Article Figures & Data
Figures
- Fig. 1.
A, chemical structures of DHT, E2, and carnosol. B, crystal structures of AR and ER bound to DHT and E2, respectively, were acquired from the protein database bank (www.rcsb.org) and the native ligand was removed and replaced with carnosol. C, the PCa cell lines LNCaP and 22Rv1 and breast cancer cell line MCF7 that express both AR and ER-α were treated with carnosol for 48 h and evaluated for cell viability using an MTT assay. D, cells were grown to 60% to 70% confluence and treated with or without carnosol (40 μmol/L) for 48 h. The effect of carnosol on cellular morphology in LNCaP and MCF7 cells can be visualized.
- Fig. 2.
A, TR-FRET AR competitive binding assay with carnosol. B, TR-FRET ER-α competitive binding assay with carnosol. A 100× stock concentration of carnosol in 100% DMSO was diluted to 4× concentration (4% DMSO) in assay buffer and challenged with a control competitor. C, to verify if carnosol is an inhibitor of native ligand-binding properties (i.e., antagonist) or binding properties (i.e., agonist), we stimulated AR-UAS-bla GripTite HEK293 cells with R1881, a known agonist of AR, using cells that express the LBD of AR. A standard curve was done using an agonist (R1881) and an antagonist (cyproterone acetate). The EC50 of R1881 was 0.127 nmol/L and the EC50 of cyproterone acetate was 11.5 nmol/L. The standard curve for R1881 was done in triplicate spanning 10 doses ranging from 0.001 to 30 nmol/L. The standard curve for cyproterone acetate was done in triplicate spanning 10 doses ranging from 0.1 nmol/L to 3 μmol/L. D, to verify if carnosol is an inhibitor of native ligand-binding properties (i.e., antagonist) or binding properties (i.e., agonist), we stimulated ER-α-UAS-bla GripTite HEK293 cells with E2, a known agonist of ER-α, using cells that express the LBD of ER-α. A standard curve was done using an agonist (E2) and an antagonist (4-hydroxytamoxifen). The EC50 of E2 was 0.027 nmol/L and the EC50 of 4-hydroxytamoxifen was 1.77 nmol/L. The standard curve for E2 was done in triplicate spanning 10 doses ranging from 0.0002 to 10 nmol/L. The standard curve for 4-hydroxytamoxifen was done in triplicate spanning 10 doses ranging from 0.002 to 100 nmol/L.
- Fig. 3.
Cells were grown to 60% to 70% confluence and treated with carnosol up to 24 h, and whole-cell lysates were prepared. Protein (40 μg) was subjected to SDS-PAGE followed by Western blot analysis and chemiluminescence detection as described in Materials and Methods. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. A, LNCaP, 22Rv1, and MCF7 cells were treated with carnosol for 24 h and whole-cell lysates were collected and prepared as described in Materials and Methods. Intracellular protein expression of AR, ER-α, and PSA was evaluated by Western blot after carnosol treatment. B, LNCaP cells were treated with cycloheximide, carnosol, or carnosol/cycloheximide, and whole-cell lysates were prepared as described above with lysates collected at 0, 6, 12, and 24 h. AR, ER-α, and PSA intracellular protein expression was evaluated by Western blot analysis.
- Fig. 4.
A and B, LNCaP cells were transiently transfected with an ∼6-kb AR promoter-luciferase reporter plasmid in a pGL3-Basic vector or an ∼5.8-kb ER-α promoter-luciferase reporter plasmid in a pGL2-Basic vector. Eighteen hours after transfection, cells were treated with DMSO (vehicle) or 30 μmol/L carnosol and analyzed for luciferase activity after 24-h treatment with carnosol. Columns, mean of three individual samples; bars, SD. *, P < 0.01. C and D, LNCaP cells were grown in chamber slides at 100,000 per chamber overnight and treated with carnosol (30 μmol/L) for 24 h. For labeling, anti-AR antibody (Santa Cruz Biotechnology) and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) were used as primary and secondary antibodies after 24-h carnosol treatment, respectively. For labeling, anti-ER-α (Cell Signaling Technology) and Alexa Fluor 594 goat anti-mouse IgG were used after 24-h treatment. DAPI was used to counterstain the nucleus.
- Fig. 5.
A, PrECs were treated with DMSO (i.e., vehicle control), carnosol (30 μmol/L), flutamide (30 μmol/L), or tamoxifen (30 μmol/L). Phase-contrast images at ×40 magnification were taken 48 h after treatment. B, PrEC cells were treated with increasing concentrations of carnosol, tamoxifen, or flutamide, and MTT assay was done to determine the effect on cell viability. Columns, mean of three individual samples; bars, SD. C, cleaved (activated) caspase-3 was detected by ELISA. 22Rv1 cells were treated with vehicle, carnosol (30 μmol/L), and carnosol (60 μmol/L) for 24 h, and protocol was followed per the manufacturer's directions. Columns, mean of three individual samples; bars, SD. *, P < 0.01, carnosol-treated samples versus control. D, LNCaP cells were grown to 60% to 70% confluence and medium was replaced containing carnosol, flutamide, and tamoxifen, or combinations of carnosol/flutamide, carnosol/tamoxifen, or flutamide/tamoxifen. Total cell lysates were prepared, and 40 μg protein was subjected to SDS-PAGE followed by Western blot analysis. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin.
- Fig. 6.
Effect of carnosol on 22Rv1 tumor growth and PSA secretion in athymic nude mice. Fourteen animals were s.c. injected in each flank of the mouse with ∼1 × 106 22Rv1 cells to initiate tumor growth. The animals were divided into two cohorts, with seven animals in each cohort receiving either cottonseed oil (control) or carnosol. Twenty-four hours after cell implantation, the animals in cohort 1 received cottonseed oil (100 μL) by oral gavage five times weekly and served as control. The animals in cohort 2 received carnosol (30 mg/kg) by oral gavage in cottonseed oil five times weekly. Once tumors started to grow, tumors were measured three times weekly and tumor volumes were calculated. A, body weights were recorded 5 d weekly and plotted over days after tumor cell inoculation. B, the average tumor volume of control and carnosol-treated mice was plotted over days after tumor cell inoculation. Points, mean of 14 tumors in seven mice; bars, SD. *, P < 0.05. C, representative tumors of control and carnosol-treated mice. Total cell lysates were prepared, and 10 μg protein was subjected to SDS-PAGE followed by Western blot analysis. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. D, serum PSA levels were analyzed by ELISA at the conclusion of the study. Columns, mean of seven animals per cohort; bars, SD. *, P < 0.01.
Volume 3, Issue 9
