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Biochimie Nov 2021Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally... (Review)
Review
Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 (Apoptotic protease activating factor 1) protein suggesting that down-regulation of Apaf-1 is an important event contributing to the resistance of these cells to apoptosis. Nonetheless, upregulation of Apaf-1 has not emerged as a common phenomenon in pathologies associated with enhanced neuronal cell death, i.e., neurodegenerative diseases. In cancer, on the other hand, Apaf-1 downregulation is a common phenomenon, which occurs through various mechanisms including mRNA hyper-methylation, gene methylation, Apaf-1 localization in lipid rafts, inhibition by microRNAs, phosphorylation, and interaction with specific inhibitors. Due to the diversity of these mechanisms and involvement of other factors, defining the exact contribution of Apaf-1 to the development of cancer in general and neurodegenerative disorders, in particular, is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to the pathologies observed in cancer and neurodegenerative diseases with the emphasis on the therapeutic aspects of Apaf-1 as an important target in these pathologies.
Topics: Animals; Apoptosomes; Apoptotic Protease-Activating Factor 1; Humans; Neoplasms; Neurodegenerative Diseases
PubMed: 34298080
DOI: 10.1016/j.biochi.2021.07.004 -
Medical Oncology (Northwood, London,... Jun 2021Drug resistance is a multifactorial process involving a variety of mechanisms and genes. Taxane drug class like Docetaxel is not effective for all types' breast cancers...
Drug resistance is a multifactorial process involving a variety of mechanisms and genes. Taxane drug class like Docetaxel is not effective for all types' breast cancers and presents a huge clinical challenge. To improve cancer treatment outcome, it is important to distinguish which proteins can kill the cancer cells and whether the expression levels of these proteins affect treatment. Cancer cells are wildly known to be protected from apoptosis, due to low level of apoptotic protease activating factor-1 (Apaf-1) compared with normal cells. Apaf-1 is an essential protein that defines whether cytochrome c released form mitochondria remains stable or degrades. According to this hypothesis, increasing of Apaf-1 expression in MCF7 breast cancer cells was performed and Docetaxel efficacy examined. The immunoassay techniques were used to investigate Apaf-1 and cytochrome c levels, and different apoptosis assay methods applied to better understand the effect of Apaf-1 expression levels in cellular response to apoptotic stimuli by Docetaxel. Our results determined that cytoplasmic cytochrome c level elevated along with increasing Apaf-1 and MCF7 cells were sensitised to Docetaxel, suggesting that loss of Apaf-1 may cause Docetaxel-resistance in breast cancer cells through less apoptosome formation. ROS level increased in cells transfected with Apaf-1 and induced mitochondrial permeability for cytochrome c release, which subsequently promoted apoptosome formation, intrinsic apoptosis and ATP depletion.
Topics: Antineoplastic Agents; Apoptosis; Apoptotic Protease-Activating Factor 1; Bridged-Ring Compounds; Docetaxel; Dose-Response Relationship, Drug; Humans; MCF-7 Cells; Taxoids; Up-Regulation
PubMed: 34181104
DOI: 10.1007/s12032-021-01532-8 -
Biochemical Society Transactions Jun 2021Molecular chaperones are essential components of the protein quality control system and maintenance of homeostasis. Heat Shock Protein 70 (HSP70), a highly... (Review)
Review
Molecular chaperones are essential components of the protein quality control system and maintenance of homeostasis. Heat Shock Protein 70 (HSP70), a highly evolutionarily conserved family of chaperones is a key regulator of protein folding, oligomerisation and prevents the aggregation of misfolded proteins. HSP70 chaperone function depends on the so-called 'HSP70-cycle', where HSP70 interacts with and is released from substrates via ATP hydrolysis and the assistance of HSP70 co-factors/co-chaperones, which also provide substrate specificity. The identification of regulatory modules for HSP70 allows the elucidation of HSP70 specificity and target selectivity. Here, we discuss how the HSP70 cycle is functionally linked with the cycle of the Ubiquitin-like molecule NEDD8. Using as an example the DNA damage response, we present a model where HSP70 acts as a sensor of the NEDD8 cycle. The NEDD8 cycle acts as a regulatory module of HSP70 activity, where conversion of poly-NEDD8 chains into mono-NEDD8 upon DNA damage activates HSP70, facilitating the formation of the apoptosome and apoptosis execution.
Topics: Adenosine Triphosphate; Animals; DNA; DNA Damage; DNA Repair; HSP70 Heat-Shock Proteins; Humans; Hydrolysis; Molecular Chaperones; NEDD8 Protein
PubMed: 34156462
DOI: 10.1042/BST20200381 -
Cells May 2021Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer...
Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method.
Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500-3000 nm) reminiscent of apoptosomes, (ii) EVs (100-500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80-300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20-200 nm) containing unidentified vesicles/particles, and (v) EVs (10-70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producinga GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.
Topics: Cell Differentiation; Exosomes; Extracellular Vesicles; Humans; Macrophages; Mouth Neoplasms; Tumor Microenvironment; Tumor-Associated Macrophages
PubMed: 34071980
DOI: 10.3390/cells10061328 -
Methods in Molecular Biology (Clifton,... 2021Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational...
Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.
Topics: Apoptosis; Blotting, Western; Caspases; Cytochromes c; Cytoplasm; Female; Humans; Mitochondria; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured
PubMed: 34033091
DOI: 10.1007/978-1-0716-1162-3_3 -
Journal of Theoretical Biology Sep 2021Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs,...
Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs, especially in the intrinsic pathway, which can be activated by a great variety of signals. In addition, the conditions in which a point of no return could be reached remain elusive. In this work, we use differential equations models to approach these issues. Our starting point was the model for caspase activation of Legewie et al. (Legewie S, et al., PLoS Computational Biology 2006, 2(9): e120), which exhibits irreversible bistability. We added an activation module to this model, with the main events related to mitochondrial outer membrane permeabilization, which includes cytochrome C release by the mitochondria and its effects on caspase activation and respiratory chain disruption. This "Extended Legewie Model" (ELM) uses BAK as the apoptotic stimulus and active caspase 3 as a measure of apoptosis activation. Unexpectedly, in the extended model, BAK cannot trigger apoptosis activation using physiologically sound initial values of the variables, due to limitations in apoptosome concentration increase. Therefore, the next step was to find a regulatory mechanism, allowing apoptosis activation in the ELM, starting from physiological initial concentrations. For this aim, we performed a sensitivity analysis on the 61 parameters of the system, finding that those producing the most relevant changes in the qualitative behaviour were the rates of synthesis of caspase 3, caspase 9 and XIAP. Based on these results, the transcription factor E2F was included in the ELM because it directly regulates the rate of synthesis of caspase 3 and 9. Depending on the concentration of E2F, the ELM shows different qualitative behaviours. On one hand, for low E2F apoptosis is impossible and for high E2F apoptosis is inevitable. Therefore, if E2F is sufficiently increased, the point of no return is crossed. On the other hand, for intermediate values of E2F there is a bistable region where the fate of the system also depends on the concentration of BAK and other signalling species.
Topics: Apoptosis; Caspases; Cytochromes c; Mitochondria; Mitochondrial Membranes
PubMed: 34019850
DOI: 10.1016/j.jtbi.2021.110765 -
Trends in Microbiology Dec 2021The programmed cell death pathways of pyroptosis and apoptosis protect mammals from infections. The activation of host cell death signaling depends on cell surface and... (Review)
Review
The programmed cell death pathways of pyroptosis and apoptosis protect mammals from infections. The activation of host cell death signaling depends on cell surface and cytosolic receptors that bind bacterial molecules or sense their activity. The formation of cytosolic protein complexes, such as the inflammasome and apoptosome, activates caspases, pore-forming proteins, and inflammatory cytokines. These pathways respond to bacteria and their released membrane vesicles. Outer membrane vesicles (OMVs) that emerge from the outer membrane of Gram-negative bacteria deliver a range of bacterial molecules, including lipids, proteins, polysaccharides and nucleic acids to host cells. Recent findings describe how OMV-associated molecules activate pyroptosis, apoptosis, and other inflammatory pathways. We discuss here how OMV-associated molecules are sensed by the immune system and how this contributes to infections and inflammatory diseases.
Topics: Animals; Apoptosis; Bacterial Outer Membrane; Bacterial Outer Membrane Proteins; Cell Membrane; Extracellular Vesicles; Gram-Negative Bacteria; Inflammasomes; Mammals
PubMed: 34001418
DOI: 10.1016/j.tim.2021.04.003 -
Medical Oncology (Northwood, London,... Apr 2021Chemoresistance is the leading cause of limiting long-term treatment success in cancer cells. Anticancer drugs usually kill cells through apoptosis induction and defects...
Chemoresistance is the leading cause of limiting long-term treatment success in cancer cells. Anticancer drugs usually kill cells through apoptosis induction and defects in this signaling pathway lead to chemoresistance. Apoptotic protease activating factor 1 regulates cellular stress evoked by chemotherapeutic agents through facilitating apoptosome assembling but can be degraded by proteasome. This study examined the role of proteasome inhibitor Bortezomib in the cytotoxic effects of Docetaxel on MCF7 cells response and its correlation with Apaf-1 expression level. MTT assay, caspase 3/7 activity assay, propidium iodide staining, adenosine triphosphate and reactive oxygen species amount measurements were utilized to demonstrate the role of Bortezomib in Docetaxel efficacy with and without Apaf-1 overexpressing. Meanwhile, two-dimensional cell migration assay was performed by scratch wound assay. The combination of Docetaxel with Bortezomib was significantly more cytotoxic compared single drug, more effectively delayed cell growth, reduced ATP level and increased ROS production. In Apaf-1 overexpressing, Docetaxel was more efficient in preventing cell migration, however, Docetaxel plus Bortezomib were not significantly effective; and fluorescence images supported the interpretation. Our findings demonstrated MCF7 resistance to Docetaxel is due in part to low Apaf-1 level and Apaf-1 overexpression resulted in the increase of cell susceptibility to Docetaxel stimulus. We assume that proteasome inhibitor may restore apoptotic proteins like Apaf-1 and prevent the degradation of cytosolic cytochrome c released by Docetaxel, consequently triggering intrinsic apoptosis and promoting cancer cell death. Collectively, treating MCF7 breast cells with proteasome inhibitor sensitizes cells to Docetaxel-induced apoptosis and possibly overcomes chemoresistance.
Topics: Adenosine Triphosphate; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptotic Protease-Activating Factor 1; Bortezomib; Caspase 3; Caspase 7; Cell Movement; Cell Survival; DNA Fragmentation; Docetaxel; Enzyme Activation; Humans; MCF-7 Cells; Proteasome Inhibitors; Reactive Oxygen Species
PubMed: 33904968
DOI: 10.1007/s12032-021-01509-7 -
Angewandte Chemie (International Ed. in... May 2021Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short...
Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short DNA elements as modular recruitment domains. Caspase-9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase-9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.
Topics: Caspase 9; DNA; Humans; Kinetics; Models, Molecular
PubMed: 33725379
DOI: 10.1002/anie.202102160 -
Current Osteoporosis Reports Jun 2021In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers. (Review)
Review
PURPOSE OF REVIEW
In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers.
RECENT FINDINGS
Crosstalk between tumor and bone has been demonstrated to promote tumor and metastatic progression. In addition to direct cell-to-cell contact and soluble factors, such as cytokines, EVs mediate crosstalk between tumor and bone. EVs are composed of a heterogenous group of membrane-delineated vesicles of varying size range, mechanisms of formation, and content. These include apoptotic bodies, microvesicles, large oncosomes, and exosomes. EVs derived from primary tumors have been shown to alter bone remodeling and create formation of a pre-metastatic niche that favors development of bone metastasis. Similarly, EVs from marrow stromal cells have been shown to promote tumor progression. Additionally, EVs can act as therapeutic delivery vehicles due to their low immunogenicity and targeting specificity. EVs play critical roles in intercellular communication. Multiple classes of EVs exist based on size on mechanism of formation. In addition to a role in pathophysiology, EVs can be exploited as therapeutic delivery vehicles.
Topics: Bone Neoplasms; Cell Communication; Disease Progression; Extracellular Vesicles; Humans; Signal Transduction; Tumor Microenvironment
PubMed: 33638774
DOI: 10.1007/s11914-021-00668-w