Figure 4.

Effects of DT-010 on expression and activity of P-gp. (A) Relative messenger RNA expression of ABCB1, ABCC1, ABCG2, and SLC22A16 in MCF-7 and MCF-7/ADR cells. Data are expressed as mean ± SD. *p < 0.05, compared with MCF-7 cells. (B) Multidrug resistance (MDR) transporter activity was detected using a fluorometric MDR assay kit. MCF-7/ADR cells were treated with DT-010 (5, 10, and 20 μM) for 1 h, followed by the addition of 100 μl MDR dye-loading solution and incubated at 37°C for another 1 h. Data are expressed as mean ± SD. *p < 0.05, or **p < 0.01, compared with control. (C) DT-010 (20 μM), Dox (2 μM), or their combination did not affect P-gp expression in MCF-7/ADR cells. Western blot was performed, with anti-GAPDH as loading control. (D and E) The effect of DT-010 on P-gp substrate efflux activity was assessed. MCF-7/ADR cells (5.0 × 10⁴/well) were incubated with the P-gp-specific fluorescent substrate Rh123 at 0.5 μg/ml with or without DT-010 (20 μM) for 1 h, then washed twice with ice-cold phosphate-buffered saline (PBS), and incubated in Rh123-free medium at 37°C for additional 1 h with or without DT-010. Cells were analyzed on flow cytometry to detect Rh123 fluorescence. The PSC-833 (0.1 μM) was used as a positive control. MCF-7 cells were used for confirmation of overexpression of P-gp (increased Rh123 efflux) in MCF-7/ADR cells.