Down-Regulation of CYP3A4 by the KCa1.1 Inhibition Is Responsible for Overcoming Resistance to Doxorubicin in Cancer Spheroid Models

Abstract

The large-conductance Ca2+-activated K+ channel, KCa1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of KCa1.1 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 KCa1.1 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 KCa1.1 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 KCa1.1 in cancer spheroid models, and KCa1.1-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 KCa1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model

“…As shown in Figure 3 A, the expression level of CEBPD was increased by the 3D spheroid formation of LNCaP cells. In a previous study, CEBPB was also up-regulated together with 3D spheroid formation [ 2 ]. Therefore, we examined the effects of the siRNA-mediated inhibition of CEBPB and CEBPD on the expression level of FBXW7 transcripts.…”

“…Balamurugan et al reported the involvement of the transcriptional regulator CE in the inhibition of FBXW7 expression in cancer cells [15]. As shown in Figure 3A expression level of CEBPD was increased by the 3D spheroid formation of LNCaP In a previous study, CEBPB was also up-regulated together with 3D spheroid form [2]. Therefore, we examined the effects of the siRNA-mediated inhibition of CEBPB CEBPD on the expression level of FBXW7 transcripts.…”

“…We recently reported that the inhibition of K Ca 1.1 suppressed the Akt-Nrf2 signaling pathway by reducing the phosphorylation level of Akt in the LNCaP spheroid model [ 2 ]. Wang et al indicated that activated Akt down-regulated FBXW7 in cancer cells [ 11 ].…”

“…Nanog is a possible substrate of FBXW7-mediated protein degradation and leads to chemoresistance in cancer [ 8 , 9 ]. Multidrug resistance proteins (MRPs), which are involved in the acquisition of DOX resistance, were up-regulated together with the spheroid formation of LNCaP cells through the Akt-Nrf2-Nanog axis [ 2 , 5 ]. Similarly, previous studies indicated the impact of Akt-Nrf2-Nanog-mediated MRPs on chemoresistance and cancer stemness.…”

“…The large-conductance Ca 2+ -activated K + channel, K Ca 1.1, encoded by the KCNMA1 gene is activated by membrane depolarization and by an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [ 1 ]. In non-excitable cells, including cancer cells, K Ca 1.1 activation-induced hyperpolarization increases the electrochemical gradient for Ca 2+ through voltage-independent, store-operated Ca 2+ channels, resulting in increases in the [Ca 2+ ] i , and K Ca 1.1 activation also reduces [K + ] i , resulting in the modulation of intracellular signaling [ 1 , 2 ]. K Ca 1.1 plays a critical role in the proliferation, migration, apoptosis, and metastasis of cancer cells by controlling Ca 2+ signaling and cell volume [ 3 ].…”