Xanthohumol Induces Apoptosis in B-CLL In Vitro by Sustained Endoplasmic Reticulum Stress and Inhibition of NFκ-B.

Introduction

Correct folding of new proteins is supervised in the endoplasic reticulum (ER) unfolded protein response (UPR). Misfolded proteins recruit the chaperone Grp78 that is thereby released from the transcription factors ATF6, IRE-1 leading to compensatory increase in Grp78, and PERK, leading to phosphorylation of eIF2α and block of further protein translation. UPR overload leads to ER stress and cell death. Targeting the endoplasmic reticulum (ER) is a new strategy explored in B-CLL. The hop-derived chalcone Xanthohumol (X) has been characterized as a ‘broad-spectrum’ cancer chemopreventive agent. Recently, we demonstrated that X induces dose- and time-dependent cell death of MCF7/6 breast cancer cells accompanied by ER stress. X induces apoptosis and cleavage of poly(ADP)-ribose-polymerase (PARP) in B-CLL in vitro. The present study investigates the branches of the UPR in relation to X induced apoptosis of B-CLL cells.

Materials and methods. Lymphocytes were isolated by Lymphoprep from 15 patients with B-CLL after informed consent. CD19 positive cells were selected by EasySep positive selection kit. Apoptosis was assessed by flow-cytometry (AnnexinV-PI). Western Blotting was used for Grp78, ATF6, XBP1, phospho-eIF2a, eIF2a, ATF4, CHOP, phospho-IKK, IKK, PARP, caspase-9, -8, -7, -4, cleaved caspase-3, mcl-1, bcl-x_L, bax, bak, and bid. NF-kB activity was assessed by EMSA. Quantitative RT-PCR was performed to analyze Grp78 mRNA levels. Bcl-2 protein level was detected by flow cytometry and reactive oxygen species (ROS) by fluorescence microscopy.

Results and conclusion

X induced an upregulation of Grp78 mRNA levels which was not translated in an increase in protein. X treatment stimulated a rapid and sustained phosphorylation of eIF2a, suggesting the involvement of PERK. In contrast, the ER-stress transducers ATF6 and IRE1 were not activated. X-induced ER stress was associated with strong induction of the pro-apoptotic protein CHOP and inhibition of the NF-kB pathway. Furthermore, the pro-apoptotic effect of X was accompanied by an accumulation of ROS, a downregulation of the anti-apoptotic proteins mcl-1, bcl-x_L, bcl-2 and processing of caspase-3, -7 and -9.In conclusion, the chalcone X is capable of inducing cell death with down-regulation of bcl-2, mcl-1, bcl-xL, and activation of the caspase cascade. This is accompanied by ER-stress as evidenced by the upregulation of Grp78 mRNA levels, induction of a rapid and sustained phosphorylation of eIF2a, upregulation of CHOP, and inhibition of the NF-kB signaling.