Kinetics of doxorubicin handling in the LLC-PK1 kidney epithelial cell line is mediated by both vesicle formation and P-glycoprotein drug transport

Abstract

The subcellular distribution of doxorubicin was evaluated in living non-fixed LLC-PK1 cells, which maintain the structural and functional characteristics of the kidney proximal tubule epithelium and also express P-glycoprotein. After 10 min incubation, doxorubicin fluorescence was detectable in the nucleus. The intensity of nuclear fluorescence progressively increased, reaching the maximum at the end of the first hour. Then, the nuclear signal started to decrease and, at 2 h, doxorubicin fluorescence disappeared almost completely from the cell nucleus. Cytoplasmic fluorescent vesicles first appeared in the perinuclear region after 10 min doxorubicin exposure and increased in number and size over a period of 2 h. From 2 to 5 h, fluorescent vesicles moved unidirectionally to the cell periphery. Disappearance of doxorubicin punctate fluorescence in LLC-PK1 cells treated with methylamine or monensin demonstrated that drug accumulation occurred inside acidic compartments. In addition, the cytoplasmic pattern of doxorubicin fluorescence was very similar to that observed upon exposure to the acidotropic tracer LysoSensor Blue. Involvement of P-glycoprotein in doxorubicin handling by LLC-PK1 cells was suggested by modified intracellular doxorubicin distribution after cell incubation with verapamil and vinblastine. Moreover, the fluorescent P-glycoprotein substrate Bodipy FL Verapamil was shown to accumulate in LLC-PK1 cells in a manner that is quite similar to that observed for doxorubicin. P-glycoprotein expression was evaluated by immunoblot using the JSB-1 and C219 monoclonal antibodies. Immunofluorescence analysis was performed using the JSB-1 monoclonal antibody. P-glycoprotein immuno-reactivity was found both on the plasma membrane and intracytoplasmically in a perinuclear position. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that MDR1 gene was expressed. This study indicates that a rapid intracellular redistribution accompanies the process of doxorubicin uptake by LLC-PK1 cells. Although these cells are non-tumour cells derived from the normal epithelium of the proximal renal tubule, they display a model of doxorubicin redistribution which is characteristic of doxorubicin-resistant tumour cells.