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5-Fluorouracil has demonstrated certain efficiency in patients with colorectal cancer. However, significant side effects of use by injection are common. To address this issue defects, a reengineered 5'-deoxy-5-fluorocytidine (DFCR) based drug delivery system (POACa) is developed as a prominent tumor-selective nano-activator. Investigations demonstrate that the constructed nano-activator exhibits good biocompatibility and high therapeutic efficiency in mice with subcutaneous and orthotopic SW-480 colorectal tumors, as its activity is strictly dependent on the tumor-associated acid environment and thymidine phosphorylase. These strategies diminish the off-target toxicity and improve the specificity and sensitivity of human colorectal cancer cells to 5-Fu, obtaining potent efficiency by the combination of H2O2 mediated oxidative stress, calcium overload and 5-Fu-induced chemotherapy (the combination index is 0.11). Overall, the engineered nano-activator exhibits a high therapeutic index in vitro and in vivo. STATEMENT OF SIGNIFICANCE: In this study, we designed and prepared a pH-responsive polymer to synchronously deliver DFCR (5'-deoxy-5-fluorocytidine, a prodrug of 5-Fu), Ca2+ and H2O2. The constructed nano-activator was denoted as POACa. (1) To address the problem of premature leakage of cargo by physical embedding, our research modified the inactive prodrug DFCR through chemical bonding. (2) The activation of the prepared nano-activator was strictly dependent on the tumor-associated acid environment and thymidine phosphorylase, providing the drug delivery system with inherent safety. (3) A distinctly low combination index value (0.11) of CaO2 and DFCR indicated that POACa has a prominent tumor suppression effect by tumor calcium overload sensitized chemotherapy and H2O2 mediated cytotoxicity.
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Copper, as an essential trace nutrient for human, plays a crucial role in numerous cellular activities, and is vital for maintaining homeostasis in organisms. Deviations from normal intracellular copper concentration range can disrupt the cellular homeostasis and lead to cell death. Cell death is the process in which cells lose their vitality and cannot sustain normal metabolism, which has various forms. The recently discovered cuproptosis mechanism differs from the previously recognized forms, which is triggered by intracellular copper accumulation. The discovery of cuproptosis has sparked interest among researchers, and this mechanism has been applied in the treatment of various intractable diseases, including different types of cancer. However, the developed cuproptosis-based therapies have revealed certain limitations, such as low immunostimulatory efficiency, poor tumor targeting, and inhibition by the tumor microenvironment. Therefore, researchers are devoted to combining cuproptosis with existing cancer therapies to develop more effective synergistic cancer therapies. This review summarizes the latest research advancements in the cuproptosis-based therapies for various types of cancer, with a focus on the synergistic cancer therapies. Finally, it provides an outlook on the future development of cuproptosis in anti-tumor therapy.
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Colorectal carcinoma (CRC) has become one of the most prevalent malignant tumors and exploring a potential therapeutic strategy with diminished drug-associated adverse effects to combat CRC is urgent. Herein, we designed a pH-responsive polymer to efficiently encapsulate a stimulator of interferon genes (STING) agonist (5,6- dimethylxanthenone-4-acetic acid, termed ASA404) and a common clinically used chemotherapeutic agent (1-hexylcarbamoyl-5-fluorouracil, termed HCFU). Investigations in vitro demonstrated that polymer encapsulation endowed the system with a pH-dependent disassembly behavior (pHt 6.37), which preferentially selected cancerous cells with a favorable dose reduction (dose reduction index (DRI) of HCFU was 4.09). Moreover, the growth of CRC in tumor-bearing mice was effectively suppressed, with tumor suppression rates up to 94.74%, and a combination index (CI) value of less than one (CI = 0.41 for CT26 cell lines), indicating a significant synergistic therapeutic effect. Histological analysis of the tumor micro-vessel density and enzyme-linked immunosorbent assay (ELISA) tests indicated that the system increased TNF-α and IFN-ß levels in serum. Therefore, this research introduces a pH-responsive polymer-based theranostic platform with great potential for immune-chemotherapeutic and anti-vascular combination therapy of CRC.
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Neoplasias Colorretais , Fluoruracila , Camundongos Endogâmicos BALB C , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Concentração de Íons de Hidrogênio , Fluoruracila/administração & dosagem , Linhagem Celular Tumoral , Xantonas/administração & dosagem , Xantonas/uso terapêutico , Polímeros/química , Polímeros/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Camundongos , Imunoterapia/métodos , Feminino , Fator de Necrose Tumoral alfaRESUMO
Both hydrogen (H2 ) and copper ions (Cu+ ) can be used as anti-cancer treatments. However, the continuous generation of H2 molecules and Cu+ in specific sites of tumors is challenging. Here we anchored Cu2+ on carbon photocatalyst (Cu@CDCN) to allow the continuous generation of H2 and hydrogen peroxide (H2 O2 ) in tumors using the two-electron process of visible water splitting. The photocatalytic process also generated redox-active Cu-carbon centers. Meanwhile, the Cu2+ residues reacted with H2 O2 (the obstacle to the photocatalytic process) to accelerate the two-electron process of water splitting and cuprous ion (Cu+ ) generation, in which the Cu2+ residue promoted a pro-oxidant effect with glutathione through metal-reducing actions. Both H2 and Cu+ induced mitochondrial dysfunction and intracellular redox homeostasis destruction, which enabled hydrogen therapy and cuproptosis to inhibit cancer cell growth and suppress tumor growth. Our research is the first attempt to integrate hydrogen therapy and cuproptosis using metal-enhanced visible solar water splitting in nanomedicine, which may provide a safe and effective cancer treatment.
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Carbono , Cobre , Humanos , Transformação Celular Neoplásica , Hidrogênio , Água , ApoptoseRESUMO
Photodynamic therapy (PDT) has become an emerging cancer treatment method. Choosing the photosensitizer (PS) compounds is one of the essential factors that can influence the PDT effect and action. Carbon dots (CDs) have shown great potential as photosensitizers in PDT of cancers due to their excellent biocompatibility and high generation of reactive oxygen species (ROS). Here, we used tea polyphenol as raw material for synthesized tea polyphenol carbon dots (T-CDs) that show dual emission bands of red and blue fluorescence and can efficiently generate hydroxyl radicals (OH) under mildly visible irradiation with a LED light (400-500 nm, 15 mW cm-2). The extremely low cytotoxicity and excellent biocompatibility of T-CDs without light irradiation were tested using MTT and hemolytic assay. Further, T-CDs have been shown by in vivo experiments, using a mouse breast cancer cell line (4T1) subcutaneously injected in the back of the mouse buttock as a model, to effectively inhibit the tumor cell proliferation in solid tumors and show an excellent PDT effect. In addition, pathological sections of the mice tissues after further treatment showed that the T-CDs had no apparent impact on the major organs of the mice and did not produce any side effect lesions. This work demonstrates that the as-synthesized T-CDs has the potential to be used as a PS in cancer treatment.
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Neoplasias , Fotoquimioterapia , Humanos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Carbono/farmacologia , Polifenóis/farmacologia , Polifenóis/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
A single-atom metal doped on carbonaceous nanomaterials has attracted increasing attention due to its potential applications as high-performance catalysts. However, few studies focus on the applications of such nanomaterials as nanotheranostics for simultaneous bioimaging and cancer therapy. Herein, it is pioneeringly demonstrated that the single-atom Gd anchored onto graphene quantum dots (SAGd-GQDs), with dendrite-like morphology, was successfully prepared. More importantly, the as-fabricated SAGd-GQDs exhibits a robustly enhanced longitudinal relaxivity (r1 = 86.08 mM-1 s-1) at a low Gd3+ concentration of 2 µmol kg-1, which is 25 times higher than the commercial Gd-DTPA (r1 = 3.44 mM-1 s-1). In vitro and in vivo studies suggest that the obtained SAGd-GQDs is a highly potent and contrast agent to obtain high-definition MRI, thereby opening up more opportunities for future precise clinical theranostics.
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Materiais Biocompatíveis/química , Gadolínio/química , Grafite/química , Imageamento por Ressonância Magnética , Pontos Quânticos/química , Animais , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Teste de Materiais , Camundongos , Tamanho da PartículaRESUMO
Carbon dots (CDs), with excellent optical property and cytocompatibility, are an ideal class of nanomaterials applied in the field of biomedicine. However, the weak response of CDs in the near-infrared (NIR) region impedes their practical applications. Here, UV-vis-NIR full-range responsive fluorine and nitrogen doped CDs (N-CDs-F) are designed and synthesized that own a favorable donor-π-acceptor (D-π-A) configuration and exhibit excellent two-photon (λex = 1060 nm), three-photon (λex = 1600 nm), and four-photon (λex = 2000 nm) excitation upconversion fluorescence. D-π-A-conjugated CDs prepared by solvothermal synthesis under the assistance of ammonia fluoride are reported and are endowed with larger multiphoton absorption (MPA) cross sections (3PA: 9.55 × 10-80 cm6 s2 photon-2 , 4PA: 6.32 × 10-80 cm8 s3 photon-3 ) than conventional organic compounds. Furthermore, the N-CDs-F show bright deep-red to NIR fluorescence both in vitro and in vivo, and can even stain the nucleoli of tumor cells. A plausible mechanism is proposed on the basis of the strong inter-dot and intra-dot hydrogen bonds through NH···F that can facilitate the expanding of conjugated sp2 domains, and thus not only result in lower highest occupied molecular orbital-lowest unoccupied molecular orbital energy level but also larger MPA cross sections than those of undoped CDs.
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Carbono , Pontos Quânticos , Fluorescência , Nitrogênio , FótonsRESUMO
Commercial gadolinium-based materials have been widely used as contrast agents for magnetic resonance imaging (MRI), but the high toxicity of leaking free Gd3+ ions still raises biosafety concerns. Here, we develop a novel, safe, and efficient MRI contrast agent based on a stable Fe(III) complex of fluorine and nitrogen co-doped carbon dots (F,N-CDs) that was prepared from glucose and levofloxacin by a simple microwave-assisted thermal decomposition method. The obtained Fe3+@F,N-CD complex exhibits higher longitudinal relaxivity ( r1 = 5.79 mM-1·s-1) than that of the control samples of the Fe3+@CD complex ( r1 = 4.23 mM-1 s-1) and free Fe3+ ( r1 = 1.59 mM-1 s-1) in aqueous solution, as assessed by a 1.5 T NMR analyzer. More importantly, the Fe3+@F,N-CD complex is very stable with a large coordination constant of 1.06 × 107 in aqueous medium. While incubated with HeLa cells, the Fe3+@F,N-CD complex shows clear MR images, demonstrating that it has potential to be an excellent MRI contrast agent. Furthermore, in vivo MRI experiments indicate that the Fe3+@F,N-CD complex provides high-resolution MRI pictures of 4T1 tumor bearing BALB/c mice 15 min after injection and can be completely excreted 2 h after administration. No cytotoxicity was observed with F,N-CDs and Fe concentration up to 0.2 mg/mL and 0.3 mM in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, respectively. The possible mechanism of the enhanced MRI effect of the Fe3+@F,N-CD complex is therefore proposed. The extremely low toxicity, high r1 relaxivity, strong photoluminescence, and low synthetic cost enable the Fe3+@F,N-CD complex to be a safe and promising T1-weighted MRI contrast agent for clinical applications.
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Carbono , Meios de Contraste , Compostos Férricos , Flúor , Imageamento por Ressonância Magnética , Nanopartículas , Neoplasias Experimentais/diagnóstico por imagem , Nitrogênio , Animais , Carbono/química , Carbono/farmacologia , Meios de Contraste/química , Meios de Contraste/farmacologia , Compostos Férricos/química , Compostos Férricos/farmacologia , Flúor/química , Flúor/farmacologia , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Nitrogênio/química , Nitrogênio/farmacologiaRESUMO
Pyridoxal 5'-phosphate (PLP) functions as a coenzyme in many enzymatic processes, including decarboxylation, deamination, transamination, racemization, and others. Enzymes, requiring PLP, are commonly termed PLP-dependent enzymes, and they are widely involved in crucial cellular metabolic pathways in most of (if not all) living organisms. The chemical mechanisms for PLP-mediated reactions have been well elaborated and accepted with an emphasis on the pure chemical steps, but how the chemical steps are processed by enzymes, especially by functions of active site residues, are not fully elucidated. Furthermore, the specific mechanism of an enzyme in relation to the one for a similar class of enzymes seems scarcely described or discussed. This discussion aims to link the specific mechanism described for the individual enzyme to the same types of enzymes from different species with aminotransferases, decarboxylases, racemase, aldolase, cystathionine ß-synthase, aromatic phenylacetaldehyde synthase, et al. as models. The structural factors that contribute to the reaction mechanisms, particularly active site residues critical for dictating the reaction specificity, are summarized in this review.
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BACKGROUND: Kynurenine aminotransferase 3 (KAT3) catalyzes the transamination of Kynurenine to kynurenic acid, and is identical to cysteine conjugate beta-lyase 2 (CCBL2) and glutamine transaminase L (GTL). GTL was previously purified from the rat liver and considered as a liver type glutamine transaminase. However, because of the substrate overlap and high sequence similarity of KAT3 and KAT1, it was difficult to assay the specific activity of each KAT and to study the enzyme localization in animals. METHODS: KAT3 transcript and protein levels as well as enzyme activity in the liver and kidney were analyzed by regular reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, biochemical activity assays combined with a specific inhibition assay, and western blotting using a purified and a highly specific antibody, respectively. RESULTS: This study concerns the comparative biochemical characterization and localization of KAT 3 in the mouse. The results showed that KAT3 was present in both liver and kidney of the mouse, but was much more abundant in the kidney than in the liver. The mouse KAT3 is more efficient in transamination of glutamine with indo-3-pyruvate or oxaloacetate as amino group acceptor than the mouse KAT1. CONCLUSIONS: Mouse KAT3 is a major glutamine transaminase in the kidney although it was named a liver type transaminase. GENERAL SIGNIFICANCE: Our data highlights KAT3 as a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney. This suggests tissue localizations of KAT3/GTL/CCBL2 in other animals may be carefully checked.
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Animal aspartate decarboxylase (ADC), glutamate decarboxylase (GDC) and cysteine sulfinic acid decarboxylase (CSADC) catalyze the decarboxylation of aspartate, glutamate and cysteine sulfinic acid to ß-alanine, γ-aminobutyric acid and hypotaurine, respectively. Each enzymatic product has been implicated in different physiological functions. These decarboxylases use pyridoxal 5-phosphate (PLP) as cofactor and share high sequence homology. Analysis of the activity of ADC in the presence of different amino determined that beta-alanine production from aspartate was diminished in the presence of cysteine. Comparative analysis established that cysteine also inhibited GDC and CSADC in a concentration-dependent manner. Spectral comparisons of free PLP and cysteine, together with ADC and cysteine, result in comparable spectral shifts. Such spectral shifts indicate that cysteine is able to enter the active site of the enzyme, interact with the PLP-lysine internal aldimine, form a cysteine-PLP aldimine and undergo intramolecular nucleophilic cyclization through its sulfhydryl group, leading to irreversible ADC inactivation. Cysteine is the building block for protein synthesis and a precursor of cysteine sulfinic acid that is the substrate of CSADC and therefore is present in many cells, but the presence of cysteine (at comparable concentrations to their natural substrates) apparently could severely inhibit ADC, CSADC and GDC activity. This raises an essential question as to how animal species prevent these enzymes from cysteine-mediated inactivation. Disorders of cysteine metabolism have been implicated in several neurodegenerative diseases. The results of our study should promote research in terms of mechanism by which animals maintain their cysteine homeostasis and possible relationship of cysteine-mediated GDC and CSADC inhibition in neurodegenerative disease development.
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Anopheles/enzimologia , Carboxiliases/metabolismo , Cisteína/metabolismo , Drosophila/enzimologia , Glutamato Descarboxilase/metabolismo , Proteínas de Insetos/metabolismo , Animais , Anopheles/química , Anopheles/genética , Carboxiliases/química , Carboxiliases/genética , Domínio Catalítico , Drosophila/química , Drosophila/genética , Ativação Enzimática , Retroalimentação Fisiológica , Glutamato Descarboxilase/química , Glutamato Descarboxilase/genética , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Cinética , Modelos MolecularesRESUMO
This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to ß-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no ß-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although ß-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in ß-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).
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Carboxiliases/fisiologia , Glutamato Descarboxilase/metabolismo , Taurina/biossíntese , Animais , Carboxiliases/genética , Carboxiliases/metabolismo , Linhagem Celular , Ácido Cisteico/metabolismo , Cisteína/análogos & derivados , Cisteína/metabolismo , Rim/metabolismo , Cinética , Camundongos , Modelos Biológicos , Músculos/metabolismo , Mioblastos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Taurina/análogos & derivados , Taurina/metabolismo , Distribuição Tecidual , beta-Alanina/metabolismoRESUMO
Insect aspartate 1-decarboxylase (ADC) catalyzes the decarboxylation of aspartate to ß-alanine. Insect ADC proteins share high sequence identity to mammalian cysteine sulfinic acid decarboxylase (CSADC), but there have been no reports indicating any CSADC activity in insect ADC or any ADC activity in mammalian CSADC. Substrate screening of Aedes aegypti ADC (AeADC), however, demonstrates that other than its activity to aspartate, the mosquito enzyme catalyzes the decarboxylation of cysteine sulfinic acid and cysteic acid as efficiently as those of mammalian CSADC under the same testing conditions. Further analysis of Drosophila melanogaster ADC also demonstrated its CSADC activity, suggesting that all insect ADC likely has CSADC activity. This represents the first identification of CSADC activity of insect ADC. On the other hand, HuCSADC displayed no detectable activity to aspartate. Homology modeling of AeADC and substrate docking suggest that residue Q377, localized at the active site of AeADC, could better interact with aspartate through hydrogen bonding, which may play a role in aspartate selectivity. A leucine residue in mammalian CSADC occupies the same position. A mutation at position 377 from glutamine to leucine in AeADC diminished its decarboxylation activity to aspartate with no major effect on its CSADC activity. Comparison of insect ADC sequences revealed that Q377 is stringently conserved among the available insect ADC sequences. Our data clearly established the CSADC activity of mosquito and Drosophila ADC and revealed the primary role Q377 plays in aspartate selectivity in insect ADC.
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Aedes/enzimologia , Cisteína/análogos & derivados , Glutamato Descarboxilase/metabolismo , Proteínas de Insetos/metabolismo , Animais , Ácido Aspártico/metabolismo , Cisteína/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
Aedes aegypti kynurenine aminotransferase (AeKAT) catalyzes the irreversible transamination of kynurenine to kynurenic acid, the natural antagonist of NMDA and 7-nicotinic acetycholine receptors. Here, we report the crystal structure of AeKAT in its PMP and PLP forms at 1.90 and 1.55 A, respectively. The structure was solved by a combination of single-wavelength anomalous dispersion and molecular replacement approaches. The initial search model in the molecular replacement method was built with the result of single-wavelength anomalous dispersion data from the Br-AeKAT crystal in combination with homology modeling. The solved structure shows that the enzyme is a homodimer, and that the two subunits are stabilized by a number of hydrogen bonds, salts bridges, and hydrophobic interactions. Each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to the prototypical fold type, aminotransferase subgroup I. The three-dimensional structure shows a strictly conserved 'PLP-phosphate binding cup' featuring PLP-dependent enzymes. The interaction between Cys284 (A) and Cys284 (B) is unique in AeKAT, which might explain the cysteine effect of AeKAT activity. Further mutation experiments of this residue are needed to eventually understand the mechanism of the enzyme modulation by cysteine.