-
Bioscience Reports May 2019Human breast cancer is a malignant form of tumor with a relatively high mortality rate. Although esophageal cancer-related gene 4 (ECRG4) is thought to be a possible...
Human breast cancer is a malignant form of tumor with a relatively high mortality rate. Although esophageal cancer-related gene 4 (ECRG4) is thought to be a possible potent tumor suppressor gene that acts to suppress breast cancer, its precise role in this disease is not understood. Herein, we assess the correlation between ECRG4 expression and DNA methylation, probing the potential epigenetic regulation of ECRG4 in breast cancer. We analyzed ECRG4 promoter methylation via methylation-specific PCR (MSPCR), bisulfite sequencing, and a promoter reporter assay in human breast cancer cell lines and samples. Gene expression was assessed by quantitative real-time PCR (qPCR), while protein levels were assessed by Western blotting. CCK8 assays were used to quantify cell growth; Esophageal cancer-related gene 4 wound healing assays were used to assess cellular migration, while flow cytometry was used to assess apoptosis and cell cycle progression. Apoptosome formation was validated via CO-IP and Western blotting. We found that human breast cancer samples exhibited increased methylation of the ECRG4 promoter and decreased ECRG4 expression. Remarkably, the down-regulation of ECRG4 was highly associated with promoter methylation, and its expression could be re-activated via 5-aza-2'-deoxycytidine treatment to induce demethylation. ECRG4 overexpression impaired breast cancer cell proliferation and migration, and led to G0/G1 cell cycle phase arrest. Moreover, ECRG4 induced the formation of the Cytc/Apaf-1/caspase-9 apoptosome and promoted breast cancer cell apoptosis. ECRG4 is silenced in human breast cancer cells and cell lines, likely owing to promoter hypermethylation. ECRG4 may act as a tumor suppressor, inhibiting proliferation and migration, inducing G0/G1 phase arrest and apoptosis via the mitochondrial apoptotic pathway.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Base Sequence; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Methylation; Decitabine; Epigenesis, Genetic; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; Tumor Suppressor Proteins
PubMed: 30918105
DOI: 10.1042/BSR20190087 -
Cancer Research Apr 2019Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with...
Although African-American (AA) patients with prostate cancer tend to develop greater therapeutic resistance and faster prostate cancer recurrence compared with Caucasian-American (CA) men, the molecular mechanisms of this racial prostate cancer disparity remain undefined. In this study, we provide the first comprehensive evidence that cytochrome deficiency in AA primary tumors and cancer cells abrogates apoptosome-mediated caspase activation and contributes to mitochondrial dysfunction, thereby promoting therapeutic resistance and prostate cancer aggressiveness in AA men. In AA prostate cancer cells, decreased nuclear accumulation of nuclear respiration factor 1 (Nrf1) and its subsequent loss of binding to the cytochrome promoter mediated cytochrome deficiency. The activation of cellular Myc (c-Myc) and NF-κB or inhibition of AKT prevented nuclear translocation of Nrf1. Genetic and pharmacologic inhibition of c-Myc and NF-κB or activation of AKT promoted Nrf1 binding to cytochrome promoter, cytochrome expression, caspase activation, and cell death. The lack of p-Drp1 in AA prostate cancer cells contributed to defective cytochrome release and increased resistance to apoptosis, indicating that restoration of cytochrome alone may be insufficient to induce effective apoptosis. Cytochrome deficiency promoted the acquisition of glycolytic phenotypes and mitochondrial dysfunction, whereas cytochrome restoration via inhibition of c-Myc and NF-κB or activation of AKT attenuated glycolysis in AA prostate cancer cells. Inhibition of c-Myc and NF-κB enhanced the efficacy of docetaxel in tumor xenografts. Therefore, restoring cytochrome may overcome therapeutic resistance and prostate cancer aggressiveness in AA men. Overall, this study provides the first comprehensive experimental, mechanistic, and clinical evidence for apoptosome and mitochondrial dysfunction in prostate cancer racial disparity. SIGNIFICANCE: Mechanistic insights on prostate cancer health disparity among American men provide novel approaches to restore mitochondrial function, which can address therapeutic resistance and aggressiveness in African-American men with prostate cancer.
Topics: Black or African American; Animals; Apoptosomes; Cell Line, Tumor; Cytochromes c; Humans; Male; Mice; Mice, SCID; Mitochondria; Mitochondrial Membranes; NF-kappa B; Nuclear Respiratory Factor 1; Oxidative Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc
PubMed: 30765600
DOI: 10.1158/0008-5472.CAN-18-2383 -
Expert Review of Precision Medicine and... 2018Despite decades of focused research efforts, cancer remains a significant cause of morbidity and mortality. Tumor necrosis factor(TNF)-related apoptosis-inducing ligand...
INTRODUCTION
Despite decades of focused research efforts, cancer remains a significant cause of morbidity and mortality. Tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) is capable of inducing cell death selectively in cancer cells while sparing normal cells.
AREAS COVERED
In this review, the authors cover TRA therapy and strategies that have been undertaken to improve their efficacy, as well as unconventional approaches to TRAIL pathway activation including TRAIL-inducing small molecules. They also discuss mechanisms of resistance to TRAIL and the use of combination strategies to overcome it.
EXPERT COMMENTARY
Targeting the TRAIL pathway has been of interest in oncology, and although initial clinical trials of TRAIL receptor agonists (TRAs) showed limitations, novel approaches represent the future of TRAIL-based therapy.
PubMed: 30740527
DOI: 10.1080/23808993.2018.1476062 -
Gene 2019Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder, characterized by the overproduction of myeloid cells in all stages of maturation. It is usually defined...
INTRODUCTION
Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder, characterized by the overproduction of myeloid cells in all stages of maturation. It is usually defined by three sequential stages (Chronic, Accelerated and Blast-crisis) where the transition from chronic to accelerated to blast phases is presumed to occur due to secondary genetic changes, viz. accumulation of mutations, activation of downstream pathways and failure of apoptosis. Caspase 9 is the initiator caspase involved in mitochondrial-mediated apoptotic pathway. Polymorphisms in the promoter (-1263 A>G, -712C>T, -293 del) and coding (Ex5 +32G>A) regions of CASP9 gene are found to influence the expression levels by either impairing the activation or loss of expression of CASP9 or insufficient formation of apoptosome.
METHODS
The present case-control study was carried out on 999 individuals, comprised of 485CML cases reported at Nizams Institute of Medical Sciences (NIMS), Hyderabad and 514 age and gender-matched healthy individuals from local population. DNA was isolated by non-enzymatic/salting-out method and was genotyped using RFLP technique.
RESULTS
It was observed that the presence of G allele of CASP9 -1263A>G polymorphism enhanced the risk for CML while CASP9 -712C>T and CASP9 -293del SNPs conferred protection against development of CML. Haplotype analysis of promoter and exonic polymorphisms had revealed increased risk associated with two haplotypes G_C_del (+)_G (OR-1.61, 95% CI-0.97-2.65, p-0.06#) and G_C_del (-)_G (OR-2.09, 95% CI-0.94-4.66, p-0.07#) suggesting the role of G allele of CASP9 -1263A>G in conferring risk independent of other SNPs. Pairwise LD analysis performed for all the four SNPs revealed the presence of LD among the SNPs.
CONCLUSION
The results of the present study therefore concludes the role of CASP9 -1263A>G polymorphism in increasing the risk for the development and progression while CASP9 -712C>T and CASP9 -293del SNPs conferred protection and CASP9 Ex5 +32G>A was involved in conferring resistance which could be in combination with other SNPs or factors affecting them.
PubMed: 34530993
DOI: 10.1016/j.gene.2018.100002 -
Hypoxia increases neutrophil-driven matrix destruction after exposure to Mycobacterium tuberculosis.Scientific Reports Jul 2018The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how...
The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how neutrophils in hypoxia influence lung tissue damage is unknown. We investigated the effect of hypoxia on neutrophil-derived enzymes and tissue destruction in TB. Human neutrophils were stimulated with M. tuberculosis (M.tb) or conditioned media from M.tb-infected monocytes (CoMTB). Neutrophil matrix metalloproteinase-8/-9 and elastase secretion were analysed by luminex array and gelatin zymography, gene expression by qPCR and cell viability by flow cytometry. Matrix destruction was investigated by confocal microscopy and functional assays and neutrophil extracellular traps (NETs) by fluorescence assay. In hypoxia, neutrophil MMP-8 secretion and gene expression were up-regulated by CoMTB. MMP-9 activity and neutrophil elastase (NE) secretion were also increased in hypoxia. Hypoxia inhibited NET formation and both neutrophil apoptosis and necrosis after direct stimulation by M.tb. Hypoxia increased TB-dependent neutrophil-mediated matrix destruction of Type I collagen, gelatin and elastin, the main structural proteins of the human lung. Dimethyloxalylglycin (DMOG), which stabilizes hypoxia-inducible factor-1α, increased neutrophil MMP-8 and -9 secretion. Hypoxia in our cellular model of TB up-regulated pathways that increase neutrophil secretion of MMPs that are implicated in matrix destruction.
Topics: Apoptosomes; Cell Line; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lung; Matrix Metalloproteinase 8; Matrix Metalloproteinase 9; Monocytes; Mycobacterium tuberculosis; Neutrophils; Pancreatic Elastase; Signal Transduction; Tuberculosis; Up-Regulation
PubMed: 30065292
DOI: 10.1038/s41598-018-29659-1 -
Molecular Neurobiology Mar 2019Hypothermia is currently the only approved therapy for global cerebral ischemia (GCI) after cardiac arrest; however, it unfortunately has multiple adverse effects. As a...
Hypothermia is currently the only approved therapy for global cerebral ischemia (GCI) after cardiac arrest; however, it unfortunately has multiple adverse effects. As a noninvasive procedure, photobiomodulation (PBM) therapy has emerged as a potential novel treatment for brain injury. PBM involves the use of low-level laser light therapy to influence cell behavior. In this study, we evaluated the therapeutic effects of PBM treatment with an 808-nm diode laser initiated 6 h after GCI. It was noted that PBM dose-dependently protected against GCI-induced neuronal death in the vulnerable hippocampal CA1 subregion. Functional assessments demonstrated that PBM markedly preserved both short-term (a week) and long-term (6 months) spatial learning and memory function following GCI. Further mechanistic studies revealed that PBM post-treatment (a) preserved healthy mitochondrial dynamics and suppressed substantial mitochondrial fragmentation of CA1 neurons, by reducing the detrimental Drp1 GTPase activity and its interactions with adaptor proteins Mff and Fis1 and by balancing mitochondrial targeting fission and fusion protein levels; (b) reduced mitochondrial oxidative damage and excessive mitophagy and restored mitochondrial overall health status and preserved mitochondrial function; and (c) suppressed mitochondria-dependent apoptosome formation/caspase-3/9 apoptosis-processing activities. Additionally, we validated, in an in vitro ischemia model, that cytochrome c oxidase served as a key PBM target for mitochondrial function preservation and neuroprotection. Our findings suggest that PBM serves as a promising therapeutic strategy for the functional recovery after GCI, with mechanisms involving PBM's preservation on mitochondrial dynamics and functions and the inhibition of delayed apoptotic neuronal death in GCI.
Topics: Animals; Brain Ischemia; Cell Death; Hippocampus; Low-Level Light Therapy; Male; Maze Learning; Mitochondria; Mitochondrial Dynamics; Neurons; Rats; Rats, Sprague-Dawley
PubMed: 29951942
DOI: 10.1007/s12035-018-1191-9 -
Oncotarget Apr 2018Triptolide (TL) is a potent anti-tumor, anti-inflammatory and immunosuppressive natural compound. Mechanistic studies revealed that TL inhibits tumor growth and triggers...
Triptolide (TL) is a potent anti-tumor, anti-inflammatory and immunosuppressive natural compound. Mechanistic studies revealed that TL inhibits tumor growth and triggers programmed cell death. Studies further suggested that TL inhibits heat shock response in cancer cells to induce apoptosis. HSP90β is the major component of heat shock response and is overexpressed in different types of cancers. Given almost all identified HSP90β inhibitors are either N or C-terminal inhibitors, small molecules attacking cysteine(s) in the middle domain might represent a new class of inhibitors. In the current study, we showed that TL inhibits HSP90β in triple manner. Characterization suggests that TL inhibits ATPase activity by preventing ATP binding thus blunts the chaperone activity. TL disrupts HSP90β-CDC37 (co-chaperone) complex through middle domain Cys366 of HSP90β and causes kinase client protein degradation. At the cellular level, the TL-mediated decrease in CDK4 protein levels in HeLa cells causes reduced phosphorylation of Rb resulting in cell cycle arrest at the G1 phase. Furthermore, our results demonstrated that TL triggers programmed cell death in an HSP90β-dependent manner as knockdown of HSP90β further sensitized TL-mediated cell cycle arrest and apoptotic effect. Surprisingly, our data showed that TL is the first drug to be reported to induce site-specific phosphorylation of HSP90β to drive apoptosome formation in the early phase of the treatment. In summary, our study established that TL is a novel middle domain HSP90β inhibitor with bi-phasic multi-mechanistic inhibition. The unique regulatory mechanism of TL on HSP90β makes it an effective inhibitor.
PubMed: 29854279
DOI: 10.18632/oncotarget.24737 -
Cell Death and Differentiation Jul 2018The apoptosome is a platform that activates apical procaspases in response to intrinsic cell death signals. Biochemical and structural studies in the past two decades... (Review)
Review
The apoptosome is a platform that activates apical procaspases in response to intrinsic cell death signals. Biochemical and structural studies in the past two decades have extended our understanding of apoptosome composition and structure, while illuminating the requirements for initiator procaspase activation. A number of studies have now provided high-resolution structures for apoptosomes from C. elegans (CED-4), D. melanogaster (Dark), and H. sapiens (Apaf-1), which define critical protein interfaces, including intra and interdomain interactions. This work also reveals interactions of apoptosomes with their respective initiator caspases, CED-3, Dronc and procaspase-9. Structures of the human apoptosome have defined the requirements for cytochrome c binding, which triggers the conversion of inactive Apaf-1 molecules to an extended, assembly competent state. While recent data have provided a detailed understanding of apoptosome formation and procaspase activation, they also highlight important evolutionary differences with functional implications for caspase activation. Comparison of the CARD/CARD disks and apoptosomes formed by CED-4, Dark and Apaf-1. Cartoons of the active states of the CARD-CARD disks, illustrating the two CED-4 CARD tetrameric ring layers (CED4a and CED4b; top row) and the binding of 8 Dronc CARDs and between 3-4 pc-9 CARDs, to the Dark and Apaf-1 CARD disk respectively (middle and lower rows). Ribbon diagrams of the active CED-4, Dark and Apaf-1 apoptosomes are shown (right column).
Topics: Animals; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Caspase 9; Drosophila Proteins; Drosophila melanogaster; Humans
PubMed: 29765111
DOI: 10.1038/s41418-017-0025-z -
PloS One 2018Apaf1 is a critical component of the apoptosome and initiates apoptosis downstream mitochondrial damages. Although the importance of Apaf1 in embryonic development was...
Apaf1 is a critical component of the apoptosome and initiates apoptosis downstream mitochondrial damages. Although the importance of Apaf1 in embryonic development was shown, the role of Apaf1 in immune responses, especially T cell responses, has yet to be elucidated. We generated T cell-specific Apaf1-deficient mice (Lck-Cre-Apaf1f/f mice) and examined the antigen-specific delayed-type hypersensitivity (DTH). Lck-Cre-Apaf1f/f mice exhibited exacerbation of DTH responses as compared with Apaf1-sufficient control mice. In Lck-Cre-Apaf1f/f mice, antigen-specific T cells proliferated more, and produced more inflammatory cytokines than control T cells. Apaf1-deficient T cells from antigen-immunized mice showed higher percentages of activation phenotypes upon restimulation in vitro. Apaf1-deficient T cells from naive (non-immunized) mice also showed higher proliferation activity and cytokine production over control cells. The impact of Apaf1-deficiency in T cells, however, was not restored by a pan-caspase inhibitor, suggesting that the role of Apaf1 in T cell responses was caspase-independent/non-apoptotic. These data collectively demonstrated that Apaf1 is a negative regulator of T cell responses and implicated Apaf1 as a potential target for immunosuppressive drug discovery.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Cells, Cultured; Hypersensitivity, Delayed; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; T-Lymphocytes
PubMed: 29596528
DOI: 10.1371/journal.pone.0195119 -
Cell Death & Disease Mar 2018Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure...
Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure in the activation of the apoptotic intrinsic pathway, thereby activating the caspase cascade through its interaction with Apaf-1. Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.
Topics: 14-3-3 Proteins; Amino Acid Motifs; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Cytochromes c; Cytosol; Enzyme Activation; Humans; Protein Binding
PubMed: 29511177
DOI: 10.1038/s41419-018-0408-1