Article Figures & Data
Figures
- Figure 1.
Effect of BITC, PEITC, and/or SFN treatment on survival of MDA-MB-231 (A), MCF-7 (B), and MCF-10A (C) cells as determined by trypan blue dye exclusion assay. The desired cell line was treated with DMSO (control) or indicated concentrations of the specified ITC compound for 24 h. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Cell survival assay was performed twice in each cell line, and the results were comparable.
- Figure 2.
A, representative histograms depicting cell cycle distribution in MDA-MB-231 cultures following a 24-h treatment with DMSO (control) or 2.5 μmol/L BITC. B, cell cycle distribution in MDA-MB-231 cultures following a 24-h treatment with DMSO (control) or 0.5, 1, or 2.5 μmol/L BITC. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Similar results were observed in two independent experiments.
- Figure 3.
A time course study for effect of 2.5 μmol/L BITC on percentage of sub-G0-G1 population and (A) G2-M fraction (B) in MDA-MB-231 cells. Control cells were exposed to DMSO. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with corresponding DMSO-treated control by paired t test. C, immunoblotting for cyclin B1, Cdk1, and Cdc25C using lysates from MDA-MB-231 cells cultured in the presence of 2.5 μmol/L BITC for the indicated times. D, immunoblotting for cyclin B1, Cdk1, and Cdc25C using lysates from MCF-7 cells cultured in the presence of 10 μmol/L BITC for the indicated times. The blots were stripped and reprobed with anti-actin antibody as a loading control.
- Figure 4.
A, representative histograms depicting cell cycle distribution in MCF-10A cultures following a 24-h treatment with DMSO (control) or 20 μmol/L BITC. B, cell cycle distribution in MCF-10A cultures following a 24-h treatment with DMSO (control) or 5, 10, or 20 μmol/L BITC. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Similar results were observed in two independent experiments.
- Figure 5.
A, DAPI staining for MDA-MB-231 cultures treated for 24 h with DMSO or 2.5 μmol/L BITC. Apoptotic cells with condensed chromatin (arrows) were clearly visible in BITC-treated culture but much less frequent in DMSO-treated controls. B, quantitation of apoptotic cells with condensed chromatin (DAPI assay) in MDA-MB-231 cultures following a 24-h treatment with DMSO (control) or BITC (1 or 2.5 μmol/L). Columns, mean of six to nine determinations; bars, SE. a, P < 0.05, significantly different compared with DMSO-treated control; b, P < 0.05, significantly different compared with 1 μmol/L BITC by one-way ANOVA followed by Bonferroni's multiple comparison test. Cytoplasmic histone-associated DNA fragmentation in MDA-MB-231 (C), MCF-7 (D), and MCF-10A (E) cells following a 24-h treatment with DMSO (control) or the indicated concentrations of BITC. C to E, columns, mean (n = 3 for MDA-MB-231 and MCF-7 cells and n = 2 for MCF-10A cells; the error bars in MCF-10A are shown to indicate range of values). *, P < 0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Similar results were observed in two independent experiments.
- Figure 6.
A, immunoblotting for Bcl-2 family proteins using lysates from MDA-MB-231 cells cultured in the presence of 2.5 μmol/L BITC for the indicated times. B, immunoblotting for Bcl-2 family proteins using lysates from MCF-7 cells cultured in the presence of 10 μmol/L BITC for the indicated times. The blots were stripped and reprobed with anti-actin antibody as a loading control. C, percentage of subdiploid fraction in cultures of WT, Bak-KO, and DKO following a 16-h treatment with DMSO (control) or the indicated concentrations of BITC. Columns, mean (n = 3); bars, SE. a, P < 0.05, significantly different compared with corresponding DMSO-treated control; b, P < 0.05, significantly different compared with WT MEFs by one-way ANOVA followed by Bonferroni's multiple comparison test. D, cytoplasmic histone-associated DNA fragmentation in WT, Bak-KO, and DKO MEFs following a 24-h treatment with DMSO (control) or the indicated concentrations of BITC. Columns, mean (n = 3); bars, SE. a, P < 0.05, significantly different compared with corresponding DMSO-treated control; b, P < 0.05, significantly different compared with WT MEFs by one-way ANOVA followed by Bonferroni's multiple comparison test. Similar results were observed in two independent experiments.
- Figure 7.
A, representative flow histograms depicting JC-1 fluorescence in MDA-MB-231 cells following a 6-h treatment with DMSO (control) or 2.5 μmol/L BITC or 25 μmol/L FCCP (positive control). Note an increase in green fluorescence in FCCP-treated (positive control) and BITC-treated MDA-MB-231 cells. B, percentage of cells with green fluorescence in MDA-MB-231 cultures treated with DMSO or 2.5 μmol/L BITC for the indicated times. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with corresponding DMSO-treated control by paired t test. Similar results were observed in two independent experiments. C, percentage of cells with green fluorescence in MCF-7 cultures treated with DMSO or BITC (5 or 10 μmol/L) for the indicated times. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with corresponding DMSO-treated control by one-way ANOVA followed by Dunnett's test. D, immunoblotting for cytochrome c and Smac/DIABLO using mitochondria-free cytosolic fractions prepared from MDA-MB-231 cells following treatment with 2.5 μmol/L BITC for the indicated times. Blots were stripped and reprobed with anti-actin antibody as a loading control.
- Figure 8.
Representative transmission electron micrographs (×10,000 magnification) of MDA-MB-231 following a 6-h treatment with DMSO or 2.5 μmol/L BITC. Note the abundance of membranous vacuoles (arrows) in BITC-treated MDA-MB-231 cells, which were rarely seen in DMSO-treated controls. Some of these vacuoles resembled autophagosomes and contained remnants of mitochondria. The BITC-treated cells also exhibited chromatin condensation and membrane blebbing (arrowhead), which are characteristic features of cells undergoing apoptosis.
- Figure 9.
A, representative histograms depicting ethidium bromide fluorescence (an indicator of superoxide anion production) and DCF fluorescence (an indicator of peroxide production) in MDA-MB-231 cells following a 2-h treatment with DMSO (control) or 2.5 μmol/L BITC. B, percentage of cells with DCF fluorescence in MDA-MB-231 cultures treated with DMSO (control) or 2.5 μmol/L BITC for the indicated times. Columns, mean (n = 3); bars, SE. *, P < 0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. The experiment was repeated twice, and representative data from a single experiment are shown.
- Figure 10.
Effect of combined superoxide dismutase and catalase mimetic EUK134 on BITC-induced ROS generation (A and D), cytoplasmic histone-associated DNA fragmentation (B and E), and cell killing (C) in MDA-MB-231 cells (A–C) and MCF-7 cells (D–E). Desired cell line was allowed to attach overnight and exposed to either DMSO or 30 μmol/L EUK134 for 1 to 2 h. The cells were then either left untreated (DMSO and EUK134 alone treatment group) or exposed to BITC (2.5 μmol/L for MDA-MB-231 and 10 μmol/L for MCF-7) for desired time point (2 or 4 h for ROS production assay and 16 or 24 h for cytoplasmic histone-associated DNA fragmentation and cell survival assays). Columns, mean (n = 3); bars, SE. a, P < 0.05, significantly different compared with control; b, P < 0.05, significantly different compared with BITC alone treatment group by one-way ANOVA followed by Bonferroni's multiple comparison test.
- Figure 11.
Immunoblotting for cleavage of procaspase-3 (A) and procaspase-9 and procaspase-8 (B) using lysates from MDA-MB-231 cells exposed to 2.5 μmol/L BITC for the indicated times. The blots were stripped and reprobed with anti-actin antibody as a loading control. Immunoblotting for each protein was done at least twice using independently prepared lysates, and the results were comparable.
- Figure 12.
A, immunoblotting for cleaved caspase-3 using lysates from MDA-MB-231 cells following treatment with BITC in the presence or absence of pan-caspase inhibitor zVAD-fmk, caspase-9-specific inhibitor zLEHD-fmk, and caspase-8-specific inhibitor zIETD-fmk. The MDA-MB-231 cells were exposed to either DMSO (control) or 40 μmol/L caspase inhibitor for 2 h before BITC treatment (2.5 μmol/L, 24 h). The blot was stripped and reprobed with anti-actin antibody as a loading control. B, cytoplasmic histone-associated DNA fragmentation in MDA-MB-231 cells following a 24-h treatment with either DMSO (control) or 2.5 μmol/L BITC in the presence or absence of 40 μmol/L pan-caspase inhibitor zVAD-fmk, caspase-9-specific inhibitor zLEHD-fmk, and caspase-8-specific inhibitor zIETD-fmk. Similar results were observed in two independent experiments. Columns, mean (n = 3); bars, SE. a, P < 0.05, significantly different compared with DMSO-treated control; b, P < 0.05, significantly different compared with BITC alone treatment group by one-way ANOVA followed by Tukey's multiple comparison test.
Tables
- Table 1.
Effect of BITC treatment on MCF-7 cell cycle distribution
BITC concentration Percent cells in phase Sub-G0-G1 G0-G1 S G2-M Control (DMSO) 3 ± 0.6 58 ± 0.6 19 ± 0.2 21 ± 1.0 2.5 μmol/L 16 ± 0.2* 20 ± 1.3* 9 ± 0.6* 55 ± 1.9* 5 μmol/L 12 ± 0.6* 19 ± 0.7* 8 ± 0.1* 61 ± 0.8* NOTE: Results are mean ± SE (n = 3).
↵* P < 0.05, significantly different compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Similar results were observed in two independent experiments.
Volume 5, Issue 11
