This effect is selleck dose dependent, being observed at 300 500 M DCA but not at lower concentrations. Similarly, DCA induced a 1. 5 fold increase in DNA fragmentation after 6 hr, which increased after 24 hr. DNA fragmen tation with low concentrations of DCA was similar to resting cells, while increasing concentra tions resulted in a steady rise in DNA damage. Taken together, these data show that DCA induces a reduction in cell proliferation, which is accompanied by low levels of apoptosis. These effects are sustained and dose dependent, being observed at high DCA concentrations similar to those found in patients with erosive esophagitis and Barretts esophagus. DCA induced PARP cleavage is caspase dependent PARP cleavage can occur via caspase dependent and inde pendent mechanisms.

The broad spectrum caspase inhibitor, Z Val Ala Asp CH2F, and the specific caspase 3 inhibitor, Z Asp Glu Val Asp FMK, were employed to assess the role of caspases in DCA induced PARP cleavage. SKGT4 cells were pretreated for 1 hr with 50 M of either Z VAD FMK or Z DEVD FMK and stimulated with 400 M DCA, a concentration which induced significant levels of PARP cleavage for 6 hr. Unstimulated SKGT4 cells showed negligible levels of PARP cleavage and DNA fragmentation. Both Z VAD FMK and Z DEVD FMK com pletely abolished DCA induced PARP cleavage while par tially inhibiting DNA fragmentation. These data indicate that DCA induced PARP cleavage is caspase 3 dependent, while DNA fragmentation is only partially dependent on this pathway.

DCA induces COX 2 expression via Erk1 2 and p38 dependent mechanisms Interestingly, the levels of DCA induced PARP cleavage plateau and do not increase progressively. This suggests that a compensatory survival mechanism might be con comitantly regulated by DCA. Enhanced protein expres sion of COX 2 has been correlated with cellular proliferation and resistance to apoptosis in various cell types. Therefore the induction of COX 2 protein expression by DCA in SKGT4 cells was examined using Western blot analysis. COX 2 is not expressed in unstim ulated cells, but it is readily induced after 4 hr of DCA stimulation. Maximal induction is achieved at 6 hours with 300 M DCA. In agreement with previous reports, COX 1 protein is not constitutively expressed in this cell line.

COX 2 protein expression can be regulated at transcrip tional and posttranscriptional levels by MAPKs and by AP 1 through binding to the CREB site in the COX 2 gene promoter in various cell types. Since DCA induces AP 1 activity through the activation of Erk1 2 and p38, we Drug_discovery explored the involvement of these path ways in the regulation of COX 2 protein expression in our system. SKGT4 cells were pre treated with 10 M PD98059, 2 M SB203580 or 1 M Go6976 for 30 min utes prior to addition of 300 M DCA for 6 hr.