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Bcl-2 overexpression attenuates apoptosis induction by sulindac sulfide in SW480 human colon cancer cells.

3195 Background: Sulindac sulfide is a NSAID and an inhibitor of cyclooxygenase enzymes that regulate prostaglandin synthesis. Sulindac is a potent chemopreventive agent in animal models of colon cancer and in patients with familial adenomatous polyposis. Sulindac sulfide induces apoptosis in colon cancer cells which may be an important mechanism of its anti-tumor effects. Previously, we found that sulindac sulfide can modulate both the caspase-8-depedent and cytochrome c-dependent apoptotic pathways. We determined whether Bcl-2 overexpression, an inhibitor of the latter apoptotic pathway, can inhibit apoptosis induction by sulindac sulfide.


MATERIALS AND METHODS
SW480 cells were stably transfected with the pC3-Bcl-2 plasmid (SW480/Bcl-2) or vector alone (SW480/neo). The level of Bcl-2 protein expression was then determined by immunoblotting (anti-Bcl-2 antibody, Upstate). Cells were incubated with sulindac sulfide (0-120 μM) for 72 hr and then floating and attached cells were harvested and assayed for Annexin V-FITC (BD PharMingen) labeling by flow cytometry. The effect of a caspase-9 inhibitor (Z-LEHD-FMK, R & D Systems) or a caspase-8 inhibitor (Z-IETD-FMK, R & D Systems) upon sulindac sulfide-induced apoptosis was also studied.


RESULTS
SW480/Bcl-2 cells showed overexpression of Bcl-2 proteins relative to SW480/neo cells. Bcl-2 overexpression produced an anti-apoptotic effect (>50% reduction) compared to SW480/neo control cells, as assayed by Annexin V binding after incubation with sulindac sulfide (72 hr). Furthermore, co-treatment with a caspase-9 inhibitor significantly reduced the Annexin V binding (>50%) triggered by sulindac sulfide treatment. A caspase-8 inhibitor was also found to significantly reduce Annexin V binding (>50%) in sulindac sulfide-treated parental cells.


CONCLUSION
Sulindac sulfide engages both the caspase-8- and caspase-9-dependent apoptotic signaling pathways in SW480 cells. Bcl-2-mediated inhibition of apoptosis by sulindac sulfide indicates that Bcl-2 is an important modulator of the therapeutic efficacy of this agent used for the prevention or treatment of human colon cancer. No significant financial relationships to disclose.