Xanthohumol: A New Potential Proteasome Inhibitor That Activates the Pro-Apoptotic Arm of the Unfolded Protein Response in B-CLL Cells.

B-Chronic Lymphocytic Leukemia (B-CLL) is incurable by current methods because of increasing resistance to chemotherapy. Therefore, new options are needed for the treatment of CLL. The proteasome-inhibitor bortezomib was described to promote apoptosis in CLL cells and to induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in refractory multiple myeloma. The UPR is mainly a self-protective mechanism activated when protein folding is disrupted and misfolded proteins are accumulating in the ER. However, sustained ER stress eventually leads to cell death. Three major ER sensors are involved during UPR, namely ATF6, PERK and IRE1. In inactivated state, they are bound to the ER-chaperone BiP/GRP78. Upon ER stress, BiP is released from the luminal domain of the ER stress transducers resulting in their activation and downstream signaling. Pro-survival signals are delivered by IRE1 (splicing of XBP1) and ATF6 (cleavage), while pro-apoptotic signals are generated by PERK (upregulation of CHOP). Previously, we demonstrated that the xanthohumol (X) is able to kill B-CLL cells in a dose- and time-dependent way as evidenced by PARP cleavage and Annexin V staining (Lust et al., 2005). In this study, we first demonstrated that X induces apoptosis of B-CLL cells in part via activation of ER stress and the UPR and identified the associated molecular markers. Treatment of freshly isolated B-CLL cells with X, stimulated the expression BiP and Hsp70 (suggestive for ER stress), the phosphorylation of eIF2a (suggestive for PERK activation), and the splicing of XBP1 mRNA (indicative for IRE activation). In contrast, ATF6 activation seemed not to be implicated since no cleaved ATF6 could be demonstrated. Induction of UPR was associated with a pro-apoptotic outcome evidenced by upregulation of CHOP, generation of ROS, downregulation of the anti-apoptotic proteins Mcl-1, Bcl-x_L, Bcl-2, cleavage of PARP, and processing of caspase-3. Next, we showed that X inhibited the 20S proteasome activity in reticulocyte lysates but also functionally in B-CLL as demonstrated from the accumulation of polyubiquitin-conjugates under X treatment. The activation of UPR and influence on the anti-apoptotic proteins can mostly be explained by this proteasome inhibitory activity. In conclusion, we identified proteasome inhibitory capacities of xanthohumol in B-CLL in vitro. Proteasome-inhibition was accompanied by ER stress, UPR and apoptosis. Our results further suggest that tackling organelles like the proteasome and the ER is a valuable strategy in treatment of B-CLL.