Article Figures & Data
Figures
- Figure 1.
A, time course response for effect of PEITC and PITC on proliferation of PC-3 cells as determined by trypan blue dye exclusion assay. B, dose response for effect of PEITC on PC-3 cell proliferation as determined by trypan blue dye exclusion assay. PC-3 cells were plated, allowed to attach overnight, and treated with DMSO (control) or desired concentration of the test compound (PITC or PEITC) for specified time intervals. Both floating and adherent cells were collected and used for counting of dead and live cells. Columns, mean; bars, SE (n = 3). *, P < 0.05, significantly different compared with DMSO-treated control (one-way ANOVA).
- Figure 2.
Effect of PEITC and PITC on PC-3 cell cycle distribution. PC-3 cells were treated with DMSO (control) or 10 μm PEITC or PITC for 24 h. The cells were then stained with propidium iodide and analyzed using a Coulter Epics XL flow cytometer.
- Figure 3.
A, ELISA-based quantitation of cytoplasmic histone-associated DNA fragments in PC-3 cells following a 24 h exposure to DMSO (control) or different concentrations of PEITC or PITC. Cytoplasmic histone-associated DNA fragments were quantified using Cell Death Detection ELISA kit from Roche Diagnostics according to the manufacturer's instructions. Columns, mean; bars, SE (n = 3). *, P < 0.05, significantly different compared with control (one-way ANOVA). B, Western blot analysis for the effect of PEITC and PITC on cleavage of PARP. Cells were treated with 10 μm PEITC or PITC for the indicated time intervals and harvested for preparation of cell lysates. The lysate proteins were subjected to Western blotting using antibodies that recognize cleaved PARP. Blots were stripped and reprobed with anti-actin antibodies to ensure equal protein loading. The experiment was repeated twice using independently prepared lysates, and the results were comparable.
- Figure 4.
A, representative Western blots for the effects of PEITC and PITC on protein levels of cyclin B1, Cdk1, Cdc25B, Cdc25C, and phospho-Cdk1 (Tyr15). Blots were stripped and reprobed with anti-actin antibodies to correct for differences in protein loading. The experiment was repeated two or more times using independently prepared lysates to ensure reproducibility. B, effect of lactacystin, a specific inhibitor of proteasome, on PEITC-induced decline in Cdc25C and Cdk1 protein levels. Cells were pretreated with DMSO or 40 μm lactacystin for 2 h at 37°C and exposed to 10 μm PEITC for 24 h. Cell lysates were prepared and subjected to Western blotting using antibodies against Cdc25C or Cdk1. The experiment was repeated twice, and the results were comparable. C, effect of lactacystin on PEITC-induced cell cycle arrest. Cells were pretreated with DMSO or 40 μm lactacystin for 2 h at 37°C and exposed to 10 μm PEITC for 24 h. Both floating and attached cells were collected and processed for cell cycle distribution analysis following staining with propidium iodide. Columns, mean of three determinations; bars, SE. a, significantly different compared with DMSO-treated control; b, significantly different compared with PEITC alone group.
- Figure 5.
A, representative Western blots for the effects of PEITC and PITC on levels of Bcl-2 family proteins. PC-3 cells were cultured in the presence of 10 μm PEITC or PITC for the indicated time intervals and processed for preparation of cell lysate and Western blotting. Blots were stripped and reprobed with antibodies against actin to correct for differences in protein loading. B, results of densitometric scanning of the immunoreactive bands corresponding to Bcl-2, Bcl-XL, and Bax for PEITC samples. Columns, mean; bars, SE (n = 3). *, P < 0.05, significantly different compared with control (one-way ANOVA).
- Figure 6.
A, Western blot analysis for Bcl-2 protein expression using lysates from PC-3 cells stably transfected with empty vector (PC-3/neo) and Bcl-2 (PC-3/Bcl-2). B, effect of PEITC on proliferation of PC-3/neo and PC-3/Bcl-2 cells as determined by trypan blue dye exclusion assay. PC-3/neo or PC-3/Bcl-2 cells were exposed to DMSO or PEITC (5 or 10 μm) for 24 or 48 h. Both floating and attached cells were collected and used for trypan blue dye exclusion assay. Columns, mean; bars, SE (n = 3). The percentage of surviving cells did not differ significantly between PC-3/neo and PC-3/Bcl-2 cells at either concentration of PEITC at both time points. C, analysis of sub-G0-G1 cells in cultures of PC-3/neo and PC-3/Bcl-2 cells following a 24 h exposure to DMSO or PEITC (5 or 10 μm PEITC). Cells with sub-G0-G1 DNA content were quantified by flow cytometry following staining with propidium iodide. Columns, mean; bars, SE (n = 3). The fraction of sub-G0-G1 cells did not differ significantly between PC-3/neo and PC-3/Bcl-2 cells either at 5 or 10 μm PEITC.
- Figure 7.
Western blot analysis for (A) cleaved caspase-3 and (B) procaspase-8 and procaspase-9 using lysates from PC-3 cells exposed to 10 μm PEITC or PITC for different time intervals. Data are representative of at least two independent experiments with similar results. C, effect of general caspase inhibitor z-VADfmk, caspase-8 specific inhibitor z-IETDfmk, and caspase-9 specific inhibitor z-LEHDfmk on PEITC-induced apoptosis as determined by quantitation of cytoplasmic histone-associated DNA fragments. Columns, mean; bars, SE (n = 3). a, P < 0.05, significantly different compared with DMSO-treated control; b, P < 0.05, significantly different compared with PEITC alone group (ANOVA followed by Bonferroni's multiple comparison test).
Tables
- Table 1.
Effects of PEITC and PITC on PC-3 cell cycle distribution
Treatment % Cells Sub-G0-G1 G0-G1 S G2-M Control (DMSO) 4 ± 0.5 56 ± 1 15 ± 0.5 19 ± 0.5 5 μm PEITC 14 ± 3* 42 ± 3* 14 ± 0.5 31 ± 1* 10 μm PEITC 16 ± 1* 34 ± 5* 13 ± 1 36 ± 4* 5 μm PITC 4 ± 0.5 59 ± 1 14 ± 0.5 20 ± 1 10 μm PITC 4 ± 0.5 56 ± 0.5 15 ± 0.5 21 ± 0.5 Note: Cells were treated with DMSO (control) or different concentrations of PEITC or PITC for 24 h at 37°C. Both floating and attached cells were collected and processed for analysis of cell cycle distribution by flow cytometry following staining with propidium iodide. Data are means ± SE (n = 3).
↵* ,P < 0.05, significantly different compared with control (one-way ANOVA).
Volume 3, Issue 5
