Afshin Samali | National University of Ireland, Galway (original) (raw)

Papers by Afshin Samali

Biochemical and biophysical research communications, Jan 2, 2015

Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein res... more Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectivel... more The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and-resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

Journal of Cellular and Molecular Medicine, 2011

Cancer Research

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF death lig... more Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF death ligand family. It is best known for its ability to induce apoptosis selectively in cancer cells but not in untransformed cells. TRAIL is recognized by four distinct membrane-bound receptors: DR4, DR5, DcR1 and DcR2. Of the four receptors, only DR4 and DR5 contain a death domain, which is required for induction of apoptosis. DcR1 and DcR2 do not have a functional death domain and thus they are thought to act as decoy receptors. Currently the role of the decoy receptors in TRAIL-resistance of tumour cells is controversial, but their ubiquitous expression on the surface of almost all somatic cells may limit the in vivo efficiency of TRAIL. We previously described the successful design of DR5-selective TRAIL variants using a computational design strategy. We found that double mutation at positions D269H/E195R resulted in enhanced affinity for DR5, and an 8-12 fold reduced affinity for DR4. This...

Endoplasmic reticulum is a major cellular organelle consisting of a vast reticular network spanni... more Endoplasmic reticulum is a major cellular organelle consisting of a vast reticular network spanning from nuclear envelope to the plasma membrane. It plays a major role in various cellular processes including protein synthesis and glycosylation, the secretory pathway and membrane biogenesis. It is sensitive to changes in its luminal conditions. Loss of ER luminal homeostasis leads to a condition referred to as ER stress. Cells response to ER stress is to orchestrate the activation of an evolutionarily conserved trascritional program termed the unfolded protein response (UPR). A consequence of UPR is the upregulation the components of the autophagic machinery and increased autophagic flux. Over the past decade much research investigating the onset and progression of autophagy following activation of UPR has been carried out. Owing to this we now have a better understanding of the signaling pathways leading to ER stress-mediated autophagy and have begun to appreciate the importance of ...

Cancer Discovery, 2015

Stress induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is obse... more Stress induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is observed in many human diseases, including cancers. Cellular adaptation to ER stress is mediated by the unfolded protein response (UPR), which aims at restoring ER homeostasis. The UPR has emerged as a major pathway in remodeling cancer gene expression, thereby either preventing cell transformation or providing an advantage to transformed cells. UPR sensors are highly regulated by the formation of dynamic protein scaffolds, leading to integrated reprogramming of the cells. Herein, we describe the regulatory mechanisms underlying UPR signaling upon cell intrinsic or extrinsic challenges, and how they engage cell transformation programs and/or provide advantages to cancer cells, leading to enhanced aggressiveness or chemoresistance. We discuss the emerging cross-talk between the UPR and related metabolic processes to ensure maintenance of protein homeostasis and its impact on cell transformation and tumor growth. ER stress signaling is dysregulated in many forms of cancer and contributes to tumor growth as a survival factor, in addition to modulating other disease-associated processes, including cell migration, cell transformation, and angiogenesis. Evidence for targeting the ER stress signaling pathway as an anticancer strategy is compelling, and novel agents that selectively inhibit the UPR have demonstrated preliminary evidence of preclinical efficacy with an acceptable safety profile. Cancer Discov; 5(6); 586-97. ©2015 AACR.

Methods in Molecular Biology, 2015

Many experimentally induced or disease-related cellular dysfunctions stress the endoplasmic retic... more Many experimentally induced or disease-related cellular dysfunctions stress the endoplasmic reticulum, commonly resulting in an accumulation of unfolded proteins in the ER lumen which is sensed by three ER-resident transmembrane proteins, PERK, ATF6, and IRE1. Their activation by such ER stress affects the unfolded protein response, which consists of a shutoff of protein translation and at the same time the switching-on of specific transcription factors that control genes which function to reduce the burden of unfolded proteins to the ER. Here, we describe two sets of methods for monitoring the occurrence of ER stress and UPR signaling in human cells by analyzing markers of activation of all three ER stress sensor proteins. The first set of methods is based on the qualitative and quantitative analysis of UPR-induced transcripts by qPCR. The second set of methods consists of Western blot-based analysis of UPR-induced proteins or protein modifications. Their combined analysis allows assessment of activation of all three ER stress-activated signaling pathways that in combination are characteristic for the UPR.

Autophagy, Jan 2, 2014

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of th... more Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex ...

Proceedings of the National Academy of Sciences, 2006

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug th... more Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that selectively induces apoptosis in a variety of cancer cells by interacting with death receptors DR4 and DR5. TRAIL can also bind to decoy receptors (DcR1, DcR2, and osteoprotegerin receptor) that cannot induce apoptosis. The occurrence of DR5-responsive tumor cells indicates that a DR5 receptor-specific TRAIL variant will permit tumor-selective therapies. By using the automatic design algorithm FOLD-X, we successfully generated DR5-selective TRAIL variants. These variants do not induce apoptosis in DR4-responsive cell lines but show a large increase in biological activity in DR5-responsive cancer cell lines. Even wild-type TRAIL-insensitive ovarian cancer cell lines could be brought into apoptosis. In addition, our results demonstrate that there is no requirement for antibody-mediated cross-linking or membrane-bound TRAIL to induce apoptosis through DR5.

Journal of Cellular and Molecular Medicine, 2007

Digestion, 2000

... kD Protein(s) of Helicobacter pylori Stimulate DNA Synthesis in Epithelial Cell Lines without... more ... kD Protein(s) of Helicobacter pylori Stimulate DNA Synthesis in Epithelial Cell Lines without Affecting Apoptosis Vadim Neopikhanov a , Afshin Samali b ... Cahill RJ, Xia H, Kilgallen C, Beatti S, Hamilton H, O &;grave;Morain C: Effect of eradication of Helicobacter pylori infection ...

Diabetes/Metabolism Research and Reviews, 2006

Gene transfer to pancreatic islets may prove useful in preventing islet cell destruction and prol... more Gene transfer to pancreatic islets may prove useful in preventing islet cell destruction and prolonging islet graft survival after transplantation in patients with type 1 diabetes mellitus (T1DM). Potentially, a host of therapeutically relevant transgenes may be incorporated into an appropriate gene delivery vehicle and used for islet modification. An increasing understanding of the molecular pathogenesis of immune-mediated beta cell death has served to highlight molecules which have become suitable candidates for promoting islet cell survival in the face of oxidative stress. This review aims to give an overview of some conventional gene transfer strategies aimed at promoting islet cell survival in the face of cytokine onslaught. These strategies target three aspects of islet cell physiology: redox status and antioxidant defence, anti-apoptotic gene expression and mediators of cytokine signal transduction pathways.

Biochemical Journal, 2005

Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular component... more Exogenously added ROS (reactive oxygen species) cause generalized oxidation of cellular components, whereas endogenously generated ROS induced by physiological stimuli activate discrete signal transduction pathways. Compartmentation is an important aspect of such pathways, but little is known about its role in redox signalling. We measured the redox states of cytosolic and nuclear Trx1 (thioredoxin-1) and mitochondrial Trx2 (thioredoxin-2) using redox Western blot methodologies during endogenous ROS production induced by EGF (epidermal growth factor) signalling. The glutathione redox state was measured by HPLC. Results showed that only cytosolic Trx1 undergoes significant oxidation. Thus EGF signalling involves subcellular compartmental oxidation of Trx1 in the absence of a generalized cellular oxidation.