RESUMO
Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Quimiocina CCL20/metabolismo , Estresse do Retículo Endoplasmático , Imunomodulação , Interleucina-8/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular Tumoral , Quimiocina CCL20/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunomodulação/efeitos dos fármacos , Interleucina-8/genética , Janus Quinases/metabolismo , Modelos Biológicos , Comunicação Parácrina/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
In spite of the impressing cytotoxicity of thapsigargin (Tg), this compound cannot be used as a chemotherapeutic drug because of general toxicity, causing unacceptable side effects. Instead, a prodrug targeted towards tumors, mipsagargin, was brought into clinical trials. What substantially reduces the clinical potential is the limited access to Tg and its derivatives and cost-inefficient syntheses with unacceptably low yields. Laser trilobum, which contains a structurally related sesquiterpene lactone, trilobolide (Tb), is successfully cultivated. Here, we report scalable isolation of Tb from L. trilobum and a transformation of Tb to 8-O-(12-aminododecanoyl)-8-O-debutanoylthapsigargin in seven steps. The use of cultivated L. trilobum offers an unlimited source of the active principle in mipsagargin.
Assuntos
Antineoplásicos Fitogênicos/química , Apiaceae/química , Butiratos/química , Técnicas de Química Sintética , Furanos/química , Tapsigargina/análogos & derivados , Antineoplásicos Fitogênicos/isolamento & purificação , Apiaceae/metabolismo , Butiratos/isolamento & purificação , Dióxido de Carbono/química , Cromatografia com Fluido Supercrítico/métodos , Frutas/química , Frutas/metabolismo , Furanos/isolamento & purificação , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Extratos Vegetais/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tapsigargina/isolamento & purificaçãoRESUMO
A sesquiterpene lactone, thapsigargin, is a phytochemical found in the roots and fruits of Mediterranean plants from Thapsia L. species that have been used for centuries in folk medicine to treat rheumatic pain, lung diseases, and female infertility. More recently thapsigargin was found to be a potent cytotoxin that induces apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, which is necessary for cellular viability. This biological activity encouraged studies on the use of thapsigargin as a novel antineoplastic agent, which were, however, hampered due to high toxicity of this compound to normal cells. In this review, we summarized the recent knowledge on the biological activity and molecular mechanisms of thapsigargin action and advances in the synthesis of less-toxic thapsigargin derivatives that are being developed as novel anticancer drugs.
Assuntos
Antineoplásicos/uso terapêutico , Medicina Tradicional/métodos , Neoplasias/tratamento farmacológico , Thapsia/química , Tapsigargina/uso terapêutico , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Tapsigargina/química , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Endoplasmic reticulum stress (ER stress) plays a main role in pancreatic [Formula: see text]-cell dysfunction and death because of intracellular Ca[Formula: see text] turbulence and inflammation activation. Although several drugs are targeting pancreatic [Formula: see text]-cell to improve [Formula: see text]-cell function, there still lacks agents to alleviate [Formula: see text]-cell ER stress conditions. Therefore we used thapsigargin (THAP) or high glucose (HG) to induce ER stress in [Formula: see text]-cell and aimed to screen natural molecules against ER stress-induced [Formula: see text]-cell dysfunction. Through screening the Traditional Chinese drug library ([Formula: see text] molecules), luteolin was finally discovered to improve [Formula: see text]-cell function. Cellular viability results indicated luteolin reduced the THAP or HG-induced [Formula: see text]-cell death and apoptosis through MTT and flow cytometry assay. Moreover, luteolin improved [Formula: see text]-cell insulin secretion ability under ER stress conditions. Also ER stress-induced intracellular Ca[Formula: see text] turbulence and inflammation activation were inhibited by luteolin treatment. Mechanically, luteolin inhibited HNF4[Formula: see text] signaling, which was induced by ER stress. Moreover, luteolin reduced the transcriptional level of HNF4[Formula: see text] downstream gene, such as Asnk4b and HNF1[Formula: see text]. Conversely HNF4[Formula: see text] knockdown abolished the effect of luteolin on [Formula: see text]-cell using siRNA. These results suggested the protective effect of luteolin on [Formula: see text]-cell was through HNF4[Formula: see text]/Asnk4b pathway. In conclusion, our study discovered that luteolin improved [Formula: see text]-cell function and disclosed the underlying mechanism of luteolin on [Formula: see text]-cell, suggesting luteolin is a promising agent against pancreatic dysfunction.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Luteolina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tapsigargina/efeitos adversos , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/fisiologia , Glucose/efeitos adversos , Células Secretoras de Insulina/metabolismo , Luteolina/isolamento & purificaçãoRESUMO
Heat stress induces apoptosis in various cells. Selenium, an essential micronutrient, has beneficial effects in maintaining the cellular physiological functions. However, its potential protective action against chronic heat stress (CHS)-induced apoptosis in granulosa cells and the related molecular mechanisms are not fully elucidated. In this study, we investigated the roles of selenium in CHS-induced apoptosis in mouse granulosa cells and explored its underlying mechanism. The heat treatment for 6-48 h induced apoptosis, potentiated caspase 3 activity, increased the expression levels of apoptosis-related gene BAX and ER stress markers, glucose-regulated protein 78 (GRP78), and CCAAT/enhancer binding protein homologous protein (CHOP) in mouse granulosa cells. The treatment with ER stress inhibitor 4-PBA significantly attenuated the adverse effects caused by CHS. Selenium treatment significantly attenuated the CHS- or thapsigargin (Tg, an ER stress activator)-induced apoptosis, potentiation of caspase 3 activity, and the increased protein expression levels of BAX, GRP78, and CHOP. Additionally, treatment of the cells with 5 ng/mL selenium significantly ameliorated the levels of estradiol, which were decreased in response to heat exposure. Consistently, administering selenium supplement alleviated the hyperthermia-caused reduction in the serum estradiol levels in vivo. Together, our findings indicate that selenium has protective effects on CHS-induced apoptosis via inhibition of the ER stress pathway. The current study provides new insights in understanding the role of selenium during the process of heat-induced cell apoptosis.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células da Granulosa/citologia , Selênio/administração & dosagem , Tapsigargina/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Butilaminas/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Camundongos , Selênio/farmacologia , Fator de Transcrição CHOP/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.
Assuntos
Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/genética , Quinases Proteína-Quinases Ativadas por AMP , Agonistas Adrenérgicos beta/metabolismo , Animais , Aripiprazol/metabolismo , Aripiprazol/farmacologia , Cálcio/agonistas , Cálcio/metabolismo , Interações Medicamentosas , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Espectrometria de Massas , Camundongos , Fosfoproteínas/química , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sistemas de Translocação de Proteínas/genética , Sistemas de Translocação de Proteínas/metabolismo , Proteoma/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Estresse Fisiológico/fisiologia , Tapsigargina/metabolismo , Tapsigargina/farmacologiaRESUMO
Specialized plant terpenoids have found fortuitous uses in medicine due to their evolutionary and biochemical selection for biological activity in animals. However, these highly functionalized natural products are produced through complex biosynthetic pathways for which we have a complete understanding in only a few cases. Here we review some of the most effective and promising plant terpenoids that are currently used in medicine and medical research and provide updates on their biosynthesis, natural occurrence, and mechanism of action in the body. This includes pharmacologically useful plastidic terpenoids such as p-menthane monoterpenoids, cannabinoids, paclitaxel (taxol®), and ingenol mebutate which are derived from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, as well as cytosolic terpenoids such as thapsigargin and artemisinin produced through the mevalonate (MVA) pathway. We further provide a review of the MEP and MVA precursor pathways which supply the carbon skeletons for the downstream transformations yielding these medically significant natural products.
Assuntos
Vias Biossintéticas , Ácido Mevalônico/metabolismo , Monoterpenos/metabolismo , Terpenos/metabolismo , Animais , Canabinoides/metabolismo , Diterpenos/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Medicina Herbária , Humanos , Monoterpenos/uso terapêutico , Paclitaxel/metabolismo , Fosfatos Açúcares/metabolismo , Terpenos/uso terapêutico , Tapsigargina/metabolismoRESUMO
Intracellular calcium ion content is tightly regulated for the maintenance of cellular functions and cell survival. Calbindin-D9k (CaBP-9k) is responsible for regulating the distribution of cytosolic free-calcium ions. In this study, we aimed to investigate the effect of CaBP-9k on cell survival in pancreatic beta cells. Six-month-old wildtype CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice were used to compare the pathological phenotypes of calcium-binding protein-deleted mice. Subsequently, the endoplasmic reticulum (ER) stress reducer tauroursodeoxycholic acid (TUDCA) was administered to wildtype and CaBP-9k KO mice. In vitro assessment of the role of CaBP-9k was performed following CaBP-9k overexpression and treatment with the ER stress inducer thapsigargin. Six-month-old CaBP-9k KO mice showed reduced islet volume and up-regulation of cell death markers resulting from ER stress, which led to pancreatic beta cell death. TUDCA treatment recovered islet volume, serum insulin level, and abdominal fat storage by CaBP-9k ablation. CaBP-9k overexpression elevated insulin secretion and recovered thapsigargin-induced ER stress in the INS-1E cell line. The results of this study show that CaBP-9k can protect pancreatic beta cell survival from ER stress and contribute to glucose homeostasis, which can reduce the risk of type 1 diabetes and provide the molecular basis for calcium supplementation to diabetic patients.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/prevenção & controle , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Knockout , Proteína G de Ligação ao Cálcio S100/genética , Ácido Tauroquenodesoxicólico/farmacologia , Tapsigargina/farmacologiaRESUMO
Pathophysiological states cause misfolded protein accumulation in the endoplasmic reticulum (ER). Then, ER stress and the unfolded protein response (UPR) are activated. Targeting ER stress may enhance the adaptive UPR and then protect the cell against pathogenic environments. In the present study, we utilized nanotechnology to synthesize thapsigargin nanoparticles (TG NPs) which induced ER stress and the UPR pathway, to study the role of ER stress and autophagy in chronic kidney disease (CKD). We found that the mRNA levels of ER stress- and autophagy-related molecules were elevated in the renal tissue of CKD patients compared to those of healthy individuals. Furthermore, TG NPs induced the UPR pathway and autophagy in HK-2 human kidney tubular epithelial cells. TG NPs protected HK-2 cells against oxidative stress-induced cell death through the activation of Nrf2 and FoxO1. The siRNA-mediated inhibition of Nrf2 or FoxO1 resulted in enhanced oxidative stress-induced cytotoxicity in HK-2 cells. In a mouse model of adenine diet-induced CKD, TG NPs and KIM-1-TG NPs ameliorated renal injury through the stimulation of ER stress and its downstream pathways. Our findings suggest that the induction of ER stress using pharmacological agents may offer a promising therapeutic strategy for preventing or interfering with CKD progression.
Assuntos
Proteína Forkhead Box O1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Nanopartículas/química , Insuficiência Renal Crônica/tratamento farmacológico , Tapsigargina/administração & dosagem , Adenina , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/uso terapêutico , Nanopartículas/ultraestrutura , Insuficiência Renal Crônica/metabolismoAssuntos
Autofagia/efeitos dos fármacos , Berberina/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fígado Gorduroso/tratamento farmacológico , Transplante de Fígado , Animais , Autofagossomos/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Tapsigargina/farmacologiaRESUMO
In proteinuric nephropathies of chronic kidney disease, the epithelial cells of the nephron including the collecting duct are exposed to high concentrations of luminal albumin. Albumin is taken up from collecting duct cells by endocytosis causing excessive reactive oxygen species (ROS) production and a proinflammatory response. Curcumin used in the traditional medicine possesses anti-inflammatory and antioxidant effects. ROS and ADP-ribose (ADPR) activate the cation channel TRPM2. We hypothesize, that albumin-induced cell stress and proinflammatory response are mediated by Ca2+ and can be reduced by curcumin. The cortical collecting duct (CCD) cells mpkCCDc14 exhibit spontaneous and inducible Ca2+ oscillations, which can be blocked by pre-treatment with curcumin. Curcumin accumulates in plasma membrane and intracellular vesicles, where it interferes with TRPM2 and decreases the influx of Ca2+. Albumin reduces cell viability and increases apoptosis, NF-κB activation, and mitochondrial membrane depolarization via Ca2+-dependent signaling, which results in increased ROS production. Albumin-induced cell stress is diminished by the inhibition of TRPM2 after administration of curcumin and ADPR (PARP1) inhibitors. Curcumin did not reduce the Ca2+ elevation induced by thapsigargin in Ca2+-free medium, but it reduced the function of store-operated Ca2+ channels and ATP-evoked Ca2+ response. In conclusion, albumin-induced oxidative stress is mediated by Ca2+-dependent signaling via TRPM2 and leads to cell damage and a proinflammatory response, strengthening the role of CCD cells in the progression of chronic kidney disease.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Curcumina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Soroalbumina Bovina/farmacologia , Canais de Cátion TRPM/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPM/genética , Tapsigargina/farmacologiaRESUMO
Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an â¼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Glucose/farmacologia , Pâncreas/citologia , Células Secretoras de Somatostatina/efeitos dos fármacos , Somatostatina/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Membrana Celular/fisiologia , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Células Secretoras de Somatostatina/metabolismo , Tapsigargina/farmacologiaRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting more than 47.5 million people worldwide. Metabolic impairments are common hallmarks of AD, and amyloid-ß (Aß) peptide and hyperphosphorylated tau protein-the two foremost histopathological signs of AD-have been implicated in mitochondrial dysfunction. Many neurodegenerative disorders, including AD, show excessive amounts of mis-/unfolded proteins leading to an activation of the unfolded protein response (UPR). In the present study, we aimed to characterize the link between ER stress and bioenergetics defects under normal condition (human SH-SY5Y neuroblastoma cells: control cells) or under pathological AD condition [SH-SY5Y cells overexpressing either the human amyloid precursor protein (APP) or mutant tau (P301L)]. More specifically, we measured UPR gene expression, cell viability, and bioenergetics parameters, such as ATP production and mitochondrial membrane potential (MMP) in basal condition and after an induced ER stress by thapsigargin. We detected highly activated UPR and dysregulated bioenergetics in basal condition in both AD cellular models. Strikingly, acute-induced ER stress increased the activity of the UPR in both AD cellular models, leading to up-regulation of apoptotic pathways, and further dysregulated mitochondrial function.
Assuntos
Doença de Alzheimer/patologia , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Resposta a Proteínas não Dobradas , Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Tapsigargina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
We have previously identified a novel endoplasmic reticulum (ER) stress-inducible protein, namely, cysteine-rich with EGF-like domains 2 (CRELD2), which is predominantly regulated by ATF6. However, few studies on intrinsic CRELD2 have been published. In the present study, we elucidated the expression of intrinsic CRELD2 in mouse tissues and ER stress- treated Neuro2a cells. Among nine tissues we tested, CRELD2 protein in the heart and skeletal muscles was negligible. CRELD2 expression in Neuro2a cells was induced at the late phase after treatment with tunicamycin (Tm) compared with rapid induction of growth arrest and DNA damage inducible gene 153 (GADD153). On the other hand, another ER stress inducer, thapsigargin, increased the intrinsic CRELD2 secretion from Neuro2a cells. We furthermore established CRELD2-deficient Neuro2a cells to evaluate their features. In combination with the NanoLuc complementary reporter system, which was designed to detect protein-protein interaction in living cells, CRELD2 interacted with not only CRELD2 itself but also with ER localizing proteins in Neuro2a cells. Finally, we investigated the responsiveness of CRELD2-deficient cells against Tm-treatment and found that CRELD2 deficiency did not affect the expression of genes triggered by three canonical ER stress sensors but rendered Neuro2a cells vulnerable to Tm-stimulation. Taken together, these findings provide the novel molecular features of CRELD2, and its further characterization would give new insights into understanding the ER homeostasis and ER stress-induced cellular dysfunctions.
Assuntos
Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Animais , Linhagem Celular , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Tapsigargina/metabolismo , Fator de Transcrição CHOP/metabolismo , Transcriptoma/genética , Tunicamicina/farmacologiaRESUMO
Diets supplemented with walnuts have shown to protect brain against oxidative and inflammatory cytotoxicity and promote protective cellular and cognitive function. The current study was undertaken to test the hypothesize that whole walnut extract (WNE) inhibits lipopolysaccharide (LPS)-induced microglial activation by regulating calmodulin (CaM) expression through [Ca2+]i. To test this hypothesis, we used an in vitro model the highly aggressively proliferating immortalized cells, a rat microglial cell line, treated with various concentrations of WNEs. Treatment with WNE (1.5%, 3%, or 6%) induced a slow rise in intracellular calcium in a concentration- and time-dependent manner, and this rise became exaggerated when cells were depolarized with potassium chloride (100 mmol/L). Cells treated with WNE (1%, 3%, or 6%) upregulated CaM protein levels, with 1 hour posttreatment being the peak time, regardless of WNE concentration. Interestingly, this WNE-induced upregulation of CaM was blocked by pretreatment with thapsigargin. Additionally, treatment with WNE (1%, 3%, or 6%) 1 hour prior to LPS treatment was found to be effective in preventing LPS-induced upregulation of inducible nitric oxide synthase expression, upregulation of ionized Ca2+-binding adaptor-1, and downregulation of CaM. These findings suggest that bioactive compounds in walnut are capable of modulating microglial activation through regulation of intracellular calcium and CaM expression. Nutritional interventions using walnuts may be effective in the amelioration of chronic inflammation and neurodegeneration.
Assuntos
Encéfalo/efeitos dos fármacos , Cálcio/metabolismo , Calmodulina/metabolismo , Juglans , Microglia/efeitos dos fármacos , Nozes , Extratos Vegetais/farmacologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Lipopolissacarídeos , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Cloreto de Potássio/farmacologia , Ratos , Tapsigargina/farmacologia , Regulação para CimaRESUMO
Tacrolimus is an immunosuppressive drug that inhibits the release of inflammatory cytokines involved in rheumatoid arthritis development by blocking T cell activation. "Endoplasmic reticulum stress," an imbalance between protein folding load and capacity leading to the accumulation of unfolded proteins in the endoplasmic reticulum lumen, has been implicated in rheumatoid arthritis and other inflammatory and metabolic diseases. We aimed to investigate the effect of tacrolimus on endoplasmic reticulum stress-mediated osteoclastogenesis and inflammation and elucidate the underlying mechanisms. In vitro studies were performed using mouse bone marrow cells that were cultured with or without interleukin-1ß, thapsigargin, or tacrolimus to induce osteoclast differentiation. A mouse model of arthritis was established by immunizing mice with bovine type II collagen. Tacrolimus was orally administered to mice from day 20 to 45 following the initial immunization, and histopathological changes and expression of specific biomarkers of endoplasmic reticulum stress-mediated inflammatory signaling pathways were examined. In vitro, tacrolimus inhibited receptor activator of nuclear factor-κB ligand-mediated osteoclast formation augmented by interleukin-1ß, thapsigargin, or both. Furthermore, tacrolimus inhibited glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase, inositol-requiring enzyme 1 (IRE 1), and activating transcription factor 6 (ATF6) augmented by interleukin-1ß, thapsigargin, or both. Tacrolimus significantly ameliorated osteolysis and endoplasmic reticulum stress intensity in mice. Simultaneously, it reduced inflammatory cell infiltration, osteoclastogenesis, and inflammatory responses by inhibiting GRP78, IRE 1, and ATF6. These findings suggest that tacrolimus exhibits an anti-inflammation effect in rheumatoid arthritis and might inhibit joint damage progression by inhibiting endoplasmic reticulum stress.
Assuntos
Artrite/metabolismo , Osteogênese/efeitos dos fármacos , Tacrolimo/farmacologia , Animais , Artrite/induzido quimicamente , Artrite/fisiopatologia , Artrite Experimental , Colágeno , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inflamação/tratamento farmacológico , Interleucina-1beta/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/metabolismo , Tapsigargina/farmacologiaRESUMO
The store-operated calcium entry (SOCE) pathway is an important route for generating cytosolic Ca2+ signals that regulate a diverse array of biological processes. Abnormal SOCE seem to underlie several diseases that notably include allergy, inflammation and cancer. Therefore, any modulator of this pathway is likely to have significant impact in cell biology under both normal and abnormal conditions. In this study, we screened the FDA-approved drug library for agents that share significant similarity in 3D shape and surface electrostatics with few, hitherto best known inhibitors of SOCE. This has led to the identification of five drugs that showed dose-dependent inhibition of SOCE in cell-based assay, probably through interacting with the Orai1 protein which effectively mediates SOCE. Of these drugs, leflunomide and teriflunomide could suppress SOCE significantly at clinically-relevant doses and this provides for an additional mechanism towards the therapeutic utility of these drugs as immunosuppressants. The other three drugs namely lansoprazole, tolvaptan and roflumilast, were less potent in suppressing SOCE but were more selective and thus they may serve as novel scaffolds for future development of new, more efficacious SOCE inhibitors.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Aprovação de Drogas , United States Food and Drug Administration , Anilidas/farmacologia , Animais , Bioensaio , Sinalização do Cálcio/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Células HeLa , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Molécula 1 de Interação Estromal/metabolismo , Tapsigargina/farmacologia , Tiadiazóis/farmacologia , Estados Unidos , Interface Usuário-ComputadorRESUMO
AIM: To investigate the effects of a water extract of Hwangryunhaedok-tang (HHTE) on the pacemaker potentials of mouse interstitial cells of Cajal (ICCs). METHODS: We dissociated ICCs from small intestines and cultured. ICCs were immunologically identified using an anti-c-kit antibody. We used the whole-cell patch-clamp configuration to record the pacemaker potentials generated by cultured ICCs under the current clamp mode (I = 0). All experiments were performed at 30 °C-32 °C. RESULTS: HHTE dose-dependently depolarized ICC pacemaker potentials. Pretreatment with a 5-HT3 receptor antagonist (Y25130) or a 5-HT4 receptor antagonist (RS39604) blocked HHTE-induced pacemaker potential depolarizations, whereas pretreatment with a 5-HT7 receptor antagonist (SB269970) did not. Intracellular GDPßS inhibited HHTE-induced pacemaker potential depolarization and pretreatment with a Ca2+-free solution or thapsigargin abolished the pacemaker potentials. In the presence of a Ca2+-free solution or thapsigargin, HHTE did not depolarize ICC pacemaker potentials. In addition, HHTE-induced pacemaker potential depolarization was unaffected by a PKC inhibitor (calphostin C) or a Rho kinase inhibitor (Y27632). Of the four ingredients of HHT, Coptidis Rhizoma and Gardeniae Fructus more effectively inhibited pacemaker potential depolarization. CONCLUSION: These results suggest that HHTE dose-dependently depolarizes ICC pacemaker potentials through 5-HT3 and 5-HT4 receptors via external and internal Ca2+ regulation and via G protein-, PKC- and Rho kinase-independent pathways.
Assuntos
Relógios Biológicos/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Células Intersticiais de Cajal/efeitos dos fármacos , Intestino Delgado/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cálcio/metabolismo , Células Cultivadas , Medicamentos de Ervas Chinesas/química , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Células Intersticiais de Cajal/fisiologia , Intestino Delgado/citologia , Camundongos , Camundongos Endogâmicos ICR , Oxazinas/farmacologia , Técnicas de Patch-Clamp , Fenóis/farmacologia , Piperidinas/farmacologia , Extratos Vegetais/química , Propano/análogos & derivados , Propano/farmacologia , Proteína Quinase C/metabolismo , Receptores de Serotonina/metabolismo , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT4 de Serotonina/farmacologia , Sulfonamidas/farmacologia , Tapsigargina/farmacologia , Quinases Associadas a rho/metabolismoAssuntos
Arabidopsis/fisiologia , Chlamydomonas reinhardtii/efeitos da radiação , Criptocromos/metabolismo , Lipídeos/fisiologia , Thapsia/química , Tapsigargina/metabolismo , Tylenchoidea/fisiologia , Animais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/parasitologia , Chlamydomonas reinhardtii/genética , Coproporfirinogênios/metabolismo , Criptocromos/genética , Metilação de DNA , Flores/crescimento & desenvolvimento , Heme/biossíntese , Homeostase , Interações Hospedeiro-Parasita , Luz , Mitocôndrias/metabolismo , Óvulo Vegetal/crescimento & desenvolvimento , Dormência de Plantas/fisiologia , Pólen/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Tetrapirróis/biossínteseRESUMO
The antiplasmodial active extract of Xanthium cavanillesii contains 3,4-dicaffeoyl quinic acid (3,4-DCQA), 3,5-dicaffeoyl quinic acid (3,5-DCQA) and 1,3,5-tricaffeoyl quinic acid (1,3,5-TCQA). These results inspired us to investigate the interaction of these molecules with a promising validated target of Plasmodium, PfATP6 orthologue of mammalian Ca+2-ATPase. Models of this receptor were obtained through comparative modelling. Afterwards, molecular docking studies were used to identify possible interaction modes of these caffeoyl quinic derivatives on the binding site. The 1,3,5-TCQA had the best energy, but all of these had better energy than thapsigargin, a non-competitive inhibitor of the sarco/endoplasmatic reticulum Ca+2-ATPase (SERCA).