Activation of CAR enhances CPA anticancer activity in the human primary hepatocyte-HL-60 cell coculture system. The anticancer activity of CPA was analyzed in a unique hepatocyte-cancer cell coculture model. (A) Illustration of the HPH-HL-60 coculture model and experimental scheme. (B) Effects of CAR activation on the concentration-dependent anticancer activity of CPA in HL-60 cells. As detailed in “Methods,” the HPH-HL-60 cocultures were treated with vehicle control (0.1% DMSO) or CPA (250, 500, and 1000μM) in the presence and absence of CITCO (1μM). Cell viability was analyzed 36 hours after the cotreatment. (C) Effects of CAR activation on the temporal changes of CPA-mediated anticancer activity in HL-60 cells. In the cocultures, cells were treated with vehicle control (0.1% DMSO) or CPA (500μM) with or without CITCO (1μM). Cell viability was measured at 0, 12, 24, 36, and 48 hours after the cotreatment. All viability data represent mean ± SD from 3 independent experiments and are expressed as percent viability of vehicle control (**P < .01). (D) The multiple-reaction monitoring chromatogram demonstrates separation and retention of 4-OH-CPA, CPA, and the internal standard from LC-MS/MS detection. (E) Effects of CAR activation on the concentration-dependent formation of 4-OH-CPA in the coculture under the treatment as outlined in panel B. (F) Effects of CAR activation on the temporal changes of 4-OH-CPA formation in cocultures under the treatment described in panel C. 4-OH-CPA concentrations represent mean ± SD of 3 LC-MS measurements (**P < .01).