RESUMO
OBJECTIVE: Minimally invasive surgery for spontaneous intracerebral hemorrhage is impeded by inadequate lysis of the target blood clot. Ultrasound is thought to expedite intravascular thrombolysis, thereby facilitating vascular recanalization. However, the impact of ultrasound on intracerebral blood clot lysis remains uncertain. This study aimed to explore the feasibility of combining ultrasound with urokinase to enhance blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage. METHODS: The blood clots were divided into four groups: control group, ultrasound group, urokinase group, and ultrasound + urokinase group. Using our experimental setup, which included a key-shaped bone window, we simulated a minimally invasive puncture and drainage procedure for spontaneous intracerebral hemorrhage. The blood clot was then irradiated using ultrasound. Blood clot lysis was assessed by weighing the blood clot before and after the experiment. Potential adverse effects were evaluated by measuring the temperature variation around the blood clot in the ultrasound + urokinase group. RESULTS: A total of 40 blood clots were observed, with 10 in each experimental group. The blood clot lysis rate in the ultrasound group, urokinase group, and ultrasound + urokinase group (24.83 ± 4.67%, 47.85 ± 7.09%, 61.13 ± 4.06%) was significantly higher than that in the control group (16.11 ± 3.42%) (p = 0.02, p < 0.001, p < 0.001). The blood clot lysis rate in the ultrasound + urokinase group (61.13 ± 4.06%) was significantly higher than that in the ultrasound group (24.83 ± 4.67%) (p < 0.001) or urokinase group (47.85 ± 7.09%) (p < 0.001). In the ultrasound + urokinase group, the mean increase in temperature around the blood clot was 0.26 ± 0.15°C, with a maximum increase of 0.38 ± 0.09°C. There was no significant difference in the increase in temperature regarding the main effect of time interval (F = 0.705, p = 0.620), the main effect of distance (F = 0.788, p = 0.563), or the multiplication interaction between time interval and distance (F = 1.100, p = 0.342). CONCLUSIONS: Our study provides evidence supporting the enhancement of blood clot lysis in an in vitro model of spontaneous intracerebral hemorrhage through the combined use of ultrasound and urokinase. Further animal experiments are necessary to validate the experimental methods and results.
Assuntos
Hemorragia Cerebral , Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Hemorragia Cerebral/diagnóstico por imagem , Hemorragia Cerebral/terapia , Terapia por Ultrassom/métodos , Humanos , Trombose , Animais , Terapia Trombolítica/métodos , Fibrinólise/efeitos dos fármacos , Coagulação Sanguínea/efeitos dos fármacosRESUMO
BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus vector-based COVID-19 vaccines. VITT is associated with markedly raised levels of D-dimer; yet, how VITT modulates the fibrinolytic system is unknown. OBJECTIVES: We aimed to compare changes in fibrinolytic activity in plasma from patients with VITT, patients diagnosed with venous thromboembolism (VTE) after vaccination but without VITT (VTE-no VITT), and healthy vaccinated controls. METHODS: Plasma levels of plasmin-antiplasmin (PAP) complexes, plasminogen, and alpha-2-antiplasmin (α2AP) from 10 patients with VITT, 10 patients with VTE-no VITT, and 14 healthy vaccinated controls were evaluated by enzyme-linked immunosorbent assay and/or Western blotting. Fibrinolytic capacity was evaluated by quantitating PAP levels at baseline and after ex vivo plasma stimulation with 50-nM tissue-type plasminogen activator (tPA) or urokinase for 5 minutes. RESULTS: Baseline PAP complex levels in control and VTE-no VITT individuals were similar but were â¼7-fold higher in plasma from patients with VITT (P < .0001). VITT samples also revealed consumption of α2AP and fibrinogenolysis consistent with a hyperfibrinolytic state. Of interest, VITT plasma produced significantly higher PAP levels after ex vivo treatment with tPA, but not urokinase, compared to the other groups, indicative of increased fibrinolytic potential. This was not due to D-dimer as addition of D-dimer to VTE-no VITT plasma failed to potentiate tPA-induced PAP levels. CONCLUSION: A marked hyperfibrinolytic state occurs in patients with VITT, evidenced by marked elevations in PAP, α2AP consumption, and fibrinogenolysis. An unidentified plasma cofactor that selectively potentiates tPA-mediated plasminogen activation also appears to exist in the plasma of patients with VITT.
Assuntos
Antifibrinolíticos , Transtornos da Coagulação Sanguínea , Trombocitopenia , Trombose , Tromboembolia Venosa , Humanos , Antifibrinolíticos/farmacologia , Vacinas contra COVID-19/efeitos adversos , Fibrinolisina/metabolismo , Fibrinólise , Plasminogênio , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Cell invasion is an important process in cancer progression and recurrence. Invasion and implantation of cancer cells from their original place to other tissues, by disabling vital organs, challenges the treatment of cancer patients. Given the importance of the matter, many molecular treatments have been developed to inhibit cancer cell invasion. Because of their low production cost and ease of production, peptides are valuable therapeutic molecules for inhibiting cancer cell invasion. In recent years, advances in the field of computational biology have facilitated the design of anti-cancer peptides. In our investigation, using computational biology approaches such as evolutionary analysis, residue scanning, protein-peptide interaction analysis, molecular dynamics, and free energy analysis, our team designed a peptide library with about 100 000 candidates based on A6 (acetyl-KPSSPPEE-amino) sequence which is an anti-invasion peptide. During computational studies, two of the designed peptides that give the highest scores and showed the greatest sequence similarity to A6 were entered into the experimental analysis workflow for further analysis. In experimental analysis steps, the anti-metastatic potency and other therapeutic effects of designed peptides were evaluated using MTT assay, RT-qPCR, zymography analysis, and invasion assay. Our study disclosed that the IK1 (acetyl-RPSFPPEE-amino) peptide, like A6, has great potency to inhibit the invasion of cancer cells.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tipo Uroquinase , Humanos , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/uso terapêutico , Peptídeos/farmacologia , Invasividade NeoplásicaRESUMO
Cathepsin L plays physiological and pathological roles in immune responses, cancer, metamorphosis, and oogenesis in several species. However, the function of Cathepsin L in medaka ovaries remains unclear. Therefore, here, we examined the physiological functions of Cathepsin L in the medaka ovaries. Cathepsin L mRNA transcripts and proteins were found to be constitutively expressed in the ovaries of Oryzias latipes over a 24-h spawning cycle. Expression was localized within the oocyte cytoplasm of growing follicles and the follicle layer of preovulatory and postovulatory follicles. Moreover, the active form of Cathepsin L was highly expressed in the follicle layer of periovulatory follicles and the ovaries 2-6 h after ovulation. Recombinant Cathepsin L was activated under acidic conditions and exhibited enzymatic activity in acidic and neutral pH conditions. However, extracellular matrix proteins were degraded by recombinant Cathepsin L under acidic, not neutral pH conditions. Cathepsin L was secreted from preovulatory follicles, while active recombinant Cathepsin L was detected in the conditioned medium of a medaka cell line, OLHNI-2. Mechanistically, recombinant Cathepsin L activates recombinant urokinase-type plasminogen activator-1, which is expressed within the follicle layers post-ovulation. Meanwhile, the treatment of medakas with an E-64 or anti-Cathepsin L antibody effectively blocked follicular layer degeneration and degradation after ovulation, whereas in vitro ovulation was not inhibited by either. Collectively, the findings of this study indicate that although Cathepsin L does not impact ovulation in medakas, it contributes to the degeneration and degradation of the follicle layers following ovulation via activation of urokinase-type plasminogen activator-1, and not via the degradation of extracellular matrix proteins.
Assuntos
Oryzias , Ovário , Feminino , Animais , Ovário/fisiologia , Oryzias/fisiologia , Catepsina L/genética , Catepsina L/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Ovulação/fisiologia , Proteínas da Matriz ExtracelularRESUMO
The safe administration of thrombolytic agents is a challenge for the treatment of acute thrombosis. Lipid-based nanoparticle drug delivery technologies present opportunities to overcome the existing clinical limitations and deliver thrombolytic therapy with enhanced therapeutic outcomes and safety. Herein, lipid cubosomes are examined as nanocarriers for the encapsulation of thrombolytic drugs. The lipid cubosomes are loaded with the thrombolytic drug urokinase-type plasminogen activator (uPA) and coated with a low-fouling peptide that is incorporated within a metal-phenolic network (MPN). The peptide-containing MPN (pep-MPN) coating inhibits the direct contact of uPA with the surrounding environment, as assessed by an in vitro plasminogen activation assay and an ex vivo whole blood clot degradation assay. The pep-MPN-coated cubosomes prepared with 22 wt% peptide demonstrate a cell membrane-dependent thrombolytic activity, which is attributed to their fusogenic lipid behavior. Moreover, compared with the uncoated lipid cubosomes, the uPA-loaded pep-MPN-coated cubosomes demonstrate significantly reduced nonspecific cell association (<10% of the uncoated cubosomes) in the whole blood assay, a prolonged circulating half-life, and reduced splenic uPA accumulation in mice. These studies confirm the preserved bioactivity and cell membrane-dependent release of uPA within pep-MPN-coated lipid cubosomes, highlighting their potential as a delivery vehicle for thrombolytic drugs.
Assuntos
Fibrinolíticos , Trombose , Camundongos , Animais , Portadores de Fármacos , Polifenóis , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/uso terapêutico , Lipídeos , Peptídeos/uso terapêuticoRESUMO
The alpha7 nicotinic acetylcholine receptor (α7-nAChR) is considered a promising pharmacological target for the carcinoma therapy. We have previously shown that the recombinant analogue of the human protein SLURP-1 (rSLURP-1) effectively inhibits the growth of carcinomas of various origins via the interaction with α7-nAChR and down-regulation of expression of this receptor. Expression of α7-nAChR is increased in gliomas compared to healthy human brain tissues; however, the role of this receptor in the gliomas development is poorly understood. It was shown for the first time that rSLURP-1 significantly inhibits the growth of glioma model cells U251 MG and A172 up to â¼70%, which is comparable with the effect of α-bungarotoxin, a selective α7-nAChR inhibitor. The half-maximum effective concentrations of rSLURP-1 for U251 MG and A172 cells were 2.82 ± 0.2 and 8.9 ± 0.3 nM, respectively. Coincubation of U251 MG cells with rSLURP-1 and the nAChR inhibitor mecamylamine attenuates the antiproliferative activity of rSLURP-1, indicating nAChR as a molecular target for the rSLURP-1 action in gliomas.
Assuntos
Antígenos Ly/farmacologia , Bungarotoxinas/farmacologia , Glioma/tratamento farmacológico , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Glioma/genética , Glioma/metabolismo , Humanos , Proteínas Recombinantes/farmacologiaRESUMO
The development of personalized therapies represents an urgent need owing to the high rate of cancer recurrence and systemic toxicity of conventional drugs. So far, targeted toxins have shown promising results as potential therapeutic compounds. Specifically, toxins conjugated to antibodies or fused to growth factors/enzymes have been largely demonstrated to selectively address and kill cancer cells. We investigated the anti-tumor potential of a chimeric recombinant fusion protein formed by the Ribosome Inactivating Protein saporin (SAP) and the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), whose receptor has been shown to be over-expressed on the surface of aggressive tumors. ATF-SAP was recombinantly produced by the P. pastoris yeast and its activity was assessed on a panel of bladder and breast cancer cell lines. ATF-SAP resulted to be highly active in vitro, as nano-molar concentrations were sufficient to impair viability on tumor cell lines. In contrast to untargeted toxins, the chimeric fusion protein displayed a significantly improved toxic effect in uPAR-expressing cells, demonstrating that the selective activity was due to the presence of the targeting moiety. Fibroblasts were not sensitive to ATF-SAP despite uPAR expression, indicating that cell-specific receptor-mediated internalization pathway(s) might be considered. The in vivo anti-tumor effect of the chimera was shown in a bladder cancer xenograft model. Current findings indicate ATF-SAP as a suitable anti-tumoral therapeutic option to cope with cancer aggressiveness, as a single treatment or in combination with traditional therapeutic approaches, to appropriately address the intra- and inter- tumor heterogeneity.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Saporinas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/análise , Proteínas Recombinantes de Fusão/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologiaRESUMO
pH-sensitive polyethylene glycol-conjugated urokinase nanogels (PEG-UK) is a new form of urokinase (UK) nanogels that could release UK at certain pH values. In our former study, we demonstrated that the pH value in the infarcted brain significantly declined to the level that could trigger the delivery of UK from PEG-UK. Thrombolysis is recommended as the first choice for ischemic stroke within the time window. However, it is common for the patients to miss the thrombolysis time window, which is one of the major causes of bad prognosis from ischemic stroke. It remains promising for seeking therapeutic approaches for ischemic stroke by investigating potential protective reagents delivered out of the usually thrombolysis time window. In this study, the protective effect of administration of PEG-UK outside the usual time window and the underlying mechanisms were investigated. PEG-UK was administrated 2 h and a half after ischemic stroke Delayed administration of PEG-UK significantly ameliorated the severity of neurological deficits of permanent middle cerebral occlusion (pMCAO) rats and reduced the infiltration of inflammatory cells and the concentration of interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the brain tissues. The content of water and the leakage of Evans Blue (EB) in the PEG-UK group were also decreased. Maintenance of the expression of platelet-derived growth factor-C (PDGF-C) and inhibition of the upregulation of metalloproteinase proteins, low-density lipoprotein receptor-related protein (LRP), nuclear factor κB (NF-κB) p65 and cyclooxygenase-2 (Cox-2) were observed through western blotting and realtime PCR in the PEG-UK group. Besides, delayed administration of PEG-UK attenuated the up regulation of Caspase8 and Caspase9 and the cleavage of Caspase3 and poly (ADP-ribose) polymerase 1 (PARP1) in ischemic lesion sites. Moreover, PEG-UK treatment also inhibited the upregulation and phosphorylation of N-methyl-D-aspartic acid receptors (NMDARs), which has been revealed to play a vital role in mediating excito-neurotoxicity in ischemic stroke. In conclusion, through the inhibition of LRP/NF-κB/Cox-2 pathway, the Caspase cascade and activation of NMDARs, administration of PEG-UK outside the usual time window could still exert protective effects in pMCAO rats through the maintenance of the integrity of BBB and the inhibition of apoptosis and excito-neurotoxicity.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Nanogéis/química , Fármacos Neuroprotetores/uso terapêutico , Polietilenoglicóis/química , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tipo Uroquinase/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/complicações , Caspases/metabolismo , Ciclo-Oxigenase 2/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Infarto da Artéria Cerebral Média/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/toxicidade , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/complicações , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).
Assuntos
Injúria Renal Aguda/sangue , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Angiografia Coronária/efeitos adversos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/prevenção & controle , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores/sangue , Estado Terminal , Modelos Animais de Doenças , Feminino , Humanos , Unidades de Terapia Intensiva , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Razão de Chances , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/etiologia , Medição de Risco/métodos , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
TNFα mediates the expression of MMP-9 in THP-1 monocytes induced by urokinase (uPA). Upregulation of MMP-9 caused by uPA and TNFα is suppressed by etanercept, a TNFα inhibitor. In addition, uPA stimulates TNFα mRNA expression. Both uPA and TNFα induce ROS generation in monocytes, while MMP-9 secretion induced by uPA and TNFα is inhibited by antioxidants. Inhibitors of NFκB, ligands of PPARα and PPARγ receptors, and SIRT1 activators negatively affect MMP-9 secretion induced by uPA. MMP-9 secretion during monocyte differentiation into macrophages is downregulated by etanercept and antioxidants. These factors as well as MMP inhibitor GM6001 reduce the number of macrophages attached to substrate during cell differentiation, indicating the role of urokinase, TNFα, and ROS in MMP expression in monocytes and MMP involvement in macrophage maturation.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Linhagem Celular , Humanos , Monócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo , Células THP-1RESUMO
Chemotherapy is widely used in the clinic though its benefits are controversial owing to low cancer specificity. Nanovehicles capable of selectively transporting drugs to cancer cells have been energetically pursued to remodel cancer treatment. However, no active targeting nanomedicines have succeeded in clinical translation to date, partly due to either modest targetability or complex fabrication. CD44-specific A6 short peptide (KPSSPPEE) functionalized polymersomal epirubicin (A6-PS-EPI), which boosts targetability and anticancer efficacy toward human multiple myeloma (MM) in vivo, is described. A6-PS-EPI encapsulating 11 wt% EPI is small (≈55 nm), robust, reduction-responsive, and easy to fabricate. Of note, A6 decoration markedly augments the uptake and anticancer activity of PS-EPI in CD44-overexpressing LP-1 MM cells. A6-PS-EPI displays remarkable targeting ability to orthotopic LP-1 MM, causing depleted bone damage and striking survival benefits compared to nontargeted PS-EPI. Overall, A6-PS-EPI, as a simple and intelligent nanotherapeutic, demonstrates high potential for clinical translation.
Assuntos
Epirubicina/química , Epirubicina/farmacologia , Receptores de Hialuronatos/metabolismo , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Sinergismo Farmacológico , Humanos , Camundongos , Mieloma Múltiplo/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/uso terapêuticoRESUMO
Immuno-positron emission tomography (immuno-PET) is expected to improve the specificity of small chemical tracers such as 18F-fluorodeoxyglucose. Whole antibodies significantly accumulate in target molecule-expressing tumors but frequently persist too long in the blood circulation for imaging purposes. We investigated the utility of whole antibodies, 64Cu-labeled via a urokinase-substrate linker, and their exogenous urokinase-responsive cleavage to enhance clearance of immuno-PET probes from the blood and shorten the time required to develop adequate imaging contrast. Specifically, we used 64Cu-labeled trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive tumor-bearing mice. 64Cu-labeled trastuzumab with a urokinase-cleavage site (64Cu-CB-TE1A1P-USL-trastuzumab) was synthesized using a bifunctional chelator incorporating an urokinase substrate peptide. Urokinase cleavage was analyzed in vitro by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and radio-gel permeation-high-performance liquid chromatography. Improvements in radioisotope clearance and HER2-imaging by urokinase injection were evaluated by PET imaging and ex vivo biodistribution studies in A431 tumor-bearing mice. 64Cu-CB-TE1A1P-USL-trastuzumab was cleaved into smaller radioactive fragments by 20â¯000 IU/mL urokinase treatment in vitro at an efficacy of â¼95%. The probe targeted HER2 in A431 tumors in mice within 24 h post-injection, and approximately two-thirds of the probe in the blood circulation was eliminated via renal clearance of radioactive fragments after three urokinase injections. Therefore, the tumor/blood ratio increased 3.0-fold. Without urokinase injection, the tumor accumulation of 64Cu-CB-TE1A1P-USL-trastuzumab slowly increased, and the blood radioactivity decreased over 72 h. However, the tumor/blood ratios in mice after three urokinase injections were higher at 24 h than those in mice without injections at 72 h. The results indicate that our approach shortened the time required to develop adequate imaging contrast of immuno-PET by >2 days. Therefore, this approach can benefit high-sensitivity imaging under lower radioactive decay conditions and can decrease patient radiation exposure. In addition, it could reduce other adverse effects of radioimmunotherapy.
Assuntos
Radioisótopos de Cobre/química , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Linhagem Celular Tumoral , Quelantes/química , Fluordesoxiglucose F18 , Compostos Heterocíclicos com 2 Anéis/química , Xenoenxertos , Humanos , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias/patologia , Organofosfonatos/química , Distribuição Tecidual , Trastuzumab/química , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagemRESUMO
In the present study we showed that the recombinant analogue of the SLURP-1 protein effectively inhibits the growth of a 3D model of tumors-multicellular spheroids reconstructed from human epidermoid carcinoma A431 cells and human lung adenocarcinoma A549 cells. The combined application of rSLURP-1 with gefitinib (inhibitor of epidermal growth factor receptor (EGFR)) leads to the synergistic antiproliferative effect on spheroids from A431 cells. The results obtained suggest the possibility for design of first-in-class anticancer drugs based on recombinant SLURP-1.
Assuntos
Antígenos Ly/farmacologia , Proteínas Recombinantes/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Células A549 , Proliferação de Células/efeitos dos fármacos , HumanosRESUMO
BACKGROUND AND OBJECTIVE: Urokinase-plasminogen activator (uPA) is a serine protease expressed at high basal level in normal gingival cervical fluid. Despite its known pathologic role in tissue proteolysis in periodontitis, little is known concerning uPA physiological function in oral tissue. Recent evidence in cancer cells has implicated the uPA system in DNA repair and anti-apoptotic pathways. This study is aimed to evaluate the protective function of urokinase against oxidative DNA damage in periodontal ligament (PDL) fibroblast, and to propose a new biological role for uPA in oral cavity. MATERIAL AND METHODS: PDL cells were isolated from human wisdom teeth obtained from healthy donors. An oxidative stress model was created in which PDL cells were incubated with 20, 30, 40 and 60 µmol/L hydrogen peroxide. Twenty-four hours before and after peroxide treatment, cells were treated with uPA and amiloride. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, apoptosis by DAPI-staining and annexin V/propidium iodide assay, and DNA breaks by alkaline comet assay. For estimating DNA damage level, γ-H2AX expression was studied using flow cytometry and immunostaining. RESULTS: The incubation of the peroxide-treated cells with uPA significantly increased cell viability and decreased apoptosis. A significant decrease in the number of γ-H2AX foci was seen at 30 µmol/L hydrogen peroxide in uPA-treated cells. uPA inhibition as a result of amiloride treatment, in turn, induced a reduction in cell viability. In addition, there was a significant decrease in the levels of DNA damage in uPA-treated groups as measured by the comet assay. CONCLUSION: The present study brings support to the theory that uPA may have a protective role for periodontal tissue and could protect PDL fibroblasts from oxidative DNA damage and apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ligamento Periodontal/citologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Amilorida/farmacologia , Sobrevivência Celular , Células Cultivadas , Citometria de Fluxo , HumanosRESUMO
Thrombosis, a critical event in blood vessels, not only is associated with myocardial infarction and stroke, but also accounts for considerable morbidity and mortality. Thrombolytic drugs are usually applied to the treatment of acute myocardial infarction, acute cerebral infarction and pulmonary embolism. However, thrombolytic drugs show limited efficacy in clinical practice because of the short half-life in plasma and systemic side effects. In this study, the cyclic RGD (cRGD) functionalized liposomes were prepared to encapsulate urokinase, a cheap and widely used thrombolytic drug in clinic and better thrombolysis efficacy was achieved. The flow cytometry analysis showed that the cRGD liposomes could bind to the activated platelets while not to the resting platelets. In vitro release study revealed that the release percentage reached plateau in about 5â¯h with 60% urokinase being released from liposomes. Results from the in vitro thrombolysis experiments demonstrated a good thrombolysis potential of the cRGD urokinase liposomes. The in vivo thrombolysis study demonstrated that the cRGD liposomes could significantly reduce the dose of urokinase by 75% while achieving the equivalent thrombolysis effect as the free urokinase in mouse mesenteric thrombosis model. In conclusion, the cRGD liposomes encapsulating urokinase hold great promise in clinic for better thrombolytic efficacy. STATEMENT OF SIGNIFICANCE: In this paper, the cRGD liposomes were prepared to encapsulate urokinase for targeted thrombolysis therapy. The cRGD liposomes could specifically bind to the activated platelets and could stably and continuously release its loaded urokinase. The mouse mesenteric thrombosis model was established to evaluate the thrombolysis effect of the cRGD urokinase liposomes. The results demonstrated that the cRGD liposomes could improve the thrombolytic efficacy by almost 4-fold over free urokinase. In conclusion, the cRGD liposomes encapsulating urokinase had great potential for the clinical treatment of thrombosis.
Assuntos
Enzimas Imobilizadas , Peptídeos Cíclicos , Terapia Trombolítica , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tipo Uroquinase , Animais , Modelos Animais de Doenças , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Humanos , Lipossomos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Trombose/metabolismo , Trombose/patologia , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
Previously, we have reported that the Secreted Ly6/uPAR related protein-1 (SLURP1) serves an important immunomodulatory function in the ocular surface. Here, we examine the involvement of SLURP1 in regulating corneal angiogenic privilege. Slurp1 expression detected by QPCR, immunoblots and immunofluorescent stain, was significantly decreased in mouse corneas subjected to alkali burn-induced corneal neovascularization (CNV). Addition of exogenous SLURP1 (6XHis-tagged, E. coli expressed and partially purified using Ni-ion columns) significantly suppressed the tumor necrosis factor-α (TNF-α)-stimulated human umbilical cord vascular endothelial cell (HUVEC) tube formation. SLURP1 suppressed the HUVEC tube length, tube area and number of branch points, without affecting their viability and/or proliferation. Exogenous SLURP1 in HUVEC also suppressed the TNF-α-induced (i) interleukin-8 (IL-8) and TNF-α production, (ii) adhesion to different components of the extracellular matrix, (iii) migration, and (iv) nuclear localization of NFκB. Together, these results demonstrate that SLURP1 suppresses HUVEC tube formation by blocking nuclear translocation of NFκB, and suggest a potential role for SLURP1 in promoting corneal angiogenic privilege.
Assuntos
Inibidores da Angiogênese/farmacologia , Antígenos Ly/farmacologia , Neovascularização da Córnea/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , NF-kappa B/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Animais , Antígenos Ly/fisiologia , Queimaduras Químicas/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Lesões da Córnea/metabolismo , Modelos Animais de Doenças , Queimaduras Oculares/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Fator de Necrose Tumoral alfa/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
INTRODUCTION: Chronic inflammatory diseases such as rheumatoid arthritis and periodontitis frequently cause bone destruction. Inflammation-induced bone loss results from the increase of bone-resorbing osteoclasts. Recently, we demonstrated that urokinase type plasminogen activator (uPA) suppressed lipopolysaccaride (LPS)-inflammatory osteoclastogenesis through the adenosine monophosphate-activated protein kinase (AMPK) pathway, whereas its receptor (uPAR) promoted that through the Akt pathway. METHODS: We investigated the effects of uPA-derived peptide (Å6) in the LPS-induced inflammatory osteoclastogenesis and bone destruction. RESULTS: We found that Å6 attenuated inflammatory osteoclastogenesis and bone loss induced by LPS in mice. We also showed that Å6 attenuated the LPS-promoted inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 mouse monocyte/macrophage lineage cells. Furthermore, we showed that Å6 attenuated the Akt phosphorylation, and promoted the AMPK phosphorylation. CONCLUSION: Å6 is involved in the suppression of LPS-promoted inflammatory osteoclastgensis and bone destruction by regulating the AMPK and Akt pathways. These findings provide a basis for clinical strategies to improve the bone loss caused by inflammatory diseases.
Assuntos
Reabsorção Óssea/prevenção & controle , Lipopolissacarídeos/toxicidade , Osteoclastos/imunologia , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Proteínas Quinases Ativadas por AMP/imunologia , Animais , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/imunologia , Reabsorção Óssea/patologia , Camundongos , Osteoclastos/patologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: The big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types. RESULTS: To selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity. CONCLUSION: Our results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.
Assuntos
Proteínas Recombinantes de Fusão/biossíntese , Proteínas Inativadoras de Ribossomos Tipo 1/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Reatores Biológicos , Ensaios de Seleção de Medicamentos Antitumorais , Fermentação , Humanos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Células U937 , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
We studied the effect of urokinase, its recombinant forms, and domain fragments on migration and proliferation of adipose tissue mesenchymal stromal cells (MSCs) and MMP secretion by these cells. Urokinase, but not its recombinant forms, slightly induced directed migration of MSCs. Spontaneous migration of MSCs increased under the action of urokinase or its isolated kringle domain. Migration induced by platelet-derived growth factor was inhibited by proteolytically inactive form of urokinase, the kringle domain, and blocking antibody to urokinase receptor. Urokinase, its proteolytically inactive form, and kringle domain produced no effect on MSC proliferation. In contrast to platelet-derived growth factor, all urokinase forms induced secretion of MMP-9 by MSCs.
Assuntos
Movimento Celular/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Isoenzimas/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células , Domínios Proteicos , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
PURPOSE: To examine a novel technique for simplified placement and removal of plaque brachytherapy by fibrin glue and urokinase (medac Gmbh, Hamburg, Germany). MATERIALS AND METHODS: In six enucleated porcine eyes, plaques were placed on the episclera and fibrin glue was applied to cover it. Urokinase was used to dissolve the glue in three eyes and saline was used in three eyes. Adhesion strength was measured further on 15 plaques affixed to porcine eyes (glued in five with intact conjunctiva, glued in five with removed conjunctiva, and sutured in five). RESULTS: Saline had no effect on the glue-plaque-eye complex, whereas the urokinase (0.38 mL ± 0.08 mL) easily dissolved the adhesion between the glue layer and surrounding tissues. The weight required to detach the plaques was 0.349 kg ± 0.173 kg for glued eyes with intact conjunctiva, 0.405 kg ± 0.083 kg for sutured eyes (P = .59), and 0.032 kg ± 0.004 kg for glued eyes without intact conjunctiva (P ≤ .015). CONCLUSIONS: The usage of the biological adhesive and dissolvent system was applicable for plaque surgery in an ex vivo animal model.