BACKGROUND: Parthenolide, an inhibitor of NF-κB, and NS398, an inhibitor of COX-2, have been shown in our lab to provide synergistic growth inhibition of hepatocellular carcinoma (HCC) in vitro. The aim of the present study was to identify the mechanism in which these inhibitors effected growth inhibition. METHODS: Three human HCC lines (Hep3B, HepG2, and PLC) were treated with parthenolide and NS398, alone and in combination. HCC growth was determined by colorimetric assay and confirmed by trypan blue excluded cell counts 72 hours after treatment. A DNA fragmentation ELISA was used to measure apoptosis 24 hours after treatment. Cell cycle alterations were determined by flow cytometry in synchronized (serum starved) cells. The expression of cyclin D1, which promotes G1-to-S phase progression, was determined by Western blot. RESULTS: Parthenolide (0.1-10μM) and NS398 (1-100 μM) caused dose-dependent growth inhibition in all cell lines (P<0.01). Combination treatment caused synergistic growth inhibition in Hep3B and PLC cells; in HepG2 cells, inhibition was additive. Parthenolide caused a significant increase in apoptosis in all cell lines (P<0.05). NS398 did not cause a significant increase in apoptosis except at the high dose in Hep3B and PLC cells (100μM; P<0.05). Combination treatment did not induce more apoptosis than parthenolide alone in Hep3B and PLC cells; in HepG2 cells, combination treatment did not differ significantly from control. Treatment with NS398 caused G0/G1 cell cycle arrest in all cell lines. Parthenolide caused G0/G1 arrest in HepG2 and PLC cells, but not in Hep3B cells. Combination treatment caused greater G0/G1 arrest than single agent treatment in all cell lines. Cyclin D1 expression was decreased by NS398 in all cell lines. Parthenolide treatment led to decreased cyclin D1 expression in HepG2 and PLC cells but did not affect expression in Hep3B cells. Combination treatment caused greater decreases in cyclin D1 expression than single agent treatment in all cell lines. CONCLUSION: The combination of parthenolide and NS398 provides synergistic or complementary growth inhibition of HCC in vitro. Treatment with parthenolide, but not NS398, induces apoptosis in HCC cells. Combination treatment causes greater G0/G1 arrest than that which is induced by either agent alone, correlating with growth inhibitory effects. The G0/G1 arrest caused by these agents may be mediated through their effects on cyclin D1 expression.