1-Methoxy-Canthin-6-One Induces c-Jun NH2-Terminal Kinase–Dependent Apoptosis and Synergizes with Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Activity in Human Neoplastic Cells of Hematopoietic or Endodermal Origin
We investigated the effects of 1-methoxy-canthin-6-one, isolated from the medicinal plant Ailanthus altissima Swingle, on apoptosis in human leukemia (Jurkat), thyroid carcinoma (ARO and NPA), and hepatocellular carcinoma (HuH7) cell lines. Cultures incubated with the compound showed >50% of sub-G 1 (hypodiploid) elements in flow cytometry analysis; the apoptosis-inducing activity was evident at <10 Mmol/L and half-maximal at about 40 Mmol/L 1-methoxy-canthin-6-one. The appearance of
... rance of hypodiploid elements was preceded by mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and Smac/DIABLO and procaspase-3 cleavage. We subsequently investigated the effect of 1-methoxy-canthin-6-one in combination with human recombinant tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) in the four cell lines. Suboptimal concentrations (10 Mmol/L 1-methoxycanthin-6-one and 0.25 ng/mL TRAIL, respectively) of the two agents, unable to elicit apoptosis when used alone, induced mitochondrial depolarization, activation of caspase-3, and 45% to 85% of sub-G 1 elements when added together to the cells. The synergism seemed to rely partly on the enhanced expression of TRAIL receptor 1 (TRAIL-R1; DR4), analyzed by immunofluorescence, by 1-methoxycanthin-6-one. Cell incubation with 1-methoxy-canthin-6one resulted in activating c-Jun NH 2 -terminal kinase (JNK), as revealed by Western blotting; induction of apoptosis and TRAIL-R1 up-regulation by 1-methoxy-canthin-6-one were >80% prevented by the addition of the JNK inhibitor (JNKI) SP600125JNKI, indicating that both effects were almost completely mediated by JNK activity. On the other hand, synergism with TRAIL was reduced by about 50%, suggesting that besides up-regulating TRAIL-R1, 1-methoxy-canthin-6-one could influence other factor(s) that participated in TRAIL-induced apoptosis. These findings indicate that 1-methoxy-canthin-6-one can represent a candidate for in vivo studies of monotherapies or combined antineoplastic therapies. (Cancer Res 2006; 66(8): 4385-93) . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Figure 6 . Effect of 1-methoxycanthin-6-one on JNK activation. A, cells (1 Â 10 6 per mL in 10% FCS-RPMI) were incubated with 1-methoxycanthin-6-one (40 Amol/L) for the indicated times. Then cell lysates were obtained and analyzed with anti-phospho-JNK, anti-phospho-c-Jun, or anti-a-tubulin antibodies in Western blot. B, cells (1 Â 10 6 per mL in 10% FCS-RPMI) were incubated with 1-methoxycanthin-6-one (40 Amol/L) for the indicated times, with or without overnight pretreatment with JNKI or ERKI (20 Amol/L). Then cell lysates were obtained and analyzed with anti-phospho-JNK or anti-a-tubulin antibodies in Western blot. C, cells (1 Â 10 6 per mL in 10% FCS-RPMI), preincubated with medium alone or JNKI or ERKI (20 Amol/L) overnight, were incubated with 1-methoxycanthin-6-one (40 Amol/L) for 16 hours. Then apoptosis was measured as percentage of sub-G 1 nuclei. Representative results. Columns, means of triplicate determinations; bars, SD.