Hinokiflavone is a novel CK2 inhibitor promoting apoptosis and synergizing with chemotherapeutic agents in cisplatin resistant bladder cancer cells.

Bladder cancer (BC) remains a major therapeutic challenge, particularly in patients with acquired resistance to platinum-based chemotherapy. In this study, we investigated the potential of hinokiflavone (HNK), a natural biflavonoid, as a therapeutic agent against cisplatin-resistant BC. Our results demonstrate that HNK differentially inhibited the proliferation of cisplatin-resistant BC cells while sparing normal uroepithelial cells. Mechanistically, HNK induced apoptosis through both intrinsic and extrinsic pathways, as evidenced by caspase activation and Annexin V staining. Next-generation sequencing and gene set enrichment analysis revealed that HNK modulates genes involved in biosynthesis, metabolism, DNA replication and DNA repair. Additionally, HNK downregulated the transcription of MUTYH, OGG1, and XRCC1, which are key genes in base excision repair. For the first time, we identified that HNK as a novel inhibitor of CK2α via in vitro kinase assays, substrate phosphorylation assays, and molecular docking analysis. HNK treatment reduced the phosphorylation of known CK2α targets, including Akt, Stat3, and XRCC1, in cisplatin-resistant BC cells. Time-course analysis revealed that the inhibition of Akt phosphorylation coincided with PARP cleavage, and genetic rescue experiments confirmed the involvement of the CK2α/Akt axis in HNK-induced apoptosis. Furthermore, combination treatment with HNK and chemotherapeutic agents such as doxorubicin or mitomycin C resulted in enhanced cytotoxic effects, suggesting a potential role of HNK as a chemosensitizing agent. HNK, by targeting both DNA repair pathways and CK2-mediated survival signaling, may serve as a promising therapeutic candidate for cisplatin-resistant BC.