Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL

Abstract

Apoptosis-based anticancer therapies require an in-depth understanding of the apoptotic machinery components of malignant cells, not only apoptotic regulators but also apoptotic executioners, given that their expression levels and functional integrity strongly determine the efficacy of therapeutic treatments. 2,3 Apoptosis is mediated by caspases, a conserved family of aspartate-specific cysteine proteases that are activated by several pathways. 4 The intrinsic mitochondrial pathway relies on caspase-9 as initiator caspase and is controlled by Bcl-2 family members. 5 The extrinsic pathway relies on caspase-8 and -10 as initiator caspases, is triggered by direct engagement of cell-surface death receptors, and can either converge on mitochondria or circumvent mitochondria to directly activate effector caspases. 6 Recently, increasing evidence has identified another intrinsic apoptotic pathway regulated by the endoplasmic reticulum (ER). 7,8 Besides controlling Ca 2ϩ homeostasis and protein trafficking, ER senses stress stimuli that lead to unfolded protein accumulation, triggering a signaling cascade termed unfolded protein response (UPR). 9 UPR is mediated by the activation of 3 ER transmembrane receptors, pancreatic ER kinase-like kinase, activating transcription factor 6, and inositol-requiring enzyme-1, which under physiologic conditions are maintained in an inactive state by the ER chaperone Binding Immunoglobulin Protein/Glucose-regulated protein78 (BiP/ GRP78). UPR is initially a self-protective response aimed at restoring the normal ER function and maintaining cell survival, with BiP/GRP78 playing a key role as prosurvival factor. 8,10 However, when ER stress is severe or prolonged, it overrides the salvage mechanisms of the initial UPR leading to apoptosis. [7][8][9] Several mechanisms link ER stress to apoptosis, including a direct caspase activation, caspase-4 in humans 11 and caspase-12 in mice, 12 which can initiate a specific caspase cascade independent of mitochondria, and a cross-talk with the mitochondria mediated by various factors, including Ca 2ϩ , Bcl-2 family proteins, and stressassociated kinases. [13][14][15] Because tumor cells can adapt to ER stress stimuli by triggering an altered protective UPR, 16,17 specific targeting of the prosurvival UPR molecules or the use of agents able to induce an ER stress-mediated apoptosis may contrast tumor development and growth, thus defining new types of cancer therapy. In this context, ER has been proposed as a critical target for anticancer therapy in several tumor types, both solid and hematologic. 8,[16][17][18][19] Apoptosis induction mediated by ER stressinducing agents is just beginning to be exploited also in B-CLL cells. Recent studies have shown that brefeldin A, an inhibitor of ER/Golgi protein transport, Tetrocarcin A, an antibiotic isolated from actinomycetes, and Xanthohumol, a plant-derived flavonoid, increase B-CLL cell apoptosis by different ER stress-mediated mechanisms. [20][21][22] Considering that B-CLL cells resist chemostimulation...

ABT-737 accelerates butyrate-induced death of HL-60 cells. Involvement of mitochondrial apoptosis pathway

“…The function of GRP78 in the hematopoietic system is just emerging, however, recent study clearly revealed involvement of GRP78 in hematopoietic stem cell survival and lymphogenesis (Wey et al 2012a). In addition, it has been shown that GRP78 expression is up-regulated in various forms of human leukaemia and implicated as causative factor for therapeutic resistance and early relapse (Tanimura et al 2009;Rosati et al 2010;Uckun et al 2011;Wey et al 2012b). In our experiments, we have detected GRP78 to be expressed in non-treated HL-60 cells and treatment of the cells with NaBu for 48 hours was associated with decrease of GRP78 level.…”

γ‐Secretase inhibitor I induces apoptosis in chronic lymphocytic leukemia cells by proteasome inhibition, endoplasmic reticulum stress increase and notch down‐regulation

“…23 Importantly, CLL cells have a functional ER stress-associated apoptotic signaling, and are susceptible to both pharmacologic and genetic ER targeting strategies. 24,25 Based on all the above observations and our previous findings that GSI I increased CLL cell apoptosis and inhibited Notch activation, 9 we investigated whether an inhibited proteasome activity and an increased ER stress signaling are also involved in GSI I-induced CLL cell apoptosis, and examined how these events and Notch inhibition contribute to this apoptosis. Clarifying the effects of GSI I on these three critical regulators of CLL cell survival may be important to design more effective therapies for this leukemia.…”

“…CD38 surface expression and ZAP-70 intracellular expression in CLL cells were examined as previously described. 24,29 Supporting Information Table 1 gives clinical laboratory characteristics and the Binet stage of CLL patients.…”

“…Based on our recent findings that ex vivo CLL cells have a functional ER stress-triggered caspase cascade initiated by caspase-4 and involving caspase-8 and -3, 24 we first investigated whether GSI I potentiates the activation of these caspases (patients [1][2][3][4][5][6][7][8][9][10][11][12]. Time-course analysis shows that the cleavage of caspase-4 into its active 20-kDa subunit was increased by GSI I, compared with controls, already after 1 hr and continued to be increased until 24 hr (Fig.…”

“…24 Equal amounts of proteins were separated by 7.5 to 15% SDS-PAGE and transferred to nitrocellulose membranes. Blots, after blocking, were incubated with primary antibodies to: caspase-4 (Medical & Biological Laboratories, Woburn, MA); cytochrome c (BD Biosciences, San Jose, CA); CHOP/GADD153, Bap31, Bax and Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA); caspase-8, IRE1a, caspase-3, caspase-9, PARP, phospho-SAPK/ JNK (Thr183/Tyr185) and total SAPK/JNK (Cell Signaling Technology, Beverly, MA); Smac/DIABLO (Upstate Biotechnology, Lake Placid, NY); Omi/HtrA2 (R&D Systems, Minneapolis, MN); cytochrome oxidase IV subunit II (COX IV; Invitrogen); Bim S/EL/L (Alexis Biochemicals, San Diego, CA); Noxa (Imgenex, San Diego, CA); Bcl-2 (DakoCytomation, Milan, Italy); GAPDH (Sigma-Aldrich); ubiquitinylated proteins (BIOMOL Research Laboratories, Plymouth Meeting, PA); Hes1 (Novus Biologicals, Littleton, CO); Notch1 (clone bTAN20) and Notch2 (clone C651.6DbHN), developed by Spiros Artavanis-Tsakonas, obtained from DSHB developed under the auspices of the NICHD, and maintained by Iowa University.…”