Cyclizing-berberine A35 induces G2/M arrest and apoptosis by activating YAP phosphorylation (Ser127)

Background: 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 top2 beta. In this study we further explored the biological effects and mechanisms of A35. Methods: 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. Results: 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 top2 alpha by A35, as top2 alpha is essential to push M phase into next phase, and targeting top2 alpha 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. Conclusions: 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.