Mistletoe extracts including Viscum Fraxini (extract of mistletoe growing on ash trees) have been used extensively in European medical clinics to treat various cancers including hepatocellular carcinoma (HCC). Results from a phase II clinical trial have shown that subcutaneous administration of Fraxini leads to either complete or partial response in 20% of chemo-naïve patients with HCC. However, how mistletoe extract exerts its antitumor activity in HCC is largely unknown. We examined the effect of Fraxini on the growth of human HCC Hep3B and HepG2 cells by MTT assay, apoptosis by PI and Annexin V staining and molecular mechanisms using Reverse Phase Proteomic Array (RPPA). All three mistletoe extracts, Fraxini, Iscador Q and M (growing on oak and maple trees, respectively), exerted relatively strong antiproliferative activity in the Hep3B cells, with IC50 values at 0.5, 6.49, and 5.7 µg/ml, respectively. A slightly weaker anticancer effect was observed in the HepG2 cells treated with the above three extracts. The antiproliferative effect of Fraxini was almost 10 and 6 times stronger than that of Iscador M and Iscador Q, respectively, in both Hep3B and HepG2 cells. Intriguingly, the water-soluble fraction of Fraxini exerted similar anticancer activity as Fraxini, but no inhibitory effect of lipid soluble fraction of Fraxini was observed. Treatment with Fraxini induced Hep3B cells to undergo apoptotic cell death, evidenced by the increased number (5-fold) of subG1/G0 phase cells in the Fraxini-treated (5 µg/ml) Hep3B cells than in the control cells and increased apoptotic cell population (by Annexin V staining). Additionally, notable ultrastructural changes i.e. extremely condensed mitochondria in perinuclear positions by transmission electron microscopy (TEM), were observed in Fraxini-treated (10 μg/ml) Hep3B cells compared to control treated cells. Proteomic array analysis suggested that Fraxini dose-dependently inhibited expression of Bcl-xl, BCl-2, Bim, and pRB proteins while increased cleaved caspase 7 and 8 as well as CHEK 1 and 2, suggesting that the apoptotic effect of Fraxini was mediated through regulation of BCL-2 family proteins and caspase activation, which correlated with the mitochondrial changes observed in the TEM study. Strikingly, c-Myc protein was strongly downregulated in a dose dependent fashion in the Hep3B cells treated with Fraxini (> 84% at 5 μg/ml), as examined by both RPPA and Western blotting, suggesting that the c-Myc protein could be an important target for Fraxini-elicited anticancer activity in HCC. Given that HCC is one of the few cancers whose death rate nearly equals its incidence and c-Myc is required for hepatocyte proliferation and liver tumorigenesis, Fraxini could have great potential in HCC treatment and therefore warrants further investigation.
Citation Format: Xiaoping Ding, Carrie Cartwright, Lin Tan, Richard Lee, Peiying Yang. Mistletoe extract inhibits the proliferation of human hepatocellular carcinoma cells by induction of apoptosis and downregulation of c-MYC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3206. doi:10.1158/1538-7445.AM2014-3206