7-Ketocholesterol induces P-glycoprotein through PI3K/mTOR signaling in hepatoma cells

Abstract

7-Ketocholesterol (7-KC) is found at an elevated level in patients with cancer and chronic liver disease. The up-regulation of an efflux pump, P-glycoprotein (P-gp) leads to drug resistance. To elucidate the effect of 7-KC on P-gp, P-gp function and expression were investigated in hepatoma cell lines Huh-7 and HepG2 and in primary hepatocyte-derived HuS-E/2 cells. At a subtoxic concentration, 48-h exposure to 7-KC reduced the intracellular accumulation and cytotoxicity of P-gp substrate doxorubicin in hepatoma cells, but not in HuS-E/2 cells. In Huh-7 cells, 7-KC elevated efflux function through the activation of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. 7-KC activated the downstream protein synthesis initiation factor 4E-BP1 and induced P-gp expression post-transcriptionally. The stimulation of efflux was reversible and could not be prevented by N-acetyl cysteine. Total cellular ATP content remained the same, whereas the lactate production was increased and fluorescence lifetime of protein-bound NADH was shortened. These changes suggested a metabolic shift to glycolysis, but glycolytic inhibitors did not eliminate 7-KC-mediated P-gp induction. These results demonstrate that 7-KC induces P-gp through PI3K/mTOR signaling and decreased the cell-killing efficacy of doxorubicin in hepatoma cells.

Keywords: 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium; 4E-BP1; 7-KC; 7-Ketocholesterol; 7-ketocholesterol; AKI; Akt kinase inhibitor; ERK; FBS; FLIM; GAPDH; Glycolysis; Hepatoma; LDH; MDR; MTT; NADH; P-Glycoprotein; P-glycoprotein; P-gp; PBMC; PCR; PI3K; PI3K/mTOR; ROS; RT; Rh123; eukaryotic translation initiation factor (eIF) 4E-binding protein 1; extracellular signal-regulated kinase; fetal bovine serum; fluorescence lifetime imaging; glyceraldehyde 3-phosphate dehydrogenase; lactate dehydrogenase; mTOR; mammalian target of rapamycin; multidrug resistance; p70 ribosomal protein S6 kinase; p70S6K; peripheral blood mononuclear cells; phosphatidylinositol 3-kinase; polymerase chain reaction; reactive oxygen species; reduced nicotinamide adenine dinucleotide; reverse transcription; rhodamine 123.