Orchestration of kaempferol nuclear translocation by phase ii metabolic enzymes and efflux transporters suppresses colorectal cancer.

Kaempferol exhibits promising anti-colorectal cancer (CRC) activity, but its intracellular exposure and efficacy are strongly influenced by phase II metabolism and efflux transport. Here, we show that UGT1A1 and UGT1A9 differentially regulate kaempferol accumulation and anti-CRC activity, and that ABCG2/BCRP and Mdr1/P-gP play distinct roles in its intracellular retention and pharmacological effects. Anti-CRC activity was evaluated using sulforhodamine B, colony formation, wound-healing, Transwell, apoptosis, and xenograft assays. Knockdown models of UGT1A1/UGT1A9 and BCRP/P-gP were combined with laser scanning confocal microscopy and LC/MS to characterize intracellular exposure, metabolite disposition, and functional consequences. SIP-based proteomics, bioinformatic analyses, molecular docking, and orthogonal validation assays were used to prioritize and validate a nuclear target of kaempferol. Kaempferol significantly suppressed tumour growth and inhibited CRC cell proliferation and migration. UGT1A1 knockdown increased intracellular accumulation of kaempferol, including nuclear exposure, but reduced cytotoxicity, whereas UGT1A9 knockdown enhanced cellular sensitivity. Similarly, BCRP knockdown increased intracellular accumulation and anti-CRC efficacy, whereas Mdr1 knockdown attenuated the antitumour effect despite increasing total intracellular accumulation. Mechanistically, these differential effects were associated with altered metabolite composition, particularly the balance between the active metabolite K-7-G and the weakly active metabolite K-3-G. BRG1 was prioritized as a functionally relevant nuclear target of kaempferol, and kaempferol promoted its ubiquitination-dependent proteasomal degradation. Together, these findings show that kaempferol efficacy is determined not simply by intracellular accumulation, but by the coordinated interplay among phase II metabolism, efflux transport, metabolite disposition, and nuclear target engagement. This study provides a mechanistic basis for improving kaempferol-based CRC therapy.