Article Figures & Data
Figures
- Figure 1.
Effect of sulindac sulfide (s. sulfide) treatment upon DR4 and DR5 expression. Incubation of SW480 cells with sulindac sulfide for the indicated doses and times induces DR4 (A) or DR4 and DR5 (B) protein levels. Protein expression was measured in whole cell lysates by immunoblotting. Expression was quantified relative to β-actin by scanning densitometry.
- Figure 2.
Sulindac sulfide activates procaspase-8 and the mitochondrial pathway as indicated by the cytosolic release of proapoptotic proteins. A, SW480 human colon carcinoma cells were incubated with 100 μmol/L sulindac sulfide for the indicated times. After isolation of the cytoplasm-enriched cell fractions, protein expression was determined by immunoblotting. B, SW480 cells with ectopic Bcl-2 expression were treated as above. Tubulin and COXIV are markers for the cytosolic and mitochondrial fractions, respectively.
- Figure 3.
Ectopic Bcl-2 expression protects SW480 cells from sulindac sulfide–induced apoptosis. A, SW480/neo (open column) and Bcl-2 transfectants (closed column, inset) were incubated with increasing doses of sulindac sulfide for 72 h. Cells were then collected and assayed for Annexin V-FITC binding and propidium iodide (PI) staining by fluorescence-activated cell sorting analysis; Annexin V-positive, PI-negative cells were considered apoptotic. B, selective inhibitors of caspase-8 (Z-IETD-FMK) and caspase-9 (Z-LEHD-FMK) attenuate sulindac sulfide (120 μmol/L)–induced Annexin V-FITC binding in SW480/Bcl-2 cells. Cells were incubated with sulindac sulfide for 72 h in the presence or absence of a caspase inhibitor. Columns, mean values of triplicate determinations; bars, ±SD.
- Figure 4.
Small-molecule Bcl-2 inhibitor, HA14-1, enhances sulindac sulfide–induced apoptosis and overcomes Bcl-2-mediated resistance to apoptotic signaling. A, SW480/Bcl-2 cells were incubated with increasing concentrations of HA14-1 for 72 h in the absence (open columns) or presence (hatched columns) of sulindac sulfide (100 μmol/L). Apoptosis was determined using the Annexin-V-FITC/PI binding assay. A nonapoptotic dose of HA14-1 (20 μmol/L) augments sulindac sulfide–induced apoptosis. B, coadministration of HA14-1 (20 μmol/L) and sulindac sulfide (100 μmol/L) enhances apoptotic signaling in SW480/Bcl-2 cells compared with treatment with sulindac (100 μmol/L) or HA14-1 (20 μmol/L) alone. Cell fractionation and immunoblotting were done as described in Materials and Methods.
- Figure 5.
A, incubation of parental SW480 cells with increasing doses of sulindac sulfide (SS) and TRAIL for 5 h produces a cooperative increase in apoptosis compared with either drug alone. B, similar results are seen in HCT-15 human colon cancer cells where sulindac was previously found to increase DR5 mRNA and protein expression (26).
- Figure 6.
Sulindac sulfide and TRAIL cooperatively increase apoptosis and enhance apoptotic signaling in SW480/Bcl-2 cells. A, SW480/Bcl-2 were incubated for 5 h with increasing doses of sulindac combined with TRAIL. Apoptosis was measured using an Annexin V-FITC, propidium iodide (PI) binding assay (B). Coadministration of sulindac and TRAIL enhanced apoptotic signaling compared to either drug alone. SW480/Bcl-2 cells were treated with sulindac sulfide (100 μmol/L), TRAIL (10 ng/mL), or their combination for indicated times. Cells were then fractionated and cytoplasm-enriched extracts were collected and analyzed by immunoblotting.
Volume 4, Issue 10
