Figure 3.

Paclitaxel-resistant NPC cells developed MDR and showed decreased intracellular drug concentrations. CNE2TR and CNE2 cells were treated with paclitaxel (a), cisplatin (b) or chlorambucil (c) at the doses as shown. MTS assays were used to test the cell viability 48 h after treatment with three repeats for each dose, and relative cell survival was calculated (treatment versus control) to compare the IC50 of drugs. Two pairs of NPC cells, CNE2TR/CNE2 (d) and CNE1TR/CNE1 (e) were used to test drug efflux. These cells were treated with 500 ng/ml paclitaxel for 2 h. The culture media were harvested for to test intracellular drug concentrations. The cells were completely washed, and then prepared by ultrasonic homogenization. The solution after spinning off cell debris was used to test intracellular drug concentrations. The drug concentrations were measured by UPLC-MS. (f) Intracellular drug concentrations were monitored by confocal microscopy. Fluorescent chlorambucil was synthesized by the conjunction with chlorambucil and the delocalized lipophilic cation probes rhodamine 123 and MKT-077. CNE2TR and CNE2 cells were treated with the fluorescent chlorambucil for 6, 12 and 24 h, and intracellular green fluorescence was monitored by confocal microscopy. (g and h) The strength of fluorescence was assessed by flow cytometry. CNE2TR/CNE2 or CNE1TR/CNE1 cells were treated with the rhodamine 123-labeled chlorambucil for 24 h, and fluorescence-positive cells were analyzed by flow cytometry. *P<0.05, **P<0.01, ***P<0.001