Inducing apoptosis of cancer cells using small-molecule plant compounds that bind to GRP78

A fundamental property of all cells is their ability to adapt and survive despite variation in environmental conditions. Minimising errors in the conformational integrity of proteins, vital to the functioning of the endoplasmic reticulum (ER), is achieved via the unfolded protein response (UPR); this homeostatic mechanism is induced by ER stress resulting from any disruption to protein production, folding or degradation. The ER-resident protein, glucose regulated protein 78 (GRP78), functions as a sensory hub and is present as complexes with three proteins: the protein kinase-like ER kinase (PERK or eukaryotic initiation factor 2 alpha kinase 3), inositol-requiring element 1 (IRE1) and activating transcription factor 6 (ATF6) (Schroder, 2005, 2006). Glucose regulated protein 78 also possesses high affinity for hydrophobic or hydrophilic regions exposed on the surface of unfolded or misfolded protein substrates; these interactions can be modulated by the binding of ADP and ATP to an N-terminal ATP-binding domain, and result in dissociation and activation of PERK, IRE1 and ATF6 (Bertolotti et al, 2000; Shen et al, 2002). Dissociation of ATF6 facilitates Golgi-mediated processing of ATF6 to an active transcription factor; the dissociation of PERK and the endoribonuclease precursor IRE1 from GRP78 allows oligomerisation and auto-phosphorylation to form active kinases (Schroder and Kaufman, 2005; Schroder, 2005, 2006). Protein kinase-like ER kinase, IRE1 and ATF6 are key UPR regulators that initiate downstream responses of ER stress including the cessation of cap-dependent mRNA translation, the induction of chaperones to assist in protein folding and the induction of apoptosis via activating transcription factor 4 (ATF4) and GADD153/CHOP (Blais et al, 2004). The expression of GRP78 may be enhanced in metastatic cells and late-stage tumours, and its role as an ER stress sensor suggests that it may be useful as a therapeutic target (Lee, 2007; Martin et al, 2010). Although siRNA-mediated knockdown of GRP78 increases cell death in vitro (Virrey et al, 2008), this may be difficult to achieve in vivo and alternative approaches to inhibiting GRP78 may be more effective as therapeutic strategies. The N-terminal ATPase domain important to GRP78 function also forms complexes with procaspases thus preventing caspase activation; this interaction can be abrogated with dATP to increase drug-induced cell death (Rao et al, 2002; Reddy et al, 2003). Treatment with nucleoside analogues that bind to the ATPase domain and facilitate dissociation of GRP78/procaspase complexes and disrupt the ability of GRP78 to bind/release substrates may, therefore, be a useful strategy to inhibit GRP78. Additional approaches may be to use natural inhibitors, such as the Green Tea C. sinensis flavonoid epigallocatechin gallate (EGCG) (Ermakova et al, 2006) or cytotoxin-mediated cleavage by AB5 Subtilase (SubAB5) (Paton et al, 2006; Paton and Paton, 2010). Epigallocatechin gallate blocks the ATPase domain of GRP78 (Ermakova et al, 2006) and enhances cell death in response to temozolamide or etoposide (Virrey et al, 2008). Epigallocatechin gallate also inhibits the 26S proteasome and NFκB signalling cascade and, while clearly not specific to GRP78, may be a candidate drug to overcome chemoresistance in solid tumours (Nam et al, 2001; Ermakova et al, 2006). There may be other natural products that could be used to inhibit GRP78. Honokiol (2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol) (HNK), a cell-wall component of M. grandiflora, is a potent antitumorigenic and neurotrophic compound (Chen et al, 2010), which induces apoptosis as a result of ER stress in chondrosarcoma cells with the concomitant upregulation of GRP78. Unpublished proteomic data have suggested that HNK binds to GRP78 and the aim of this study was to investigate the binding of HNK and EGCG to GRP78, and characterise the effect of these as single agents on neuroectodermal tumour cells and in combination with two drugs, fenretinide and bortezomib, known to induce ER stress-induced cell death.