2002;2:301C310. therapy for induction of apoptosis. Additionally, either celecoxib alone or in combination with curcumol inhibited NSCLC cell migration and invasion by suppressing FAK and matrix metalloproteinase-9 activities. Furthermore, the combined treatment reduced tumor volume and excess weight in xenograft mouse model, and significantly decreased tumor metastasis nodules in lung tissues by tail vein injection. Our results confirm and provide mechanistic insights into the prominent anti-proliferative activities of celecoxib and/or curcumol on NSCLC cells, which provide a rationale for further detailed preclinical and potentially clinical studies of this combination for the therapy of lung malignancy. < 0.05, **< 0.01. In recent years, more and more malignancy therapeutics in preclinical trails or on the market turn to natural products with low toxicity and drug resistance. Curcumol (for its structure, see Physique ?Physique1A),1A), a guaiane-type sesquiterpenoid hemiketal, is one of the major components of the essential oil of and the effect on tumor growth and metastasis < 0.05). As compared MD2-TLR4-IN-1 with A549 cells, H1299 cells were found to be more sensitive to celecoxib + curcumol treatment, once the proliferation inhibition MD2-TLR4-IN-1 rate was 25.5 3.2 (%) since 20 M of celecoxib. However, no synergistic cytotoxicity was observed in BEAS-2B cells. These results suggest that curcumol has an enhanced effect on celecoxib-inhibited proliferation of tumor cells at a subtoxic concentration without increasing cytotoxicity to normal cells. In this study, we used a subtoxic concentration at which celecoxib alone did not induce significant proliferation inhibition. Therefore, drug dosages of celecoxib (30 M) and curcumol (30 M) were chosen for combinative therapy in the following experiments. Although celecoxib is usually a COX-2 inhibitor, it has been found to exhibit potent pro-apoptotic activity in malignancy cells through a mechanism that is impartial of its COX-2 inhibitory activity [25, 26]. Therefore, in order to determine whether the synergistic effect of celecoxib and curcumol on NSCLC cell growth occur in a COX-2-dependent route, other COX inhibitors such as nimesulide (COX-2 inhibitor) or indomethacin (COX-1 inhibitor) were also used in combination with curcumol. As shown in Supplementary Physique 1A and 1B, we found that curcumol exerted no synergistic effect on the anti-proliferative action of COX-2-selective inhibitor nimesulide and the NSAID inhibitor indomethacin in A549 cells. In addition, the inhibition on COX-2 protein expression by celecoxib MD2-TLR4-IN-1 and curcumol combinative treatment in A549 cells was just similar to that by celecoxib monotherapy (Supplementary Physique 1C). Thus, our data provide definitive proof that this enhanced inhibitory effect on tumor cell growth of the combinative treatment is not a result of COX inhibition. Combined effect of celecoxib and curcumol on tumor cell apoptosis To determine whether tumor cellular MD2-TLR4-IN-1 viability decreased with celecoxib and curcumol apoptosis, we tested the externalization of phosphatidylserine around the cell membrane by Annexin V/PI staining. Two NSCLC cell lines (A549 and H1299) were exposed to celecoxib (30 M), curcumol (30 M) or a combination of both. As shown in Physique ?Physique2A,2A, after 24 h of treatment, curcumol alone had no obvious effect on tumor cell apoptosis, while monotherapy with celecoxib induced 15-25% apoptosis ratio. However, when A549 and H1299 cells were exposed to combined treatment with celecoxib and curcumol, the number of cells undergoing apoptosis significantly increased (50-65%). This effect was statistically significant as compared to single treatment with either drug alone. TUNEL assays further indicated that curcumol led to an increased apoptotic rate in NSCLC cells treated with celecoxib (Physique ?(Figure2B).2B). Additionally, colony-forming assay showed that 30 M celecoxib alone caused moderate inhibition of the clonogenic growth of A549 and H1299 cells. In contrast, combined treatment with celecoxib and curcumol markedly suppressed Notch1 the clonogenic growth of A549 and H1299 cells with inhibition rates of 92.5% and 95.8%, respectively (Determine ?(Figure2C2C). Open in MD2-TLR4-IN-1 a separate window Physique 2 Curcumol enhances celecoxib-induced cell apoptosis and their combination suppresses the clonogenic growth of NSCLC cells(A) A549 and H1299 cells were exposed to celecoxib (30 M) and/or curcumol (30 M). 18 h later, all cells were harvested for circulation cytometry analysis. Annexin V/PI-stained cells were analyzed and the percentage of apoptotic cells was decided. The experiments were carried out independently in triplicate; representative data.