The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation ...of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress‐induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.
The death ligand tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine superfamily, has long been recognized for its potential as a cancer therapeutic ...due to its low toxicity against normal cells. However, its translation into a therapeutic molecule has not been successful to date, due to its short in vivo half-life associated with insufficient tumor accumulation and resistance of tumor cells to TRAIL-induced killing. Nanotechnology has the capacity to offer solutions to these limitations. This review provides a perspective and a critical assessment of the most promising approaches to realize TRAIL’s potential as an anticancer therapeutic, including the development of fusion constructs, encapsulation, nanoparticle functionalization and tumor-targeting, and discusses the current challenges and future perspectives.
There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. ...CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represents a barrier to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently healthy donor expanded NK cells. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion achieving a mean knockdown efficiency of 84%. The resulting CD38 KD expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pre-treatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression offering a rational combination therapy. These findings support the further investigation of CD38 KD - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.
Bcl-2 family on guard at the ER Szegezdi, Eva; MacDonald, David C; Ni Chonghaile, Triona ...
American Journal of Physiology: Cell Physiology,
05/2009, Letnik:
296, Številka:
5
Journal Article
Recenzirano
Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
Submitted 21 November 2008
; accepted in final form 4 March 2009
...ABSTRACT
The endoplasmic reticulum (ER) is the main site for protein folding, lipid biosynthesis, and calcium storage in the cell. Disturbances of these critical cellular functions lead to ER stress. The ER responds to disturbances in its homeostasis by launching an adaptive signal transduction pathway, known as the unfolded protein response (UPR). The UPR strives to maintain ER function during stress; however, if the stress is not resolved, apoptotic responses are activated that involve cross talk between the ER and mitochondria. In addition, ER stress is also known to induce autophagy to counteract XBP-1-mediated ER expansion and assist in the degradation of unfolded proteins. One family of proteins involved in the regulation of apoptosis is that of B-cell lymphoma protein 2 (Bcl-2). Complex interactions among the three subgroups within the Bcl-2 family the antiapoptotic, the multidomain proapoptotic, and the Bcl-2 homology domain 3 (BH3)-only members control the signaling events of apoptosis upstream of mitochondrial outer membrane permeabilization. These proteins were found to have diverse subcellular locations to aid in the response to varied intrinsic and extrinsic stimuli. Of recent interest is the presence of the Bcl-2 family at the ER. Here, we review the involvement of proteins from each of the three Bcl-2 family subgroups in the maintenance of ER homeostasis and their participation in ER stress signal transduction pathways.
endoplasmic reticulum stress; unfolded protein response; inositol-requiring enzyme 1; apoptosis
Address for reprint requests and other correspondence: A. Samali, Cell Stress and Apoptosis Research Group, Dept. of Biochemistry, National Univ. of Ireland Galway, Galway, Ireland (e-mail: afshin.samali{at}nuigalway.ie )
APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays ...also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.
An unfractionated fucoidan was extracted from the brown alga Ascophyllum nodosum. Extraction of fucoidan from seaweed was carried out using an innovative low-chemical process. A combinational ...approach involving compositional analysis, HPAEC, IR analysis, GPC, and NMR was employed to elucidate the composition and structure of an unfractionated fucoidan from A. nodosum. This fucoidan is composed mainly of fucose (52.1%), and also galactose (6.1%), glucose (21.3%), and xylose (16.5%). Sulfate content was determined to be 19%. GPC data indicated a polydisperse fucoidan containing two main size fractions (47 and 420 kDa). NMR analyses revealed a fucoidan displaying broad, complex signals as expected for such a high molecular weight and heterogeneous polymer with resonances consistent with a fucoidan isolated previously from A. nodosum. The effects of fucoidan on the apoptosis of human colon carcinoma cells and fucoidan-mediated signaling pathways were also investigated. Fucoidan decreased cell viability and induced apoptosis of HCT116 colon carcinoma cells. Fucoidan treatment of HCT116 cells induced activation of caspases-9 and -3 and the cleavage of PARP, led to apoptotic morphological changes, and altered mitochondrial membrane permeability. These results detail the structure and biological activity of an unfractionated fucoidan from A. nodosum.
Plasma membrane (PM) and mitochondrial (mt) ion channels - particularly potassium channels - became oncological targets soon after the discovery that they are involved both in the regulation of ...proliferation and apoptosis. Some members of the Kv Shaker family, namely Kv1.1, Kv1.3, Kv1.5 and Kv11.1 (Herg), and the intermediate-conductance calcium-activated potassium KCa3.1 (IK) channels have been shown to contribute to apoptosis in various cell lines. Kv1.3, Kv1.5 and IK are located in the plasma membrane but also in the mitochondrial inner membrane, where they participate in apoptotic signalling. Interestingly, an altered protein expression of some of the channels mentioned above has been reported in neoplastic cell lines/tissues, but a systematic quantification addressing the protein expression of the above potassium channels in tumor cell lines of different origin has not been carried out yet. In the present study we investigated whether expression of specific potassium channels, at the mRNA and protein level, can be correlated with cell sensitivity to various apoptotic stimuli, including chemotherapeutic drugs, in a panel of cancer cell lines. The results show correlation between the protein expression of the Kv1.1 and Kv1.3 channels and susceptibility to death upon treatment with staurosporine, C2-ceramide and cisplatin. Furthermore, we investigated the correlation between Kv channel expression and sensitivity to three distinct membrane-permeant Kv1.3 inhibitors, since these drugs have recently been shown to be able to induce apoptosis and also reduce tumor volume in an in vivo model. Higher protein expression of Kv1.3 significantly correlated with lower cell survival upon treatment with clofazimine, one of the Kv1.3 inhibitors. These results suggest that expression of Kv1.1 and Kv1.3 sensitizes tumour cells of various origins to cytotoxins. Data reported in this work regarding potassium channel protein expression in different cancer cell lines may be exploited for pharmacological manipulation aiming to affect proliferation/apoptosis of cancer cells.
The application of mesenchymal stem cells (MSCs) for myocardial repair following ischemic injury is of strong interest, but current knowledge regarding the survival and retention of differentiation ...potency of stem cells under ischemic conditions is limited. The present study investigated the effects of ischemia and its components (hypoxia and glucose depletion) on MSC viability and multipotency. We demonstrate that MSCs have a profoundly greater capacity to survive under conditions of ischemia compared with cardiomyocytes, measured by detecting changes in cellular morphology, caspase activity and phosphatidylserine exposure. MSCs were also resistant to exposure to hypoxia (0.5% O2), as well as inhibition of mitochondrial respiration with 2,4‐dinitrophenol for 72 hours, indicating that in the absence of oxygen, MSCs can survive using anaerobic ATP production. Glucose deprivation (glucose‐free medium in combination with 2‐deoxyglucose) induced rapid death of MSCs. Depletion of cellular ATP occurred at a lower rate during glucose deprivation than during ischemia, suggesting that glycolysis has specific prosurvival functions, independent of energy production in MSCs. After exposure to hypoxic or ischemic conditions, MSCs retained the ability to differentiate into chondrocytes and adipocytes and, more importantly, retained cardiomyogenic potency. These results suggest that MSCs are characterized by metabolic flexibility, which enables them to survive under conditions of ischemic stress and retain their multipotent phenotype. These results highlight the potential utility of MSCs in the treatment of ischemic disease.
Disclosure of potential conflicts of interest is found at the end of this article.
Background
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 ...or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known.
Methods
Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes
in vitro
. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients.
Results
TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells
in vitro
. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content.
Conclusions
TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.
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