Hydrogen sulfide protects colon cancer cells from chemopreventative agent β-phenylethyl isothiocyanate induced apoptosis

Figure 1 (A) Concentration dependant loss of cell viability induced by PEITC in colon cancer cells as determined at 24 h using the MTT viability assay. Data are representative of three or more separate experiments (mean±SD).

Figure 2 A: Time dependant Induction of caspase -3 activities in HCT116 and HT29 cells treated with 40 µmol/L PEITC as determined at 0. 5, 1, 3, 6, 16 and 24 h. B: Concentration dependant activation of caspase-3 as determined at 24 h after incubation with PEITC (10-80 µmol/L). C: Z-VAD-FMK, Ac-LEHD-CHO and Ac-DEVD-CHO inhibition on PEITC mediated apoptosis as determined at 24 h. ^bP < 0.01, significant difference compared to control groups. Cells were treated with individual caspase inhibitors for 1 h prior to the addition of 40 µmol/L PEITC. SubG1 populations were determined by propodium iodide staining followed by flow cytometry analysis as described in the materials and methods. ^bP < 0.01, comparing PEITC (alone) with control groups. D: Representative flow cytometry analysis of HCT116 and HT29 cells was used to determine the extent of DNA fragmentation in cells treated with PEITC (40 µmol/L) in the presence of caspase inhibitors. Data were determined by PI staining at 24 h. Figure shows representative example of at least 3 experiments. In each analysis, 10 000 events were recorded. ^bP < 0.01, PEITC (alone) vs control and caspase inhibitor treated groups.

Figure 3 A: Effects of low molecular weight sulfur compounds on PEITC mediate loss of cell viability; B and C: Inhibition of PEITC mediated loss of cell viability in HCT116 cells following either co-treatment or pre-treatment (24 h) with H₂S or NAC (125 µmol-1 mmol/L) as determined by MTT or LDH leakage. Data are expressed as mean±SD, (n = 8); D: Co-treatment with either H₂S or NAC (125 µmol-1 mmol/L) inhibits PEITC induced caspase-3 activity as determined at 16 h and Data are representative of 3 or more separate experiments conducted on separate occasions. mean±SD, ^bP < 0.01 vs PEITC treated groups.

Figure 4 Inhibition of PEITC mediated loss in cell viability in GSH depleted cells following pre-treatment with 0. 5 mmol/L BSO for 24 h. Both H₂S and NAC inhibited PEITC induced loss of cell viability in concentration dependant manner as determined using the MTT assay (A) and (B) LDH leakage. (C) Respective