ABSTRACT
Importance: Hidradenitis suppurativa is a painful immune-mediated disorder with limited treatment options; hence, a need exists for new treatments. Objective: To evaluate the feasibility of heat shock protein 90 inhibition by RGRN-305 as a novel mechanism of action in treating moderate to severe hidradenitis suppurativa. Design, Setting, and Participants: This was a parallel-design, double-blind, proof-of-concept, placebo-controlled randomized clinical trial conducted between September 22, 2021, and August 29, 2022, at the Department of Dermatology, Aarhus University Hospital in Denmark. The study included a 1- to 30-day screening period, a 16-week treatment period, and a 4-week follow-up period. Eligibility criteria included age 18 years or older and moderate to severe hidradenitis suppurativa with 6 or more inflammatory nodules or abscesses in at least 2 distinct anatomic regions. Of 19 patients screened, 15 patients were enrolled in the study. Intention-to-treat analysis was performed. Interventions: Patients were randomly assigned (2:1) to receive oral RGRN-305, 250-mg tablet, or matching placebo once daily for 16 weeks. Main Outcomes and Measures: The primary efficacy end point was the percentage of patients achieving Hidradenitis Suppurativa Clinical Response 50 (HiSCR-50) at week 16. Secondary efficacy end points included HiSCR-75 or HiSCR-90, Hidradenitis Suppurativa Physician's Global Assessment, Dermatology Life Quality Index scores, and a pain numeric rating scale. Safety was assessed by adverse events, physical examinations, clinical laboratory measurements, and electrocardiograms. Results: A total of 15 patients were enrolled, completed the study, and were included in all analyses (10 [67%] female; median age, 29 [IQR, 23-41] years). The primary end point HiSCR-50 at week 16 was achieved by a higher percentage in the RGRN-305 group (60% [6 of 10]) than in the placebo group (20% [1 of 5]). Improvements were also observed across all secondary end points at week 16, including higher rates of the harder-to-reach HiSCR levels; 50% (5 of 10) achieved HiSCR-75 and 30% (3 of 10) achieved HiSCR-90, whereas none of the placebo-treated patients achieved HiSCR-75 or HiSCR-90. RGRN-305 was well tolerated, with no deaths or serious adverse events, and treatment-emergent adverse events were similarly frequent between the RGRN-305 and placebo groups. Conclusions and Relevance: The findings of this trial suggest that heat shock protein 90 inhibition by RGRN-305 offers a novel mechanism of action in treating hidradenitis suppurativa, warranting further evaluation in larger trials. Trial Registration: ClinicalTrials.gov Identifier: NCT05286567.
Subject(s)
Heat-Shock Proteins , Hidradenitis Suppurativa , Adult , Female , Humans , Male , Double-Blind Method , Heat-Shock Proteins/adverse effects , Heat-Shock Proteins/agonists , Hidradenitis Suppurativa/drug therapy , Severity of Illness Index , Treatment Outcome , Young AdultABSTRACT
Stress proteins (SPs) including heat-shock proteins (HSPs), RNA chaperones, and ER associated stress proteins are molecular chaperones essential for cellular homeostasis. The major functions of HSPs include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. Regarded as a double-edged sword, HSPs also cooperate with numerous viruses and cancer cells to promote their survival. RNA chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnRNPs), which are essential factors for manipulating both the functions and metabolisms of pre-mRNAs/hnRNAs transcribed by RNA polymerase II. hnRNPs involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, RNP assembly and stabilization, RNA export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. Dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., Parkinson's diseases, Alzheimer disease), stroke and infectious diseases. In this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. As SPs also attract a great interest as potential antiviral targets (e.g., COVID-19), we also discuss the present progress and challenges in this area of HSP-based drug development, as well as with compounds already under clinical evaluation.
Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Heat-Shock Proteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Host-Pathogen Interactions/drug effects , Pneumonia, Viral/drug therapy , Antiviral Agents/chemical synthesis , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 , Chromatin Assembly and Disassembly/drug effects , Coronavirus Infections/genetics , Coronavirus Infections/pathology , Coronavirus Infections/virology , Gene Expression Regulation , Heat-Shock Proteins/agonists , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/agonists , Heterogeneous-Nuclear Ribonucleoproteins/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Host-Pathogen Interactions/genetics , Humans , Molecular Targeted Therapy/methods , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , SARS-CoV-2 , Severity of Illness Index , Signal Transduction , Transcription, Genetic/drug effects , Virus Replication/drug effectsABSTRACT
Heat shock proteins (Hsp) are among highly conserved proteins across all domains of life. Though originally discovered as a cellular response to stress, these proteins are also involved in a wide range of cellular functions such as protein refolding, protein trafficking and cellular signalling. A large number of potential Hsp modulators are under clinical trials against various human diseases. As the number of modulators targeting Hsps is growing, there is a need to develop a comprehensive knowledge repository of these findings which is largely scattered. We have thus developed a web-accessible database, HSPMdb, which is a first of its kind manually curated repository of experimentally validated Hsp modulators (activators and inhibitors). The data was collected from 176 research articles and current version of HSPMdb holds 10 223 entries of compounds that are known to modulate activities of five major Hsps (Hsp100, Hsp90, Hsp70, Hsp60 and Hsp40) originated from 15 different organisms (i.e. human, yeast, bacteria, virus, mouse, rat, bovine, porcine, canine, chicken, Trypanosoma brucei and Plasmodium falciparum). HSPMdb provides comprehensive information on biological activities as well as the chemical properties of Hsp modulators. The biological activities of modulators are presented as enzymatic activity and cellular activity. Under the enzymatic activity field, parameters such as IC50, EC50, DC50, Ki and KD have been provided. In the cellular activity field, complete information on cellular activities (percentage cell growth inhibition, EC50 and GI50), type of cell viability assays and cell line used has been provided. One of the important features of HSPMdb is that it allows users to screen whether or not their compound of interest has any similarity with the previously known Hsp modulators. We anticipate that HSPMdb would become a valuable resource for the broader scientific community working in the area of chaperone biology and protein misfolding diseases. HSPMdb is freely accessible at http://bioinfo.imtech.res.in/bvs/hspmdb/index.php.
Subject(s)
Enzyme Activators , Enzyme Inhibitors , Heat-Shock Proteins , Animals , Drug Discovery , Heat-Shock Proteins/agonists , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/metabolism , HumansABSTRACT
Kaempferol is a flavonoid compound that has many functions, such as anti-inflammation and antioxidation. Acute liver failure (ALF) is a life-threatening illness accompanied by serious inflammation and extensive hepatocyte apoptosis. The aim of this study was to examine the therapeutic potential of kaempferol and its mechanism in ALF. In a murine ALF model induced by d-galactosamine (d-GalN, 700 mg/kg) / lipopolysaccharide (LPS, 10 µg/kg), mice were pretreated with kaempferol at 2 h before d-GalN/LPS administration and then sacrificed 6 h after d-GalN/LPS injection. Lethality, liver damage, endoplasmic reticulum(ER) stress, hepatocyte viability and apoptosis were evaluated. Whether pretreatment of kaempferol protected hepatocytes from ER stress-induced apoptosis was detected in vitro. Pretreatment of kaempferol decreased lethality, prolonged the survival time and significantly protected against liver injury, which was indicated by decreased transaminase levels and the well-preserved liver structure. The protective effect of kaempferol on the ALF mouse model was achieved by inhibiting hepatocyte apoptosis. Moreover, pretreatment of kaempferol increased the expression of glucose-regulated/binding immunoglobulin protein 78 (Grp78), decreased the expression of C/EBP-homologous protein (CHOP), and protected hepatocytes from ER stress-induced apoptosis in vitro. Our results showed that pretreatment of Grp78 siRNA partially negated the hepatic protection from kaempferol and reversed the inhibition of CHOP protein expression in d-GalN/LPS-induced ALF mice. In conclusion, kaempferol inhibits hepatocyte apoptosis to protect mice from liver failure by regulating the ER stress-Grp78-CHOP signaling pathway. Therefore, kaempferol may be used to treat ALF.
Subject(s)
Endoplasmic Reticulum Stress/drug effects , Galactosamine/toxicity , Heat-Shock Proteins/biosynthesis , Kaempferols/therapeutic use , Liver Failure, Acute/metabolism , Transcription Factor CHOP/biosynthesis , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/physiology , Heat-Shock Proteins/agonists , Hepatocytes/drug effects , Hepatocytes/metabolism , Kaempferols/pharmacology , Lipopolysaccharides/toxicity , Liver Failure, Acute/chemically induced , Liver Failure, Acute/prevention & control , Male , Mice , Mice, Inbred C57BL , Signal Transduction/drug effects , Signal Transduction/physiology , Transcription Factor CHOP/antagonists & inhibitorsABSTRACT
Spinocerebellar ataxia type 17 (SCA17) is caused by the expansion of translated CAG repeat in the TATA box binding protein (TBP) gene encoding a long polyglutamine (polyQ) tract in the TBP protein, which leads to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On cells with inducible SCA17 TBP/Q79-GFP expression to test five in-house NC009 indole compounds for neuroprotection. We found that both aggregation and polyQ-induced reactive oxygen species can be significantly prohibited by the tested NC009 compounds in Tet-On TBP/Q79 293 cells. Among the five indole compounds, NC009-1 up-regulated expression of heat shock protein family B (small) member 1 (HSPB1) chaperone to reduce polyQ aggregation and promote neurite outgrowth in neuronal differentiated TBP/Q79 SH-SY5Y cells. The increased HSPB1 thus ameliorated the increased BH3 interacting domain death agonist (BID), cytochrome c (CYCS) release, and caspase 3 (CASP3) activation which result in apoptosis. Knock down of HSPB1 attenuated the effects of NC009-1 on TBP/Q79 SH-SY5Y cells, suggesting that HSPB1 might be one of the major pathways involved for NC009-1 effects. NC009-1 further reduced polyQ aggregation in Purkinje cells and ameliorated behavioral deficits in SCA17 TBP/Q109 transgenic mice. Our results suggest that NC009-1â¯has a neuroprotective effect on SCA17 cell and mouse models to support its therapeutic potential in SCA17 treatment.
Subject(s)
Heat-Shock Proteins/metabolism , Indoles/therapeutic use , Mental Disorders/drug therapy , Mental Disorders/metabolism , Neoplasm Proteins/metabolism , Neuronal Outgrowth/drug effects , TATA-Box Binding Protein/metabolism , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Heat-Shock Proteins/agonists , Humans , Indoles/chemistry , Indoles/pharmacology , Mice , Mice, Transgenic , Molecular Chaperones , Neoplasm Proteins/agonists , Neuronal Outgrowth/physiology , TATA-Box Binding Protein/geneticsABSTRACT
SCOPE: We investigated the role of endoplasmic reticulum (ER) stress in the protective effects of EGCG against the neuronal apoptosis in Aß1-42 -induced SH-SY5Y cells and APP/PS1 transgenic mice. METHODS AND RESULTS: Cell viability (CCK8 assay), flow cytometry, Hoechst 33258 staining, immunohistochemistry, transmission electron microscopy (TEM), and western blotting were used. EGCG prevented Aß1-42-induced toxicity in SH-SY5Y cells, increased cell viability, and decreased apoptosis in a dose-dependent manner. In a subsequent mechanism study, it was found that this effect contributed to the down-regulation of GRP78, CHOP, cleaved-caspase-12 and -3. Moreover, EGCG also reduced the cytotoxicity induced by tunicamycin (TM) and thapsigargin (TG), two ER stress activators. Consistent with the in vitro study, EGCG inhibited neuronal apoptosis in the cortex of APP/PS1 transgenic mice, with the mitigation of ER abnormal ultrastructural swelling and the downregulation of ER-stress-associated proteins. CONCLUSION: These results indicate that EGCG attenuates the neurotoxicity in Alzheimer's disease (AD) via a novel mechanism that involves inhibition of ER-stress-associated neuronal apoptosis in vitro and in vivo, suggesting the tremendous potential of EGCG for use in a nutritional preventive strategy against AD.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Apoptosis , Catechin/analogs & derivatives , Dietary Supplements , Endoplasmic Reticulum Stress , Neurons/metabolism , Neuroprotective Agents/metabolism , Peptide Fragments/antagonists & inhibitors , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/prevention & control , Amyloid beta-Peptides/metabolism , Animals , Caspase 12/chemistry , Caspase 12/genetics , Caspase 12/metabolism , Caspase 3/chemistry , Caspase 3/genetics , Caspase 3/metabolism , Catechin/metabolism , Catechin/therapeutic use , Cell Line, Tumor , Cell Survival , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Cerebral Cortex/ultrastructure , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/agonists , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Mice, Transgenic , Microscopy, Electron, Transmission , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/pathology , Neurons/ultrastructure , Neuroprotective Agents/therapeutic use , Nootropic Agents/metabolism , Nootropic Agents/therapeutic use , Peptide Fragments/metabolism , Random Allocation , Transcription Factor CHOP/agonists , Transcription Factor CHOP/antagonists & inhibitors , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolismABSTRACT
Melatonin is a pleiotropic signal molecule that plays critical roles in regulating plant growth and development, as well as providing physiological protections against various environmental stresses. Nonetheless, the mechanisms for melatonin-mediated pollen thermotolerance remain largely unknown. In this study, we report that irrigation treatment with melatonin (20 µM) effectively ameliorated high temperature-induced inactivation of pollen and inhibition of pollen germination in tomato (Solanum lycopersicum) plants. Melatonin alleviated reactive oxygen species production in tomato anthers under high temperature by the up-regulation of the transcription and activities of several antioxidant enzymes. Transmission electron micrograph results showed that high temperature-induced pollen abortion is associated with a premature degeneration of the tapetum cells and the formation of defective pollen grains with degenerated nuclei at the early uninuclear microspore stage, whilst melatonin protected degradation of organelles by enhancing the expression of heat shock protein genes to refold unfolded proteins and the expression of autophagy-related genes and formation of autophagosomes to degrade denatured proteins. These findings suggest a novel function of melatonin to protect pollen activity under high temperature and support the potential effects of melatonin on reproductive development of plants.
Subject(s)
Antioxidants/pharmacology , Gene Expression Regulation, Plant , Melatonin/pharmacology , Plant Growth Regulators/pharmacology , Plant Proteins/genetics , Pollen/drug effects , Solanum lycopersicum/drug effects , Ascorbate Peroxidases/genetics , Ascorbate Peroxidases/metabolism , Autophagy , Autophagy-Related Proteins/agonists , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Catalase/genetics , Catalase/metabolism , Heat-Shock Proteins/agonists , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Heat-Shock Response/genetics , Hot Temperature , Solanum lycopersicum/genetics , Solanum lycopersicum/growth & development , Solanum lycopersicum/metabolism , Peroxidase/genetics , Peroxidase/metabolism , Plant Proteins/metabolism , Pollen/genetics , Pollen/growth & development , Pollen/metabolism , Protein Denaturation , Proteolysis , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Thermotolerance/drug effects , Thermotolerance/geneticsABSTRACT
SCOPE: Glutamate excitotoxicity has been observed in association with neurodegenerative disorders. This study aimed to investigate whether a phycoerythrin-derived tryptic peptide of Pyropia yezoensis (PYP) reduces glutamate-induced excitotoxicity and neuronal senescence in primary rat hippocampal neurons. METHODS AND RESULTS: Glutamate exposure (100 µm) decreased cell viability and increased expression of endoplasmic reticulum (ER) stress response protein glucose-regulated protein 78 (GRP78) starting at 60 min following glutamate exposure, which was prevented by pretreating the neurons with PYP (1 µg mL-1 ). The glutamate-induced increase in GRP78 expression was downregulated by blocking N-methyl-d-aspartate (NMDA) receptor with MK801 (10 µm) and inhibiting c-Jun N-terminal kinase (JNK) phosphorylation with SP600125 (10 µm). Moreover, phosphorylation of JNK was decreased by blockade of NMDA receptor. The PYP pretreatment downregulated glutamate-induced increase in GRP78 expression and JNK phosphorylation, and this effect was abolished by inhibiting tropomyosin-related kinase B (TrkB) receptor, phosphatidylinositiol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 using cyclotraxin B (200 nm), LY294002 (20 µm), and SL327 (10 µm), respectively. In addition, PYP downregulated increase in GRP78 expression, senescence-associated ß-galactosidase activity, and neurite degeneration in aging hippocampal neurons. CONCLUSION: These findings indicate that activation of TrkB receptor-mediated ERK1/2 by PYP attenuates glutamate-induced ER stress, which may improve the survival of hippocampal neurons with age.
Subject(s)
Endoplasmic Reticulum Stress , Hippocampus/metabolism , Neurons/metabolism , Neuroprotective Agents/metabolism , Peptide Fragments/metabolism , Phycoerythrin/metabolism , Receptor, trkB/agonists , Animals , Cell Survival/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Dietary Supplements , Embryo, Mammalian/cytology , Endoplasmic Reticulum Stress/drug effects , Enzyme Activation/drug effects , Excitatory Amino Acid Antagonists/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Gene Expression Regulation/drug effects , Glutamic Acid/poisoning , Heat-Shock Proteins/agonists , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hippocampus/cytology , Hippocampus/drug effects , MAP Kinase Signaling System/drug effects , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Nootropic Agents/metabolism , Peptide Fragments/chemistry , Phycoerythrin/chemistry , Protein Kinase Inhibitors/pharmacology , Rats , Receptor, trkB/antagonists & inhibitors , Receptor, trkB/metabolism , Rhodophyta/enzymologyABSTRACT
STUDY QUESTION: Does the flavonoid naringenin inhibit proliferation of human endometriosis cells? SUMMARY ANSWER: Naringenin suppresses proliferation and increases apoptosis via depolarization of mitochondrial membrane potential and generation of reactive oxygen species (ROS) in human endometriosis cells. WHAT IS KNOWN ALREADY: For management of endometriosis, hormonal therapy is commonly used to decrease production of estrogens by the ovaries, but that has limitations including undesirable side effects with long-term therapies. To overcome these limitations, it is important to discover novel compounds which have no adverse effects, but inhibit expression of target molecules involved in the pathogenesis of endometriosis. STUDY DESIGN SIZE, DURATION: Well-established endometriosis cell lines (VK2/E6E7 and End1/E6E7) were purchased from the American Type Culture Collection. Effects of naringenin on VK2/E6E7 and End1/E6E7 cells were assessed in diverse assays in a dose- and time-dependent manner. PARTICIPANTS/MATERIALS, SETTING, METHODS: Effects of naringenin on viability, apoptosis (Annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of ROS and endoplasmic reticulum (ER) stress proteins of VK2/E6E7 and End1/E6E7 cells were determined. Signal transduction pathways in VK2/E6E7 and End1/E6E7 cells in response to naringenin were determined by western blot analyses. MAIN RESULTS AND THE ROLE OF CHANCE: In the present study, we demonstrated that naringenin suppressed proliferation and increased apoptosis through depolarization of mitochondrial membrane potential and inducing pro-apoptotic proteins, Bax and Bak, in both endometriosis cell lines. In addition, naringenin increased ROS, ER stress, through activation of eIF2α and IRE1α, GADD153 and GRP78 proteins in a dose-dependent manner. Furthermore, the induction of apoptosis by naringenin involved activation of MAPK and inactivation of PI3K pathways in VK2/E6E7 and End1/E6E7 cells. LIMITATIONS REASONS FOR CAUTION: Lack of in vivo animal studies is a major limitation of this research. Effectiveness of naringenin to induce apoptosis of human endometriosis cells requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that naringenin is a promising therapeutic compound for treatment of endometriosis in women. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.). The authors declare that there are no conflicts of interest.
Subject(s)
Endoplasmic Reticulum Stress/drug effects , Estrogen Antagonists/pharmacology , Flavanones/pharmacology , Mitochondria/drug effects , Mitogen-Activated Protein Kinases/genetics , Stromal Cells/drug effects , Annexin A5/genetics , Annexin A5/metabolism , Apoptosis/drug effects , Cell Line , Dose-Response Relationship, Drug , Endometriosis/metabolism , Endometriosis/pathology , Endometrium/metabolism , Endometrium/pathology , Endoplasmic Reticulum Chaperone BiP , Endoribonucleases/genetics , Endoribonucleases/metabolism , Eukaryotic Initiation Factor-2/agonists , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Female , Gene Expression Regulation , Heat-Shock Proteins/agonists , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondria/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/agonists , Reactive Oxygen Species/metabolism , Signal Transduction , Stromal Cells/metabolism , Stromal Cells/pathology , Transcription Factor CHOP/agonists , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism , bcl-2 Homologous Antagonist-Killer Protein/agonists , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/agonists , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Cyclosporine-A induces gingival overgrowth with disturbance in the homeostasis of cells and connective tissue proteins. Human gingival fibroblasts were cultured with cyclosporine A, and the expression of two vital endoplasmic stress markers and two prime matrix proteins (connective tissue growth factor (CTGF and periostin) were assessed by RT-PCR. We found that expression of Glucose-Regulated Protein 78 (GRP78/BIP) and CCAAT/enhancer binding protein, C/EBP homologous protein (CHOP) were significantly increased, along with CTGF and periostin, suggesting a role for these factors in gingival overgrowth.
Subject(s)
Cell Adhesion Molecules/genetics , Connective Tissue Growth Factor/genetics , Cyclosporine/adverse effects , Endoplasmic Reticulum Stress/drug effects , Heat-Shock Proteins/genetics , Immunosuppressive Agents/adverse effects , Transcription Factor CHOP/genetics , Biomarkers/metabolism , Cell Adhesion Molecules/agonists , Cell Adhesion Molecules/metabolism , Connective Tissue Growth Factor/agonists , Connective Tissue Growth Factor/metabolism , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation , Gingiva/cytology , Gingiva/drug effects , Gingiva/metabolism , Heat-Shock Proteins/agonists , Heat-Shock Proteins/metabolism , Humans , Primary Cell Culture , Signal Transduction , Transcription Factor CHOP/agonists , Transcription Factor CHOP/metabolismABSTRACT
Neurotoxicity of 1-bromopropane (1-BP) has been reported in both human cases and animal studies. To date, neurotoxicity of 1-BP has been induced in rats but not in mice due to the lethal hepatotoxicity of 1-BP. Oxidization by cytochromes P450 and conjugation with glutathione (GSH) are two critical metabolism pathways of 1-BP and play important roles in toxicity of 1-BP. The aim of the present study was to establish a murine model of 1-BP neurotoxicity, by reducing the hepatotoxicity of 1-BP with 1-aminobenzotriazole (1-ABT); a commonly used nonspecific P450s inhibitor. The results showed that subcutaneous or intraperitoneal injection of 1-ABT at 50mg/kg body weight BID (100mg/kg BW/day) for 3days, inhibited about 92-96% of hepatic microsomal CYP2E1 activity, but only inhibited about 62-64% of CYP2E1 activity in brain microsomes. Mice treated with 1-ABT survived even after exposure to 1200ppm 1-BP for 4 weeks and histopathological studies showed that treatment with 1-ABT protected mice from 1-BP-induced hepatic necrosis, hepatocyte degeneration, and hemorrhage. After 4-week exposure to 1-BP, the brain weight of 1-ABT(+)/1200ppm 1-BP group was decreased significantly. In 1-ABT-treated groups, expression of hippocampal Ran protein and cerebral cortical GRP78 was dose-dependently increased by exposure to 1-BP. We conclude that the control of hepatic P450 activity allows the observation of effects of 1-BP on the murine brain at a higher concentration by reduction of hepatotoxicity. The study suggests that further experiments with liver-specific control of P450 activity using gene technology might provide better murine models for 1-bromopropane-induced neurotoxicity.
Subject(s)
Cerebral Cortex/drug effects , Cytochrome P-450 Enzyme System/metabolism , Disease Models, Animal , Neurons/drug effects , Neurotoxicity Syndromes/etiology , Solvents/toxicity , Animals , Atmosphere Exposure Chambers , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/mortality , Chemical and Drug Induced Liver Injury/prevention & control , Cytochrome P-450 Enzyme Inhibitors/administration & dosage , Cytochrome P-450 Enzyme Inhibitors/therapeutic use , Dose-Response Relationship, Drug , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/agonists , Heat-Shock Proteins/metabolism , Hydrocarbons, Brominated/administration & dosage , Hydrocarbons, Brominated/toxicity , Injections, Intraperitoneal , Injections, Subcutaneous , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , Mice, Inbred C57BL , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Neurons/pathology , Neurotoxicity Syndromes/metabolism , Neurotoxicity Syndromes/pathology , Organ Size/drug effects , Solvents/administration & dosage , Triazoles/administration & dosage , Triazoles/therapeutic use , ran GTP-Binding Protein/agonists , ran GTP-Binding Protein/metabolismABSTRACT
Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/drug effects , Caffeine/pharmacology , Gene Expression Regulation, Developmental/drug effects , Heat-Shock Proteins/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/agonists , Caenorhabditis elegans Proteins/antagonists & inhibitors , Caenorhabditis elegans Proteins/metabolism , Dose-Response Relationship, Drug , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Heat-Shock Proteins/agonists , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/metabolism , Larva/drug effects , Larva/genetics , Larva/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Stress, Physiological , Transcription, GeneticABSTRACT
We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies.
Subject(s)
Coccidiosis/drug therapy , Coccidiostats/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Protozoan Proteins/antagonists & inhibitors , Toxoplasmosis/drug therapy , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Protozoan/genetics , Antigens, Protozoan/metabolism , Coccidiosis/parasitology , Coccidiosis/transmission , Female , Fibroblasts/drug effects , Fibroblasts/parasitology , Gene Expression , Heat-Shock Proteins/agonists , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Infectious Disease Transmission, Vertical/prevention & control , Mice , Mice, Inbred BALB C , Neospora/drug effects , Neospora/enzymology , Neospora/genetics , Pregnancy , Primary Cell Culture , Protein Kinases/metabolism , Protozoan Proteins/agonists , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Toxoplasma/drug effects , Toxoplasma/enzymology , Toxoplasma/genetics , Toxoplasmosis/parasitology , Toxoplasmosis/transmissionABSTRACT
Avarol is a sesquiterpenoid hydroquinone with potent cytotoxicity. Although resolving endoplasmic reticulum (ER) stress is essential for intracellular homeostasis, erratic or excessive ER stress can lead to apoptosis. Here, we reported that avarol selectively induces cell death in pancreatic ductal adenocarcinomas (PDAC), which are difficult to treat owing to the availability of few chemotherapeutic agents. Analyses of the molecular mechanisms of avarol-induced apoptosis indicated upregulation of ER stress marker BiP and ER stress-dependent apoptosis inducer CHOP in PDAC cells but not in normal cells, suggesting that avarol selectively induces ER stress responses. We also showed that avarol activated the PERK-eIF2α pathway but did not affect the IRE1 and ATF6 pathways. Moreover, CHOP downregulation was significantly suppressed by avarol-induced apoptosis. Thus, the PERK-eIF2α-CHOP signaling pathway may be a novel molecular mechanism of avarol-induced apoptosis. The present data indicate that avarol has potential as a chemotherapeutic agent for PDAC and induces apoptosis by activating the PERK-eIF2α pathway.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Pancreatic Neoplasms/drug therapy , Sesquiterpenes/pharmacology , Signal Transduction/drug effects , Transcription Factor CHOP/agonists , eIF-2 Kinase/metabolism , Animals , Antineoplastic Agents/adverse effects , Apoptosis/drug effects , Biomarkers/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Cell Line , Cell Line, Tumor , Dysidea/chemistry , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Eukaryotic Initiation Factor-2/agonists , Eukaryotic Initiation Factor-2/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Heat-Shock Proteins/agonists , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Inhibitory Concentration 50 , Neoplasm Proteins/agonists , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Pancreatic Neoplasms/metabolism , RNA Interference , Sesquiterpenes/adverse effects , Transcription Factor CHOP/antagonists & inhibitors , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism , Up-Regulation/drug effects , eIF-2 Kinase/chemistryABSTRACT
Circulating TNF-α levels are elevated in a wide variety of cardiovascular pathologies including congestive heart failure (CHF). This cytokine is one of the leading mediators of the immune inflammatory response with widespread biological functions regulated by membrane receptors. The pathophysiological implication of the downstream effects of activating the TNF-α system in CHF appears to depend on its direct effects on the heart and endothelium. Evidence supporting the notion that circulating TNF-α promotes protein breakdown was initially obtained from studies utilizing transgenic animals overexpressing TNF-α, animals with experimental diseases that augment TNF-α and in animals treated with exogenous TNF-α. It was then demonstrated that TNF-α acts directly on cultured myotubes to stimulate catabolism; however, whether the effects are the same in the heart remains poorly understood. The present study shows that TNF-α treatment induces autophagy, but clearance through this pathway appears obstructed and, consequently, results in increased protein ubiquitination. Furthermore, prolonged TNF-α treatment enhanced E3 ubiquitin ligase expression but reduced activity of the proteasome. These results suggest that TNF-α induces sarcomeric dysfunction and remodeling by disrupting autophagy and elevating the degradation of myofibrillar proteins. Therefore, myocardial remodeling, as a consequence to reduced contractile proteins, contributes to contractile dysfunction, a symptom often observed in the end stages of CHF.
Subject(s)
Heat-Shock Proteins/genetics , Mitochondria/drug effects , Myocytes, Cardiac/drug effects , Proteasome Endopeptidase Complex/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation , Heat-Shock Proteins/agonists , Heat-Shock Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis/drug effects , Rats , Reactive Oxygen Species/metabolism , SKP Cullin F-Box Protein Ligases/genetics , SKP Cullin F-Box Protein Ligases/metabolism , Sequestosome-1 Protein , Signal Transduction , Tripartite Motif Proteins , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effectsABSTRACT
Leptin is a circulating hormone that plays a critical role in regulating energy expenditure and food intake. Evidence to suggest the involvement of endoplasmic reticulum (ER) stress in the development of obesity is increasing. To adapt against ER stress, cells trigger the unfolded protein response (UPR). The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that protects cells against ER stress by inducing protein folding. In the present study, we hypothesized that leptin may activate UPR and protect against ER stress associated with obesity. SH-SY5Y, a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with leptin. We demonstrated that leptin induced GRP78 expression. We then validated the mechanism responsible for the leptin-induced expression of GRP78. Interestingly, leptin-induced GRP78 expression was not dependent on IRE1-XBP1 pathway. On the other hand, the PI3K inhibitor, LY294002, and mTOR inhibitor, rapamycin, inhibited the leptin-induced expression of GRP78. These results suggested that the leptin-induced expression of GRP78 may be dependent on the PI3K-mTOR pathway. Leptin specifically induced GRP78 because the induction of the ER-apoptotic marker, CHOP, was not detected in leptin-treated cells. Therefore, leptin may upregulate the expression of GRP78, thereby protecting against ER stress associated with obesity.
Subject(s)
Heat-Shock Proteins/agonists , Leptin/pharmacology , Neurons/drug effects , Phosphatidylinositol 3-Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism , Cell Line, Tumor , Chromones/pharmacology , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Gene Expression , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Leptin/genetics , Leptin/metabolism , Morpholines/pharmacology , Neurons/cytology , Neurons/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Receptors, Leptin/genetics , Receptors, Leptin/metabolism , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Unfolded Protein Response/drug effectsABSTRACT
Although many studies have suggested that estrogen prevents postmenopausal bone loss partially due to its anti-apoptosis effects in osteoblasts, the underlying mechanism has not been fully elucidated. In the present study, we found that 17ß-estradiol (17ß-E2), one of the primary estrogens, inhibited endoplasmic reticulum (ER) stress-induced apoptosis in MC3T3-E1 cells and primary osteoblasts. Interestingly, 17ß-E2-promoted Grp78 induction, but not CHOP induction in response to ER stress. We further confirmed that Grp78-specific siRNA reversed the inhibition of 17ß-E2 on ER stress-induced apoptosis by activating caspase-12 and caspase-3. Moreover, we found that 17ß-E2 markedly increased the phosphorylated TFII-I levels and nuclear localization of TFII-I in ER stress conditions. 17ß-E2 stimulated Grp78 promoter activity in a dose-dependent manner in the presence of TFII-I and enhanced the binding of TFII-I to the Grp78 promoter. In addition, 17ß-E2 notably increased phosphorylated ERK1/2 levels and Ras kinase activity in MC3T3-E1 cells. The ERK1/2 activity-specific inhibitor U0126 remarkably blocked 17ß-E2-induced TFII-I phosphorylation and Grp78 expression in response to ER stress. Together, 17ß-E2 protected MC3T3-E1 cells against ER stress-induced apoptosis by promoting Ras-ERK1/2-TFII-I signaling pathway-dependent Grp78 induction.
Subject(s)
Apoptosis/drug effects , Bone Density Conservation Agents/pharmacology , Endoplasmic Reticulum Stress/drug effects , Estradiol/pharmacology , Heat-Shock Proteins/agonists , Osteoblasts/drug effects , Transcription Factor TFIIA/agonists , Animals , Animals, Newborn , Bone Density Conservation Agents/chemistry , Bone Density Conservation Agents/therapeutic use , Cell Line , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Estradiol/chemistry , Estradiol/therapeutic use , Estrogens/chemistry , Estrogens/pharmacology , Estrogens/therapeutic use , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Osteoblasts/cytology , Osteoblasts/metabolism , Osteoporosis/chemically induced , Osteoporosis/metabolism , Osteoporosis/prevention & control , Phosphorylation/drug effects , Promoter Regions, Genetic/drug effects , Protein Processing, Post-Translational/drug effects , RNA Interference , Random Allocation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transcription Factor TFIIA/genetics , Transcription Factor TFIIA/metabolismABSTRACT
BACKGROUND: Geranylgeranylacetone (GGA) has been reported up-regulating heat shock protein (HSP) expression, and protecting against atrial remodeling. This study aimed to investigate the effects of GGA on atrial electrophysiology and inducibility of atrial fibrillation (AF) in heart failure (HF) model. METHODS AND RESULTS: HF rabbits were created 4 weeks after coronary artery ligation. Monophasic action potential recordings and multielectrode array were used to record the electrophysiological characteristics of left atrium (LA) in normal, or HF rabbits with (HF-GGA) and without (HF-control) oral administration of GGA (200 mg/kg, 24 h before experiments). The mRNA and protein expressions of ionic channels were measured by Western blot and PCR. HF-GGA LA (n = 10), similar to normal LA (n = 10) had a shorter action potential duration (APD) and effective refractory period than HF-control LA (n = 10). HF-GGA LA had less triggered activity and APD alternans (20% vs. 100%, P = 0.001), lower maxima slope of restitution curve of APD (0.94 ± 0.04 vs.1.69 ± 0.04, P < 0.001), and less inducibility of AF (50% vs. 100%, P = 0.033) than HF-control LA. HF-GGA LA had a shorter activation time and higher conduction velocity than HF-control LA. HF-GGA LA had a higher mRNA expression of Cav1.2, Nav1.5, Kir2.1, Kv1.4, Kv7.1, Kv11.1, sarcoplasmic reticulum Ca(2+)-ATPase, and higher phosphorylation of phospholamban than HF-control LA. CONCLUSIONS: GGA decreases triggered activity, dispersion of APD and inducibility of AF in failing heart through induction of HSP, and modulation of ionic channels and calcium homeostasis.
Subject(s)
Atrial Fibrillation/prevention & control , Diterpenes/therapeutic use , Heart Failure/drug therapy , Heat-Shock Proteins/biosynthesis , Action Potentials/drug effects , Action Potentials/physiology , Animals , Atrial Fibrillation/physiopathology , Diterpenes/pharmacology , Heat-Shock Proteins/agonists , RabbitsABSTRACT
The aim of this study was to elucidate photodynamic therapy (PDT) effects mediated by hypericin and a liposomal meso-tetrahydroxyphenyl chlorin (mTHPC) derivative, with focus on their 1:1 mixture, on head and neck squamous cell carcinoma cell lines. Absorption, excitation and photobleaching were monitored using fluorescence spectrometry, showing the same spectral patterns for the mixture as measured for single photosensitizers. In the mixture mTHPC showed a prolonged photo-stability. Singlet oxygen yield for light-activated mTHPC was Φ(Δ) = 0.66, for hypericin Φ(Δ) = 0.25 and for the mixture Φ(Δ) = ~0.4. A linear increase of singlet oxygen yield for mTHPC and the mixture was found, whereas hypericin achieved saturation after 35 min. Reactive oxygen species fluorescence was only visible after hypericin and mixture-induced PDT. Cell viability was also more affected with these two treatment options under the selected conditions. Examination of death pathways showed that hypericin-mediated cell death was apoptotic, with mTHPC necrotic and the 1:1 mixture showed features of both. Changes in gene expression after PDT indicated strong up-regulation of selected heat-shock proteins. The application of photosensitizer mixtures with the features of reduced dark toxicity and combined apoptotic and necrotic cell death may be beneficial in clinical PDT. This will be the focus of our future investigations.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma, Squamous Cell/drug therapy , Head and Neck Neoplasms/drug therapy , Mesoporphyrins/pharmacology , Perylene/analogs & derivatives , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Aged , Anthracenes , Antineoplastic Combined Chemotherapy Protocols/chemical synthesis , Apoptosis/drug effects , Carcinoma, Squamous Cell/pathology , Cell Survival/drug effects , Gene Expression/drug effects , Head and Neck Neoplasms/pathology , Heat-Shock Proteins/agonists , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Light , Liposomes/chemistry , Male , Mesoporphyrins/chemical synthesis , Middle Aged , Necrosis/metabolism , Necrosis/pathology , Perylene/chemical synthesis , Perylene/pharmacology , Photochemotherapy , Photosensitizing Agents/chemical synthesis , Porphyrins/chemical synthesis , Reactive Oxygen Species/agonists , Reactive Oxygen Species/metabolism , Tumor Cells, CulturedABSTRACT
Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.