Abstract Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate co-factor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation, and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II).On the other hand, it has been reported that the expression level of VCP is correlated with progression of cancer, and VCP-overexpressing cell lines showed the resistance to apoptosis. Therefore, in this study, we examined the antitumor effects of XN on several types of human tumor cells. First we tested the sensitivity to XN in 15 human tumor cell lines. Each cell was treated with XN for 48 hrs, and XN-induced apoptosis was quantitatively analyzed by flow cytometry. As a result, XN potently induced apoptosis in 6 cell lines (SW480, SW620, HCT116, A2058, A375 and SW48 cells) as judged from subG1 population, and another 9 tumor cell lines were resistant to XN. Next, we examined whether VCP is involved in XN-induced apoptosis. Although, these 15 cell lines showed similar expression levels of VCP, another VCP inhibitor, Eeyarestatin I, also induced apoptosis in XN-sensitive tumor cells (SW480 cells and HCT116 cells) but not in VCP-insensitive tumor cells (A431 cells and EC17 cells). These findings suggest that the several human tumor cell lines require VCP function for their survival, and VCP would be a potential chemotherapeutic target molecule for subset of tumor cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C18. Citation Format: Yuki Shikata, Shuhei Kanagaki, Yukiko Sasazawa, Etsu Tashiro, Tetsuro Yoshimaru, Masato Komatsu, Toyomasa Katagiri, Masaya Imoto. Antitumor activity of xanthohumol, an inhibitor of valosin-containing protein. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C18.