Sodium salicylate reduces TNFα -induced apoptosis in SEG1 EAC cells

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Background: The anti-proliferative, anti-invasive and pro-apoptotic properties of salicylate have been documented for multiple neoplasias including colorectal adenocarcinomas as well as for neoplasia of other organs and systems. However, to our knowledge, extensive studies have not yet been performed for esophageal adenocarcinoma (EAC), one of the most rapidly increasing cancers in the Western world, caused by chronic acidic gastroesophageal refluxate. TNFα expression is increased in neoplastic and preneoplastic esophageal mucosa. We previously demonstrated that TNFα induces apoptosis in EAC cell lines in vitro, and that brief acid preexposure partially rescues cells from TNFα-induced apoptosis. We hypothesized that salicylate would counteract the rescuing effect of acid on TNFα-induced apoptosis in SEG1 EAC cells.
 Materials and Methods: To measure baseline apoptosis, cellular caspase3 activity was determined after 6h and 18h of either medium alone or medium supplemented with TNFα. To assess effects of salicylate, cells were preincubated with sodium salicylate [10 μM] for 30 minutes prior to the 6h and 18h caspase3 measurements. To further induce apoptosis, cells were exposed to TNFα [50nm] and caspase3 activity was determined atr 6h and 18 h. Finally, to assess the effects of acid, cells were pulsed with pH3 serum-free medium for 3 minutes, washed, and further incubated in normal-pH medium with or without TNFα as described above.
 Results: Incubation of cells with salicylate alone did not induce caspase3 activation at 6h or 18h. TNFα alone (vs. unsupplemented medium) increased caspase3 activity by 1.3-fold and 3.5-fold at 6h and 18h, respectively, reflecting the apoptosis-inducing properties of TNFα. Surprisingly, preincubation with salicylate reduced TNFα-induced caspase3 activation by 30% at 6h and by 40% at 18h. Acid pulsation prior to TNFα stimulation resulted in a 6% increase at 6h and a 14% decrease at 18h in TNFα-induced caspase3 activation relative to no acid pulsation. Again surprisingly, preincubation with salicylate prior to acid pulsation and TNFα stimulation completely prevented caspase3 activation at 6h and reduced caspase3 activity by 25% at18h vs. acid pulsation plus TNFα without salicylate.
 Conclusions: These data indicate, somewhat counterintuitively, that salicylate is not an apoptosis inducer in SEG1 cells. On the contrary, preincubation with salicylate increases cellular resistance to TNFα-induced apoptosis. Salicylate maintains the rescuing effect of brief acid exposure on cell death triggered by TNFα exposure. These results imply that salicylate may not constitute an effective therapeutic agent to induce tumor cell apoptosis in the EAC setting.