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1.
Sheng Li Xue Bao ; 71(5): 681-688, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31646321

RESUMO

Polyamines (putrescine, spermidine, and spermine) are essential polycations that play important roles in various physiological and pathophysiological processes in mammalian cells. The study was to investigate their role in cardioprotection against ischemia/reperfusion (I/R) injury and the underlying mechanism. Isolated hearts from male Sprague-Dawley rats were Langendorff-perfused and cardiac I/R was achieved by 30 min of global ischemia followed by 120 min of reperfusion. Different concentrations of polyamines (0.1, 1, 10, and 15 µmol/L of putrescine, spermidine, and spermine), cyclosporin A (0.2 µmol/L), or atractyloside (20 µmol/L) were given 10 min before the onset of reperfusion. The hemodynamics were monitored; the lactate dehydrogenase (LDH) levels in the coronary effluent were measured spectrophotometrically; infarct size was determined by the 2,3,5-triphenyltetrazolium chloride staining method; and mitochondrial permeability transition pore (MPTP) opening was determined spectrophotometrically by the Ca2+-induced swelling of isolated cardiac mitochondria. The results showed that compared to I/R alone, 0.1 and 1 µmol/L polyamines treatment improved heart function, reduced LDH release, decreased infarct size, and these effects were inhibited by atractyloside (MPTP activator). In isolated mitochondria from normal rats, 0.1 and 1 µmol/L polyamines treatment inhibited MPTP opening. However, 10 and 15 µmol/L polyamines treatment had the opposite effects, and these effects were inhibited by cyclosporin A (MPTP inhibitor). Our findings showed that polyamines may have either protective or damaging effects on hearts suffering from I/R by inhibiting or activating MPTP opening.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Poliaminas/metabolismo , Animais , Ciclosporina/farmacologia , Masculino , Mitocôndrias Cardíacas/fisiologia , Ratos , Ratos Sprague-Dawley
2.
Acta Cir Bras ; 34(8): e201900806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618406

RESUMO

PURPOSE: To assess Cyclosporine A (CsA) therapy at an intraperitoneal dose of 15 mg.kg -1 in a rodent model of non-septic renal ischemia. METHODS: Twenty male Wistar rats were randomized to receive CsA therapy or none therapy before undergoing 30 minutes of renal ischemia followed by reperfusion. Additionally, 10 rats were randomized to undergo the same surgical procedure of the aforementioned animals with neither ischemia nor CsA therapy. Twelve hours after kidney ischemia, the left kidneys were evaluated for histological injury according to Park's criteria. Serum creatinine (Cr), urea nitrogen (Ur) and sodium levels were obtained at different times of the experimental protocol. RESULTS: Rodents in the CsA group showed negative results (p<0.05) in serum variables (Cr: 0.41±0.05mg/dL vs . 4.17±1.25mg/dL; Ur: 40.90±3.98mg/dL vs . 187.70±22.93mg/dL) even the non CsA or control group (Cr: 0.35±0.07mg/dL vs . 3.80±1.20mg/dL; Ur: 40.10±4.70mg/dL vs . 184.50±49.80mg/dL). The negative results were also verified in histological evaluation, CsA group had 50% in the very severe grade of lesion, 10% in the severe and 40% in the moderate to severe whereas the control group had 90% in the very severe grade. CONCLUSION: CsA was incapable of preventing the deleterious effects of ischemia-reperfusion injury in rat kidneys.


Assuntos
Ciclosporina/farmacologia , Imunossupressores/farmacocinética , Rim/irrigação sanguínea , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Creatinina/sangue , Modelos Animais de Doenças , Isquemia/prevenção & controle , Rim/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Sódio/sangue , Ureia/sangue
3.
Life Sci ; 235: 116841, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31494173

RESUMO

Indanyloxyacetic acid-94 (IAA-94), an intracellular chloride channel blocker, is shown to ablate cardioprotection rendered by ischemic preconditioning (IPC), N (6)-2-(4-aminophenyl) ethyladenosine or the PKC activator phorbol 12-myristate 13-acetate and cyclosporin A (CsA) in both ex-vivo and in-vivo ischemia-reperfusion (IR) injury. Thus signifying the role of the IAA-94 sensitive chloride channels in mediating cardio-protection upon IR injury. Although IAA-94 sensitive chloride currents are recorded in cardiac mitoplast, there is still a lack of understanding of the mechanism by which IAA-94 increases myocardial infarction (MI) by IR injury. Mitochondria are the key arbitrators of cell life and death pathways. Both oxidative stress and calcium overload in the mitochondria, elicit pathways resulting in the opening of mitochondrial permeability transition pore (mPTP) leading to cell death. Therefore, in this study we explored the role of IAA-94 in MI and in maintaining calcium retention capacity (CRC) of cardiac mitochondria after IR. IAA-94 inhibited the CRC of the isolated cardiac mitochondria in a concentration-dependent manner as measured spectrofluorimetrically using calcium green-5 N. Interestingly, IAA-94 did not change the mitochondrial membrane potential. Further, CsA a blocker of mPTP opening could not override the effect of IAA-94. We also showed for the first time that IAA-94 perfusion after ischemic event augments MI by reducing the CRC of mitochondria. To conclude, our results demonstrate that the mechanism of IAA-94 mediated cardio-deleterious effects is via modulating the mitochondria CRC, thereby playing a role in mPTP opening. These findings highlight new pharmacological targets, which can mediate cardioprotection from IR injury.


Assuntos
Cálcio/metabolismo , Glicolatos/efeitos adversos , Infarto do Miocárdio/metabolismo , Animais , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Glicolatos/antagonistas & inibidores , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/induzido quimicamente , Ratos
4.
Vet Immunol Immunopathol ; 216: 109892, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31446206

RESUMO

Cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases in dogs. Cyclosporine inhibits calcineurin-dependent pathways of T cell activation and resultant T cell cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. Little work has been done comparing the effects of these agents on T cell cytokine production in dogs. Our study measured T cell interleukin-2 (IL-2) and interferon-gamma (IFN-γ) production using flow cytometry and T cell IL-2 and IFN-γ gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in activated canine T cells incubated with cyclosporine and dexamethasone in vitro. For flow cytometric assays, diluted whole blood was cultured for 7 h in the presence of cyclosporine (10, 100, 500, and 1000 ng/mL) or dexamethasone (10 ng/mL, 100 ng/mL, 1 µg/mL, and 10 µg/mL). For qRT-PCR, whole blood was cultured for 5 h with the same drugs at the same concentrations, and RNA was then extracted from leukocytes. Flow cytometry and qRT-PCR both demonstrated inhibition of IL-2 and IFN-γ that was concentration-dependent in response to cyclosporine, and was more variable for dexamethasone. Quantitative RT-PCR but not flow cytometry documented significant reduction of IL-2 expression after dexamethasone treatment, while both methods showed concentration-dependent suppression of IFN-γ. Quantitative RT-PCR also revealed additional cytokine suppression at higher cyclosporine concentrations, an effect not found using flow cytometry, and may therefore be the preferred method for cytokine determination in dogs. Suppression of IL-2 and IFN-γ in activated T cells may have potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine T cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients.


Assuntos
Ciclosporina/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Linfócitos T/efeitos dos fármacos , Animais , Ciclosporina/administração & dosagem , Dexametasona/administração & dosagem , Cães , Relação Dose-Resposta a Droga , Glucocorticoides/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunossupressores/administração & dosagem , Imunossupressores/farmacologia , Interferon gama/genética , Interleucina-2/genética
5.
J Photochem Photobiol B ; 198: 111578, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31408842

RESUMO

In recent years, biological nanomedicine-based biomaterials have an extreme attention for biomedical uses, herein we examined a novel kind advance of photoluminescent Graphene quandum dots encapsulated mesoporous nanoparticles (GND@MSNs) encapsulated by well-known anticancer drugs Doxorubicin (DOX) and Cyclosporin (CsA) for lung carcinoma. Electron microscopic technique exhibit the nanostructure and spherical morphology of GND@MSNs+DOX+CsA with mean size ≈110 nm. Moreover, Dynamic Light Scattering (DLS) exposed that blended GND@MSNs+DOX+CsA nanoparticles were highly stable with extremely negatively charged nanoparticles. Raman investigation was done on the all naturally dynamic nanoparticles containing shed graphene to survey the blend condition of the graphene inside the silica mesoporous nanoparticles. GND@MSNs+DOX+CsA provided an outstanding anti-cancer efficiency against the lung cancer cell lines (i.e., A549 and HEL-299). MTT assay monitored that GND@MSNs, GND@MSNs+DOX and GND@MSNs+DOX+CsA have a robust toxicity behaviour on the A549 and HEL-299 model lung cancer cell lines. Additionally, investigation of the cell death was found on AO-EB, Hoechst 33452 staining and flowcytometry techniques. Furthermore, the DNA damage were confirmed by cell cycle arrest and comet assay. Hence, we suggesting that these GND@MSNs+DOX+CsA could be applied as auspicious drug vesicles for novel lung cancer therapeutic potential and new openings to solve the complexity of lung cancer in the care of cancer patients.


Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/farmacologia , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Grafite/química , Nanopartículas/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclosporina/química , Ciclosporina/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Porosidade
6.
Int J Mol Sci ; 20(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108847

RESUMO

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.


Assuntos
Benzoquinonas/farmacologia , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ciclofilinas/química , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Monócitos/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteólise , Receptores de Interleucina/química , Receptores de Interleucina/genética
7.
Biochim Biophys Acta Gene Regul Mech ; 1862(8): 846-857, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31054961

RESUMO

Heat shock factor 1 (HSF1) is a transcription factor essential for tumorigenesis, and targeting HSF1 may be effective in combined therapeutics for cervical cancer. Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation. However, the roles and regulatory mechanisms by which CsA modulates HSP expression remain largely unknown. In this study, we found that CsA pretreatment prevented induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 on HSF1 and thus inhibiting its transcriptional activity. Suppression of ERK1/2, GSK3ß and CK2 activities attenuated CsA-induced down-regulation of HSP expression and up-regulation of HSF1 phosphorylation. CsA interfered with HSF1-SSBP1 complex formation and HSF1 nuclear translocation and recruitment to the HSP70 promoter. CsA clearly caused HeLa cell death during proteotoxic stress through reduced expression of HSPs. These results indicate that CsA suppresses HSP induction during heat shock by regulating the phosphorylation and nuclear translocation of HSF1. Our results provide a conceptual framework for the development of novel therapeutic strategies for cervical cancer through application of CsA during hyperthermia or chemotherapy.


Assuntos
Ciclosporina/farmacologia , Fatores de Transcrição de Choque Térmico/metabolismo , Hipertermia Induzida/métodos , Neoplasias do Colo do Útero/metabolismo , Terapia Combinada , Feminino , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Resposta ao Choque Térmico , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Serina/metabolismo , Neoplasias do Colo do Útero/terapia
8.
Drug Deliv ; 26(1): 542-550, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31090467

RESUMO

Self-nanoemulsifying drug delivery system (SNEDDS) have been considered as a promising platform for oral delivery of many BCS (biopharmaceutics classification system) class IV drugs, such as docetaxel (DTX). However, oral chemotherapy with DTX is also restricted by its active P-glycoprotein (P-gp) efflux and hepatic first-pass metabolism. To address these challenges, we developed a novel SNEDDS co-loaded with DTX and cyclosporine A (CsA) to achieve effective inhibition of P-gp efflux and P450 enzyme metabolization, improving oral bioavailability of DTX. The SNEDDS showed uniform droplet size of about 30 nm. Additionally, the prepared SNEDDS exhibited a sequential drug release trend of CsA prior to DTX. The intestinal experiments confirmed that the membrane permeability of DTX was significantly increased in the whole intestinal tract, especially in the jejunum segment. Furthermore, the oral bioavailability of co-loaded SNEDDS was 9.2-fold and 3.4-fold higher than DTX solution and DTX SNEDDS, respectively. More importantly, it exhibited a remarkable antitumor efficacy with a reduced toxicity compared with intravenously administered DTX solution. In summary, DTX-CsA co-loaded SNEDDS is a promising platform to facilitate oral docetaxel-based chemotherapy.


Assuntos
Antineoplásicos/administração & dosagem , Ciclosporina/administração & dosagem , Docetaxel/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ciclosporina/farmacocinética , Ciclosporina/farmacologia , Docetaxel/farmacocinética , Docetaxel/uso terapêutico , Liberação Controlada de Fármacos , Emulsões , Feminino , Absorção Intestinal , Camundongos Endogâmicos BALB C , Nanopartículas/química , Ratos Sprague-Dawley , Solubilidade , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Mol Cell Biochem ; 458(1-2): 159-169, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31020492

RESUMO

Cyclosporin A (CSA) is a widely used drug to prevent the immune cell function. It is well known that CSA blocks transcription of cytokine genes in activated T cells. The connection between T cells and CSA has been well established. However, the effect of CSA on natural killer (NK) cells is not thoroughly understood. Therefore, in the present study, splenocytes and peripheral blood mononuclear cells (PBMCs) were treated with CSA in the presence of concanavalin A (Con A) or interleukin-2 (IL-2). CSA at higher concentrations induces apoptosis and inhibition of proliferation, while lower concentrations showed synergistically enhanced proliferation in splenocytes and PBMCs. Further, CSA favored the in vitro conversion of CD3+CD161+ cells. Splenocytes and PBMC were found to have synergistic proliferation with Con A, and PBMC exhibited significantly higher expression of NKp30, NKp44, and granzyme B along with enhanced cytotoxicity against K-562 cells in CSA-treated animals. Proliferation assay also showed that proliferation of CD161+ cells was higher in CSA-treated animals. Collectively, our results suggest that CSA differentially influences the population, function, and expression of the NK cell phenotype.


Assuntos
Complexo CD3/imunologia , Proliferação de Células/efeitos dos fármacos , Ciclosporina/farmacologia , Células Matadoras Naturais/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Concanavalina A/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Granzimas/imunologia , Humanos , Interleucina-2/farmacologia , Células K562 , Células Matadoras Naturais/citologia , Masculino , Receptor 2 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Ratos , Ratos Sprague-Dawley
10.
Cell Physiol Biochem ; 52(5): 1075-1091, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977989

RESUMO

BACKGROUND/AIMS: Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia. METHODS: In vivo, kidneys of mice challenged daily with CsA for one week were analyzed by microarray analysis, gene ontology analysis, and qPCR. In vitro, renal cells were subjected to hypoxia (1 % O2) which was either episodic (4 h for 6 consecutive days), short-term (4 h), or sustained (24 h). Western blot analysis quantified hypoxia-inducible factor-1α (HIF-1α). 2',7'-dichlorofluorescein diacetate detected intracellular ROS. After re-oxygenation, staurosporine served to induce apoptosis, quantified by active caspase-3. RESULTS: In vivo, HIF target gene expression was suppressed by daily CsA treatment. Yet, we found up-regulation of genes involved in defence against cellular stress, notably against ROS. Renal cells in vitro behaved largely different under episodic and sustained hypoxia, while their response to short-term hypoxia oscillated between the previous two. Episodic hypoxia exhibited the highest total HIF-1α protein level, lowest nucleus-to-cytoplasm ratio, and lowest HIF target gene expression. When compared with normoxia, re-oxygenation after sustained hypoxia increased ROS by 3.04 ± 1.04 fold (p<0.001), and re-oxygenation after episodic hypoxia by 1.26 ± 0.16 fold (p<0.01). Staurosporine-induced active caspase-3 was highest after sustained, and lowest after episodic hypoxia. CONCLUSION: In vitro episodic hypoxia mimics the largely HIF-independent transcriptome observed after repetitive CsA treatment in vivo. In vitro preconditioning with episodic hypoxia protects against stress-induced apoptosis. Despite of its long-term adverse effects, CsA derived episodic hypoxia induces a unique renal hypoxia response that provides adaptation to re-oxygenation mediated ROS damage.


Assuntos
Adaptação Fisiológica , Apoptose , Hipóxia , Rim , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Rim/irrigação sanguínea , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Camundongos , Camundongos Transgênicos
11.
Artigo em Inglês | MEDLINE | ID: mdl-30995791

RESUMO

Immunosuppressive drugs are widely used to avoid graft rejection, but they are also known to be strongly hepatotoxic. The goal of the current study was to determine: (i) the immunoexpression of SOD1, CAT, GPX1; (ii) the concentration of MDA, GSH; (iii) the activity of SOD, CAT, GPX, in the native liver of a pregnant female rats undergoing immunosuppressive therapy. The study was based on archival material obtained from Department of Nephrology, Transplantology and Internal Medicine of the Independent Public Clinical Hospital No. 2 at the Pomeranian Medical University in Szczecin, Poland. The study was carried out on 32 female rats exposed to oral administration of immunosuppressants two weeks before and during pregnancy. The percentage of SOD1 immunopositive hepatocytes in rats treated with cyclosporine A, mycophenolate mofetil, everolimus, and glucocorticosteroid was significantly elevated above that of the control rats. The concentration of MDA in the liver of animals exposed to cyclosporine A, everolimus, and glucocorticosteroid was significantly higher than in other groups. Among the groups of dams treated with immunosuppressive drugs, the highest significant concentration of GSH was found in the livers of rats treated with cyclosporine A, mycophenolate mofetil and glucocorticosteroid. Immunosuppressive therapy during pregnancy affects the oxidoreductive balance in the livers of rats, depending on the regimen used.


Assuntos
Imunossupressores/farmacologia , Animais , Ciclosporina/farmacologia , Everolimo/farmacologia , Feminino , Rejeição de Enxerto , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Oxirredução , Gravidez , Ratos , Ratos Wistar
12.
Toxicol In Vitro ; 58: 230-238, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30930230

RESUMO

The alteration of thyroxine (T4) cellular uptake by an environmental chemical can serve as a contributing factor in thyroid hormone (TH) disruption. Herein, we describe a non-radiolabeled (LC-MS/MS) oil-filtration technique designed to characterize the mechanism(s) responsible for T4 cellular uptake in cryopreserved rat hepatocyte suspensions. The environmental chemicals perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) were evaluated for their effect on T4 hepatic uptake. At 37 °C, hepatic assays demonstrated saturable kinetics with increasing T4 concentrations, while a linear uptake rate consistent with passive diffusion was detected at 4 °C. Carrier-mediated (37-4 °C) transport of T4 was the predominant hepatic uptake process versus passive diffusion. Cyclosporin A (CsA) chemically inhibited T4 hepatic uptake, whereas PFOA/PFOS displayed no inhibition of T4 translocation. Increasing PFOA/PFOS concentration levels with the T4 serum carrier-protein transthyretin (TTR) present resulted in a dose-response increase in T4 hepatic uptake rates, correlating with increased T4 free fraction values. Hepatic assays conducted in the presence of PFOA/PFOS and TTR displayed an enhanced first-order T4 hepatic uptake rate consistent with carrier-mediated transport. These in vitro findings characterizing increased T4 hepatic uptake provides mechanistic insight regarding decreased T4 serum levels (hypothyroxinemia) previously observed within in vivo rodent studies following perfluorinated chemical exposure.


Assuntos
Ácidos Alcanossulfônicos/farmacocinética , Caprilatos/farmacocinética , Poluentes Ambientais/farmacocinética , Fluorcarbonetos/farmacocinética , Hepatócitos , Tiroxina/metabolismo , Animais , Criopreservação , Ciclosporina/farmacologia , Masculino , Ratos Sprague-Dawley , Suspensões
13.
Med Sci Monit ; 25: 1637-1644, 2019 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-30826814

RESUMO

BACKGROUND Accumulating evidence has indicated that S100B protein may be involved in the pathophysiology of ischemia-reperfusion brain injury. Cyclosporine has been shown to have neuroprotective functions. This study investigated the effect of cyclosporine on S100B serum levels and the severity of brain tissue damage in a rat model of cerebral ischemia-reperfusion (I/R). MATERIAL AND METHODS Twelve-week-old Wistar male rats were randomly divided into Control I/R and Cyclosporine I/R groups (n=10 each). Cyclosporine was given orally by gavage for 5 days prior to cerebral I/R, at a total volume of 15 mg/kg/day. The Control group received an equal volume of saline. Body weight was measured and all animals were subjected to 60-min focal ischemia by filament occlusion of the middle cerebral artery. ELISA was used to assess the concentrations of serum S100B and development of brain infarct size and neurological outcomes were determined at 2 and 24 h after occlusion withdrawal. RESULTS Cyclosporine improved the neurological deficit score and decreased the cerebral infarct size and body weight. S100B serum levels were significantly elevated in Cyclosporine-treated rats compared with untreated Control rats during the reperfusion phase. Total infarct size was positively associated with S100B serum levels in the Control I/R group, but no significant correlation was observed in the Cyclosporine I/R group. CONCLUSIONS Cyclosporine seems to affect both ischemia-reperfusion brain tissue damage and S100B protein serum levels. S100B serum level appears to be a state marker for the severity of the cerebral ischemia-reperfusion, rather than a trait marker for Cyclosporine responsiveness.


Assuntos
Ciclosporina/farmacologia , Traumatismo por Reperfusão/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Infarto Cerebral , Masculino , Fármacos Neuroprotetores/farmacologia , Prognóstico , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100/fisiologia
14.
Mater Sci Eng C Mater Biol Appl ; 98: 472-481, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30813049

RESUMO

Cyclosporine A (CsA) is an extremely hydrophobic immunosuppressive drug, whose systemic administration to suppress the activity of T cells and T cell-based immune responses is frequently associated with a number of adverse drug reactions. Local delivery of CsA focused on a specific target organ has been proposed as a possible solution to this problem. In this study, we developed biodegradable sol-gel drug delivery systems, consisting of HA-Ca-Alg hydrogels combining hyaluronic acid calcium complex (HA-Ca) and sodium alginate (Alg-Na) components, for the local sustained delivery of CsA. A HA-Ca complex with very high degree of substitution was prepared by the acid-base reaction of hyaluronic acid and calcium acetate. The gelation was completed within about 2-45 min without external addition of calcium salts such as CaSO4 and CaCl2, indicating the high potential of the present hydrogel systems for drug delivery by injection in vivo. The HA-Ca system was characterized by high-resolution inductively coupled plasma-optical emission spectroscopy, 1H NMR, FT-IR, and thermogravimetric analysis methods. Moreover, the scanning electron microscopy analysis of the HA-Ca-Alg hydrogels showed an irregular porous morphology, with interconnected pores of 50-300 µm width. The sol-gel transition and the maximum viscosity (about 10,000 cP) of the HA-Ca-Alg hydrogels were characterized by examining the time evolution of the viscosity at 37 °C. The hydrolytic degradation of the HA-Ca-Alg hydrogel was also examined at 37 °C. CsA-encapsulated HA-Ca-Alg hydrogels exhibited sustained in vitro release of CsA over 14 days, which was confirmed through in vitro measurements of the activity of murine T cells over 2 weeks. These results show that the present injectable HA-Ca-Alg hydrogels can be used effectively for the sustained delivery of extremely hydrophobic immunosuppressive drugs, including CsA.


Assuntos
Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos , Hidrogéis/química , Imunossupressores/administração & dosagem , Injeções , Alginatos/química , Animais , Cálcio/química , Proliferação de Células/efeitos dos fármacos , Ciclosporina/administração & dosagem , Ciclosporina/farmacologia , Feminino , Ácido Hialurônico/química , Interleucina-2/biossíntese , Camundongos Endogâmicos C57BL , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Termogravimetria , Viscosidade
15.
PLoS One ; 14(3): e0210771, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925148

RESUMO

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future.


Assuntos
Ciclofilinas/química , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ciclosporina/química , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Ureia/farmacologia
16.
EBioMedicine ; 42: 326-339, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30879923

RESUMO

BACKGROUND: Crizotinib has potent anti-tumor activity in patients with advanced MET-amplified non-small cell lung cancer (NSCLC). However, the therapeutic effect is still not satisfying. Thus, developing approaches that improve the efficacy of crizotinib remains a significant challenge. METHODS: MET-amplified NSCLC cell lines were treated with crizotinib and cyclosporine A (CsA). Cell viability was determined by MTS assay. The changes of apoptosis, cell cycle and calcineurin-Erk pathways were assessed by western blot. Xenograft mouse model, primary human NSCLC cells and hollow fiber assays were utilized to confirm the effects of CsA. FINDINGS: We demonstrated that CsA significantly increased the anti-tumor effect of crizotinib on multiple MET-amplified NSCLC cells in vitro and in vivo. Mechanistically, crizotinib treatment led to the activation of Ca2+-calcineurin (CaN)-Kinase suppressor of Ras 2 (KSR2) signaling, resulting in Erk1/2 activation and enhanced survival of cancer cells. CsA effectively blocked CaN-KSR2-Erk1/2 signaling, promoting crizotinib-induced apoptosis and G2/M arrest. Similarly, pharmacologic or genetic inhibition of Erk1/2 also enhanced crizotinib-induced growth inhibition in vitro. Xenograft studies further confirmed that CsA or Erk1/2 inhibitor PD98059 enhanced the anti-cancer activity of crizotinib through inhibition of CaN-Erk1/2 axis. The results were also validated by primary human NSCLC cells in vitro and hollow fiber assays in vivo. INTERPRETATION: This study provides preclinical evidences that combination therapy of CsA and crizotinib is a promising approach for targeted treatment of MET-amplified lung cancer patients. FUND: This work was supported by the National Natural Science Foundation of China, the Key Projects of Natural Foundation of Zhejiang Province, the Ten thousand plan youth talent support program of Zhejiang Province, the Zhejiang Natural Sciences Foundation Grant, and the Zhejiang medical innovative discipline construction project-2016.


Assuntos
Antineoplásicos/farmacologia , Calcineurina/metabolismo , Cálcio/metabolismo , Crizotinibe/farmacologia , Ciclosporina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sinergismo Farmacológico , Amplificação de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas c-met/genética
17.
Int Immunopharmacol ; 69: 358-367, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30776644

RESUMO

27-Hydroxycholesterol (27OHChol) is a bioactive molecule that induces monocytic cell activation and differentiation and thereby participates in immune responses under hypercholesterolemic condition. However, it is unknown whether cyclosporin A (CsA), an immunosuppressant, affects biological effects of 27OHChol. In this study, we investigated whether CsA alters 27OHChol-induced cellular and molecular responses using the human monocyte/macrophage THP-1 cells. Treatment of the cells with CsA resulted in decreased expression of the mDC-specific markers (CD80, CD83 and CD88) induced by 27OHChol. Reduced endocytic activity recovered in the presence of CsA. The drug also inhibited the expressions of MHC class I and II molecules and CD197, a homing molecule of mDCs. We further investigated the outcomes of CsA treatment on the expression of M1 polarization markers and CD14, a component of the innate immune system. The drug decreased transcript levels of genes associated with the M1 polarization of monocytic cells, including CCL2, as well as expression of CD14 and MMP-9 which is involved in soluble CD14 shedding. Taken together, these results indicate that CsA inhibits the 27OHChol-induced differentiation and activation of monocytic cells into a mature dendritic cell (mDC) type and an immuno-stimulatory M1 subset, respectively, thereby modifying immune responses in a milieu rich in cholesterol and oxidized cholesterol molecules.


Assuntos
Ciclosporina/farmacologia , Hidroxicolesteróis/farmacologia , Imunossupressores/farmacologia , Macrófagos/fisiologia , Monócitos/fisiologia , Diferenciação Celular , Quimiocina CCL2/genética , Citocinas/metabolismo , Humanos , Imunidade Inata , Imunização , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Células THP-1 , Células Th1/imunologia
18.
PLoS One ; 14(2): e0212818, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794682

RESUMO

BACKGROUND: Growth Arrest and DNA Damage 45γ (GADD45γ) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45γ in renal tubular cell apoptosis induced by nephrotoxic drugs. METHODS: Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45γ expression was silenced, HRE cells were transduced with a plasmid encoding GADD45γ-specific shRNA. The recombinant adenovirus containing the GADD45γ gene was synthesized to overexpress GADD45γ protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry. RESULTS: Expression of the GADD45γ gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45γ, and significantly augmented by the overexpression of GADD45γ. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45γ, and was augmented by the overexpression of GADD45γ, indicating that caspase activation is dependent on the expression of GADD45γ. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part. CONCLUSIONS: To our knowledge, this is the first study to show that GADD45γ is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.


Assuntos
Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túbulos Renais/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase/farmacologia , Linhagem Celular , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Cicloexilaminas/farmacologia , Ciclosporina/efeitos adversos , Ciclosporina/farmacologia , Células Epiteliais/patologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Túbulos Renais/patologia , Fenilenodiaminas/farmacologia
19.
Oncogene ; 38(22): 4264-4282, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30718919

RESUMO

While HER2 and EGFR are overexpressed in breast cancers and multiple other types of tumors, the use of EGFR and/or HER2 inhibitors have failed to cure many cancer patients, largely because cancers acquire resistance to HER2/EGFR-specific drugs. Cancers that overexpress the HER-family proteins EGFR, HER2, and HER3 are uniquely sensitive to agents that disrupt HER2 and EGFR protein folding. We previously showed that disruption of disulfide bond formation by Disulfide Disrupting Agents (DDAs) kills HER2/EGFR overexpressing cells through multiple mechanisms. Herein, we show that interference with proline isomerization in HER2/EGFR overexpressing cells also induces cancer cell death. The peptidyl-prolyl isomerase inhibitor Cyclosporine A (CsA) selectively kills EGFR+ or HER2+ breast cancer cells in vitro by activating caspase-dependent apoptotic pathways. Further, CsA synergizes with the DDA tcyDTDO to kill HER2/EGFR overexpressing cells in vitro and the two agents cooperate to kill HER2+ tumors in vivo. There is a critical need for novel strategies to target HER2+ and EGFR+ cancers that are resistant to currently available mechanism-based agents. Drugs that target HER2/EGFR protein folding, including DDAs and CsA, have the potential to kill cancers that overexpress EGFR or HER2 through the induction of proteostatic synthetic lethality.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
J Nanobiotechnology ; 17(1): 18, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683110

RESUMO

BACKGROUND: Cyclosporin A (CsA) is a promising therapeutic drug for myocardial ischemia reperfusion injury (MI/RI) because of its definite inhibition to the opening of mitochondrial permeability transition pore (mPTP). However, the application of cyclosporin A to treat MI/RI is limited due to its immunosuppressive effect to other normal organ and tissues. SS31 represents a novel mitochondria-targeted peptide which can guide drug to accumulate into mitochondria. In this paper, mitochondria-targeted nanoparticles (CsA@PLGA-PEG-SS31) were prepared to precisely deliver cyclosporin A into mitochondria of ischemic cardiomyocytes to treat MI/RI. RESULTS: CsA@PLGA-PEG-SS31 was prepared by nanoprecipitation. CsA@PLGA-PEG-SS31 showed small particle size (~ 50 nm) and positive charge due to the modification of SS31 on the surface of nanoparticles. CsA@PLGA-PEG-SS31 was stable for more than 30 days and displayed a biphasic drug release pattern. The in vitro results showed that the intracellular uptake of CsA@PLGA-PEG-SS31 was significantly enhanced in hypoxia reoxygenation (H/R) injured H9c2 cells. CsA@PLGA-PEG-SS31 delivered CsA into mitochondria of H/R injured H9c2 cells and subsequently increased the viability of H/R injured H9c2 cell through inhibiting the opening of mPTP and production of reactive oxygen species. In vivo results showed that CsA@PLGA-PEG-SS31 accumulated in ischemic myocardium of MI/RI rat heart. Apoptosis of cardiomyocyte was alleviated in MI/RI rats treated with CsA@PLGA-PEG-SS31, which resulted in the myocardial salvage and improvement of cardiac function. Besides, CsA@PLGA-PEG-SS31 protected myocardium from damage by reducing the recruitment of inflammatory cells and maintaining the integrity of mitochondrial function in MI/RI rats. CONCLUSION: CsA@PLGA-PEG-SS31 exhibited significant cardioprotective effects against MI/RI in rats hearts through protecting mitochondrial integrity, decreasing apoptosis of cardiomyocytes and myocardial infract area. Thus, CsA@PLGA-PEG-SS31 offered a promising therapeutic method for patients with acute myocardial infarction.


Assuntos
Ciclosporina/administração & dosagem , Ciclosporina/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Mitocôndrias/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Oligopeptídeos/química , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Ciclosporina/farmacocinética , Ciclosporina/farmacologia , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Tamanho da Partícula , Ratos
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