Figure 1.
Effect of triol on viability and proliferation of human prostate cancer cell lines.
LNCaP CDXR-3, DU-145, and PC-3 prostate cancer cells were treated with increasing concentrations of triol for 48 hrs (A)(C) or 96 hrs (B)(D). Relative viability and relative cell number of prostate cancer cells was determined by MTT (3,4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) 96-well assay (A)(B) and Hoechst dye 33258-based 96-well proliferation assay (C)(D), respectively, as described in Materials and Methods. Cell numbers were normalized to control (dimethylsulfoxide) of each cell line. Asterisk (*, **, ***) represents statistically significant difference (p<0.05, p<0.01, p<0.001) between the treated group and the control group.
Figure 2.
Effect of triol on colony formation in soft agar.
PC-3 (A) and LNCaP CDXR-3 (B) cells treated with 0, 10, or 20 µM triol for 14 and 17 days, respectively. Image is a representative result of three biological replicates.
Figure 3.
Effect of triol on the growth of PC-3 xenografts in nude mice.
Male Balb/c nu/nu mice, 6–8 weeks of age, were injected subcutaneously in both flanks with 5×105 PC-3 cells suspended in 0.5 ml of Matrigel. Tumors were measured daily using calipers and volume was calculated using the formula volume = length×width×height×0.52. Monitoring of tumor growth was started one week after tumor inoculation. Mice were separated into control and treatment groups. The control group had 5 mice carrying 10 tumors and the treatment group had 5 mice carrying 8 tumors. Three weeks after the initial inoculation, the treatment group was orally administrated triol daily at a dose of 20 mg/kg, 5 days/week. Mice in the control group were gavaged with vehicle only. Treatment started on day 22 and ended on day 42 after tumor inoculation. Tumor volumes were shown as mean ± standard error (A) while body weight of mice was shown as mean ± Standard Deviation (B).
Figure 4.
Effect of triol on cell cycle distribution of prostate cancer cells.
LNCaP CDXR-3 (A) and DU-145 cells (B) were treated with 0 or 10 µM triol for 48 hrs. PC-3 cells (C) were treated with 0, 10, or 20 µM triol for 48 hrs. (D) DU-145 cells were treated with 0, 20, and 40 µM triol for 48 hrs. Cell cycle distribution was determined by flow cytometry. Asterisk (*, **, ***) represents statistically significant difference (p<0.05, p<0.01, and p<0.001, respectively) between the treated group and the control group.
Figure 5.
Triol induces apoptosis in prostate cancer cells.
LNCaP CDXR-3, DU-145, and PC-3 cells were treated with 0, 20, and 40 µM triol for 48 hrs. Cell morphology was determined by light microscopy. TUNEL assay was performed as described in Materials and Methods to determine the apoptotic cell population. Green fluorescent light indicated apoptotic cells stained with TUNEL assay. Images were viewed at 200× with Olympus confocal microscope.
Figure 6.
Micro-Western Array analysis of protein expression in CDXR-3, DU-145, and PC-3 cells treated with 0 or 20 µM triol.
CDXR-3, DU-145, and PC-3 cells were treated with 0 or 20 µM of triol for 48 hrs. Cell lysates were collected according to Micro-Western Array (MWA) protocol [18] and MWAs were performed to measure the changes in abundance and modification of indicated proteins. The six samples printed in each well from left to right were CDXR-3 (control), CDXR-3 (20 µM triol), DU-145 (control), DU-145 (20 µM triol), PC-3 (control), PC-3 (20 µM triol) (Figure S2). The right half blot (well A7-H12) was the technical duplicate of the left half blot (well A1-H6). Antibody list is shown in Table S2. Red and green were artificial color for anti-rabbit and anti-mouse 2nd antibody, respectively. The blot is the same size as a standard 96-well microtiter plate. Relative protein abundance of each signaling protein in each cell line is listed in Table S3.
Figure 7.
Protein expression profile of Micro-Western Array results.
Protein expression profiles of CDXR-3, DU-145, and PC-3 cells treated with triol are displayed in a heat map format. Black color indicated no change, while green and red colors indicated decrease and increase of protein expression level. Grey color indicated signal below detection threshold. Protein abundance was normalized to the average of α-tubulin, GAPDH, and β-actin. Proteins in different cell lines were normalized to the control condition (no triol treatment) of that cell line.
Figure 8.
Effect of triol treatment on the abundance and phosphorylation status of signaling proteins.
Protein expression of Akt1, phospho-Akt Thr308, phospho-Akt Ser473, PDK1, phospho-p44/42 MAPK Thr202/Tyr204, c-Myc, Skp2, p27Kip1, FASN, GAPDH, β-actin, and α-tubulin in CDXR-3, DU-145, and PC-3 cells treated with 0, 10 or 20 µM triol for 48 hrs were assayed by Western blotting. Values represent relative protein abundance.
Figure 9.
Effect of overexpression of Skp2 protein on growth inhibition by triol treatment.
(A) Protein expression of Skp2 assayed by Western blotting in PC-3 LPCX control and PC-3 LPCX Skp2 cell lines. PC-3 cells overexpressing Skp2 with LPCX vector and control PC-3 cells (empty LPCX vector) were treated with increasing concentrations of triol for 48 hrs (B) or 96 hrs (C) and analyzed by the 96-well proliferation assay. Asterisk * and *** represent statistically significant difference (p<0.05 and p<0.001) between the PC-3 control cells and PC-3 overexpressing Skp2- cells.
Figure 10.
Triol treatment affected expression of Akt1 protein in PC-3 xenografts.
(A) Protein expression of Akt1, c-Myc, FASN, Ki-67, and α-tubulin was examined in 5 PC-3 tumors from control group and 8 PC-3 tumors from treatment group (Fig. 3). (B) Relative protein abundance of each signaling protein was quantified and calculated for average and standard deviation. Asterisk *** represents statistically significant difference (p<0.001) between the control and triol treatment group.
Figure 11.
Effect of triol on LXR reporter and ABCA1 gene expression.
(A) PC-3 cells were transfected with pRL-TK-Renilla luciferase plasmid, pSG5RXRα, pSG5LXRα, and 4xDR4Δ56cfos pGL3 reporter gene plasmid. PC-3 cells were treated with 0, 5, 10, 20 µM triol or 1 µM T0901317. Luciferase reporter gene assay was performed to determine the activity of LXRα receptor. (B) PC-3 and DU-145 cells were treated with 0, 10, 20 µM triol for 48 hrs. ABCA1 gene, a target gene of LXRα, was determined by qRT-PCR. Asterisk * represents statistically significant difference (p<0.05) between the control and triol treatment group.
Figure 12.
Effect of triol treatment on cytoskeleton morphology in CDXR-3 and DU-145 cells.
Imaging of α-tubulin and β-actin in CDXR-3 and DU-145 cells treated with vehicle or 10 µM triol for 48 hrs was determined by confocal microscopy.
Figure 13.
Effect of triol treatment on migration and invasion of PC-3, DU-145, and CDXR-3 cells.
PC-3, DU-145, and CDXR-3 cells were pre-treated with 0, 10, or 20 µM triol for 48 hrs. Cells were then counted and seeded at identical number. Cancer cell migration ability (A) and invasion ability (B) was determined by transwell migration assay (A) and transwell invasion assay 6 hrs (A) and 24 hrs (B) after cell seeding, respectively. (C) Migration of CDXR-3 cells was determined by transwell migration assay 24 hrs after cell seeding. There was no triol in medium during the migration or invasion assay. Asterisk *** represents statistically significant difference (p<0.001) between the control group and triol treatment group.
Figure 14.
Triol treatment inhibited migration of CDXR-3 cells determined by wound healing assay.
Wound healing assay was performed to determine the effect of (A) 0 µM (B) 10 µM or (C) 20 µM triol on migration ability of CDXR-3 cells 8 or 24 hrs after cell seeding. (D) Results of (A)(B)(C) were quantified. Asterisk * and ** represents statistically significant difference (p<0.05 and p<0.01) between the control group and the triol treatment group.
Figure 15.
Effect of triol on expression levels of EMT marker and FAK proteins in PC-3, DU-145, and CDXR-3 cells.
Protein expression of E-cadherin, N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, phospho-FAK Tyr861, β-actin, and α-tubulin in PC-3, DU-145, and CDXR-3 cells treated with 0, 10, or 20 µM triol for 48 hrs were assayed by Western blotting. Values represent relative protein abundance.
Figure 16.
Effect of triol on viability of non-cancer human cell lines.
Human prostate epithelial PZ-HPV-7 cells were treated with increasing concentrations of triol for 48 hrs. Relative cell viability was determined by MTT assay as described in Material and Methods. Cell numbers were normalized to control (dimethylsulfoxide treatment) for each cell line. Asterisk * represents statistically significant difference (p<0.05) between the treated and the control group.