细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）的失调是癌细胞增加其恶性程度的一个主要问题。从这些角度来看，HS 的硫酸化模式是由平衡位点特异性硫酸化的酶的协调活动产生的，是至关重要的。这些酶通常会因癌症中的表观遗传过程而失调，例如因 DNA 高甲基化而沉默。在这里，我们在人类乳腺癌细胞系中解决了这个问题，旨在靶向表观遗传过程以重新激活 HS 硫酸化，将 HS 转变为抗血栓表型，其中 3-O-硫酸化对此尤为重要。用无毒浓度的 5-氮杂胞苷 (azacytidine) 和 5-氟-2'-脱氧胞苷 (FdCyd) 作为 DNMT 抑制剂或靶向 HDAC 的伏立诺他 (vorinostat) 处理 MCF-7 和 MDA-MB-231 细胞，显着增加 HS3-O-硫酸化，通过荧光显微镜证实，通过上调 HS3-O-磺基转移酶，通过定量实时聚合酶链反应和蛋白质印迹检测。流式细胞术和显微方法证实，抑制剂处理后，HS3-O-硫酸化增加可改善细胞与抗凝血酶的结合，从而产生抗血栓活性。然而，只有氮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、人血小板聚集活化较低或 ATP 释放。相比之下，FdCyd 还上调了两种细胞系中的组织因子表达，掩盖了 HS 的抗凝作用，导致整体血栓前表型。我们的数据首次提供证据表明，针对 HS 硫酸化中的表观遗传过程是培养抗凝血细胞特性以降低恶性肿瘤和转移效力的宝贵手段。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动的潜在生物标志物。© 2024 美国化学会。

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-O-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-O-sulfation remarkably, as confirmed by fluorescence microscopy, by upregulating HS3-O-sulfotransferases, detected by quantitative real-time polymerase chain reaction and Western blot. Flow cytometry and microscopic approaches confirm that upon inhibitor treatment, increased HS3-O-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.© 2024 American Chemical Society.