Cyclooxygenase-2 inhibitors exert anti-tumor actions in human hepatocellular carcinoma (HCC) cells. The anti-tumor mechanism, however, is unlikely to be entirely due to COX-2 inhibition. Extracellular regulated kinases (Erk1/Erk2) play an important role in HCC mitogenesis and may be inhibited by COX-2 inhibitors. We investigated the effect of the COX-2 inhibitor, NS398, on ERK1/2 kinase (MEK) activity and Erk1/Erk2 expression in HepG2 and Hep3B cells. Total Erk1 and Erk2 expression as determined by immunoblot did not change with NS398 treatment. Surprisingly, Erk1/2 phosphorylation (MEK activity) was increased in a concentration-dependent fashion ([NS398]: 1-100 uM). We hypothesized that inhibition of this COX-2 inhibitor-induced Erk1/2 activation may improve the anti-tumor actions of COX-2 inhibitors. Human HCC cells (HepG2 and Hep3B) were treated with the COX-2 inhibitor, NS398, in the presence or absence of U0126 (MEK inhibitor). U0126 effectively suppressed Erk1/2 phosphorylation as determined by phospho-specific Erk1/2 immunoblot. In Hep3B cells, apoptosis as determined by DNA fragmentation ELISA was unchanged with NS398 (1-50 uM) or U0126 (0.1-5 uM) alone. The combination of NS398 and U0126 in Hep3B cells resulted in a synergistic increase in apoptosis (7X control). Relative apoptosis in HepG2 cells was increased with U0126 alone or in combination with NS398 (9-10X control). Relative apoptosis in both cell lines was strongly correlated with changes in the expression of the anti-apoptotic protein Bcl-xL. Cellular growth was assessed by colorimetric proliferation assay and confirmed by cell counts (trypan blue exclusion). HepG2 and Hep3B cells had concentration-dependent inhibition of cell growth with NS398 or U0126 treatment alone. The combination of NS398 and U0126 resulted in additive inhibitory effects on growth. Growth inhibitory effects in HepG2 and Hep3B cells appear to be in part secondary to the induction of G0/G1 and G2/M cell cycle arrest respectively, as determined by flow cytometry. Despite differential signaling in HepG2 and Hep3B cells, the sum effect of combining a COX-2 inhibitor and a MEK inhibitor results in enhanced anti-tumor actions. This novel combination may be useful for treating patients with HCC.