Figure 1.
CAPE suppresses proliferation and colony formation of PC-3 cells.
Proliferation of PC-3 cell treated with increasing concentration of CAPE was determined by Trypan blue staining after 72 h treatment (A) or measuring total DNA content per well using Hoechst 33258 fluorescence by 96-well proliferation assay after 24, 48, and 72 h treatment (B). Relative cell numbers were normalized to the average cell number of the control (no CAPE treatment) of each cell line in each individual experiment. Columns represent mean for 18 replicates; bars represent standard deviation. Asterisk (*) represents cell number is statistically significantly different (p<0.05) compared to the control. Columns represent mean for 5 biological replicates; bars represent standard deviation. (C) Anticancer effect of CAPE was determined by colony formation assay of PC-3 cells treated with 0, 10, 20 µM for 14 days. Image is a representative result of three biological replicates.
Figure 2.
CAPE inhibits cell cycle progression in PC-3 cells.
(A) PC-3 cells transfected with a 4X NF-κB luciferase reporter plasmid for 24 hr were treated with increasing concentrations of CAPE for additional 24 h. Relative luciferase activity was determined to compare the effect of CAPE on NF-κB transcriptional activity. (B) PC-3 cells were treated with CAPE for 24, 48, or 72 h, harvested, and stained with propidium iodide dye for flow cytometric analysis for cell cycle distribution. (*) represents statistically significant difference (p<0.05) between the two group of cells being compared.
Figure 3.
CAPE affects cell cycle regulating proteins in PC-3 cells.
Protein expression of c-Myc, cyclin D1, cyclin E, SKP2, phosho-Rb (S807/811), p27Kip1, p21Cip1, ERK1/2, pERK1/2 Thr202/Tyr204, β-tubulin, and β-actin in PC-3 cells treated with 20 µM CAPE for 24, 48, and 5, 10, 20 µM CAPE for 72 h were assayed by Western blotting.
Figure 4.
CAPE inhibits Akt signaling-related proteins in PC-3 cells.
Protein expression of Akt, Akt1, Akt2, Akt3, total Akt, phospho-Akt S473, phospho-Akt T308, mTOR, phospho-mTOR Ser2448 and Ser2481, GSK3α, GSK3β, phopho-GSK3α S21, phospho-GSK3β S9, PDK1, phospho-PDK1 Ser241, Bcl-2, KLF6, β-tubulin, and β-actin in PC-3 cells treated with 20 µM CAPE for 24, 48, and 5, 10, 20 µM CAPE for 72 h were assayed by Western blotting.
Figure 5.
A scatter plot of log2 ratio (logR) for genes whose expression were significantly affected at either 24 h or 72 h post CAPE treatment.
Genes commonly affected at both time points are in red color, while those specifically affected at either time point are in black. IPA analysis of the unique genes (n = 191) genes changed either in 24 h or 72 h CAPE treatment indicated that group of genes regulating several cell functions, including cell proliferation (p-value 9.82×10−11, 52 genes), cell growth (p-value 1.40×10−10, 41 genes), cell death (p-value 1.40×10−12, 68 genes), and cell survival (p-value 1.40×10−6, 27 genes).
Figure 6.
Validation of gene microarray result with qRT-PCR.
Gene expression level of GDF15, HIST1H2BD, CCL20, CXCL5, RND3, KLF4, DUSP5, NOV, CDKN1A, CXCL2, DUSP1, KLF6, TOP2A, PPP1R15A, CAV2, S100P, GADD45A, and TUBA1A in PC treated with 0 or 20 µM CAPE for 24 h or 72 h was determined by qRT-PCR.
Figure 7.
Combined treatment of CAPE with chemotherapy drugs shows synergistic and antagonistic inhibition on proliferation of PC-3 cells.
Proliferation of PC-3 cells treated with increasing dosage (0, 5, 10, 20 µM) of CAPE in combination with increasing concentration of etoposide (A), paclitaxol (B), vinblastine (C), mitoxantrone (D), and estramustine (E) for 72 h was determined by 96-well proliferation assay. The right part of the figure show the ratio of expected cell number/observed cell number. For example, treatment of 5 µM of CAPE or 1 nM vinblastine decreases cell number of PC-3 to 80.9% and 88.7%, respectively, compared to the control (no treatment). The expected cell number of treatment combining 5 µM of CAPE and 1 nM vinblastine is 0.809*0.887 = 71.8%. The observed cell number is 48.8% compared to the control. So the ratio is 0.718/0.488 = 1.5. Ratio larger than one represents synergy of growth inhibition, while ratio smaller than one represents antagonistic effect.
Figure 8.
Growth response to CAPE treatment of PC-3 and PC-3 p21Cip1 siRNA cells.
Protein levels of wild type PC-3, PC-3 cells transfect with scramble control (20 nM), and PC-3 cells transfected with p21Cip1 siRNA (20 nM) were determined by Western blotting assay. Proliferation of these PC-3 cells treated with 20 µM CAPE for 24 h was determined by 96-well plate proliferation assay as described in Material and Methods.
Figure 9.
Putative model of anticancer effect of CAPE in PC-3 human prostate cancer cells.
Protein abundance or activity being stimulated by CAPE treatment are labeled with red upward arrows, while those being suppressed by CAPE treatment are labeled with blue downward arrows.