苷（氮杂胞苷）和5 - 氟 - 2'-脱氧胞苷（FdCyd）作为DNA甲基转移酶抑制剂或用伏立诺他靶向组蛋白去乙酰化酶来处理MCF - 7和MDA - MB - 231细胞，显著增加了HS 3 - 硫酸化，这通过荧光显微镜得到证实，通过定量实时聚合酶链反应和蛋白质印迹检测到HS 3 - 硫酸转移酶上调。流式细胞术和显微镜方法证实，在抑制剂处理后，增加的HS 3 - 硫酸化改善了细胞与抗凝血酶的结合，导致抗血栓形成活性。然而，只有经氮杂胞苷和伏立诺他处理的细胞表现出抗凝特性，表现为凝血酶形成减弱、人血小板聚集的激活降低或ATP释放减少。相比之下，FdCyd在两种细胞系中额外上调了组织因子表达，掩盖了HS的抗凝作用，导致总体促血栓形成表型。我们的数据首次证明，靶向HS硫酸化中的表观遗传过程是增强抗凝细胞特性以降低恶性程度和转移潜能的一种有价值的手段。这些数据值得进一步研究以微调表观遗传靶向并寻找归因于这些活性的潜在生物标志物。
