Fig 1.
Lipophilic atorvastatin is more effective at suppressing cancer cell growth than hydrophilic rosuvastatin or pravastatin.
Dose response curves for (A) MCF-7 RFP, (B) MDA-MB-231 RFP, (C) MDA-MB-231 RFP/Ecad, (D) DU-145 RFP, (E) SF-295, and (F) MDA-MB-435 cancer cells when cultured with atorvastatin (red), rosuvastatin (green), or pravastatin (blue) for 72 hours. Cell number was determined by crystal violet staining. Sigmoidal curves were fit to the dose response data and (G) IC50 values of atorvastatin and rosuvastatin in each cell line were extrapolated. (H) Pharmacologic parameters of atorvastatin, rosuvastatin, and pravastatin as found in the literature [24,31]. All data are representative of at least three independent experiments.
Fig 2.
Atorvastatin decreases proliferation of breast cancer cells more potently than rosuvastatin.
(A) Experimental schematic for assessing the proliferation of breast cancer cells under treatment with atorvastatin or rosuvastatin for 48 hours. (B) MCF-7 RFP, (C) MDA-MB-231 RFP, and (D) MDA-MB-231 RFP/Ecad were cultured with atorvastatin or rosuvastatin for 48 hours; during the final 24 hours the media included 10uM EdU. Cells were fixed, EdU was detected, and cells were counterstained with DAPI to label all nuclei. Cellular proliferation was quantified by determining the percentage of EdU positive cells (green, all nuclei are blue—DAPI). (E-H) MDA-MB-231 RFP and (I-L) MDA-MB-231 RFP/Ecad cells treated with (E,I) 0μM, (F,J) 1μM, (G,K) 5μM, or (H,L) 20μM atorvastatin demonstrate both E-cadherin mediated growth suppression and atorvastatin resistance. All data are representative of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 3.
Atorvastatin decreases survival of sensitive but not resistant breast cancer cells.
(A-L) MDA-MB-231 or (M-X) MCF-7 breast cancer cells were treated with 0.01% DMSO (untx, A-D and M-P), 5μM atorvastatin (5uM Atorv, E-H and Q-T), or 1μM Doxorubicin (1uM Doxo, I-L and U-X) for 0 hours, 24 hours, 48 hours, or 72 hours in the presence of 1μg/mL propidium iodide. Cells were imaged using an inverted microscope to detect propidium iodide (red) and look at cell morphology and density using phase contrast microscopy. Scale bar = 200μm. All data are representative of at least three independent experiments.
Fig 4.
Atorvastatin treatment decreases membrane-bound Ras in statin sensitive MDA-MB-231 but not statin resistant MCF-7.
(A-C) MDA-MB-231 RFP and (D-F) MCF-7 RFP cells were treated with atorvastatin for 0, 1, 2, 4, 8, 24, or 48 hours and protein was collected in cytoplasmic and membrane fractions. (A,B) Cytoplasmic Ras increased in statin treated MDA-MB-231 RFP cells over the course of 48 hours whereas (A,C) membrane Ras decreased. (D,E) Cytoplasmic and (D,F) membrane Ras were unchanged by atorvastatin treatment in MCF-7 RFP cells. All data are representative of at least three independent experiments.
Fig 5.
Atorvastatin sensitivity correlates with blunted Akt phosphorylation in response to EGF.
(A) MDA-MB-231 RFP, (B) MDA-MB-231 RFP/Ecad, and (C) MCF-7 RFP cells were treated with 5μM atorvastatin for 24 hours and then stimulated with 5nM EGF for 5 or 30 minutes. (D) Akt phosphorylation fold change, defined as the density of pAkt under atorvastatin pretreatment divided by the density of pAkt under vehicle treatment, was quantified for 0, 5, or 30 minutes of 5nM EGF stimulation. All data are representative of at least three independent experiments.
Fig 6.
Inhibition of Akt but not Erk signaling is synergistic with atorvastatin.
(A) MCF-7 RFP, (B) MDA-MB-231 RFP, and (C) MDA-MB-231 RFP/Ecad cells were cultured with atorvastatin and either 0μM, 3μM, or 10μM PD98059 for 72 hours. (D) IC50 values for atorvastatin susceptibility were extrapolated from sigmoid curve fits to the dose response data. (E) MDA-MB-231 RFP cells were treated with 0μM, 3μM, or 10μM PD98059 with or without 5μM atorvastatin for 24 hours and probed by western blot and (F) quantified by densitometry. (G) MCF-7 RFP, (H) MDA-MB-231 RFP, and (I) MDA-MB-231 RFP/Ecad cells were cultured with atorvastatin and either 0μM, 3μM, or 10μM LY294002 for 72 hours. (J) IC50 values for atorvastatin susceptibility were extrapolated from sigmoid curve fits to the dose response data. (K) MDA-MB-231 RFP cells were treated with 0μM, 3μM, or 10μM LY294002 with or without 5μM atorvastatin for 24 hours and probed by western blot and (L) quantified by densitometry. * P < 0.05, *** P < 0.001, and **** P < 0.0001. All data are representative of at least three independent experiments.