Interaction of CJY, an Isoflavone, with ATPase of P-glycoprotein in Doxorubicin-resistant Human Myelogenous Leukemia (K562/DOX) Cells

Abstract

The previous study reported CJY, an isoflavone, can reverse P-glycoprotein (P-gp)-mediated multidrug-resistance (MDR) in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. This study will investigate the exact mechanism of CJY on P-gp. By assessment of ATPase activity, we gained further insight into the nature of the CJY interactions with P-gp. Kinetic studies on ATPase activity were applied to show the effects of Verapamil (Ver) on CJY-stimulated, CJX1 on Ver-stimulated, and CJX1 on CJY-stimulated P-gp ATPase activity. Furthermore, the combined effects of CJY with Ver, and CJY with CJX1 were also evaluated isobolographically in numerous fixed-ratio combinations of 1:1, 1:2, 1:4, 1:8, and 1:10. The results showed that basal P-gp ATPase activity was increased by CJY with half-maximal activity concentration (Km) of 2.9±0.3 μM and the maximal ATPase activity velocity (Vmax) of 265±21 nM · min-1 · mg-1. Kinetic studies on ATPase activity showed the effects of Verapamil (Ver) on CJY-stimulated, CJX1 on Ver-stimulated, and CJX1 on CJY-stimulated P-gp ATPase activity were all non-competitive inhibition, indicating that these substrates can simultaneously but independently bind to diverse sites on P-gp. The combined effects of CJY with Ver, and CJY with CJX1 show that mixtures of both drugs at these fixed-ratios displayed synergistic interactions. CJY, CJX1 and Ver bind P-gp on different sites. CJY could be applied combining with other P-gp inhibitors to get better reversal of multidrug resistance than it used alone.