Mechanism of the induction of endoplasmic reticulum stress by the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT): Activation of PERK/eIF2α, IRE1α, ATF6 and calmodulin kinase
Graphical abstract
Introduction
The endoplasmic reticulum (ER) is a crucial organelle that is responsible for the synthesis, maturation and folding of proteins [1]. Homeostasis of the ER is maintained by preventing toxic accumulation of unfolded or misfolded proteins and/or calcium (Ca2+) depletion [1]. The unfolded protein response (UPR) is an ER-specific cellular stress response that has been found to be conserved in all mammalian species [1]. Disruption of ER homeostasis triggers the UPR, which arrests protein translation and activates signaling pathways for molecular chaperones to assist protein folding and to direct the degradation of misfolded proteins [1], [2]. The prolongation of the UPR can also lead to apoptosis in a caspase-dependent manner [1], [2].
Under resting conditions, the ER chaperone, binding immunoglobulin protein (BiP; also known as glucose-related protein 78, GRP78) binds to the luminal domains of ER membrane sensor proteins to render them inactive [3], [4]. These membrane proteins trigger the main axes of the UPR and include: (1) protein kinase RNA-like endoplasmic reticulum kinase (PERK); (2) inositol-requiring enzyme 1 (IRE1); (3) activating transcription factor 6 (ATF6); and (4) the translocon [3], [4] via phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). During the UPR, BiP dissociates from these complexes, resulting in auto-phosphorylation of PERK and IRE1α, translocation of ATF6 to the Golgi for cleavage and the release of Ca2+ from the ER via the translocon into the cytosol [4]. These alterations result in activation of the downstream signaling pathways of each of these axes, which is described in turn below [2].
In terms of the PERK axis, activated PERK phosphorylates eukaryotic initiation factor-2α (eIF2α), which then results in the attenuation of protein translation to reduce protein overload in the ER [3]. However, despite attenuation of protein translation, certain essential mRNAs are selectively translated, including the transcription factor, ATF4, which induces the expression of C/EBP homologous protein (CHOP) that can lead to apoptosis [1].
The second axis, which is mediated by the activation of IRE1α, results in the increased splicing of X-box binding protein 1 (XBP1) and the activation of genes important for cell survival during ER stress e.g., the ER chaperone, p58IPK [3]. In addition, IRE1α also has a pro-apoptotic role and can bind to the tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) to promote phosphorylation of c-jun N-terminal kinase (JNK) that can induce apoptosis [3].
The third axis mediated by the activation of the transcription factor, ATF6, occurs after its proteolytic cleavage in the Golgi to generate cleaved ATF6, which then undergoes nuclear translocation to transcribe genes involved in apoptosis (e.g., DNA-damage-inducible transcript 3 [gene encoding CHOP]) and/or those responsible for cell survival (e.g., ER chaperones such as heat shock 70 kDa protein 5 [gene encoding BiP], calreticulin, calnexin, etc.; [1], [3]).
The fourth axis is activated by Ca2+ release from the ER into the cytosol and is mediated by the translocon [4]. The increased cytosolic Ca2+ then binds to calmodulin to activate CaMKII signaling, leading to ER stress-induced cell apoptosis through activation of caspase-12/caspase-3 and the mitochondrial apoptosis pathway [5]. To some extent, the cell’s fate is decided by the balance between survival and apoptotic signaling, and the specific ER stressor plays a crucial role in tuning these signals [1], [3].
Iron is a critical nutrient for cells, which is required for many processes, such as DNA synthesis and energy transduction [6]. Excessive iron on the other hand, can cause cellular damage due to the generation of cytotoxic reactive oxygen species (ROS; [6], [7]). Hence, cellular iron levels are intricately regulated by a variety of molecular mechanisms and its alteration may cause activation of cellular stress pathways [8]. Recently, some fragmentary evidence of a potential intersection between the alteration of intracellular iron levels and ER stress has emerged. For example: (i) iron-loaded cells have increased transcript levels of ER chaperones, namely, BiP, calreticulin 3 and calnexin [9]; (ii) iron-overloaded rats show increased BiP expression in the heart and liver [10]; and (iii) it has been demonstrated that the expression of several molecules integrally involved in ER stress are regulated by cellular iron levels, including PERK/eIF2α [11] and CHOP [12]. However, there has been no attempt to comprehensively investigate the ER stress pathway in relation to understanding the effects of cellular iron depletion. This is significant considering that iron chelation therapy for iron-loading diseases (e.g., β-thalassemia) is widely utilized [7], [13] and iron-deficiency affects more than ∼2 billion people worldwide [14]. Despite this, little is known regarding the response of cells to iron deficiency-induced stress.
Desferrioxamine (DFO; Fig. 1A) is an iron chelator used for the treatment of iron overload diseases [6], [7]. DFO does not show potent anti-tumor activity, as it is poorly membrane-permeant and its mechanism of action involves binding cellular iron, resulting in a redox-inert iron complex [6], [7]. On the other hand, novel chelators based on the di-2-pyridylketone thiosemicarbazone (DpT) scaffold, such as di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1B), induce iron sequestration and also form redox-active metal complexes that demonstrate potent and selective anti-tumor activity [6], [15], [16], [17], [18], [19], [20]. Notably, Dp44mT and its analogs possess broad anti-cancer and anti-metastatic activity in vitro and in vivo against a variety of aggressive solid tumors [6], [16], [17], [21], [22], [23]. In fact, clinical trials of one of these agents will soon commence [22], highlighting the importance of understanding the mechanism of action of these ligands and their effect on the ER stress pathway. Indeed, it has been reported that iron depletion induced by Dp44mT and its metal complexes causes apoptosis by generating cytotoxic ROS [17], [24], [25] and by inducing DNA strand breaks [26]. Importantly, the redox active complexes formed result in lysosomal damage that plays a significant role in tumor cell cytotoxicity [24], [25].
The alterations in gene expression after iron depletion are complex [27], [28], [29], [30]. Currently, it is unclear if ER stress-mediated apoptotic pathways play a crucial role in iron depletion-induced cell death. Investigating the molecular regulation of iron depletion on Ca2+ homeostasis and ER stress-mediated cell apoptosis is fundamental to the mechanistic understanding of the pharmacology of iron chelation, particularly for cancer treatment.
In the current investigation, we demonstrate for the first time that iron sequestration mediated by the chelator, Dp44mT, which forms redox-active metal complexes, robustly induces ER stress and thereby activates the UPR signaling pathways in multiple cell-types. In contrast, the effect of DFO, which only induces cellular iron depletion, is far less active in stimulating ER stress. This suggests using iron chelators that induce ER stress and apoptosis could serve as a novel approach for developing new chemotherapeutics. Collectively, these results are important for understanding the role of iron chelation and redox cycling in cell death mediated by the ER stress signaling response.
Section snippets
Reagents
Di-2-pyridylketone 2-methyl-3-thiosemicarbazone (Dp2mT; Fig. 1C) and Dp44mT were synthesized and characterized using standard procedures [15]. Briefly, equimolar equivalents of di-2-pyridylketone and either 2-methyl-3-thiosemicarbazide or 4,4-dimethyl-3-thiosemicarbazide (all purchased from Sigma–Aldrich, St. Louis, MO) were refluxed in EtOH in the presence of glacial acetic acid (5 drops) for 2 h [15]. After cooling, the precipitate was collected by vacuum filtration and washed in EtOH to
Iron chelation by DFO or Dp44mT leads to cellular iron depletion as demonstrated by TfR1 up-regulation and ferritin down-regulation
A common response to stress stimuli involves the accumulation of unfolded proteins in the ER, which leads to the UPR [36]. To date, there has been no comprehensive molecular assessment of the effect of iron depletion on inducing ER stress and this was the aim of the current investigation. Consequently, SK-N-MC neuroepithelioma cells, that have been very well characterized in terms of their response to iron chelators [31], [32], [33], [37], [38], were utilized to assess the effect of these
Discussion
Cellular adaptation to ER stress is achieved by the activation of the UPR, which is an integrated signal transduction pathway that modulates many aspects of ER physiology to maintain homeostasis [2]. If protein accumulation is sustained and ER stress stimuli does not resolve, UPR activation leads to apoptosis. The precise mechanisms regulating transition from an adaptive, pro-survival response to the induction of apoptosis are not fully understood [73]. Iron is a critically essential nutrient
Author contributions
A.M.M. and N.H.S. designed experiments, performed majority of the experiments, analyzed results and wrote the manuscript. Y.Y. performed some experiments and initiated the investigation. D.S.K. prepared figures and wrote the manuscript, while D.J.R.L, Z.K., P.J.J., S.S. and V.R. were involved in writing the manuscript. D.R.R. designed experiments, analyzed results and wrote the manuscript.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Acknowledgments
A.M.M. and Z.K. sincerely appreciate an Early Career Research Grant from the University of Sydney, a National Health and Medical Research Council of Australia (NHMRC) Peter Doherty Early Career Fellowship and Cancer Institute NSW Early Career Fellowship. N.H.S. thanks the Australian Government for an Endeavour Scholarship. D.R.R. and D.S.K. thank the NHMRC for a Senior Principal Research Fellowship and a RD Wright Career Development Fellowship, respectively, and Project Grant funding. S.S.
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