Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart

Abstract

P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.