K. M. Sokolowski1, S. Kunnimalaiyaan1, M. Balamurugan1, S. T. Koprowski1, T. C. Gamblin1, M. Kunnimalaiyaan1 1Medical College Of Wisconsin,Surgical Oncology/Surgery/Medical College Of Wisconsin,Milwaukee, WI, USA
Introduction: Despite improvement in therapeutic strategies, median survival in advanced hepatocellular carcinoma (HCC) remains less than one year. Therefore, molecularly targeted compounds with less toxic profiles are needed. Xanthohumol (XN), a prenylated chalcone flavonoid has been shown to have anti-proliferative effects in various cancers types in vitro. XN treatment in healthy mice and humans yielded favorable pharmacokinetics and bioavailability. Although a report providing therapeutic potential of XN in prostate cancer in transgenic mice, the effects of XN on HCC proliferation is unknown. A potential molecular target, midkine (MK- heparin-binding growth factor) is negatively correlated with HCC prognosis and readily detected in serum. Therefore, investigating the effects of XN on HCC cellular proliferation and the evaluation of midkine as a possible therapeutic biomarker is integral.
Methods: The effects of XN on a panel of HCC cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colometric and colonogenic assay. Cell lysates were analyzed via Western blotting for pro-apoptotic (c-PARP and cleaved caspase-3) and anti-apoptotic proteins (Bcl2, Survivin, and Mcl-1). Mechanism of XN on HCC cellular proliferation was also examined. Midkine expression was evaluated following XN treatment of conditioned HCC media.
Results: Up to 2 µM concentration of XN resulted in cell viabilities exceeding 90% in HCC cell lines. However, 5 µM and above of XN significantly reduced cell viability in a dose-dependent manner. Colonogenic ability also decreased with increasing concentrations of XN (Figure). Additionally, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced anti-apoptotic proteins. Importantly, MK secretion into media was significantly reduced following XN treatment. Furthermore, XN inhibited Notch1 signaling.
Conclusion: Xanthohumol effectively inhibits HCC growth in cell culture and reduces MK secretion. This may provide XN as a novel therapeutic agent and midkine as a potential biomarker. We report for the first time that XN targets Notch1 signaling in HCC cells. The favorable cytotoxic profile based on both healthy mice and human studies along with these findings warrant further preclinical in vivo analysis. Evaluation of Notch1 reduction and its effect on HCC cells following XN treatment is innovative. In addition, confirmation of MK as a therapeutic marker for HCC to XN treatment will be novel. To our knowledge, this is the first characterization of MK and Notch1 inhibition in HCC in response to XN treatment.
