Abstract 4845: Fraxini and mistletoe lectins inhibit the proliferation of hepatocellular carcinoma by promoting proteasomal degradation of c-Myc

Abstract

Abstract Mistletoe extracts, including Fraxini, have been used to treat cancer, including hepatocellular carcinoma (HCC), in Europe. However, the mechanism(s) underlying the effect of Fraxini in HCC is(are) inconclusive which hampers its optimal use for the treatment of HCC. We previously reported that Fraxini, and two other mistletoe extracts Iscador Q and M exerted strong antiproliferative and pro-apoptotic activities in Hep3B cells through targeting c-Myc protein. In current study, we examined the efficacy of Fraxini in HCC bearing animals and further investigated the mechanism by which Fraxini down-regulates c-Myc protein. How c-Myc was regulated by Fraxini was investigated by measuring the half-life of c-Myc protein with cycloheximide chase assay, proteasomal degradation and phosphorylation of c-Myc. The antitumor efficacy of the Fraxini was tested in the Hep3B mouse xenograft model and Fraxini was administered subcutaneously. We found that Fraxini dose dependently suppressed the c-Myc protein expression, but did not affect c-Myc mRNA level in Hep3B cells, suggesting Fraxini down-regulates c-Myc through post-translational regulation. Fraxini reduced the half-life of c-Myc protein from control (40 minutes) to 27 minutes. Inhibition of 26S proteasome activity using MG-132 in Hep3B cells notably antagonized Fraxini induced down-regulation of c-Myc, suggesting that Fraxini down-regulated c-Myc by promoting its proteasome degradation. Fraxini decreased phosphorylation of S62 (stabilizing c-Myc) while slightly increased p-cMycT58 (destabilized c-Myc) in Hep3B cells. When Burkitt's lymphoma Raji cells, which are known to host T58 mutation on c-Myc and result in c-Myc stabilization, were treated with Fraxini, Fraxini (up to 20 μg/ml) exerted minimum activity in cell growth and c-Myc protein expression, augmenting that Fraxini is acting on c-Myc phosphorylation sites which resulted in down-regulation c-Myc and elicited anticancer activity in HCC. Additionally, Fraxini injected (s.c.) to mice carrying Hep3B cell xenograft tumors for 2 weeks significantly reduced the average tumor volume (71.8 ± 22.2 mm3) compared to that in the control treated mice (179.0 ± 36.3 mm3, p < 0.05) and lead to reduction of c-Myc protein in the tumor tissues. Intriguingly, mistletoe lectin, a bioactive component in Fraxini, appears to be most effective in inhibiting the proliferation of c-Myc expressing Hep3B cells compared to that of PLC/RF5 cells with limited expression of c-Myc. Mistletoe lectin (1 pg/ml) in the meantime reduced almost 90% c-Myc protein expression in Hep3B cells. In conclusion, since c-MYC gene is required for hepatocyte proliferation and liver tumorigenesis, Fraxini and mistletoe lectin through targeting c-Myc may have great therapeutic potential for patients with advanced HCC and warrant further investigation. The study is supported by the start-up fund of UT MDACC. Citation Format: Yan Jiang, Yong Pan, Patrea R. Rhea, Richard Lee, Zhimin Lu, Peiying Yang. Fraxini and mistletoe lectins inhibit the proliferation of hepatocellular carcinoma by promoting proteasomal degradation of c-Myc. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4845.