Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Tiantan Xili, Beijing, 100050, China.
J Exp Clin Cancer Res. 2018 May 4;37(1):98. doi: 10.1186/s13046-018-0759-6.
A35 is a novel synthetic cyclizing-berberine recently patented as an antitumor compound. Based on its dual targeting topoisomerase (top) activity, A35 might overcome the resistance of single-target top inhibitors and has no cardiac toxicity for not targeting top2β. In this study we further explored the biological effects and mechanisms of A35.
Antitumor activity of A35 was evaluated by SRB and colony formation assay. G2/M phase arrest (especially M) and first damage of M-phase cells were investigated by flow cytometry, cytogenetic analysis, immunofluorescence, co-immunoprecipitation and WB. The key role of phospho-YAP (Ser127) in decreasing YAP nuclear localization, subsequent G2/M arrest and proliferation inhibition were explored by YAP1 cells, mutated Ser127 YAP construct (Ser127A) and TUNEL.
G2/M arrest induced by A35 was independent of p53. M phase cells at low dose were firstly damaged and most damaged-cells accumulated in M phase, and that was a result of preferring targeting top2α by A35, as top2α is essential to push M phase into next phase, and targeting top2α induced cells arrested at M phase. A35 decreased YAP1 nuclear localization by activating YAP phosphorylation (Ser127) which subsequently regulated the transcription of YAP target genes associated with growth and cycle regulation to induce G2/M arrest and growth inhibition.
Our studies suggested the mechanism of G2/M arrest induced by A35 and a novel role of YAP1 (Ser127) in G2/M arrest. As a dual topoisomerase inhibitor characterized by no cardiac toxicity, A35 is a promising topoisomerase anticancer agent and worthy of further development in future.
A35 是一种新型的合成环化小檗碱，最近被专利作为一种抗肿瘤化合物。基于其双重靶向拓扑异构酶（top）活性，A35 可能克服单靶点拓扑异构酶抑制剂的耐药性，并且由于不靶向 top2β，因此没有心脏毒性。在这项研究中，我们进一步探讨了 A35 的生物学效应和机制。
通过 SRB 和集落形成测定评估 A35 的抗肿瘤活性。通过流式细胞术、细胞遗传学分析、免疫荧光、共免疫沉淀和 WB 研究 G2/M 期阻滞（尤其是 M 期）和 M 期细胞的首次损伤。通过 YAP1 细胞、突变的 Ser127 YAP 构建体（Ser127A）和 TUNEL 探讨了磷酸化-YAP（Ser127）在降低 YAP 核定位、随后的 G2/M 期阻滞和增殖抑制中的关键作用。
A35 诱导的 G2/M 期阻滞不依赖于 p53。低剂量的 M 期细胞首先受损，大多数受损细胞积累在 M 期，这是因为 A35 优先靶向 top2α，因为 top2α 对于推动 M 期进入下一阶段至关重要，而靶向 top2α 诱导细胞停滞在 M 期。A35 通过激活 YAP 磷酸化（Ser127）来减少 YAP1 的核定位，随后调节与生长和周期调节相关的 YAP 靶基因的转录，从而诱导 G2/M 期阻滞和生长抑制。
我们的研究表明了 A35 诱导的 G2/M 期阻滞的机制以及 YAP1（Ser127）在 G2/M 期阻滞中的新作用。作为一种以无心脏毒性为特征的双重拓扑异构酶抑制剂，A35 是一种有前途的拓扑异构酶抗肿瘤药物，值得在未来进一步开发。
