Fig 1.
Chemical structures of enantiopure forms of statins.
Four individual enantiomers of atorvastatin, rosuvastatin and fluvastatin are shown in the figure. Clinically used enantiopure forms are circled.
Fig 2.
Cytotoxicity of statin enantiomers in human cancer cell lines.
AZ-AHR, AZ-GR and LS180 cells were seeded in 96-well plates, stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10−10 M to 10−4 M. The vehicle was dimethylsulfoxide (DMSO; 0.1% v/v). After the treatment, a conventional MTT test was performed and absorbance was measured at 540 nm. Treatments were performed in triplicates. The data are the mean ± SD from experiments performed in three consecutive passages of cells and are expressed as percentage of viability of control cells. The values of IC50 were calculated where appropriate and they are indicated in plots. Student´s t-test, One-way ANOVA followed by Dunnett's post test and IC50 values were calculated using GraphPad Prism.
Fig 3.
Effects of statin enantiomers on trancriptional activity of human aryl hydrocarbon receptor.
AZ-AHR cells were seeded in 96-well plates and stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10−10 M to 10−4 M in the absence (agonist mode–upper panels) or presence (antagonist mode–lower panels) of dioxin (TCDD; 5 nM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates. Data are expressed as a fold induction of luciferase activity over control cells (agonist mode) or as a percentage of maximal activation attained by TCDD (antagonist mode). The values of EC50 and IC50 from n independent cell passages were calculated where appropriate and the average values are indicated in plots. Representative gene reporter assays are shown. Student´s t-test, One-way ANOVA followed by Dunnett's post test and EC50/IC50 values were calculated using GraphPad Prism.
Fig 4.
Effects of statin enantiomers on transcriptional activity of human glucocorticoid receptor.
AZ-GR cells were seeded in 96-well plates and stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10−10 M to and 10−4 M in the absence (agonist mode–upper panels) or presence (antagonist mode–lower panels) of dexamethasone (DEX; 100 nM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates. Data are expressed as a fold induction of luciferase activity over control cells (agonist mode) or as a percentage of maximal activation attained by DEX (antagonist mode). The values of EC50 and IC50 from n independent cell passages were calculated where appropriate and the average values are indicated in plots. Representative gene reporter assays are shown. Student´s t-test, One-way ANOVA followed by Dunnett's post test and EC50/IC50 values were calculated using GraphPad Prism.
Fig 5.
Effects of statin enantiomers on transcriptional activity of human pregnane X receptor.
LS180 cells, transiently transfected with p3A4-luc reporter, were seeded in 96-well plates and stabilized for 16 h and then incubated for 24 h with enantiopure forms of atorvastatin, rosuvastatin and fluvastatin in concentration ranging from 10−10 M to 10−4 M in the absence (agonist mode–upper line) or presence (antagonist mode–lower line) of rifampicin (RIF; 10 μM). The vehicle was DMSO (0.1% v/v). After the treatments, cells were lysed and luciferase activity was measured. Treatments were performed in triplicates. Data are expressed as a fold induction of luciferase activity over control cells (agonist mode) or as a percentage of maximal activation attained by RIF (antagonist mode). The values of EC50 and IC50 from n independent cell passages were calculated where appropriate and the average values are indicated in plots. Representative gene reporter assays are shown. Student´s t-test, One-way ANOVA followed by Dunnett's post test and EC50/IC50 values were calculated using GraphPad Prism.
Fig 6.
Effects of statin enantiomers on the expression of drug-metabolizing cytochromes P450, PXR and tyrosin aminotransferase TAT at mRNA level in primary human hepatocytes.
Primary human hepatocytes from three different donors (HH59, HH61, HH63) were used. Cells were incubated for 24 h with vehicle (DMSO; 0.1% v/v), dioxin (TCDD; 5 nM), rifampicin (RIF; 10 μM) and individual enantiomers of statins (1 μM, 10 μM, 30 μM). Bar graphs of RT-PCR analyses of CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP3A4, PXR and TAT mRNAs are shown. The data are the mean ± SD from triplicate measurements and are expressed as a fold induction over vehicle-treated cells. The data were normalized to GAPDH mRNA levels. Student´s t-test and One-way ANOVA followed by Dunnett's post test were calculated using GraphPad Prism.
Fig 7.
Effects of statin enantiomers on the expression of drug-metabolizing cytochromes P450 at protein level in primary human hepatocytes.
Primary human hepatocytes from three different donors (HH59, HH61 and HH63) were used. Cells were incubated for 48 h with vehicle (DMSO; 0.1% v/v), dioxin (TCDD; 5 nM), rifampicin (RIF; 10 μM) and individual enantiomers of statins (1 μM, 30 μM). Simple Western blots of CYP1A1, CYP1A2, CYP2A6, CYP2B6 and CYP3A4 are shown. The data are expressed as a fold induction over vehicle-treated cells and normalized to β-actin levels. Note: analyses of 780 samples are contained in a figure.
Fig 8.
Effect of statin enantiomers on the binding of PXR/RXR complex to the DR3 motif of human CYP3A4 gene promoter.
Nuclear fractions of LS174T cells from three independent cell passages were incubated for 2 h at 30°C with DMSO (0.1% v/v), RIF (10 μM) and individual enantiomers of atorvastatin, rosuvastatin and fluvastatin at concentration 10 μM. Treated nuclear extracts were incubated with a biotin-labeled CYP3A4-DR3 probe and electrophoresed on 5% polyacrylamide gel as described under ‘‘Materials and methods section.” A. The complex formation of CYP3A4 DR3 response element with PXR-RXRα heterodimer. B. Simple western blot showing equal expression levels of PXR nuclear extracts used for EMSA (normalized to β-actin levels).