RESUMO
The attenuated Salmonella typhimurium VNP20009, enriched in tumors, is known to have antitumor effects and recruit macrophages. Little is known, however, about whether VNP will lead to specific changes in macrophages, e.g., cell temperature. Here, using a real-time wireless multicell thermometry system, we reported for the first time that VNP20009 increases the macrophage temperature by 0.2 °C. Nigericin, recognized as an inducer of pyroptosis, was found to induce macrophage warming. Moreover, the ΔsipD-VNP20009 strain failed to induce macrophage pyroptosis and simultaneously failed to warm macrophages, and the Gsdmd-/- macrophages that were unable to achieve pyroptosis were no longer warmed following VNP20009 induction. These results suggested that the occurrence of macrophage pyroptosis is the key to VNP20009-mediated cell warming. With the aid of a single-cell thermometry system, it was further confirmed that cell warming occurred in pyroptosis-like macrophages. Cellular warming was not detected after the induction of pyroptosis in macrophages with loss of mitochondrial biological function, suggesting a critical role of mitochondria in warming. Moreover, we found that VNP20009 caused local tumor temperature increases. The local tumor warming induced by VNP20009 was significantly reduced after macrophage clearance. Notably, this temperature increase contributed to M1-type polarization. These findings expanded our knowledge of the cellular biological changes induced by the strain on macrophages, as well as the biochemical phenomena accompanying pyroptosis, and provide a reference for the study of biochemical signals transduced to biothermal signals with a combined cell-level temperature detector.
Assuntos
Salmonella typhimurium , Termometria , Piroptose , Nigericina , Macrófagos , FenótipoRESUMO
Hydrogen sulfide (H2S) is widely recognized as the third endogenous gas signaling molecule and may play a key role in cancer biological processes. ADT-OH (5-(4-hydroxyphenyl)-3H-1,2-dithiocyclopentene-3-thione) is one of the most widely used organic donors for the slow release of H2S and considered to be a potential anticancer compound. In this study, we investigated the antimetastatic effects of ADT-OH in highly metastatic melanoma cells. A tail-vein-metastasis model was established by injecting B16F10 and A375 cells into the tail veins of mice, whereas a mouse footpad-injection model was established by injecting B16F10 cells into mouse footpads. We showed that administration of ADT-OH significantly inhibited the migration and invasion of melanoma cells in the three different animal models. We further showed that ADT-OH dose-dependently inhibited the migration and invasion of B16F10, B16F1 and A375 melanoma cells as evaluated by wound healing and Transwell assays in vitro. LC-MS/MS and bioinformatics analyses revealed that ADT-OH treatment inhibited the EMT process in B16F10 and A375 cells by reducing the expression of FAK and the downstream response protein Paxillin. Overexpression of FAK reversed the inhibitory effects of ADT-OH on melanoma cell migration. Moreover, after ADT-OH treatment, melanoma cells showed abnormal expression of the H2S-producing enzymes CSE/CBS and the AKT signaling pathways. In addition, ADT-OH significantly suppressed the proliferation of melanoma cells. Collectively, these results demonstrate that ADT-OH inhibits the EMT process in melanoma cells by suppressing the CSE/CBS and FAK signaling pathways, thereby exerting its antimetastatic activity. ADT-OH may be used as an antimetastatic agent in the future.
Assuntos
Melanoma , Tionas , Animais , Linhagem Celular Tumoral , Movimento Celular , Cromatografia Líquida , Quinase 1 de Adesão Focal/metabolismo , Melanoma/tratamento farmacológico , Camundongos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Paxilina , Transdução de Sinais , Neoplasias Cutâneas , Espectrometria de Massas em Tandem , Melanoma Maligno CutâneoRESUMO
BACKGROUND: Ulcerative colitis (UC) is a major type of inflammatory bowel disease (IBD), which could induce bloody stool, diarrhea, colon atrophy and eventually lead to colorectal cancer. The conventional daily oral administration of drugs only relieve the inflammatory response of colon in the short term, Biological agents such as antibody drugs has proven its efficiency in inhibiting colitis, while the low drug bioavailability means that large doses of antibodies are required, ultimately causing systemic toxicity. Small interfering RNA (siRNA) has significant advantages over antibody drugs in terms of safety and efficacy, and it have been widely applied as potential candidates for a variety of inflammation-related diseases. However, oral delivery of siRNA fails to overcome the degradation of the gastrointestinal environment to produce a significant therapeutic effect in ulcerative colitis. Herein, we design the hybrid delivery system that the siRNA loaded MOF encapsulated in the sodium alginate particles to overcome the barriers in the oral process. RESULTS: The hybrid delivery system (SA@MOF-siRNATNFα) was successfully constructed, and it could not only survive the low pH environment in the stomach and small intestine, but also taken up more by inflammatory macrophages, as well as released much more MOF-siRNATNFα. Moreover, SA@MOF-siRNATNFα tended to enriched and infiltrated into local colon tissues. As a result, SA@MOF-siRNATNFα significantly reduced the progression of colitis, of which the treated mice did not experience significant weight loss, bloody stools and diarrhea. CONCLUSION: We confirmed that the formulation of hydrogel-metal-organic framework hybrids could improve the protection of incorporated payload in the gastric and early small intestine, enhancing the delivery of MOF-siRNA to colon.
Assuntos
Colite Ulcerativa , Colite , Estruturas Metalorgânicas , Animais , Colite/tratamento farmacológico , Colite Ulcerativa/tratamento farmacológico , Diarreia , Hidrogéis , Camundongos , RNA Interferente Pequeno , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Previous studies have indicated that Fas-FasL pathway and its downstream caspase-8 can regulate islet mass and insulin secretion. As a classical adaptor in Fas-FasL signaling, Fas-associated death domain-containing protein (FADD) takes part in many non-apoptosis processes regulated by its phosphorylation. However, its role in islets has not been evaluated to date. Here, through comparative proteomics and bioinformatic analysis on FADD phosphorylated (FADD-D) and wild-type (WT) MEFs, we found three proteins involved in islet differentiation and function were dysregulated due to FADD phosphorylation. The mouse model of FADD-D, which mimics constitutive phosphorylated FADD expression in mice, was further analyzed to address this issue. We confirmed the proteomic results by immunohistological analyses on pancreatic islets. In addition, we found that FADD-D mice displayed decreased islet area, and the glucose stimulated insulin secretion (GSIS) of FADD-D islets was impaired. These data suggest a novel role of FADD in islet development and insulin secretion.
Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Ilhotas Pancreáticas/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/genética , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Camundongos , FosforilaçãoRESUMO
Fas-associated death domain-containing protein (FADD), a classical apoptotic signaling adaptor, participates in different nonapoptotic processes regulated by its phosphorylation. However, the influence of FADD on metabolism, especially glucose homeostasis, has not been evaluated to date. Here, using both two-dimensional electrophoresis and liquid chromatography linked to tandem mass spectrometry (LC/MS/MS), we found that glycogen synthesis, glycolysis, and gluconeogenesis were dysregulated because of FADD phosphorylation, both in MEFs and liver tissue of the mice bearing phosphorylation-mimicking mutation form of FADD (FADD-D). Further physiological studies showed that FADD-D mice exhibited lower blood glucose, enhanced glucose tolerance, and increased liver glycogen content without alterations in insulin sensitivity. Moreover, investigations on the molecular mechanisms revealed that, under basal conditions, FADD-D mice had elevated phosphorylation of Akt with alterations in its downstream signaling, leading to increased glycogen synthesis and decreased gluconeogenesis. Thus, we uncover a novel role of FADD in the regulation of glucose homeostasis by proteomic discovery and physiological validation.
Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Glucose/metabolismo , Animais , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas/genética , Fibroblastos , Expressão Gênica , Glicogênio/metabolismo , Homeostase , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: The urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein. METHODS: We performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay. RESULTS: HSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P < 0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved. All the biological function experiments in cell level showed an additive effect when HSP70 and MRJ were regulated simultaneously indicating their collaborated regulation effects on uPAR. CONCLUSIONS: These findings may offer a novel insight into the interactions between uPAR and HSP70/MRJ and their functions in cell adhesion and migration may provide more understanding of the roles in regulating cancer metastasis.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Células HeLa , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Transdução de Sinais , Vitronectina/metabolismoRESUMO
High basal autophagy and enhanced mitochondrial fission in triple-negative breast cancer (TNBC) cells support cell migration and promote plasticity of cancer cell metabolism. Here, we suggest a novel combination therapy approach for the treatment of TNBC that targets Drp1-mediated mitochondrial fission and autophagy pathways. Hydrogen sulfide (H2S) mediates a myriad of biological processes, including autophagy and mitochondrial function. In this study, we demonstrated that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most widely utilized sustained-release H2S donors, effectively suppresses metastasis of TNBC cells in the absence of proliferation inhibition in vitro and in vivo. ADT-OH treatment ameliorated autophagy flux by suppressing autophagosome formation and induced mitochondrial elongation through decreasing expression of dynamin-related protein 1 (Drp1) and increasing expression of mitochondrial fusion protein (Mfn2). At the same time, ADT-OH downregulated mitophagy flux and inhibited mitochondrial function, eventually leading to the inhibition of migration and invasion in TNBC cells. In vivo, intraperitoneal administration of ADT-OH revealed a potent anti-metastatic activity in three different animal models, the MDA-MB-231 orthotopic xenograft model, the 4T1-Luci orthotopic model and the 4T1-Luci tail vein metastasis model. However, ADT-OH has an extremely low water solubility, which is a significant barrier to its effectiveness. Thus, we demonstrated that the solubility of ADT-OH in water can be improved significantly by absorption with hydroxypropyl-ß-cyclodextrin (CD). Remarkably, the obtained CD-ADT-OH demonstrated superior anti-cancer effect to ADT-OH in vivo. Altogether, this study describes a novel regulator of mammalian mitochondrial fission and autophagy, with potential utility as an experimental therapeutic agent for metastatic TNBC.
Assuntos
Autofagia , Dinâmica Mitocondrial , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Humanos , Animais , Autofagia/efeitos dos fármacos , Feminino , Linhagem Celular Tumoral , Camundongos , Movimento Celular/efeitos dos fármacos , Camundongos Nus , Tionas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Metástase Neoplásica , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Dinaminas/metabolismo , Tiofenos/farmacologiaRESUMO
Ferroptosis is iron-dependent and regulates necrosis caused by lipid peroxidation and mitochondrial damage. Recent evidence has revealed an emerging role for ferroptosis in the pathophysiology of acute kidney injury (AKI). Sulfide:quinone oxidoreductase (SQOR) is a mitochondrial inner membrane protein highly expressed in the renal cortex. However, the effects of SQOR on ferroptosis and AKI have not been elucidated. In this study, we evaluated the effects of SQOR in several AKI models. We observed a rapid decrease in SQOR expression after cisplatin stimulation in both in vivo and in vitro models. SQOR-deletion mice exhibit exacerbated kidney impairment and ferroptosis in renal tubular epithelial cells following cisplatin injury. Additionally, our results showed that the overexpression of SQOR or ADT-OH (the slow-releasing H2S donor) preserved renal function in the three AKI mouse models. These effects were evidenced by lower levels of serum creatinine (SCr), blood urea nitrogen (BUN), renal neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (KIM-1). Importantly, SQOR knockout significantly aggravates cisplatin-induced ferroptosis by promoting mitochondrial dysfunction in renal tubular epithelial cells (RTECs). Moreover, online database analysis combined with our study revealed that SYVN1, an upregulated E3 ubiquitin ligase, may mediate the ubiquitin-mediated degradation of SQOR in AKI. Consequently, our results suggest that SYVN1-mediated ubiquitination degradation of SQOR may induce mitochondrial dysfunction in RTECs, exacerbating ferroptosis and thereby promoting the occurrence and development of AKI. Hence, targeting the SYVN1-SQOR axis could be a potential therapeutic strategy for AKI treatment.
Assuntos
Injúria Renal Aguda , Ferroptose , Doenças Mitocondriais , Camundongos , Animais , Cisplatino/efeitos adversos , Injúria Renal Aguda/etiologia , Oxirredutases , Quinonas , SulfetosRESUMO
Overweight and obesity affect almost 2 billion adults worldwide, and food restriction (FR) is commonly used to reduce body fat. Whether refeeding (Re) after FR at different ages and to different degrees leads to overweight and its possible mechanisms are uncertain. In this study, adult and young mice were both restricted to 15% and 40% of their casual food intake, and then were fed 60% high-fat chow (FR15%-Re, FR40%-Re), whereas the control groups(CON) consumed high-fat or normal food throughout, respectively. The results of the study suggest that mild FR-heavy feeding may lead to more significant abnormal fat accumulation, liver damage, and increased recruitment of intestinal inflammatory factors and immune cells in mice of different ages and involves multiple types of alterations in the gut microbiota. Further fecal transplantation experiments as well as serum and liver enzyme-linked immunosorbent assay experiments preliminarily suggest that the link between lipid metabolism and inflammatory responses and the gut microbiota may be related to the regulation of the gut and live by Lipopolysaccharides(LPS) and Peroxisome Proliferator-Activated Receptor-Alpha(PPAR-α). In addition, our study may also serve as a reference for studying obesity prevention and treatment programs at different ages.
RESUMO
Fas-associated death domain-containing protein (FADD) is a classical apoptotic pathway adaptor. Further studies revealed that it also plays essential roles in nonapoptotic processes, which is assumed to be regulated by its phosphorylation. However, the exact mechanisms are still poorly understood. To study the nonapoptotic effects of FADD, a comprehensive strategy of proteomics identification combined with bioinformatic analysis was undertaken to identify proteins differentially expressed in three cell lines containing FADD and its mutant, FADD-A and FADD-D. The cell lines were thought to bear wild-type FADD, unphosphorylated FADD mimic and constitutive phosphorylated FADD mimic, respectively. A total of 47 proteins were identified to be significantly changed due to FADD phosphorylation. Network analysis using MetaCore™ identified a number of changed proteins that were involved in cellular metabolic process, including lipid metabolism, fatty acid metabolism, glycolysis, and oxidative phosphorylation. The finding that FADD-D cell line showed an increase in fatty acid oxidation argues that it could contribute to the leaner phenotype of FADD-D mice as reported previously. In addition, six proteins related to the ubiquitin-proteasome pathway were also specifically overexpressed in FADD-D cell line. Finally, the c-Myc gene represents a convergent hub lying at the center of dysregulated pathways, and was upregulated in FADD-D cells. Taken together, these studies allowed us to conclude that impaired mitochondrial function and proteolysis might play pivotal roles in the dysfunction associated with FADD phosphorylation-induced disorders.
Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/metabolismo , Mapas de Interação de Proteínas , Análise de Variância , Animais , Células Cultivadas , Eletroforese em Gel Bidimensional , Metabolismo Energético , Proteína de Domínio de Morte Associada a Fas/análise , Proteína de Domínio de Morte Associada a Fas/genética , Fibroblastos/química , Camundongos , Camundongos Transgênicos , Oxirredução , Fosforilação , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
Protein kinase C (PKC) plays important roles in diverse cellular processes. PKC has been implicated in regulating Fas-associated protein with death domain (FADD), an important adaptor protein involved in regulating death receptor-mediated apoptosis. FADD also plays an important role in non-apoptosis processes. The functional interaction of PKC and FADD in non-apoptotic processes has not been examined. In this study, we show that FADD is involved in maintaining the phosphorylation of the turn motif and hydrophobic motif in the activated conventional PKC (cPKC). A phosphoryl-mimicking mutation (S191D) in FADD (FADD-D) abolished the function of FADD in the facilitation of the turn motif and hydrophobic motif dephosphorylation of cPKC, suggesting that phosphorylation of Ser-191 negatively regulates FADD. We show that FADD interacts with PP2A, which is a major phosphatase involved in dephosphorylation of activated cPKC and FADD deficiency abolished PP2A mediated dephosphorylation of cPKC. We show that FADD deficiency leads to increased stability and activity of cPKC, which, in turn, promotes cytoskeleton reorganization, cell motility, and chemotaxis. Collectively, these results reveal a novel function of FADD in a non-apoptotic process by modulating cPKC dephosphorylation, stability, and signaling termination.
Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Animais , Movimento Celular , Citoesqueleto/metabolismo , Estabilidade Enzimática , Proteína de Domínio de Morte Associada a Fas/genética , Células HEK293 , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Proteólise , Transdução de SinaisRESUMO
BACKGROUND: Urokinase (uPA) and its receptor (uPAR) play an important role in tumour growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumours. Targeting the excessive activation of this system as well as the proliferation of the tumour vascular endothelial cell would be expected to prevent tumour neovasculature and halt tumour development. The amino terminal fragment (ATF) of urokinase has been confirmed effective to inhibit the proliferation, migration and invasiveness of cancer cells via interrupting the interaction of uPA and uPAR. Triptolide (TPL) is a purified diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook F that has shown antitumor activities in various cancer cell types. However, its therapeutic application is limited by its toxicity in normal tissues and complications caused in patients. In this study, we attempted to investigate the synergistic anticancer activity of TPL and ATF in various solid tumour cells. METHODS: Using in vitro and in vivo experiments, we investigated the combined effect of TPL and ATF at a low dosage on cell proliferation, cell apoptosis, cell cycle distribution, cell migration, signalling pathways, xenograft tumour growth and angiogenesis. RESULTS: Our data showed that the sensitivity of a combined therapy using TPL and ATF was higher than that of TPL or ATF alone. Suppression of NF-κB transcriptional activity, activation of caspase-9/caspase-3, cell cycle arrest, and inhibition of uPAR-mediated signalling pathway contributed to the synergistic effects of this combination therapy. Furthermore, using a mouse xenograft model, we demonstrated that the combined treatment completely suppressed tumour growth by inhibiting angiogenesis as compared with ATF or TPL treatment alone. CONCLUSIONS: Our study suggests that lower concentration of ATF and TPL used in combination may produce a synergistic anticancer efficacy that warrants further investigation for its potential clinical applications.
Assuntos
Antineoplásicos/farmacologia , Diterpenos/farmacologia , Peptídeos/farmacologia , Fenantrenos/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/química , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Diterpenos/administração & dosagem , Sinergismo Farmacológico , Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/farmacologia , Células HCT116 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Peptídeos/administração & dosagem , Fenantrenos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis is a major feature of tumor growth and metastasis. As such, targeting the tumor neovasculature is an attractive strategy for effective cancer therapy. Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. Our laboratory previously reported a strategy for expression and purification of human vasostatin120-180 (VAS) in a GST-tagged fusion form using Escherichia coli expression system. However, the yield of 7.2 mg per liter of culture was relatively low and the protein activity was also limited. In this study, the biologically active and soluble VAS was cloned and expressed in Pichia pastoris. The yield of the active VAS was about 20 mg/L of the P. pastoris culture medium. The recombinant protein was purified to homogeneity, and confirmed to be biologically active. The recombinant VAS could efficiently inhibit angiogenesis and endothelial cell proliferation in vitro. Moreover, the P. pastoris-derived VAS showed relatively higher protein activity than E. coli-derived VAS. Furthermore, it can inhibit in vivo xenograft tumor growth and prolong the tumor doubling time significantly by inhibiting angiogenesis. Taken together, this is the first report on the heterologous expression of VAS in P. pastoris, and P. pastoris is a highly efficient and cost-effective expression system for large amount production of biologically active recombinant VAS for potential therapeutic application.
Assuntos
Calreticulina/metabolismo , Calreticulina/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Análise de Variância , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sequência de Bases , Calreticulina/genética , Calreticulina/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis is essential for the survival and growth of most tumors. As such, targeting the tumor neovasculature is an attractive strategy for effective cancer therapy. Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. The functional domain within amino acid residues 120-180 of vasostatin (VAS) has been confirmed to be effective in inhibiting the proliferation, migration, and invasiveness of cancer cells by its suppressive capacity against angiogenesis. Triptolide (TPL) is an active component extracted from the traditional Chinese herbal medicine Tripterygium wilfordii Hook F that has shown antitumor activities in various cancer cell types. However, its therapeutic application is limited by its toxicity in normal tissues and complications caused in patients. In this study, we attempted to investigate the synergistic antitumor activity of TPL and VAS in solid tumor cells. Our results showed that the sensitivity of combined therapy using TPL and VAS was higher than that of monotherapy using TPL or VAS. Apoptosis induced by the combined treatment was accompanied by activation of caspase-9, caspase-8, and caspase-3. Upregulation of proapoptotic protein (Bax, Bak, and Bad) expression and suppression of NF-κB transcriptional activity and its targeting antiapoptotic genes (c-FLIP, cIAP, Bcl-2, Bcl-xl, and Mcl-1) may contribute to the synergistic effects of this combination therapy. Further, using a mouse xenograft model, we demonstrated that combined treatment completely suppressed tumor growth as compared with treatment with TPL or VAS alone. These results suggest that the combination of TPL and VAS at lower concentrations may produce a synergistic antitumor effect that warrants further investigation for its potential clinical applications.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Calreticulina/uso terapêutico , Diterpenos/uso terapêutico , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Fragmentos de Peptídeos/uso terapêutico , Fenantrenos/uso terapêutico , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Calreticulina/administração & dosagem , Calreticulina/genética , Calreticulina/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Diterpenos/administração & dosagem , Diterpenos/efeitos adversos , Diterpenos/farmacologia , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/química , Compostos de Epóxi/administração & dosagem , Compostos de Epóxi/efeitos adversos , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Etnofarmacologia , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Fenantrenos/administração & dosagem , Fenantrenos/efeitos adversos , Fenantrenos/farmacologia , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Tripterygium/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.
Assuntos
Inibidores da Angiogênese/metabolismo , Terapia Biológica/métodos , Endostatinas/metabolismo , Regiões Promotoras Genéticas , Proteoma/genética , Salmonella typhimurium/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Anaerobiose , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/terapia , Morte Celular , Hipóxia Celular , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Endostatinas/genética , Feminino , Estimativa de Kaplan-Meier , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Organismos Geneticamente Modificados , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonella typhimurium/genética , Carga Tumoral , Regulação para CimaRESUMO
FADD, a classical apoptotic signaling adaptor, has recently been reported to exhibit a series of non-apoptotic functions. Here, we report that FADD may play a critical role in the development of renal fibrosis. Neutrophil infiltration in the renal interstitial part, glomerular mesangial cell proliferation, and base-membrane thickening were observed in FADD-D mice by H&E, PAS, and PASM staining. Immunofluorescence analysis revealed that macrophage infiltration was significantly enhanced in FADD-D mice. Renal fibrosis might be induced by IgA nephritis in FADD-D mice as evidenced by increased Ki67 and type IV collagen. Additionally, the levels of α-SMA, Fibronectin, and Vimentin were also found to be elevated. Mechanism study indicated that the TLR4/myD88/NF-κB signaling pathway was activated in FADD-D mice. Moreover, FADD phosphorylation activated the mTOR and TGF-ß/Smad pathway and accelerated the process of epithelial mesenchymal transition. Further studies indicated that the TGF-ß1 pathway was also activated and the process of EMT was accelerated in both FADD-disrupted HEK293 cells and FADD-deficient MES cells. Thus, we concluded that FADD phosphorylation could lead to IgA nephritis and eventually result in renal fibrosis. Taken together, our study provides evidence, for the first time, that FADD, especially in its phosphorylated form, has an effect on the development of renal fibrosis.Abbreviations: FADD: FAS-associated protein with death domain; DED: death effector domain; DD: death domain; CKD: chronic kidney disease; ECM: extracellular matrix; ESRD: end-stage renal disease; RRT: renal replacement therapy; H&E: hematoxylin and eosin; PASM: periodic acid silver methenamine.
Assuntos
Nefropatias , Nefrite , Camundongos , Humanos , Animais , Transição Epitelial-Mesenquimal , Fosforilação , Células HEK293 , Nefropatias/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fibrose , Imunoglobulina A/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/farmacologiaRESUMO
BACKGROUND: Colorectal cancer is one of the most prevalent cancers in the world, but the research on its prevention, early diagnosis and treatment is still a major challenge in clinical oncology. Thus, there is a pressing requirement to find effective strategies to improve the survival of colon cancer patients. METHODS: Celecoxib has been accounted to be an effective antitumor drug, but may exhibit significant side effects. In recent studies, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), one of the most commonly used reagents for the synthesis of sustained-release H2 S donors, has also been reported to inhibit cancer progression by affecting processes such as cell cycle, angiogenesis, and apoptosis. Therefore, we evaluated the therapeutic effect of the combination of ADT-OH and celecoxib on colorectal cancer through in vitro and in vivo, hoping to achieve better therapeutic effect and reduce the effect of celecoxib on gastric injury through exogenous administration of H2 S. RESULTS: Our results demonstrated that ADT-OH combined with celecoxib synergistically inhibited the proliferation and migration ability of human colorectal cancer HCT116 cells, altered cell cycle and cytoskeleton, increased intracellular reactive oxygen species (ROS), and promoted cell apoptosis. Noteworthy, in vivo studies also indicated the excellent antitumor therapeutic effect of the combination therapy without apparent toxicity. CONCLUSIONS: In general, our results provide a reasonable combination strategy of low-dose ADT-OH and celecoxib in the preclinical application of colorectal cancer.
Assuntos
Neoplasias do Colo , Tionas , Humanos , Celecoxib/farmacologia , Celecoxib/uso terapêutico , Tionas/farmacologia , Tionas/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Apoptose , Proliferação de Células , Linhagem Celular TumoralRESUMO
Owing to the increasing incidence and prevalence of inflammatory bowel disease (IBD) worldwide, effective and safe treatments for IBD are urgently needed. Hydrogen sulfide (H2S) is an endogenous gasotransmitter and plays an important role in inflammation. To date, H2S-releasing agents are viewed as potential anti-inflammatory drugs. The slow-releasing H2S donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), known as a potent therapeutic with chemopreventive and cytoprotective properties, has received attention recently. Here, we reported its anti-inflammatory effects on dextran sodium sulfate (DSS)-induced acute (7 days) and chronic (30 days) colitis. We found that ADT-OH effectively reduced the DSS-colitis clinical score and reversed the inflammation-induced shortening of colon length. Moreover, ADT-OH reduced intestinal inflammation by suppressing the nuclear factor kappa-B pathway. In vivo and in vitro results showed that ADT-OH decreased intestinal permeability by increasing the expression of zonula occludens-1 and occludin and blocking increases in myosin II regulatory light chain phosphorylation and epithelial myosin light chain kinase protein expression levels. In addition, ADT-OH restored intestinal microbiota dysbiosis characterized by the significantly increased abundance of Muribaculaceae and Alistipes and markedly decreased abundance of Helicobacter, Mucispirillum, Parasutterella, and Desulfovibrio. Transplanting ADT-OH-modulated microbiota can alleviate DSS-induced colitis and negatively regulate the expression of local and systemic proinflammatory cytokines. Collectively, ADT-OH is safe without any short-term (5 days) or long-term (30 days) toxicological adverse effects and can be used as an alternative therapeutic agent for IBD treatment.
Assuntos
Colite , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Humanos , Camundongos , Animais , Função da Barreira Intestinal , Camundongos Endogâmicos C57BL , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Inflamação , Anti-Inflamatórios/farmacologia , Modelos Animais de DoençasRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing, transactivation of gene expression, and regulation of protein translation. As a core component of the hnRNP complex in mammalian cells, heterogeneous nuclear ribonucleoprotein A2B1 (hnRNP A2B1) participates in and coordinates various molecular events. Given its regulatory role in inflammation and cancer progression, hnRNP A2B1 has become a novel player in immune response, inflammation, and cancer development. Concomitant with these new roles, a surprising number of mechanisms deemed to regulate hnRNP A2B1 functions have been identified, including post-translational modifications, changes in subcellular localization, direct interactions with multiple DNAs, RNAs, and proteins or the formation of complexes with them, which have gradually made hnRNP A2B1 a molecular target for multiple drugs. In light of the rising interest in the intersection between cancer and inflammation, this review will focus on recent knowledge of the biological roles of hnRNP A2B1 in cancer, immune response, and inflammation.