H89 Reverses Multidrug Resistance in Colorectal Cancer by Inhibiting the ATPase Activity of ABCB1

<b>Background:</b> Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy, and overexpression of ABCB1 plays a critical role in the pathogenesis of MDR. Despite decades of research, significant clinical progress in the development of ABCB1 inhibitors has yet to be achieved. The small-molecule H89 is originally identified as an inhibitor of protein kinase A (PKA), but it also exhibits various functions unrelated to the PKA. This study investigates H89 as a novel ABCB1-inhibitor to reverse MDR in colorectal cancer (CRC). <b>Methods:</b> Cytotoxicity assays were performed on ABCB1-overexpressing MDR cell line HCT-8/V and parental CRC cell line HCT-8. Drug accumulation was quantified via flow cytometry, and cell cycle effects were analyzed using propidium iodide staining. The ATPase activity of ABCB1 was detected using an ATPase activity assay kit. Molecular docking utilized the ABCB1 crystal structure. <b>Results:</b> Both 3 μM and 10 μM H89 significantly reverses resistance to two ABCB1 substrate drugs (doxorubicin and vincristine) in HCT-8/V cells in a dose-dependent manner, with no such effect observed inHCT-8 cells. The combination of H89 and doxorubicin or vincristine resulted in a significant increase in the proportion ofHCT-8/Vcells in the sub-G1 and G2/M phases. Further mechanistic studies reveal that H89 exerts its effect by inhibiting the drug efflux function of ABCB1, thereby increasing the intracellular accumulation of the substrate drugs and reversing multidrug resistance. Furthermore, H89 did not alter the expression of ABCB1. H89 effectively inhibited the ATPase activity of ABCB1. Molecular docking simulations revealed the binding mode of H89 with ABCB1. <b>Conclusions:</b> The combination of H89 with ABCB1 substrate drugs significantly reverses multidrug resistance in colorectal cancer. These findings provide a strong theoretical and experimental foundation for the development of novel MDR-reversing agents targeting ABCB1.