DataSheet_2_Combination Treatment of CI-994 With Etoposide Potentiates Anticancer Effects Through a Topoisomerase II-Dependent Mechanism in Atypical Teratoid/Rhabdoid Tumor (AT/RT).docx

PurposeAtypical teratoid/rhabdoid tumor (AT/RT) is arising typically in young children and is associated with a dismal prognosis which there is currently no curative chemotherapeutic regimen. Based on previous studies showing high histone deacetylase 1 (HDAC1) expression in AT/RT, the HDAC1 inhibitor CI-994 was used as a novel treatment strategy in this study. We assessed the anticancer effects of CI-994 and conventional drugs (etoposide, cisplatin or 4-HC) in AT/RT cells.
MethodsAT/RT patient-derived primary cultured cells and cell lines were prepared. HDAC1 was estimated by real-time quantitative polymerase chain reaction (RT-qPCR). The interaction of the drugs was analyzed using isobologram analysis. Cell viability, apoptosis, HDAC enzyme activity and western blot assays were carried out.
ResultsHDAC1 was overexpressed in AT/RT compared to medulloblastoma. The combination index (CI) of CI-994 with etoposide revealed a synergistic effect in all AT/RT cells, but no synergistic effect was observed between CI-994 and cisplatin or 4-HC. CI-994 effectively reduced not only Class I HDAC gene expression but also HDAC enzyme activity. The combination treatment of CI-994 with etoposide significantly increased apoptosis compared to the single treatment. The enhanced effect of apoptosis by this combination treatment is related to a signaling pathway which decreases topoisomerase (Topo) II and increases histone H3 acetylation (Ac-H3).
ConclusionWe demonstrate that the combination treatment of CI-994 with etoposide exerts a synergistic anticancer effect against AT/RT by significantly inducing apoptosis through Topo II and Ac-H3 regulation.
Clinical RelevanceThis combination treatment might be considered a viable therapeutic strategy for AT/RT patients.

研究目的：非典型畸胎瘤/横纹肌样瘤（Atypical teratoid/rhabdoid tumor, AT/RT）好发于幼儿群体，预后极差，目前尚无治愈性化疗方案。既往研究显示，AT/RT组织中组蛋白去乙酰化酶1（histone deacetylase 1, HDAC1）呈高表达，因此本研究选用HDAC1抑制剂CI-994作为新型治疗策略，评估其与常规化疗药物（依托泊苷（etoposide）、顺铂（cisplatin）或4-HC）对AT/RT细胞的抗癌效应。
实验方法：本研究制备了AT/RT患者来源的原代培养细胞及细胞系。采用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）检测HDAC1的表达水平；采用等效图分析（isobologram analysis）分析药物间的相互作用；此外还开展了细胞活力检测、细胞凋亡检测、HDAC酶活性检测及蛋白质印迹（western blot）实验。
实验结果：与髓母细胞瘤（medulloblastoma）相比，AT/RT组织中HDAC1呈现过表达状态。CI-994与依托泊苷（etoposide）的联合指数（combination index, CI）显示，二者在所有AT/RT细胞系中均表现出协同抗癌效应；而CI-994分别与顺铂（cisplatin）或4-HC联合给药时，未观察到协同作用。CI-994不仅可有效下调I类HDAC基因的表达，还能显著抑制HDAC酶活性。与单药治疗相比，CI-994联合依托泊苷可显著提升细胞凋亡率。该联合疗法增强细胞凋亡的作用机制与一条信号通路相关：该通路可下调拓扑异构酶（topoisomerase, Topo）II的表达，并上调组蛋白H3乙酰化（Ac-H3）水平。
研究结论：本研究证实，CI-994与依托泊苷（etoposide）联合治疗可通过调控Topo II与Ac-H3的表达水平，显著诱导细胞凋亡，从而对AT/RT发挥协同抗癌效应。
临床相关性：该联合疗法或可成为AT/RT患者的可行治疗策略。