ABSTRACT
Clinical studies have shown that traumatic brain injury (TBI) increases the onset of Parkinson's disease (PD) in later life by >50%. Oxidative stress, endoplasmic reticulum (ER) stress, and inflammation are the major drivers of both TBI and PD pathologies. We presently evaluated if curtailing oxidative stress and ER stress concomitantly using a combination of apocynin and tert-butylhydroquinone and salubrinal during the acute stage after TBI in mice reduces the severity of late-onset PD-like pathology. The effect of multiple low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on post-TBI neurodegeneration was also evaluated. The combo therapy elevated the level of phosphorylation at serine 129 (pS129) of α-Syn in the pericontusional cortex of male mice at 72 h post-TBI. Motor and cognitive deficits induced by TBI lasted at least 3 months and the combo therapy curtailed these deficits in both sexes. At 3 months post-TBI, male mice given combo therapy exhibited significantly lesser α-Syn aggregates in the SN and higher TH+ cells in the SNpc, compared to vehicle control. However, the aggregate number was not significantly different between groups of female mice. Moreover, TBI-induced loss of TH+ cells was negligible in female mice irrespective of treatment. The MPTP treatment aggravated PD-like pathology in male mice but had a negligible effect on the loss of TH+ cells in female mice. Thus, the present study indicates that mitigation of TBI-induced oxidative stress and ER stress at the acute stage could potentially reduce the risk of post-TBI PD-like pathology at least in male mice, plausibly by elevating pS129-α-Syn level.
Subject(s)
Antioxidants , Brain Injuries, Traumatic , Endoplasmic Reticulum Stress , Mice, Inbred C57BL , Animals , Male , Mice , Brain Injuries, Traumatic/pathology , Brain Injuries, Traumatic/metabolism , Brain Injuries, Traumatic/drug therapy , Female , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/physiology , Phosphorylation/drug effects , Antioxidants/pharmacology , Sex Characteristics , Acetophenones/pharmacology , Acetophenones/therapeutic use , Acetophenones/administration & dosage , Thiourea/analogs & derivatives , Thiourea/pharmacology , Thiourea/therapeutic use , Thiourea/administration & dosage , Serine/metabolism , Hydroquinones/pharmacology , Hydroquinones/administration & dosage , Hydroquinones/therapeutic use , Drug Therapy, Combination , Oxidative Stress/drug effectsABSTRACT
The increasing resistance of bacteria to commercially available antibiotics threatens patient safety in healthcare settings. Perturbation of ion homeostasis has emerged as a potential therapeutic strategy to fight against antibacterial resistance and other channelopathies. This study reports the development of 8-aminoquinoline (QN) derivatives and their transmembrane Zn2+ transport activities. Our findings showed that a potent QN-based Zn2+ transporter exhibits promising antibacterial properties against Gram-positive bacteria with reduced hemolytic activity and cytotoxicity to mammalian cells. Furthermore, this combination showed excellent in vivo efficacy against Staphylococcus aureus. Interestingly, this combination prevented bacterial resistance and restored susceptibility of gentamicin and methicillin-resistant S. aureus to commercially available ß-lactam and other antibiotics that had lost their activity against the drug-resistant bacterial strain. Our findings suggest that the transmembrane transport of Zn2+ by QN derivatives could be a promising strategy to combat bacterial infections and restore the activity of other antibiotics.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Quinolines , Staphylococcal Infections , Animals , Humans , Zinc , Ionophores/therapeutic use , Thiourea/pharmacology , Thiourea/therapeutic use , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Staphylococcal Infections/drug therapy , Quinolines/pharmacology , Quinolines/therapeutic use , Microbial Sensitivity Tests , MammalsABSTRACT
AIMS: The relationship between stress to endoplasmic reticulum (ER) and periodontitis has been known, and ER stress induced by Porphyromonas gingivalis results in the loss of alveolar bone. Salubrinal is a small synthetic compound and attenuates ER stress through inhibition of de-phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). In this study, we examined whether salubrinal attenuates periodontitis in a mouse model of experimental periodontal disease. MATERIALS AND METHODS: We evaluated loss of alveolar bone and attachment levels in periodontium using micro-computed tomography (µCT) and hematoxylin-eosin (HE) staining, respectively. Furthermore, we measured osteoclast numbers using tartrate-resistant acid phosphatase (TRAP) staining and osteoblast numbers using HE staining for bone resorption and for bone formation, respectively. To examine the inhibitory effects of salubrinal against pro-inflammatory cytokines, we measured TNF-α and IL1-ß score in periodontium using immunohistostaining. KEY FINDINGS: The results revealed that salubrinal suppressed loss of alveolar bone and attachment levels in periodontium induced by periodontitis. It decreased osteoclast numbers and increased osteoblasts. It also suppressed the expression levels of TNF-α in periodontium. SIGNIFICANCE: These results show that salubrinal alleviates periodontitis through suppression of alveolar bone resorption and the pro-inflammatory cytokine, and promotion of the bone formation. Since salubrinal has been shown to have these beneficial effects for periodontal disease, it may provide a novel therapeutic possibility for the disease.
Subject(s)
Alveolar Bone Loss/drug therapy , Cinnamates/therapeutic use , Thiourea/analogs & derivatives , Alveolar Bone Loss/complications , Alveolar Bone Loss/diagnostic imaging , Alveolar Bone Loss/pathology , Animals , Cell Count , Cinnamates/administration & dosage , Cinnamates/pharmacology , Disease Models, Animal , Endoplasmic Reticulum Stress/drug effects , Interleukin-1beta/metabolism , Male , Mice, Inbred C57BL , Osteoblasts/drug effects , Osteoblasts/pathology , Periodontitis/complications , Periodontitis/drug therapy , Periodontitis/pathology , Thiourea/administration & dosage , Thiourea/pharmacology , Thiourea/therapeutic use , Transcription Factor CHOP/metabolism , Tumor Necrosis Factor-alpha/metabolism , X-Ray MicrotomographyABSTRACT
Growth arrest and DNA damage-inducible protein 34 (GADD34), one of the key effectors of negative feedback loops, is induced by stress and subsequently attempts to restore homeostasis. It plays a critical role in response to DNA damage and endoplasmic reticulum stress. GADD34 has opposing effects on different stimulus-induced cell apoptosis events in many nervous system diseases, but its role in ischemic stroke is unclear. In this study, we evaluated the role of GADD34 and its distribution in a rat cerebral ischemic model. The results showed that GADD34 was increased in the cortex and contributed to brain injury in ischemic rats. Furthermore, treatment with a GADD34 inhibitor reduced the infarct volume, improved functional outcomes, and inhibited neuronal apoptosis in the cortical penumbra after ischemia. The role of GADD34 in ischemic stroke was associated with the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and phosphorylation of p53. In addition, the GADD34 level was increased in plasma exosomes of cerebral ischemic rats. These findings indicate that GADD34 could be a potential therapeutic target and biomarker for ischemic stroke.
Subject(s)
Antigens, Differentiation/metabolism , Cinnamates/pharmacology , Infarction, Middle Cerebral Artery/diagnosis , Proto-Oncogene Proteins/metabolism , Reperfusion Injury/prevention & control , Thiourea/analogs & derivatives , Animals , Antigens, Differentiation/blood , Biomarkers/blood , Biomarkers/metabolism , Cinnamates/therapeutic use , Disease Models, Animal , Eukaryotic Initiation Factor-2/blood , Eukaryotic Initiation Factor-2/metabolism , Exosomes/metabolism , Humans , Infarction, Middle Cerebral Artery/blood , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/drug therapy , Male , Phosphorylation/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/blood , Rats , Reperfusion Injury/etiology , Thiourea/pharmacology , Thiourea/therapeutic use , Tumor Suppressor Protein p53/blood , Tumor Suppressor Protein p53/metabolismABSTRACT
Cdc42 is a member of the Rho family of small GTPases and a key regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. It signals downstream of the master regulator Ras and is essential for cell transformation by this potent oncogene. Overexpression of Cdc42 is observed in several cancers, where it is linked to poor prognosis. As a regulator of both cell architecture and motility, deregulation of Cdc42 is also linked to tumour metastasis. Like Ras, Cdc42 and other components of the signalling pathways it controls represent important potential targets for cancer therapeutics. In this review, we consider the progress that has been made targeting Cdc42, its regulators and effectors, including new modalities and new approaches to inhibition. Strategies under consideration include inhibition of lipid modification, modulation of Cdc42-GEF, Cdc42-GDI and Cdc42-effector interactions, and direct inhibition of downstream effectors.
Subject(s)
Actin Cytoskeleton/metabolism , Neoplasms/metabolism , Signal Transduction/physiology , cdc42 GTP-Binding Protein/metabolism , Aminoquinolines/therapeutic use , Animals , Benzamides/therapeutic use , Benzazepines/therapeutic use , Humans , Molecular Targeted Therapy/methods , Neoplasms/drug therapy , Neoplasms/genetics , Oximes/therapeutic use , Protein Binding/drug effects , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Signal Transduction/drug effects , Signal Transduction/genetics , Sulfonamides/therapeutic use , Thiourea/analogs & derivatives , Thiourea/therapeutic use , cdc42 GTP-Binding Protein/antagonists & inhibitors , cdc42 GTP-Binding Protein/geneticsABSTRACT
Background osteosarcoma is a rare, primary malignant bone tumour with limited available treatments for advanced or recurrent disease, resulting in a poor prognosis for patients. TAS-115 is a novel tyrosine kinase inhibitor under investigation in a phase I study in patients with solid tumours. We report data of osteosarcoma patients in the expansion cohort of this ongoing study. Patients and methods an analysis of this multicentre, open-label study was performed 6 months after the final patient was enrolled, and included patients aged ≥15 years, with unresectable or recurrent osteosarcoma, and who had refractory to standard therapy or for whom no standard therapy was available. TAS-115 650 mg/day was orally administered in a 5 days on/2 days off schedule. Results a total of 20 patients with osteosarcoma were enrolled. The most common adverse drug reactions (ADRs) were neutrophil count decreased (75%), aspartate aminotransferase increased (50%), and platelet count decreased (50%); 85% of patients had grade ≥ 3 ADRs. Long-term disease control (>1 year) with TAS-115 was achieved in three patients. The best overall response was stable disease (50%); no patient achieved a complete or partial response. Median progression-free survival was 3 months; 4-month and 12-month progression-free rates were 42% and 31%, respectively. Conclusion the safety and tolerability of TAS-115 and long-term disease stability for patients with unresectable or recurrent osteosarcoma were confirmed in this study, suggesting that TAS-115 is a promising novel therapy for advanced osteosarcoma patients. Trial registration number: JapicCTI-132333 (registered on November 8, 2013).
Subject(s)
Antineoplastic Agents/therapeutic use , Osteosarcoma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Quinolines/therapeutic use , Thiourea/analogs & derivatives , Adolescent , Adult , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/adverse effects , Biomarkers, Tumor , Dose-Response Relationship, Drug , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Protein Kinase Inhibitors/adverse effects , Quinolines/administration & dosage , Quinolines/adverse effects , Survival Analysis , Thiourea/administration & dosage , Thiourea/adverse effects , Thiourea/therapeutic use , Young AdultABSTRACT
The previous virtual screening of ten million compounds yielded two novel nonlipopeptide-like chemotypes as TLR2 agonists. Herein, we present the chemical optimization of our initial hit, 1-phenyl-3-(thiophen-2-yl)urea, which resulted in the identification of SMU-C80 (EC50 = 31.02 ± 1.01 nM) as a TLR2-specific agonist with a 370-fold improvement in bioactivity. Mechanistic studies revealed that SMU-C80, through TLR1/2, recruits the adaptor protein MyD88 and triggers the NF-κB pathway to release cytokines such as TNF-α and IL-1ß from human, but not murine, cells. To the best of our knowledge, it is the first species-specific TLR1/2 agonist reported until now. Moreover, SMU-C80 increased the percentage of T, B, and NK cells ex vivo and activated the immune cells, which suppressed cancer cell growth in vitro. In summary, we obtained a highly efficient and specific human TLR1/2 agonist that acts through the MyD88 and NF-κB pathway, facilitating cytokine release and the simultaneous activation of immune cells that in turn affects the apoptosis of cancer cells.
Subject(s)
Drug Design , Thiourea/analogs & derivatives , Toll-Like Receptor 1/agonists , Toll-Like Receptor 2/agonists , Animals , Apoptosis/drug effects , Cytokines/metabolism , Humans , Immunotherapy , Killer Cells, Natural/cytology , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/metabolism , Neoplasms/therapy , Signal Transduction/drug effects , Structure-Activity Relationship , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Thiourea/metabolism , Thiourea/therapeutic use , Toll-Like Receptor 1/metabolism , Toll-Like Receptor 2/metabolismABSTRACT
BACKGROUND: Unlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC. METHODS: Breast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice. RESULTS: In large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity. CONCLUSIONS: Our results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.
Subject(s)
Breast/pathology , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/metabolism , Oxadiazoles/pharmacology , Tetrazoles/pharmacology , Thiourea/analogs & derivatives , Triple Negative Breast Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Datasets as Topic , Female , G2 Phase Cell Cycle Checkpoints/drug effects , Humans , Intravital Microscopy , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/agonists , Membrane Potentials/drug effects , Mice , Oxadiazoles/therapeutic use , Patch-Clamp Techniques , Tetrazoles/therapeutic use , Thiourea/pharmacology , Thiourea/therapeutic use , Triple Negative Breast Neoplasms/pathologyABSTRACT
Synthetic ion transporters have attracted tremendous attention for their therapeutic potential against various ion-transport-related diseases, including cancer. Inspired by the structure and biological activities of natural products, we synthesized a small series of squaramide and thiourea derivatives of quinine and investigated their ion transport activities. The involvement of a quinuclidine moiety for the cooperative interactions of Cl- and H+ ions with the thiourea or squaramide moiety resulted in an effectual transport of these ions across membranes. The interference of ionic equilibrium by the potent Cl- ion carrier selectively induced cancer cell death by endorsing caspase-arbitrated apoptosis. In vivo assessment of the potent ionophore showed an efficient reduction in tumor growth with negligible immunotoxicity to other organs.
Subject(s)
Antineoplastic Agents/therapeutic use , Cell Proliferation/drug effects , Ion Transport/drug effects , Neoplasms/drug therapy , Quinine/analogs & derivatives , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Chlorides/metabolism , Humans , Mice , Microbial Sensitivity Tests , Protons , Quinine/pharmacology , Quinine/therapeutic use , Thiourea/analogs & derivatives , Thiourea/pharmacology , Thiourea/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.
Subject(s)
Brain Ischemia/metabolism , Catecholamines/metabolism , Extracellular Fluid/metabolism , Hypothermia/metabolism , Sodium-Calcium Exchanger/metabolism , Stroke/metabolism , Adult , Aged , Animals , Brain Ischemia/prevention & control , Catecholamines/antagonists & inhibitors , Extracellular Fluid/drug effects , Frontal Lobe/drug effects , Frontal Lobe/metabolism , Humans , Middle Aged , Rats , Rats, Wistar , Sodium-Calcium Exchanger/antagonists & inhibitors , Stroke/therapy , Thiourea/analogs & derivatives , Thiourea/pharmacology , Thiourea/therapeutic useABSTRACT
Endoplasmic reticulum (ER) stress has been indicated to be involved in the pathogenesis of epilepsy. Sodium valproate (VPA), one of the most commonly used antiepileptic drugs, is reported to regulate ER stress in many neurological diseases. However, the effect of VPA on ER stress in epilepsy remains unclear. The current study was performed to investigate the role of ER stress in the neuroprotection of VPA against seizure induced by pentylenetetrzole (PTZ). Our results showed that VPA treatment could inhibit the increased expressions of ER stress proteins (GRP78 and CHOP), and significantly reduce neuronal apoptosis in the PTZ-induced experimental seizure model. In addition, Salubrinal, an ER stress inhibitor, was used as a positive control, and exhibited neuroprotective effects via inhibiting excessive ER stress in the seizure model, which further supported that the inhibition in ER stress by VPA treatment could exert neuroprotection in seizures. In summary, our work demonstrated for the first time that ER stress was involved in the neuroprotective potential of VPA for seizures.
Subject(s)
Anticonvulsants/therapeutic use , Apoptosis/drug effects , Endoplasmic Reticulum Stress/drug effects , Neuroprotective Agents/therapeutic use , Seizures/drug therapy , Valproic Acid/therapeutic use , Animals , Cinnamates/therapeutic use , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/metabolism , Hippocampus/metabolism , Hippocampus/pathology , Male , Mice, Inbred C57BL , Pentylenetetrazole , Seizures/chemically induced , Thiourea/analogs & derivatives , Thiourea/therapeutic use , Transcription Factor CHOP/metabolismABSTRACT
Loss of galactose-1 phosphate uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT-/-) mouse mimics the patient condition. GalT-/- ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT-/- adults. Here, we further characterized galactosemic stress in GalT-/- mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT-/- ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.
Subject(s)
Cinnamates/therapeutic use , Eukaryotic Initiation Factor-2/metabolism , Galactosemias/complications , Infertility, Female/drug therapy , Infertility, Female/etiology , Thiourea/analogs & derivatives , Animals , Cinnamates/pharmacology , Disease Models, Animal , Female , Fertility/drug effects , Galactosemias/metabolism , Infertility, Female/metabolism , Mice , Ovary/drug effects , Ovary/metabolism , Phosphorylation/drug effects , Thiourea/pharmacology , Thiourea/therapeutic use , Unfolded Protein Response/drug effectsABSTRACT
Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
Subject(s)
Brain Ischemia/pathology , Brain Ischemia/prevention & control , Cinnamates/therapeutic use , Disease Models, Animal , Necroptosis/drug effects , Neuroprotective Agents/therapeutic use , Thiourea/analogs & derivatives , Animals , Brain Ischemia/metabolism , Cell Survival/drug effects , Cell Survival/physiology , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Cinnamates/pharmacology , Male , Necroptosis/physiology , Neuroprotective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Thiourea/pharmacology , Thiourea/therapeutic useABSTRACT
BACKGROUND: Endoplasmic reticulum (ER) stress in the pancreas is closely associated with the development of acute pancreatitis. However, the role of the protein kinase RNA-like ER kinase (PERK) in this disease is not fully understood. We investigated whether an inhibitor of the dephosphorylation of eukaryotic initiation factor 2α, salubrinal, could improve murine experimental pancreatitis through the amelioration of ER stress. METHODS: Acute pancreatitis was induced by the intraperitoneal administration of cerulein (50⯵g/kg) six times at 1-h intervals followed by lipopolysaccharide (10â¯mg/kg). Salubrinal was administered intraperitoneally immediately after lipopolysaccharide injection and 3â¯h later. Mice were sacrificed 24â¯h after the first injection of cerulein, and serum amylase and proinflammatory cytokines were measured. The severity of pancreatitis was evaluated histologically using a scoring system. The expression levels of ER stress-related proteins were evaluated by Western blotting. RESULTS: The administration of salubrinal significantly attenuated the increase in serum amylase levels and improved histologically assessed pancreatitis. The serum levels of proinflammatory cytokines were significantly suppressed in salubrinal-treated mice, as was the expression of glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein, and cleaved caspase-3. CONCLUSIONS: The amelioration of ER stress through augmentation of the PERK-signaling pathway may be a therapeutic target for the treatment of acute pancreatitis.
Subject(s)
Cinnamates/therapeutic use , Eukaryotic Initiation Factor-2/drug effects , Eukaryotic Initiation Factor-2/metabolism , Pancreatitis/drug therapy , Thiourea/analogs & derivatives , Acute Disease , Amylases/blood , Animals , Apoptosis/drug effects , Ceruletide , Cytokines/blood , Endoplasmic Reticulum Stress/drug effects , Injections, Intraperitoneal , Lipopolysaccharides , Male , Mice , Mice, Inbred C57BL , Pancreatitis/chemically induced , Phosphorylation/drug effects , Thiourea/therapeutic useABSTRACT
Hepatocellular carcinoma (HCC) is the one of the most common cancers worldwide. Because the side effects of current treatments are severe, new effective therapeutic strategies are urgently required. Pterostilbene (PT), a natural analogue of resveratrol, has diverse pharmacologic activities, including antioxidative, anti-inflammatory and antiproliferative activities. Here we demonstrated that PT inhibits HCC cell growth without the induction of apoptosis in an endoplasmic reticulum (ER) stress- and autophagy-dependent manner. Mechanistic studies indicated that the combination of salubrinal and PT modulates ER stress-related autophagy through the phospho-eukaryotic initiation factor 2α/activating transcription factor-4/LC3 pathway, leading to a further inhibition of eIF2α dephosphorylation and the potentiation of cell death. An in vivo xenograft analysis revealed that PT significantly reduced tumour growth in mice with a SK-Hep-1 tumour xenograft. Taken together, our results yield novel insights into the pivotal roles of PT in ER stress- and autophagy-dependent cell death in HCC cells.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Carcinoma, Hepatocellular/drug therapy , Endoplasmic Reticulum Stress/drug effects , Eukaryotic Initiation Factor-2/metabolism , Liver Neoplasms/drug therapy , Stilbenes/pharmacology , Activating Transcription Factor 4/metabolism , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Autophagosomes/drug effects , Autophagosomes/ultrastructure , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cinnamates/pharmacology , Cinnamates/therapeutic use , Eukaryotic Initiation Factor-2/chemistry , Eukaryotic Initiation Factor-2/genetics , Female , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Microtubule-Associated Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Stilbenes/chemistry , Stilbenes/therapeutic use , Thiourea/analogs & derivatives , Thiourea/pharmacology , Thiourea/therapeutic use , Transplantation, HeterologousABSTRACT
Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of ß-hemoglobinopathies like ß-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562â¯cell line. Erythroid induction of K562â¯cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562â¯cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562â¯cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including ß-thalassemia and sickle cell anemia.
Subject(s)
Fetal Hemoglobin/biosynthesis , Thiourea/analogs & derivatives , Thiourea/pharmacology , beta-Thalassemia/drug therapy , beta-Thalassemia/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Erythroid Cells/drug effects , Erythroid Cells/pathology , Gene Expression Regulation/drug effects , Humans , K562 Cells , Thiourea/therapeutic use , beta-Thalassemia/pathologyABSTRACT
Sirtuin 2 (SIRT2) is a protein lysine deacylase that has been indicated as a therapeutic target for cancer. To further establish the role of SIRT2 in cancers, it is necessary to develop selective and potent inhibitors. Here, we report the facile synthesis of novel lysine-derived thioureas as mechanism-based SIRT2 inhibitors with anticancer activity. Compounds AF8, AF10, and AF12 selectively inhibited SIRT2 with IC50 values of 0.06, 0.15, and 0.08 µM, respectively. Compounds AF8 and AF10 demonstrated broad cytotoxicity amongst cancer cell lines, but minimal toxicity in noncancerous cells. AF8 and AF10 inhibited the anchorage-independent growth of human colorectal cancer cell line HCT116 with GI50 values of â¼7 µM. Furthermore, AF8 potently inhibited tumor growth in a HCT116 xenograft murine model, supporting that SIRT2 is a viable therapeutic target for colorectal cancer.
Subject(s)
Antineoplastic Agents/chemistry , Lysine/chemistry , Sirtuin 2/antagonists & inhibitors , Thiourea/chemistry , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Disease Models, Animal , Drug Design , Drug Screening Assays, Antitumor , Female , Humans , Male , Mice , Mice, Transgenic , Molecular Docking Simulation , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Sirtuin 2/metabolism , Structure-Activity Relationship , Thiourea/chemical synthesis , Thiourea/pharmacology , Thiourea/therapeutic useABSTRACT
Increasing evidence suggests that Ras-related in brain 7 (Rab7), an endosome-localized small GTPase contributes to cerebral ischemic brain injury. In the present study, we investigated the role of Rab7 in ischemic stroke-induced formation of astrogliosis and glial scar. Rats were subjected to transient middle cerebral artery occlusion (tMCAO); the rats were injected with the Rab7 receptor antagonist CID1067700 (CID). Primary astrocytes were subjected to an oxygen and glucose deprivation and reoxygenation (OGD/Re) procedure; CID was added to the cell culture media. We found that Rab7 was significantly elevated over time in both the in vivo and in vitro astrocytic injury models, and administration of CID significantly down-regulated the glial scar markers such as glial fibillary acidic protein (GFAP), neurocan and phosphacan. Moreover, administration of CID significantly attenuated the brain atrophy and improved neurologic deficits in tMCAO rats, and protected astrocytes against OGD/Re-induced injury. Further, CID downregulated the protein levels of Lamp1 and active cathepsin B in astrocytes after OGD/Re or tMCAO injury; CID inhibited the co-localization of cathepsin B and Rab7, Lamp1 and Rab7; CID decreased OGD/Re-induced increase in lysosomal membrane permeability and blocked OGD/Re-induced release of cathepsin B from the lysosome into the cytoplasm in astrocytes. Taken together, these results suggest that Rab7 is involved in ischemic stroke-induced formation of astrogliosis and glial scar. CID administration attenuates brain atrophy and improves neurologic deficits and inhibits astrogliosis and glial scar formation after ischemic stroke via reducing the activation and release of cathepsin B from the lysosome into the cytoplasm.
Subject(s)
Atrophy/drug therapy , Brain/drug effects , Gliosis/drug therapy , Heterocyclic Compounds, 2-Ring/therapeutic use , Infarction, Middle Cerebral Artery/drug therapy , Neuroprotective Agents/therapeutic use , Thiourea/analogs & derivatives , rab GTP-Binding Proteins/antagonists & inhibitors , Animals , Astrocytes/drug effects , Astrocytes/pathology , Brain/pathology , Cathepsin B/metabolism , Chondroitin Sulfate Proteoglycans/metabolism , Glial Fibrillary Acidic Protein/metabolism , Lysosomal Membrane Proteins/metabolism , Lysosomes/metabolism , Male , Neurocan , Rats, Sprague-Dawley , Receptor-Like Protein Tyrosine Phosphatases, Class 5/metabolism , Thiourea/therapeutic use , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
BACKGROUND: Pneumonia is a major cause of high morbidity and mortality in critically illness, and frequently requires support with mechanical ventilation. The latter can lead to ventilator-induced lung injury characterized by neutrophil infiltration. The cationic human neutrophil peptides (HNP) stored in neutrophils can kill microorganisms, but excessive amount of HNP released during phagocytosis may contribute to inflammatory responses and worsen lung injury. Based on our previous work, we hypothesized that blocking the cell surface purinergic receptor P2Y6 will attenuate the HNP-induced inflammatory responses while maintaining their antimicrobial activity in pneumonia followed by mechanical ventilation. METHODS: Plasma HNP levels were measured in patients with pneumonia who received mechanical ventilation and in healthy volunteers. FVB littermate control and HNP transgenic (HNP+) mice were randomized to receive P. aeruginosa intranasally. The P2Y6 antagonist (MRS2578) or vehicle control was given after P. aeruginosa instillation. Additional mice underwent mechanical ventilation at either low pressure (LP) or high pressure (HP) ventilation 48 h after pneumonia, and were observed for 24 h. RESULTS: Plasma HNP concentration increased in patients with pneumonia as compared to healthy subjects. The bacterial counts in the bronchoalveolar lavage fluid (BALF) were lower in HNP+ mice than in FVB mice 72 h after P. aeruginosa instillation. However, upon receiving HP ventilation, HNP+ mice had higher levels of cytokines and chemokines in BALF than FVB mice. These inflammatory responses were attenuated by the treatment with MRS2578 that did not affect the microbial effects of HNP. CONCLUSIONS: HNP exerted dual effects by exhibiting antimicrobial activity in pneumonia alone condition while enhancing inflammatory responses in pneumonia followed by HP mechanical ventilation. Blocking P2Y6 can attenuate the inflammation without affecting the antibacterial property of HNP. The P2Y6 receptor may be a novel therapeutic target in attenuation of the leukocyte-mediated excessive host responses in inflammatory lung diseases.
Subject(s)
Disease Models, Animal , Isothiocyanates/therapeutic use , Neutrophils , Pneumonia, Ventilator-Associated/drug therapy , Receptors, Purinergic P2 , Thiourea/analogs & derivatives , Ventilator-Induced Lung Injury/drug therapy , Aged , Animals , Female , Humans , Isothiocyanates/pharmacology , Male , Mice , Mice, Transgenic , Middle Aged , Pneumonia, Ventilator-Associated/metabolism , Pneumonia, Ventilator-Associated/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/isolation & purification , Receptors, Purinergic P2/metabolism , Thiourea/pharmacology , Thiourea/therapeutic use , Treatment Outcome , Ventilator-Induced Lung Injury/metabolism , Ventilator-Induced Lung Injury/microbiologyABSTRACT
Purinergic signaling in spinal cord microglia plays an important role in the pathogenesis of neuropathic pain. Among all P2 receptors, P2Y6 receptor is expressed in rat dorsal spinal cord. However, it's not clear that the role of P2Y6 receptor in the chronic constriction injury (CCI) model of neuropathic pain rats. We evaluated the effect of repeated intrathecal administration of MRS2578 (selective P2Y6 receptor antagonist) on CCI-induced nociceptive behaviors in rats. After CCI, MRS2578 (10-11-10-4â¯M) was administration. The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were assessed. The expression of P2Y6 receptor and Iba-1 at rat dorsal spinal cord was observed by using RT-PCR. We found that intrathecal injection of MRS2578 suppressed CCI-induced mechanical allodynia and thermal hyperalgesia with a dose-dependent manner. The CCI rats presented increased expression of P2Y6 receptor and Iba-1 at the mRNA level in the ipsilateral dorsal spinal cord than that in sham group. Treatment with either minocycline or SB203580 effectively inhibited P2Y6 receptor expression compared to CCI rats. Intrathecal injection of UDP enhanced mechanical and thermal allodynia than that in CCI group. To the further study, intrathecal injection of UDP causes mechanical allodynia and thermal hyperalgesia in naive rats. The increased expression of P2Y6 receptor and Iba-1 were observed in UDP-treated rats. Intrathecal injection of MRS2578 alleviates pain response in UDP-treated rats. These observations suggested that P2Y6 receptor in dorsal spinal cord contribute to mechanical allodynia and thermal hyperalgesia in CCI-induced neuropathic pain.