Down-Regulation of CYP3A4 by the K_Ca1.1 Inhibition Is Responsible for Overcoming Resistance to Doxorubicin in Cancer Spheroid Models

The large-conductance Ca²⁺-activated K⁺ channel, K_Ca1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of K_Ca1.1 overcame resistance to doxorubicin (DOX) by down-regulating multidrug resistance-associated proteins in the three-dimensional spheroid models of human prostate cancer LNCaP, osteosarcoma MG-63, and chondrosarcoma SW-1353 cells. Investigations have recently focused on the critical roles of intratumoral, drug-metabolizing cytochrome P450 enzymes (CYPs) in chemoresistance. In the present study, we examined the involvement of CYPs in the acquisition of DOX resistance and its overcoming by inhibiting K_Ca1.1 in cancer spheroid models. Among the CYP isoforms involved in DOX metabolism, CYP3A4 was up-regulated by spheroid formation and significantly suppressed by the inhibition of K_Ca1.1 through the transcriptional repression of CCAAT/enhancer-binding protein, CEBPB, which is a downstream transcription factor of the Nrf2 signaling pathway. DOX resistance was overcome by the siRNA-mediated inhibition of CYP3A4 and treatment with the potent CYP3A4 inhibitor, ketoconazole, in cancer spheroid models. The phosphorylation levels of Akt were significantly reduced by inhibiting K_Ca1.1 in cancer spheroid models, and K_Ca1.1-induced down-regulation of CYP3A4 was reversed by the treatment with Akt and Nrf2 activators. Collectively, the present results indicate that the up-regulation of CYP3A4 is responsible for the acquisition of DOX resistance in cancer spheroid models, and the inhibition of K_Ca1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis.