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BACKGROUND: Sorafenib improves the overall survival in patients with advanced hepatocellular carcinoma (HCC). Dickkopf-1 (DKK1) is commonly overexpressed in HCC. In this study, we investigated whether the inhibition of DKK1 enhances the anti-tumor efficacy of sorafenib in HCC. METHODS: HCC cells were treated with sorafenib and WAY-262611, which is an inhibitor of DKK1. Transgenic mouse models were also developed using hydrodynamic tail vein injection. Mice were orally administered with sorafenib (32 mg/kg), WAY-262611 (16 mg/kg), or sorafenib + WAY-262611 for 10 days. Mechanisms of sorafenib and WAY-262611 were explored via western blotting, immunostaining, and RNA sequencing. RESULTS: DKK1 was significantly overexpressed in patients with HCC than in the healthy controls and patients with liver diseases except HCC (all P < 0.05). Compared with sorafenib alone, sorafenib + WAY-262611 significantly inhibited the cell viability, invasion, migration, and colony formation by promoting apoptosis and altering the cell cycles in HCC cells (all P < 0.05). Moreover, sorafenib + WAY-262611 decreased the p110α, phospho-Akt (all P < 0.05), active ß-catenin (all P < 0.05) and phospho-GSK-3ß (Ser9) expression levels, while increasing the phospho-GSK-3ß (Tyr216) expression levels compared with those in the sorafenib alone in vitro and in vivo. In addition, sorafenib + WAY-262611 inhibited tumor progression by regulating cell proliferation and apoptosis, significantly better than sorafenib alone in mouse models. CONCLUSIONS: Our results indicate that DKK1 inhibition significantly enhances the anti-tumor efficacy of sorafenib by inhibiting the PI3K/Akt and Wnt/ß-catenin pathways via regulation of GSK3ß activity, suggesting a novel therapeutic strategy for HCC. Video Abstract.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Ratones , Animales , Carcinoma Hepatocelular/genética , Sorafenib/farmacología , Glucógeno Sintasa Quinasa 3 beta , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias Hepáticas/metabolismo , beta Catenina/metabolismo , Proliferación Celular , Línea Celular TumoralRESUMEN
Histone acetylation involves the transfer of two-carbon units to the nucleus that are embedded in low-concentration metabolites. We found that lactate, a high-concentration metabolic byproduct, can be a major carbon source for histone acetylation through oxidation-dependent metabolism. Both in cells and in purified nuclei, 13C3-lactate carbons are incorporated into histone H4 (maximum incorporation: ~60%). In the purified nucleus, this process depends on nucleus-localized lactate dehydrogenase (LDHA), knockout (KO) of which abrogates incorporation. Heterologous expression of nucleus-localized LDHA reverses the KO effect. Lactate itself increases histone acetylation, whereas inhibition of LDHA reduces acetylation. In vitro and in vivo settings exhibit different lactate incorporation patterns, suggesting an influence on the microenvironment. Higher nuclear LDHA localization is observed in pancreatic cancer than in normal tissues, showing disease relevance. Overall, lactate and nuclear LDHA can be major structural and regulatory players in the metabolism-epigenetics axis controlled by the cell's own status or the environmental status.
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Histonas , Ácido Láctico , Histonas/metabolismo , Ácido Láctico/metabolismo , Acetilación , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Epigénesis GenéticaRESUMEN
The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. STATEMENT OF SIGNIFICANCE: This study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.
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Osteogénesis por Distracción , Profármacos , Ratas , Humanos , Animales , Tibia/metabolismo , Osteogénesis por Distracción/métodos , Profármacos/farmacología , Microtomografía por Rayos X , Proteínas Morfogenéticas Óseas , Proteína Morfogenética Ósea 2/farmacología , Osteogénesis , Proteína Morfogenética Ósea 7/farmacologíaRESUMEN
Decreased angiogenesis contributes to delayed wound healing in diabetic patients. Recombinant human bone morphogenetic protein-2 (rhBMP2) has also been demonstrated to promote angiogenesis. However, the short half-lives of soluble growth factors, including rhBMP2, limit their use in wound-healing applications. To address this limitation, we propose a novel delivery model using a protein transduction domain (PTD) formulated in a lipid nanoparticle (LNP). We aimed to determine whether a gelatin hydrogel dressing loaded with LNP-formulated PTD-BMP2 (LNP-PTD-BMP2) could enhance the angiogenic function of BMP2 and improve diabetic wound healing. In vitro, compared to the control and rhBMP2, LNP-PTD-BMP2 induced greater tube formation in human umbilical vein endothelial cells and increased the cell recruitment capacity of HaCaT cells. We inflicted large, full-thickness back skin wounds on streptozotocin-induced diabetic mice and applied gelatin hydrogel (GH) cross-linked by microbial transglutaminase containing rhBMP2, LNP-PTD-BMP2, or a control to these wounds. Wounds treated with LNP-PTD-BMP2-loaded GH exhibited enhanced wound closure, increased re-epithelialization rates, and higher collagen deposition than those with other treatments. Moreover, LNP-PTD-BMP2-loaded GH treatment resulted in more CD31- and α-SMA-positive cells, indicating greater neovascularization capacity than rhBMP2-loaded GH or GH treatments alone. Furthermore, in vivo near-infrared fluorescence revealed that LNP-PTD-BMP2 has a longer half-life than rhBMP2 and that BMP2 localizes around wounds. In conclusion, LNP-PTD-BMP2-loaded GH is a viable treatment option for diabetic wounds.
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Background: Approximately 30% of diabetic patients develop diabetic nephropathy, a representative microvascular complication. Although the etiological mechanism has not yet been fully elucidated, renal tubular damage by hyperglycemia-induced expression of transforming growth factor-ß (TGF-ß) is known to be involved. Recently, a new type of cell death by iron metabolism called ferroptosis was reported to be involved in kidney damage in animal models of diabetic nephropathy, which could be induced by TGF-ß. Bone morphogenetic protein-7 (BMP7) is a well-known antagonist of TGF-ß inhibiting TGF-ß-induced fibrosis in many organs. Further, BMP7 has been reported to play a role in the regeneration of pancreatic beta cells in diabetic animal models. Methods: We used protein transduction domain (PTD)-fused BMP7 in micelles (mPTD-BMP7) for long-lasting in vivo effects and effective in vitro transduction and secretion. Results: mPTD-BMP7 successfully accelerated the regeneration of diabetic pancreas and impeded progression to diabetic nephropathy. With the administration of mPTD-BMP7, clinical parameters and representative markers of pancreatic damage were alleviated in a mouse model of streptozotocin-induced diabetes. It not only inhibited the downstream genes of TGF-ß but also attenuated ferroptosis in the kidney of the diabetic mouse and TGF-ß-stimulated rat kidney tubular cells. Conclusion: BMP7 impedes the progression of diabetic nephropathy by inhibiting the canonical TGF-ß pathway, attenuating ferroptosis, and helping regenerate diabetic pancreas.
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Diabetes Mellitus Experimental , Nefropatías Diabéticas , Ferroptosis , Animales , Ratones , Ratas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Páncreas/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
A structural protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), nucleocapsid (N) protein is phosphorylated by glycogen synthase kinase (GSK)-3 on the serine/arginine (SR) rich motif located in disordered regions. Although phosphorylation by GSK-3ß constitutes a critical event for viral replication, the molecular mechanism underlying N phosphorylation is not well understood. In this study, we found the putative alpha-helix L/FxxxL/AxxRL motif known as the GSK-3 interacting domain (GID), found in many endogenous GSK-3ß binding proteins, such as Axins, FRATs, WWOX, and GSKIP. Indeed, N interacts with GSK-3ß similarly to Axin, and Leu to Glu substitution of the GID abolished the interaction, with loss of N phosphorylation. The N phosphorylation is also required for its structural loading in a virus-like particle (VLP). Compared to other coronaviruses, N of Sarbecovirus lineage including bat RaTG13 harbors a CDK1-primed phosphorylation site and Gly-rich linker for enhanced phosphorylation by GSK-3ß. Furthermore, we found that the S202R mutant found in Delta and R203K/G204R mutant found in the Omicron variant allow increased abundance and hyper-phosphorylation of N. Our observations suggest that GID and mutations for increased phosphorylation in N may have contributed to the evolution of variants.
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Glucógeno Sintasa Quinasa 3 , Proteínas de la Nucleocápside , SARS-CoV-2 , Humanos , Fosforilación , Proteínas de la Nucleocápside/genéticaRESUMEN
PURPOSE: Limited treatment options are currently available for glioblastoma (GBM), an extremely lethal type of brain cancer. For a variety of tumor types, bioenergetic deprivation through inhibition of cancer-specific metabolic pathways has proven to be an effective therapeutic strategy. Here, we evaluated the therapeutic effects and underlying mechanisms of dual inhibition of carnitine palmitoyltransferase 1A (CPT1A) and glucose-6-phosphate dehydrogenase (G6PD) critical for fatty acid oxidation (FAO) and the pentose phosphate pathway (PPP), respectively, against GBM tumorspheres (TSs). METHODS: Therapeutic efficacy against GBM TSs was determined by assessing cell viability, neurosphere formation, and 3D invasion. Liquid chromatography-mass spectrometry (LC-MS) and RNA sequencing were employed for metabolite and gene expression profiling, respectively. Anticancer efficacy in vivo was examined using an orthotopic xenograft model. RESULTS: CPT1A and G6PD were highly expressed in GBM tumor tissues. Notably, siRNA-mediated knockdown of both genes led to reduced viability, ATP levels, and expression of genes associated with stemness and invasiveness. Similar results were obtained upon combined treatment with etomoxir and dehydroepiandrosterone (DHEA). Transcriptome analyses further confirmed these results. Data from LC-MS analysis showed that this treatment regimen induced a considerable reduction in the levels of metabolites associated with the TCA cycle and PPP. Additionally, the combination of etomoxir and DHEA inhibited tumor growth and extended survival in orthotopic xenograft model mice. CONCLUSION: Our collective findings support the utility of dual suppression of CPT1A and G6PD with selective inhibitors, etomoxir and DHEA, as an efficacious therapeutic approach for GBM.
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Glioblastoma , Animales , Humanos , Ratones , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Línea Celular Tumoral , Deshidroepiandrosterona/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
INTRODUCTION: The importance of fatty acid oxidation (FAO) in the bioenergetics of glioblastoma (GBM) is being realized. Etomoxir (ETO), a carnitine palmitoyltransferase 1 (CPT1) inhibitor exerts cytotoxic effects in GBM, which involve interrupting the FAO pathway. We hypothesized that FAO inhibition could affect the outcomes of current standard temozolomide (TMZ) chemotherapy against GBM. METHODS: The FAO-related gene expression was compared between GBM and the tumor-free cortex. Using four different GBM tumorspheres (TSs), the effects of ETO and/or TMZ was analyzed on cell viability, tricarboxylate (TCA) cycle intermediates and adenosine triphosphate (ATP) production to assess metabolic changes. Alterations in tumor stemness, invasiveness, and associated transcriptional changes were also measured. Mouse orthotopic xenograft model was used to elucidate the combinatory effect of TMZ and ETO. RESULTS: GBM tissues exhibited overexpression of FAO-related genes, especially CPT1A, compared to the tumor-free cortex. The combined use of ETO and TMZ further inhibited TCA cycle and ATP production than single uses. This combination treatment showed superior suppression effects compared to treatment with individual agents on the viability, stemness, and invasiveness of GBM TSs, as well as better downregulation of FAO-related gene expression. The results of in vivo study showed prolonged survival outcomes in the combination treatment group. CONCLUSION: ETO, an FAO inhibitor, causes a lethal energy reduction in the GBM TSs. When used in combination with TMZ, ETO effectively reduces GBM cell stemness and invasiveness and further improves survival. These results suggest a potential novel treatment option for GBM.
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Purpose: We investigated a role of bone morphogenic protein 7 (BMP7), a member of the TGF-ß superfamily on pathogenic mechanism of Graves' orbitopathy (GO). The therapeutic effects of BMP7 on inflammation and fibrosis were evaluated in cultured Graves' orbital fibroblasts. Methods: Expression of BMP7 was compared in cultured orbital tissue explants from GO (n = 12) and normal control (n = 12) subjects using real-time PCR. Orbital fibroblasts were cultured from orbital connective tissues obtained from GO (n = 3) and normal control patients (n = 3). Cells were pretreated with recombinant human BMP7 (rhBMP7) before stimulation with TGF-ß, IL-1ß, and TNF-α. Fibrosis-related proteins and inflammatory cytokines were analyzed by Western blotting. The activation of signaling molecules in inflammation and fibrosis was also analyzed. Results: The expressions of BMP7 mRNA were lower in GO orbital tissues than control. Fibrosis-related proteins, fibronectin, collagen 1α, and α-SMA induced by TGF-ß were suppressed by treating rhBMP7, and rhBMP7 upregulated TGF-ß induced SMAD1/5/8 protein expression, whereas downregulated SMAD2/3. Increased pro-inflammatory molecules, IL-6, IL-8, and intercellular adhesion molecule-1 (ICAM-1) by IL-1ß or TNF-α were blocked by rhBMP7 treatment, and the expression of phosphorylated NFκB and Akt was suppressed by rhBMP7 treatment. Conclusions: BMP7 transcript levels were downregulated in Graves' orbital tissues. Exogenous BMP7 treatment showed inhibitory effects on the production of profibrotic proteins and proinflammatory cytokines in orbital fibroblasts. Our results provide a molecular basis of BMP7 as a new potential therapeutic agent through the opposing mechanism of profibrotic TGF-ß/SMAD signaling and proinflammatory cytokine production.
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Oftalmopatía de Graves , Proteína Morfogenética Ósea 7/farmacología , Células Cultivadas , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibrosis , Oftalmopatía de Graves/metabolismo , Humanos , Inflamación/metabolismo , Órbita/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Considering the high metastatic potential of colorectal cancer (CRC), the inhibition of metastasis is important for anti-CRC therapy. Agrimonia pilosa Ledeb (A. pilosa) is a perennial herbaceous plant that is widely distributed in Asia. The extracts of A. pilosa have shown diverse pharmacological properties, such as antimicrobial, anti-inflammatory, and antitumor activities. In the present study, the antimetastatic activity of A. pilosa was evaluated. Methanol extraction from the roots of A. pilosa was performed by high-performance liquid chromatography (HPLC) and 12 fractions were obtained. Among these, fraction 4 showed the most potent inhibitory effect on the migration of colon cancer cells. Using LC-HR MS analysis, quercetin and quercitrin were identified as flavonoids contained in fraction 4. Like fraction 4, quercetin and quercitrin effectively inhibited the migration and invasion of RKO cells. While the level of E-cadherin was increased, the levels of N-cadherin and vimentin were decreased by the same agents. Although they all activate the p38, JNK, and ERK signaling pathways, only SP600125, an inhibitor of the JNK pathway, specifically inhibited the effect of fraction 4, quercetin, and quercitrin on cell migration. An in vivo experiment also confirmed the antitumor activity of quercetin and quercitrin. Collectively, these results suggest that A. pilosa and its two flavonoids, quercetin and quercitrin, are candidates for the antimetastatic treatment of CRC.
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Bone morphogenetic protein-7 (BMP-7) antagonizes transforming growth factor-ß (TGF-ß), which is critically involved in liver fibrogenesis. Here, we designed a micelle formulation consisting of a protein transduction domain (PTD) fused BMP-7 polypeptide (mPTD-BMP-7) to enhance endocytic delivery, and investigated its ability to ameliorate liver fibrosis. The mPTD-BMP-7 formulation was efficiently delivered into cells via endocytosis, where it inhibited TGF-ß mediated epithelial-mesenchymal transition. After successfully demonstrating delivery of fluorescently labeled mPTD-BMP-7 into the murine liver in vivo, we tested the mPTD-BMP-7 formulation in a murine liver fibrosis model, developed by repeated intraperitoneal injection of hepatotoxic carbon tetrachloride, twice weekly from 4 to 16 weeks. mPTD-BMP-7 effects were tested by injecting the mPTD-BMP-7 formulation (or vehicle control) into the lateral tail at a dose of 50 (n=8) or 500 µg/kg (n=10), also twice per week from 4 to 16 weeks. Vehicle-treated control mice developed fibrous septa surrounding the liver parenchyma and marked portal-to-portal bridging with occasional nodules, whereas mice treated with mPTD-BMP-7 showed only fibrous expansion of some portal areas, with or without short fibrous septa. Using the Ishak scoring system, we found that the fibrotic burden was significantly lower in mPTD-BMP-7 treated mice than in control mice (all P<0.001). Treatment with mPTD-BMP-7 protected tight junctions between hepatocytes and reduced extracellular matrix protein levels. It also significantly decreased mRNA levels of collagen 1A, smooth muscle α-actin, and connective tissue growth factor compared with that in control mice (all P<0.001). Collectively, out results indicate that mPTD-BMP-7, a prodrug formulation of BMP-7, ameliorates liver fibrosis by suppressing the TGF-ß signaling pathway in a murine liver fibrosis model.
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The epithelial-mesenchymal transition (EMT) comprises an important biological mechanism not only for cancer progression but also in the therapeutic resistance of cancer cells. While the importance of the protein abundance of EMT-inducers, such as Snail (SNAI1) and Zeb1 (ZEB1), during EMT progression is clear, the reciprocal interactions between the untranslated regions (UTRs) of EMT-inducers via a competing endogenous RNA (ceRNA) network have received little attention. In this study, we found a synchronized transcript abundance of Snail and Zeb1 mediated by a non-coding RNA network in colorectal cancer (CRC). Importantly, the trans-regulatory ceRNA network in the UTRs of EMT inducers is mediated by competition between tumor suppressive miRNA-34 (miR-34) and miRNA-200 (miR-200). Furthermore, the ceRNA network consisting of the UTRs of EMT inducers and tumor suppressive miRs is functional in the EMT phenotype and therapeutic resistance of colon cancer. In The Cancer Genome Atlas (TCGA) samples, we also found genome-wide ceRNA gene sets regulated by miR-34a and miR-200 in colorectal cancer. These results indicate that the ceRNA networks regulated by the reciprocal interaction between EMT gene UTRs and tumor suppressive miRs are functional in CRC progression and therapeutic resistance.
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Neoplasias Colorrectales/metabolismo , Genes Supresores de Tumor , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Neoplásico/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/terapia , Femenino , Células HCT116 , Humanos , Ratones , Ratones Desnudos , MicroARNs/genética , Proteínas de Neoplasias/genética , ARN Neoplásico/genética , Factores de Transcripción de la Familia Snail/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
African swine fever virus (ASFV) causes a highly contagious and severe hemorrhagic viral disease with high mortality in domestic pigs of all ages. Although the virus is harmless to humans, the ongoing ASFV epidemic could have severe economic consequences for global food security. Recent studies have found a few antiviral agents that can inhibit ASFV infections. However, currently, there are no vaccines or antiviral drugs. Hence, there is an urgent need to identify new drugs to treat ASFV. Based on the structural information data on the targets of ASFV, we used molecular docking and machine learning models to identify novel antiviral agents. We confirmed that compounds with high affinity present in the region of interest belonged to subsets in the chemical space using principal component analysis and k-means clustering in molecular docking studies of FDA-approved drugs. These methods predicted pentagastrin as a potential antiviral drug against ASFVs. Finally, it was also observed that the compound had an inhibitory effect on AsfvPolX activity. Results from the present study suggest that molecular docking and machine learning models can play an important role in identifying potential antiviral drugs against ASFVs.
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Virus de la Fiebre Porcina Africana/efectos de los fármacos , Fiebre Porcina Africana/tratamiento farmacológico , Antivirales/química , Antivirales/farmacología , Aprendizaje Automático/normas , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Secuencia de Aminoácidos , Animales , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Pentagastrina/química , Pentagastrina/farmacología , Porcinos , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
The Wnt and Hippo pathways are tightly coordinated and understanding their reciprocal regulation may provide a novel therapeutic strategy for cancer. Anti-helminthic niclosamide is an effective inhibitor of Wnt and is now in a phase II trial for advanced colorectal cancer (CRC) patients. We found that Axin2, an authentic target gene of canonical Wnt, acts as aYAP phosphorylation activator in APC-mutated CRC. While niclosamide effectively suppresses Wnt, it also inhibits Hippo, limiting its therapeutic potential for CRC. To overcome this limitation, we utilized metformin, a clinically available AMPK activator. This combinatory approach not only suppresses canonical Wnt activity, but also inhibits YAP activity in CRC cancer cells and in patient-derived cancer organoid through the suppression of cancer stemness. Further, combinatory oral administration suppressed in vivo tumorigenesis and the cancer progression of APC-MIN mice models. Our observations provide not only a reciprocal link between Wnt and Hippo, but also clinically available novel therapeutics that are able to target Wnt and YAP in APC-mutated CRC.
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Although protein-protein interactions (PPIs) have emerged as the basis of potential new therapeutic approaches, targeting intracellular PPIs with small molecule inhibitors is conventionally considered highly challenging. Driven by increasing research efforts, success rates have increased significantly in recent years. In this study, we analyze the physicochemical properties of 9351 non-redundant inhibitors present in the iPPI-DB and TIMBAL databases to define a computational model for active compounds acting against PPI targets. Principle component analysis (PCA) and k-means clustering were used to identify plausible PPI targets in regions of interest in the active group in the chemical space between active and inactive iPPI compounds. Notably, the uniquely defined active group exhibited distinct differences in activity compared with other active compounds. These results demonstrate that active compounds with regions of interest in the chemical space may be expected to provide insights into potential PPI inhibitors for particular protein targets.
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Proteínas/química , Bibliotecas de Moléculas Pequeñas/química , Simulación por Computador , Descubrimiento de Drogas/métodos , Humanos , Aprendizaje Automático , Análisis de Componente Principal/métodos , Mapeo de Interacción de Proteínas/métodosRESUMEN
Ischemia-reperfusion injury is a major cause of acute kidney injury. Recent studies on the pathophysiology of ischemia-reperfusion-induced acute kidney injury showed that immunologic responses significantly affect kidney ischemia-reperfusion injury and repair. Nuclear factor (NF)-ĸB signaling, which controls cytokine production and cell survival, is significantly involved in ischemia-reperfusion-induced acute kidney injury, and its inhibition can ameliorate ischemic acute kidney injury. Using EXPLOR, a novel, optogenetically engineered exosome technology, we successfully delivered the exosomal super-repressor inhibitor of NF-ĸB (Exo-srIĸB) into B6 wild type mice before/after kidney ischemia-reperfusion surgery, and compared outcomes with those of a control exosome (Exo-Naïve)-injected group. Exo-srIĸB treatment resulted in lower levels of serum blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin in post-ischemic mice than in the Exo-Naïve treatment group. Systemic delivery of Exo-srIĸB decreased NF-ĸB activity in post-ischemic kidneys and reduced apoptosis. Post-ischemic kidneys showed decreased gene expression of pro-inflammatory cytokines and adhesion molecules with Exo-srIĸB treatment as compared with the control. Intravital imaging confirmed the uptake of exosomes in neutrophils and macrophages. Exo-srIĸB treatment also significantly affected post-ischemic kidney immune cell populations, lowering neutrophil, monocyte/macrophage, and T cell frequencies than those in the control. Thus, modulation of NF-ĸB signaling through exosomal delivery can be used as a novel therapeutic method for ischemia-reperfusion-induced acute kidney injury.
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Lesión Renal Aguda , Exosomas , Daño por Reperfusión , Lesión Renal Aguda/etiología , Lesión Renal Aguda/prevención & control , Animales , Riñón , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa , Daño por Reperfusión/prevención & controlRESUMEN
Fibrosis is presented in various physiologic and pathologic conditions of the salivary gland. Transforming growth factor beta (TGF-ß) pathway has a pivotal role in the pathogenesis of fibrosis in several organs, including the salivary glands. Among the TGF-ß superfamily members, TGF-ß1 and 2 are pro-fibrotic ligands, whereas TGF-ß3 and some bone morphogenetic proteins (BMPs) are anti-fibrotic ligands. TGF-ß1 is thought to be associated with the pro-fibrotic pathogenesis of sialadenitis, post-radiation salivary gland dysfunction, and Sjögren's syndrome. Potential therapeutic strategies that target multiple levels in the TGF-ß pathway are under preclinical and clinical research for fibrosis. Despite the anti-fibrotic effect of BMPs, their in vivo delivery poses a challenge in terms of adequate clinical efficacy. In this article, we will review the relevance of TGF-ß signaling in salivary gland fibrosis and advances of potential therapeutic options in the field.
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Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Susceptibilidad a Enfermedades , Fibrosis , Humanos , Radiación , Síndrome de Sjögren/etiología , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patologíaRESUMEN
Tubulointerstitial renal fibrosis is a chronic disease process affecting chronic kidney disease (CKD). While the etiological role of transforming growth factor-beta (TGF-ß) is well known for epithelial-mesenchymal transition (EMT) in chronic kidney disease, effective therapeutics for renal fibrosis are largely limited. As a member of the TGF-ß superfamily, bone morphogenetic protein-7 (BMP-7) plays an important role as an endogenous antagonist of TGF-ß, inhibiting fibrotic progression in many organs. However, soluble rhBMP-7 is hardly available for therapeutics due to its limited pharmacodynamic profile and rapid clearance in clinical settings. In this study, we have developed a novel therapeutic approach with protein transduction domain (PTD) fused BMP-7 in micelle (mPTD-BMP-7) for long-range signaling in vivo. Contrary to rhBMP-7 targeting its cognate receptors, the nano-sized mPTD-BMP-7 is transduced into cells through an endosomal pathway and secreted to the exosome having active BMP-7. Further, transduced mPTD-BMP-7 successfully activates SMAD1/5/8 and inhibits the TGF-ß-mediated epithelial-mesenchymal transition process in vitro and in an in vivo unilateral ureter obstruction model. To determine the clinical relevance of our strategy, we also developed an intra-arterial administration of mPTD-BMP-7 through renal artery in pigs. Interestingly, mPTD-BMP-7 through renal artery intervention effectively delivered into Bowman's space and inhibits unilateral ureter obstruction-induced renal fibrosis in pigs. Our results provide a novel therapeutic targeting TGF-ß-mediated renal fibrosis and other organs as well as a clinically available approach for kidney.
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Recent findings indicate that (a) mitochondria in proliferating cancer cells are functional, (b) cancer cells use more oxygen than normal cells for oxidative phosphorylation, and (c) cancer cells critically rely on cytosolic NADH transported into mitochondria via the malate-aspartate shuttle (MAS) for ATP production. In a spontaneous lung cancer model, tumor growth was reduced by 50% in heterozygous oxoglutarate carrier (OGC) knock-out mice compared with wild-type counterparts. To determine the mechanism through which OGC promotes tumor growth, the effects of the OGC inhibitor N-phenylmaleimide (NPM) on mitochondrial activity, oxygen consumption, and ATP production were evaluated in melanoma cell lines. NPM suppressed oxygen consumption and decreased ATP production in melanoma cells in a dose-dependent manner. NPM also reduced the proliferation of melanoma cells. To test the effects of NPM on tumor growth and metastasis in vivo, NPM was administered in a human melanoma xenograft model. NPM reduced tumor growth by approximately 50% and reduced melanoma invasion by 70% at a dose of 20 mg/kg. Therefore, blocking OGC activity may be a useful approach for cancer therapy.
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While the bone morphogenetic protein-7 (BMP-7) is a well-known therapeutic growth factor reverting many fibrotic diseases, including peritoneal fibrosis by peritoneal dialysis (PD), soluble growth factors are largely limited in clinical applications owing to their short half-life in clinical settings. Recently, we developed a novel drug delivery model using protein transduction domains (PTD) overcoming limitation of soluble recombinant proteins, including bone morphogenetic protein-7 (BMP-7). This study aims at evaluating the therapeutic effects of PTD-BMP-7 consisted of PTD and full-length BMP-7 on epithelial-mesenchymal transition (EMT)-related fibrosis. Human peritoneal mesothelial cells (HPMCs) were then treated with TGF-ß1 or TGF-ß1 + PTD-BMP-7. Peritoneal dialysis (PD) catheters were inserted into Sprague-Dawley rats, and these rats were infused intra-peritoneally with saline, peritoneal dialysis fluid (PDF) or PDF + PTD-BMP-7. In vitro, TGF-ß1 treatment significantly increased fibronectin, type I collagen, α-SMA and Snail expression, while reducing E-cadherin expression in HPMCs (P < .001). PTD-BMP-7 treatment ameliorated TGF-ß1-induced fibronectin, type I collagen, α-SMA and Snail expression, and restored E-cadherin expression in HPMCs (P < .001). In vivo, the expressions of EMT-related molecules and the thickness of the sub-mesothelial layer were significantly increased in the peritoneum of rats treated with PDF, and these changes were significantly abrogated by the intra-peritoneal administration of PTD-BMP-7. PTD-BMP-7 treatment significantly inhibited the progression of established PD fibrosis. These findings suggest that PTD-BMP-7, as a prodrug of BMP-7, can be an effective therapeutic agent for peritoneal fibrosis in PD patients.