Xanthohumol activates the proapoptotic arm of the unfolded protein response in chronic lymphocytic leukemia

Abstract

Background: Chronic lymphocytic leukemia (CLL) is an incurable disease with a natural history of increasing resistance to chemotherapy. A novel approach to overcome chemotherapy resistance may be targeting the endoplasmic reticulum (ER).

Patients and methods: The involvement of the unfolded protein response (UPR) in the cell killing effect of xanthohumol (X) was examined in 18 patient samples.

Results: X-induced apoptosis of CLL cells was accompanied by the induction of glucose-regulated protein of 78 kDa (GRP78) and heat-shock protein of 70 kDa (Hsp70) protein levels and by sustained phosphorylation of the eukaryotic translation initiation factor 2 (eIF2alpha), suggesting the involvement of the ER stress transducer, the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK). The X-box-binding protein 1 (XBP1) mRNA was spliced but no clear activation of activating transcription factor 6 (ATF6) was observed. The proapoptotic outcome was further demonstrated by the up-regulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), down-regulation of myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2), cleavage of poly-(ADP)-ribose polymerase (PARP) and processing of caspase-3, -4 and -9. Furthermore, X showed proteasome inhibitory activity.

Conclusion: X stimulates the proapoptotic arm of the UPR in ex vivo CLL cells, suggesting that ER stress may play an important role during X-induced apoptosis.

Figure 1.

Effect of X on apoptosis in CLL patient samples. CLL cells from 14 patients were treated with 25 μM X or 0.1% EtOH solvent control E. Western blot evaluation using tubulin as loading control and mean relative intensity (% ) assessed by quantification of the radiograms. (A-B) Caspase-3 (n=8), -4 (n=4), -7 (n=4), -8 (n=4) and -9 (n=8). (C-D) Mcl-1 (n=9), Bcl-xL (n=7), Bak (n=7), Bax (n=8) and Bid (n=6). (E) CLL cells stained with Bcl-2-FITC after 24 h treatment and evaluated by flow cytometry. (F) Relative percentage living cells of CLL cells treated with 25 μM X or 0.1% EtOH, E, and pretreated with 20 μM or 50 μM Z-VAD-fmk (Z) after 24 or 48 h incubation time. Error bars: standard deviation, *p<0.05.

Figure 2.

Effect of X on GRP78, GRP94, Hsp70 and Hsp90 expression in CLL cells. CLL cells were treated for 3, 6, 12 or 24 h with 25 μM X or 0.1% EtOH solvent control (S) while 10 μg/ml brefeldin A (Ba) and corresponding DMSO solvent control (S) was used as positive control. (A) GRP78 mRNA expression in CLL cells (n=6) analyzed by real-time quantitative RT-PCR. (B-C) Effect of X on GRP78, GRP94, Hsp70 and Hsp90 protein levels evaluated by Western blot with tubulin as loading control (n=4) and quantified by densitometric analysis. Error bars: standard deviation, *p<0.05.

Figure 3.

Activation of the proapoptotic arm of the UPR in CLL. CLL cells were exposed for 3, 6, 12 or 24 h to 25 μM X with solvent 0.1% EtOH, E, or 10 μg/ml brefeldin A (Ba) with solvent 2% DMSO, D, used as positive control. Bands detected after RT-PCR and Western blotting were quantified by densitometric analysis (Solvents = S). (A-B) Splicing of ER stress-induced XBP1 analyzed by RT-PCR (n=4 for X; n=2 for Ba). Unspliced XBP1 (XBP1(u)) contained 312 bp, spliced XBP1 (XBP1(s)) 286 bp. Actin was used as loading control. Western blot analysis with tubulin as loading control for (C-D) phospho-eIF2α and eIF2α (n=9 for X, n=2 for Ba) and (E-F) CHOP (for X, n=7 and for Ba, n=2). (G) Effect of X on the formation of ROS determined by fluorescence microscopy, ×20 original magnification. Error bars: standard deviation, *p<0.05, **p<0.1.

Figure 4.

Proteasome inhibitory effect of X in CLL. (A) Western blot of CLL cells treated for 3, 6, 12 or 24 h with 25 μM X, 1 μM MG132 (MG) or 10 nM bortezomib (Bo) (both used as positive control) and corresponding solvent controls [distilled water (AD), EtOH (E) or DMSO (D)=solvent (S)]. (B) Mean relative intensity after 3, 6, 12 or 24 h (n=3). Error bars: standard deviation **p<0.1. (C) The 20S proteasomal activity of different concentrations of Bo and of X.

Figure 5.

The NF-κB inhibitory activity of X in CLL. CLL cells were left untreated (U) or treated with 0.1% EtOH (E), 25 μM X, 100 ng/ml PMA or cotreated with 100 ng/ml PMA and 25 μM X for 10, 30 or 60 min (indicated as separate dots in the figure). (A) The relative NF-κB DNA binding activity by EMSA (n=4). Western blot analysis for (B) IκBα and (C) IKK phosphorylation levels.