Dose response of prostate cancer cells to individual drugs.

<p>(A-B) Cells were grown in either control regular media or media supplemented with DHT of increasing concentrations. Cell growth was assessed using Alamar Blue assay as described in Materials and Methods. Results are representative of 3 independent experiments. (C) Immunoblotting was performed from equal amount of total protein using the phospho-AKT, AKT, and β-actin antibodies as described in Materials and Methods. (D-I) Cytotoxic effects of increasing doses of docetaxel (D, G), rapamycin (E, H), and 17-AAG (F, I) in PTEN-P2 cells (D, E, F) and PTEN-CaP2 cells (G, H, I). Cells were exposed to the indicated concentration of drug-loaded micelle or empty micelle for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. * indicates statistically significant differences compared to micelle control (ANOVA, p < 0.05). Results are representative of 3 independent experiments.</p

Dose response of prostate cancer cells to multi-drug loaded micelles.

<p>Cytotoxic effects of DR17 in PTEN-P2 (A, C, E) and PTEN-CaP2 cells (B, D, F). Dose responses of PTEN-P2 (A, C) and PTEN-CaP2 (B, D) to DR17 free form or DR17 delivered by micelle (DR17). Cells were exposed to the indicated concentrations of free form DR17 or DMSO, or DR17 loaded micelle or empty micelle for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. E-F, Comparison of cytotoxic effects of three-drug combination DR17 to individual drug loaded micelles (C, D). For C and D, DR17 was added to the media to the final concentration of 105μM. Final concentrations of docetaxel, rapamycin, 17-AAG were calculated based on molarity ratio of each individual drug in the DR17 formula [docetaxel:rapamycin:17-AAG (1:1:8.5)], which are 12.38μM, 10.94μM, and 85.37μM, respectively. Cells were exposed to the indicated drug conditions for 72 hours. Cell viability was assessed using Alamar Blue assay as described in Materials and Methods. * indicates statistically significant differences compared to DR17 treatment (ANOVA, p < 0.05). Results are representative of 3 independent experiments.</p

Histology of DR17 and micelle control treated prostates.

<p>Pten het N = 9 (A, B, E, F, I, J) and Pten null N = 6 (C, D, G, H, K, L) mice were treated with empty micelles (Ctrl) or DR17 loaded micelles for 3 weeks. Animals were sacrifice two days after the last injection. Prostates were dissected and fixed in formalin. Prostate sections were used for H&E staining to assess overall morphology (A-D). Immunohistochemistry was performed to examine stromal smooth muscle actin (SMA, E-H) and epithelial cytokeratin (I-L) markers. Pictures were taken at 40X magnification. The black bar in each picture represents 30 microns.</p

Effects of DR17 on cell proliferation, apoptosis, and prostate basal cells.

<p>Pten het N = 9 (A, B, E, F, I, J) and Pten null N = 6 (C, D, G, H, K, L) mice were treated with empty micelles or DR17 loaded micelles for 3 weeks. Animals were sacrifice two days after the last injection. Prostates were dissected and fixed in formalin. Immunohistochemistry staining for proliferation marker Ki67 (A-D), apoptosis marker cleaved caspase 3 (E-H), and basal cell marker p63 (I-L) was performed on prostate tissue sections. Pictures were taken at 100X magnification. The black bar in each picture represents 10 microns. Quantification of Ki67 (M), cleaved caspase 3 (N), and p63 (O) labeling index was conducted as described in Materials and Methods. Differences from control were statistically significant as indicated by * (Student’s <i>t</i> test, p < 0.05).</p

Expression of proteins in PI3K/Akt and AR pathways in PTEN-P2 and PTEN-CaP2 cells in response to DR17, docetaxel (DTX), 17AAG, and rapamycin (Rap) treatments.

<p>DR17, docetaxel, 17AAG, and rapamycin were added to media to the final concentration of 105μM, 12.38μM, 85.37μM, and 10.94μM, respectively, in which concentrations of individual drug were calculated based on their molarity ratio in DR17 formula [docetaxel:rapamycin:17-AAG (1:1:8.5)]. Cells were exposed to drug treatments for 24h, after which total protein was collected. Immunoblotting was performed from equal amount of total protein using the indicated antibodies as described in Materials and Methods. Results are representative of 3 independent experiments.</p