Isovalerylspiramycin I inhibits proliferation, migration and invasion of osteosarcoma cells by targeting Topoisomerase 1 and suppressing the ataxia telangiectasia and Rad3-related/checkpoint kinase 1 pathway

PurposeTopoisomerase 1 (TOP1) plays a crucial role in various cell cycle processes and its dysregulation can lead to the development of multiple tumours. However, conventional TOP1 inhibitors such as topotecan and irinotecan have poor clinical efficacy in osteosarcoma (OS) patients. This is partly due to the activation of the ataxia telangiectasia and Rad3-related/checkpoint kinase 1 (ATR/CHEK1) DNA damage repair pathway, which repairs TOP1 poison-induced DNA lesions, compromises the cytotoxicity of TOP1 inhibitors and contributes to drug resistance. Therefore, there is a need to develop more effective TOP1 inhibitors for OS.Experimental designIn this study, we evaluated the antitumor effects of isovalerylspiramycin I (ISP-I), a novel macrolide antibiotic, using various assays including CCK-8 proliferation assays, wound healing migration assays, Transwell invasion assays, apoptosis, cell cycle, DNA replication and damage analyses on OS cells. We also performed a surface plasmon resonance-high-performance liquid chromatography-mass spectrometry assay to identify ISP-I's direct target protein in OS. Molecular docking analysis, thermoshift assays, enzyme activity assays and reverse tests were used to confirm ISP-1 ' s target. Finally, we tested the efficacy of ISP-I in vivo using a tumour xenograft model.ResultsOur results showed that ISP-I significantly suppressed the growth of OS cells both in vitro and in vivo. Furthermore, ISP-I dose-dependently inhibited cell migration and invasion, and induced apoptosis and cell cycle arrest in OS cells. Mechanistically, ISP-I directly bound to TOP1 and inhibited DNA replication. Additionally, ISP-I significantly downregulated the ATR/CHEK1 pathway, which led to the suppression of DNA damage repair, ultimately augmenting DNA damage and triggering cell death.ConclusionsIn conclusion, our study suggests that ISP-I could be a novel TOP1 inhibitor that does not activate the ATR/CHEK1 DNA damage repair pathway. This characteristic allows ISP-I to synergistically inhibit OS cell proliferation, migration and invasion. ISP-I may represent a promising candidate for the treatment of OS. The target protein of ISP-I was determined by surface plasmon resonance high-performance liquid chromatography-mass spectrometry (SPR-HPLC-MS). ISP-I targeting TOP1 causes DNA damage. ISP-I inhibits the DNA damage repair pathway, which prevents DNA repair and leads to cell death. image