Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation

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Abstract
Recently, we demonstrated that the cyclooxygenase-2 (COX-2) inhibitor celecoxib acts to significantly suppress the growth of rat C611B cholangiocarcinoma (ChC) cells in vitro. To establish a molecular mechanism for this growth suppression, we investigated the effects of celecoxib on apoptotic signaling pathways in cultured rat C611B ChC cells. Celecoxib and another COX-2 inhibitor, rofecoxib, at 5 μM were almost equally effective in inhibiting prostaglandin E2 (PGE2) production by these cells, but at this low concentration, neither inhibitor suppressed growth or induced apoptosis. Celecoxib at 50 μM induced prominent apoptosis in these cells, whereas rofecoxib at 50 μM was without effect in either suppressing growth or inducing apoptosis. Celecoxib (50 μM) did not alter Bcl-2, Bcl-xL, or COX-2 protein levels, nor did it inhibit p42/44 mitogen-activated protein kinase (MAPK) phosphorylation; however, it significantly suppressed serine/threonine kinase Akt/PKB (Akt) phosphorylation and kinase activity in cultured C611B cells. This effect, in turn, directly correlated with Bax translocation to mitochondria, cytochrome c release into cytosol, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Addition of 25 μM PGE2 to C611B cell cultures blocked the apoptotic actions of celecoxib. Rofecoxib (50 μM) was without effect in suppressing Akt phosphorylation and caspase-3 activation. In vivo, celecoxib partially suppressed tumorigenic growth of C611B ChC cells. In conclusion, our results indicate that celecoxib preferentially acts in vitro to induce apoptosis in ChC cells through a mechanism involving Akt inactivation, Bax translocation, and cytochrome c release. Our in vivo results further suggest celecoxib might have potential therapeutic or chemopreventive value against ChC. (Hepatology 2004;39:1028-1037.)