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CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.
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Quinases Ciclina-Dependentes , Neoplasias , Animais , Humanos , Camundongos , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina/metabolismo , Fosforilação , Pirimidinas/farmacologia , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologiaRESUMO
Mild photothermal therapy (mPTT), which circumvents the limitations of conventional photothermal therapy, is emerging and exhibits remarkable potential in clinical applications. Nevertheless, mPTT is not able to efficiently eradicate tumors because its therapeutic efficacy is dramatically diminished by stress-induced heat shock proteins (HSP). Herein, a core-shell structured Au@Pd (AP) bimetallic nanozyme was fabricated for reactive oxygen species (ROS) augmentation-induced mPTT. The nanocatalytic AP nanozymes with photothermal conversion performance harbor multienzymatic (catalase, oxidase, and peroxidase) activities to induce ROS storm formation. The generated ROS could suppress the heat-defense response of tumor cells by cleaving HSP. Overall, our work highlights a ROS-regulating strategy to counteract hyperthermia-associated resistance in mPTT.
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Neoplasias , Terapia Fototérmica , Humanos , Espécies Reativas de Oxigênio , Neoplasias/terapia , Peroxidase , Peroxidases , Linhagem Celular Tumoral , Microambiente Tumoral , Peróxido de HidrogênioRESUMO
In photothermal treatments (PTTs), normal tissues around cancerous tumors get injured by excessive heat, whereas damaged cancer cells are easily restored by stress-induced heat shock proteins (HSPs) at low temperatures. Therefore, to achieve a unique tumor microenvironment (TME), it is imperative to increase PTT efficiency and reduce normal tissue injury by adopting appropriate reactive oxygen species (ROS) and lipid peroxides (LPO) cross-linked with HSPs. In the present research, a potential strategy for mild photothermal treatments (mPTTs) was proposed by initiating localized catalytic chemical reactions in TME based on Pd nanozyme-modified hydrogenated TiO2 (H-TiO2@Pd). In vitro and in vivo evaluations demonstrated that H-TiO2@Pd had good peroxidase-like activities (POD), glutathione oxidase-like activities (GSHOx), and photodynamic properties and also satisfactory biocompatibility for 4T1 cells. Localized catalytic chemical reactions in H-TiO2@Pd significantly depleted GSH to downregulate the protein expression of GPX4 and promoted the accumulation of LPO and ROS, which consumed HSP70 or inhibited its function in 4T1 cells. Hence, the as-constructed low-temperature photothermal therapeutic platform based on Pd nanozyme-modified H-TiO2 can be a promising candidate to develop a safe and effective mPTT for cancer treatments.
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Peróxidos Lipídicos , Terapia Fototérmica , Espécies Reativas de Oxigênio , Temperatura , CatáliseRESUMO
Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice. Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface, graft necrosis and sclerosis. However, poor gap integration is a serious concern, which eventually leads to deterioration of joint function. To deal with such complications, this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl (GelMA) hydrogel (BSN-GelMA). A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty, as early as six weeks. Moreover, the International Cartilage Repair Society score, histology score, glycosaminoglycan content, subchondral bone volume, and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group. This improved outcome was due to bio-interactive materials, which acted as tissue fillers to bridge the gap, prevent cartilage degeneration, and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel. This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty. It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.
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OBJECTIVES: This study investigated the stage-specific and location-specific deposition and characteristics of minerals in human osteoarthritis (OA) cartilages via multiple nano-analytical technologies. METHODS: Normal and OA cartilages were serially sectioned for micro-CT, scanning electron microscopy with energy dispersive X-ray spectroscopy, micro-Raman spectroscopy, focused ion beam scanning electron microscopy, high-resolution electron energy loss spectrometry with transmission electron microscopy, nanoindentation and atomic force microscopy to analyse the structural, compositional and mechanical properties of cartilage in OA progression. RESULTS: We found that OA progressed by both top-down calcification at the joint surface and bottom-up calcification at the osteochondral interface. The top-down calcification process started with spherical mineral particle formation in the joint surface during early-stage OA (OA-E), followed by fibre formation and densely packed material transformation deep into the cartilage during advanced-stage OA (OA-A). The bottom-up calcification in OA-E started when an excessive layer of calcified tissue formed above the original calcified cartilage, exhibiting a calcified sandwich structure. Over time, the original and upper layers of calcified cartilage fused, which thickened the calcified cartilage region and disrupted the cartilage structure. During OA-E, the calcified cartilage was hypermineralised, containing stiffer carbonated hydroxyapatite (HAp). During OA-A, it was hypomineralised and contained softer HAp. This discrepancy may be attributed to matrix vesicle nucleation during OA-E and carbonate cores during OA-A. CONCLUSIONS: This work refines our current understanding of the mechanism underlying OA progression and provides the foothold for potential therapeutic targeting strategies once the location-specific cartilage calcification features in OA are established.
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Calcinose , Cartilagem Articular , Osteoartrite , Humanos , Cartilagem Articular/diagnóstico por imagem , Osteoartrite/diagnóstico por imagem , Calcinose/diagnóstico por imagem , Calcinose/etiologiaRESUMO
The depletion of reactive oxygen species (ROS) by glutathione (GSH) and oxidative stress induced protective autophagy severely impaired the therapeutic effect of chemodynamic therapy (CDT). Therefore, how to construct a CDT treatment nanosystem with high yield and full utilization of ROS in tumor site is the main issue of CDT. Herein, a multifunctional cascade bioreactor based on mesoporous Mo-doped Cu9S5 (m-MCS) nanozymes loaded with L-Arginine (LA), abbreviated as m-MCS@LA, is constructed for realizing enhanced CDT promoted by ultrasound (US) triggered gas therapy. The m-MCS based on the catalytic performance of multivalent metal ions, which were served as nanozymes, exhibit enhanced Fenton-like and glutathione (GSH) peroxidase-like activities in comparison to Cu9S5 nanoparticles without Mo-doping. Once placed in tumor microenvironment (TME), the existence of redox couples (Cu+/Cu2+ and Mo4+/Mo6+) in m-MCS enabled it to react with hydrogen peroxide (H2O2) to generate ·OH for achieving CDT effect via Fenton-like reaction. Meanwhile, m-MCS could consume overexpressed GSH in tumor microenvironment (TME) to alleviate antioxidant capability for enhancing CDT effect. Moreover, m-MCS with mesoporous structure could be employed as the carrier to load natural nitric oxide (NO) donor LA. US as the excitation source with high tissue penetration can trigger m-MCS@LA to produce NO. As the gas transmitter with physiological functions, NO could play dual roles to kill cancer cells through gas therapy directly, and enhance CDT effect by inhibiting protective autophagy simultaneously. As a result, this US-triggered and NO-mediated synergetic cancer chemodynamic/gas therapy based on m-MCS@LA NPs can effectively eliminate primary tumor and achieved tumor-specific treatment, which provide a possible strategy for developing more effective CDT in future practical applications. STATEMENT OF SIGNIFICANCE: The depletion of reactive oxygen species (ROS) by glutathione (GSH) and oxidative stress induced protective autophagy severely impaired the therapeutic effect of chemodynamic therapy (CDT). Herein, a multifunctional cascade bioreactor based on mesoporous Mo-doped Cu9S5 (m-MCS) nanozymes loaded with L-Arginine (m-MCS@LA) is constructed for realizing enhanced CDT promoted by ultrasound (US) triggered gas therapy. The m-MCS with double redox couples presents the enhanced enzyme-like activities to perform cascade reactions for reducing GSH and generating ROS. LA loaded by m-MCS can produce NO triggered by US to inhibit the mitochondria protective autophagy for reactivating mitochondria involved apoptosis pathway. The US-triggered and NO-mediated CDT based on m-MCS@LA can effectively eliminate primary tumor through the high yield and full utilization of ROS.
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Peróxido de Hidrogênio , Neoplasias , Antioxidantes/farmacologia , Arginina/farmacologia , Autofagia , Linhagem Celular Tumoral , Glutationa/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Óxido Nítrico/farmacologia , Peroxidases/farmacologia , Peroxidases/uso terapêutico , Espécies Reativas de Oxigênio , Microambiente TumoralRESUMO
Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly "tuned" for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose-derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high-content live-cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5'-triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs, which are mainly linked to signaling pathways associated with DNA replication and cell division. We hypothesize that increase in ATP and cyclin-dependent kinase 1 and 2 expression might be responsible for promoting enhanced proliferation, migration, and differentiation of ADSCs in vitro. More importantly, mito-transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full-thickness skin defect rat model with improved skin repair compared with nontreated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.
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Various stimuli have been applied to harvest complete cell sheets, including temperature, magnetic, pH, and electrical stimuli. Cell sheet technology is a convenient and efficient approach with beneficial effects for tissue regeneration and cell therapy. Lights of different wavelengths, such as ultraviolet (UV), visible light, and near infrared ray (NIR) light, were confirmed to aid in fabricating a cell sheet. Changes in the wettability, potential, or water content of the culturing surfaces that occur under light illumination induce conformational changes in the adhesive proteins or collagens, which then leads to cell sheet detachment. However, the current approaches face several limitations, as few standards for safe light illumination have been proposed to date, and require a careful control of the wavelength, power, and irradiation time. Future studies should aim at generating new materials for culturing and releasing cell sheets rapidly and effectively.
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Luz , Raios Ultravioleta , Tecnologia , Temperatura , MolhabilidadeRESUMO
BACKGROUND: The MER signaling pathway represents an attractive therapeutic target for human cancers. Growth arrest-specific protein 6 (GAS6)-induced MER phosphorylation is often unstable and difficult to detect without pervanadate pretreatment in human cancer cells, posing a challenge for the development of selective MER kinase inhibitors. Here, we identified phosphorylated AKT (pAKT) as a specific pharmacodynamic marker for MER kinase inhibitors in human melanoma G361 cells. METHODS: The expression of MER, TYRO3, and AXL were profiled among multiple human cancer cells. To determine whether they play a role in the activation of pAKT, MER and TYRO3 were selectively depleted by small, interfering RNA knockdown. In addition, using AKT phosphorylation as a readout, a high-throughput cell-based assay was established in G361 cells for evaluation of the potency of potential inhibitors of MER pathway activation. RESULTS: We demonstrated that high levels of MER and TYRO3, but not AXL, were expressed in G361 cells. In these cells, pAKT was induced by GAS6 treatment, which could be reversed by AXL/MER inhibitors. We showed that GAS6-induced pAKT is only dependent on MER kinase, but not TYRO3, in G361 cells. Furthermore, we observed a correlation in potency between inhibition of pAKT in G361 cells and pMER in MER-overexpressing Ba/F3 cells by these inhibitors. CONCLUSIONS: In summary, we have demonstrated that GAS6-induced pAKT is a possible pharmacodynamic marker for the inhibition of MER kinase, and we have successfully developed a cell-based functional assay for screening small-molecule inhibitors of MER kinase for potential therapeutic utility in treating GAS6/MER-deregulated human cancers.
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TYRO3, AXL, and MERTK constitute the TAM family of receptor tyrosine kinases, which play important roles in tumor growth, survival, cell adhesion, as well as innate immunity, phagocytosis, and immune-suppressive activity. Therefore, targeting both AXL and MERTK kinases may directly impact tumor growth and relieve immunosuppression. We describe here the discovery of INCB081776, a potent and selective dual inhibitor of AXL and MERTK that is currently in phase 1 clinical trials. In cellular assays, INCB081776 effectively blocked autophosphorylation of AXL or MERTK with low nanomolar half maximal inhibitory concentration values in tumor cells and Ba/F3 cells transfected with constitutively active AXL or MERTK. INCB081776 inhibited activation of MERTK in primary human macrophages and partially reversed M2 macrophage-mediated suppression of T-cell proliferation, which was associated with increased interferon-γ production. In vivo, the antitumor activity of INCB081776 was enhanced in combination with checkpoint blockade in syngeneic models, and resulted in increased proliferation of intratumoral CD4+ and CD8+ T cells. Finally, antitumor activity of INCB081776 was observed in a subset of sarcoma patient-derived xenograft models, which was linked with inhibition of phospho-AKT. These data support the potential therapeutic utility of INCB081776 as an immunotherapeutic agent capable of both enhancing tumor immune surveillance and blocking tumor cell survival mechanisms.
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Graphene (Gr) presents promising applications in regulating the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Light illumination is regarded as a spatiotemporally controllable, easily applicable, and noninvasive mean to modulate material responses. Herein, Gr-transferred silicon (Gr/Si) with a Schottky junction is utilized to evaluate the visible-light-promoted osteogenic differentiation of BMSCs. Under light illumination, light-induced charges, owing to the formation of the Schottky junction at the interface of Gr and Si, accumulated on the surface and then changed the surface potential of Gr/Si. The Schottky junction and surface potential at the interface of Gr and Si was measured by photovoltaic test and scanning Kelvin probe microscopy. Alkaline phosphatase (ALP) activity and quantitative real-time polymerase chain reaction (PCR) measurement showed that such variations of surface improved the osteogenic differentiation of BMSCs, and the activation of the voltage-gated calcium channels through surface potential and accumulation of cytosolic Ca2+ could be the reason. Moreover, X-ray photoelectron spectroscopy characterization showed that surface charge could also affect BMSCs differentiation through the promotion or inhibition of the adsorption of osteogenic growth factors. Such light-promoted osteogenic differentiation of BMSCs on Gr/Si may have huge potential for biomedical materials or devices for bone regeneration application.
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Grafite/química , Células-Tronco Mesenquimais/citologia , Osteogênese , Silicones/química , Engenharia Tecidual/instrumentação , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Diferenciação Celular , Células Cultivadas , Luz , Masculino , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Extracellular matrix (ECM) provides a dynamic and complex environment to determine the fate of stem cells. In this work, light harvested cell sheets were treated with paraformaldehyde or ethanol, which eventually become ECM. Such ECM was then immobilized on titanium substrates via polydopamine chemistry. Their effects on bone marrow mesenchymal stromal cells (BMSCs) behaviors were investigated. It was found that paraformaldehyde-treated ECM coating (PT-ECM) showed a well-maintained microstructure, whereas that of ethanol-treated (ET-ECM) was completely changed. As a result, different amide structures and distributions of ECM components, such as laminin and collagen I, were exhibited. Alkaline phosphatase activity, osteocalcin secretion, related gene expression, and mineral deposition were evaluated for BMSCs cultured on both ECM coatings. PT-ECM was demonstrated to promote osteogenic differentiation much more efficiently than that of ET-ECM. That is ascribed to the preservation of native ECM milieu of PT-ECM. Such ECM acquirement and immobilization method could establish surfaces being able to direct stem cell responses on various materials. That shows promising potential in bone tissue engineering and other related biomedical applications.
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Matriz Extracelular , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Células-Tronco Mesenquimais , OsteogêneseRESUMO
In electrical stimulation (ES), daily stimulation time means the interacting duration with cells per day, and is a vital factor for mediating cellular function. In the present study, the effect of stimulation time on osteogenic differentiation of MC3T3-E1 cells was investigated under ES on polypyrrole (Ppy) planar interdigitated electrodes (IDE). The results demonstrated that only a suitable daily stimulation time supported to obviously upregulate the expression of ALP protein and osteogenesis-related genes (ALP, Col-I, Runx2 and OCN), while a short or long daily stimulation time showed no significant outcomes. These might be attributed to the mechanism that an ES induced transient change in intracellular calcium ion concentration, which was responsible for activating calcium ion signaling pathway to enhance cellular osteogenic differentiation. A shorter daily time could lead to insufficient duration for the transient change in intracellular calcium ion concentration, and a longer daily time could give rise to cellular fatigue with no transient change. This work therefore provides new insights into the fundamental understanding of cell responses to ES and will have an impact on further designing materials to mediate cell behaviors.
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Diferenciação Celular , Estimulação Elétrica , Osteoblastos/citologia , Osteogênese , Polímeros/química , Pirróis/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Eletrodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Crânio/citologia , Crânio/metabolismoRESUMO
To overcome release of silk sericin (SS) from semi-interpenetrating polymer network (semi-IPN) SS/poly(N-isopropylacrylamide) (PNIPAm) hydrogels, natural biocompatible genipin (GNP) was adopted as cross-linking agent of SS. The GNP/SS/PNIPAm hydrogels with various GNP contents were prepared by radical polymerization. Depending on GNP content, the resultant hydrogels present white, yellow, or dark blue. Required time of color change for GNP/SS mixture solution shortened with increasing GNP ratio. The GNP/SS/PNIPAm hydrogels present good oscillatory shrinking-swelling behavior between 20 and 37°C. The behaviors of L929 cell proliferation, desorption, and transshipment on the surface of hydrogels and tissue culture polystyrene were investigated by 3-(4,5-dimethy thioazol-2-yl)-2,5-di-phenytetrazoliumromide and scanning electron microscopy method. In comparison with pure SS/PNIPAm hydrogels, the introduction of certain GNP can accelerate cell adhesion and proliferation. Due to reversible change between hydrophobicity and hydrophilicity, by lowering temperature to 4°C from 37°C, L929 cells could spontaneously detach from the surface of hydrogels without the need for trypsin or ethylenediaminetetraacetic acid. The detached cells could subsequently be recultured. The prepared hydrogel and detached cells have potential applications in biomedical fields, such as organs or tissue regeneration and cancer or disease therapy.