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1.
Cancer Res ; 2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728538

RESUMO

Loss-of-function mutations of JAK1/2 impair cancer cell responsiveness to IFN-γ and immunogenicity. Therefore, an understanding of compensatory pathways to activate IFN-γ signaling in cancer cells is clinically important for the success of immunotherapy. Here we demonstrate that the transcription factor SOX10 hinders immunogenicity of melanoma cells through the IRF4-IRF1 axis. Genetic and pharmacological approaches revealed that SOX10 repressed IRF1 transcription via direct induction of a negative regulator, IRF4. The SOX10-IRF4-IRF1 axis regulated PD-L1 expression independently of JAK-STAT pathway activity, and suppression of SOX10 increased the efficacy of combination therapy with an anti-PD-1 antibody and HDAC inhibitor against a clinically relevant melanoma model. Thus, the SOX10-IRF4-IRF1 axis serves as a potential target that can bypass JAK-STAT signaling to immunologically warm up melanoma with a "cold" tumor immune microenvironment.

2.
Cancer Sci ; 2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34632664

RESUMO

From a mouse triple-negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G-protein-coupled receptor (ADGR) family, G-protein-coupled receptor 56 (GPR56)/adhesion G-protein-coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1-transduced 4T1, TS/A (mouse breast cancer cell line), or MDA-MB-231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1-induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

3.
Planta Med ; 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34474490

RESUMO

Three new flavanols, (2R,3S)-7-methoxy-flavan-3-ol (1: ), (2R,3S)-7-hydroxy-flavan-3-ol (2: ), and (2R,3S)-2'-hydroxy-7-methoxy-flavan-3-ol (3: ), together with two known flavans (4: and 5: ), were isolated from the chloroform extract of Crinum asiaticum. Their structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-MS, and CD data. The isolated compounds 1: and 3: -5: showed inhibitory activity toward LPS-induced nitric oxide (NO) production. Further investigation of the NF-κB pathway mechanisms indicated that 1: and 3: -5: inhibited the LPS-induced IL-6 production and p65 subunit phosphorylation of NF-κB in RAW264.7 cells, with an effective dose of 10 µM.

4.
J Immunother Cancer ; 9(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34215690

RESUMO

BACKGROUND: Resistance to an immune checkpoint inhibitor (ICI) is a major obstacle in cancer immunotherapy. The causes of ICI resistance include major histocompatibility complex (MHC)/histocompatibility locus antigen (HLA) class I loss, neoantigen loss, and incomplete antigen presentation. Elimination by natural killer (NK) cells would be expected to be an effective strategy for the treatment of these ICI-resistant tumors. We previously demonstrated that a lipid nanoparticle containing a stimulator of an interferon gene (STING) agonist (STING-LNP) efficiently induced antitumor activity via the activation of NK cells. Thus, we evaluated the potential of reducing ICI resistance by STING-LNPs. METHODS: Lung metastasis of a B16-F10 mouse melanoma was used as an anti-programmed cell death 1 (anti-PD-1)-resistant mouse model. The mice were intravenously injected with the STING-LNP and the mechanism responsible for the improvement of anti-PD-1 resistance by the STING-LNPs was analyzed by RT-qPCR and flow cytometry. The dynamics of STING-LNP were also investigated. RESULTS: Although anti-PD-1 monotherapy failed to induce an antitumor effect, the combination of the STING-LNP and anti-PD-1 exerted a synergistic antitumor effect. Our results indicate that the STING-LNP treatment significantly increased the expression of CD3, CD4, NK1.1, PD-1 and interferon (IFN)-γ in lung metastases. This change appears to be initiated by the type I IFN produced by liver macrophages that contain the internalized STING-LNPs, leading to the systemic activation of NK cells that express PD-1. The activated NK cells appeared to produce IFN-γ, resulting in an increase in the expression of the PD ligand 1 (PD-L1) in cancer cells, thus leading to a synergistic antitumor effect when anti-PD-1 is administered. CONCLUSIONS: We provide a demonstration to show that a STING-LNP treatment can overcome PD-1 resistance in a B16-F10 lung metastasis model. The mechanism responsible for this indicates that NK cells are activated by stimulating the STING pathway which, in turn, induced the expression of PD-L1 on cancer cells. Based on the findings reported herein, the STING-LNP represents a promising candidate for use in combination therapy with anti-PD-1-resistant tumors.

5.
Bioorg Chem ; 113: 105033, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34089945

RESUMO

STAT3 signaling is known to be associated with tumorigenesis and further cancer cell-intrinsic activation of STAT3 leads to altered regulation of several oncogenic processes. Given the importance of STAT3 in cancer development and progression particularly breast cancer, it is crucial to discover new chemical entities of STAT3 inhibitor to develop anti-breast cancer drug candidates. Herein, 4-benzyl-2-benzylthio-5-methyl-1H-imidazole (2a) and 4-benzyl-5-methyl-2-[(2,6-difluorobenzyl)thio]-1H-imidazole (2d) from a group of thirty imidazole-bearing compounds showed greater STAT3 inhibition than their lead compounds VS1 and the oxadiazole derivative MD77. Within all tested compounds, ten derivatives effectively inhibited the growth of the two tested breast cancer cells with IC50 values ranging from 6.66 to 26.02 µM. In addition, the most potent derivatives 2a and 2d inhibited the oncogenic function of STAT3 as seen in the inhibition of colony formation and IL-6 production of breast cancer cell lines. Modeling studies provided evidence for the possible interactions of the synthesized compounds with the key residues of the STAT3-SH2 domain. Collectively, our present study suggests 2-substituted-4-benzyl-5-methylimidazoles are a new class of anti-cancer drug candidates to inhibit oncogenic STAT3 function.


Assuntos
Antineoplásicos/síntese química , Desenho de Fármacos , Imidazóis/química , Fator de Transcrição STAT3/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Interleucina-6/metabolismo , Simulação de Acoplamento Molecular , Fator de Transcrição STAT3/antagonistas & inibidores , Relação Estrutura-Atividade , Domínios de Homologia de src
6.
Cancer Sci ; 112(9): 3484-3490, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34187084

RESUMO

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8+ T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8+ T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8+ cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8+ T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8+ T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8+ T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/metabolismo , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacocinética , Bromodesoxiuridina/administração & dosagem , Bromodesoxiuridina/farmacocinética , Interferon gama/deficiência , Interferon gama/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/metabolismo , Neoplasias Cutâneas/patologia , Vacinação/métodos
7.
Biomed Pharmacother ; 140: 111737, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34020249

RESUMO

Guggulsterone (GS) [4,17(20)-pregnadiene-3,16-dione], is the main active phytosterol constituent in guggul, the gum resin of Commiphora wightii (Arnott.) Bhand./Commiphora mukul Engl. tree, and is known for its medicinal effects. In this study, we report that GSD-1, a structurally-related synthetic GS derivative, strongly inhibits NF-κB activation induced by TNF-α. GSD-1 prevented the nuclear translocation of p65 through the blockade of IκBα degradation and p65 phosphorylation, and further inhibited the activation of upstream kinases, including transforming growth factor-ß activated kinase 1 (TAK1), IκB kinase (IKK) α, and IKKß. Furthermore, GSD-1 inhibited the cell-intrinsic activation of NF-κB, and exerted its direct anti-cancer and anti-metastatic effects in both murine and human breast cancer cell lines. This study demonstrated GSD-1 to be an attractive compound to target NF-κB activation that has potential for treating breast cancer growth and metastasis.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Pregnenodionas/farmacologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Cicatrização/efeitos dos fármacos
8.
J Nat Med ; 75(4): 840-853, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33988779

RESUMO

Anthraquinones are a major class of compounds naturally occurring in Asphodelus microcarpus. The pharmacological actions of anthraquinones in cancer cells are known to induce apoptosis or autophagy, and revert multidrug resistance. In this study, five anthraquinone-type analogs were isolated from the methanol extract of A. microcarpus leaves and identified as, emodin, rhein, physcion, aloe-emodin, and emodic acid. Among them, aloe-emodin and emodic-acid strongly inhibited the proliferation, cells-intrinsic NF-κB activity and metastatic ability of breast cancer. Although aloe-emodin inhibited p38 and ERK phosphorylation, emodic-acid more markedly inhibited JNK, in addition to p38 and ERK phosphorylation. Both aloe-emodin and emodic-acid inhibited the secretion of the pro-tumorigenic cytokines IL-1ß and IL-6, and VEGF and MMP expression, and subsequently inhibited the invasive and migratory potential of 4T1 cells. Thus, our study demonstrated the effects of aloe-emodin and emodin-acid in controlling the migratory and invasive ability of 4T1 breast cancer cells, in addition to inhibiting NF-κB activity and the expression of its downstream target molecules.

9.
Biol Pharm Bull ; 44(5): 686-690, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33952824

RESUMO

Although more than 400 species of Cordyceps s.l. have been identified, most have not been well explored regarding their potential for medicinal use. In this study, the profiles of constituents of ten different species of Ophiocordyceps, which is an unexplored species of Cordyceps, were analyzed and their anti-tumor effects were further examined. Although all Ophiocordyceps samples exhibited similar peak patterns, Ophiocordyceps gracilioides (O. grac) had a distinct constituent profile from the other samples. Furthermore, O. grac was the most active in suppressing the transcriptional activities of both nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription (STAT)3, and the production of interleukin (IL)-6 from breast cancer cells. This study demonstrated that O. grac is a relatively unexplored Cordyceps s.l. that may have medicinal potential to inhibit the NFκB-STAT3-IL-6 inflammatory pathway in cancer.


Assuntos
Produtos Biológicos/farmacologia , Hypocreales/química , Neoplasias/tratamento farmacológico , Animais , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Interleucina-6/metabolismo , Camundongos , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
10.
J Nat Med ; 75(4): 824-832, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33905080

RESUMO

Ophiocordyceps gracilioides is an entomoparasitic ascomycetes whose bioactivity has not been examined in detail. In this study, we identified the bioactive compounds ergosterol peroxide (EPO) and ergosterol (ERG) from the MeOH extract of O. gracilioides mycelia related to its anti-cancer effects by targeting the Nuclear Factor kappa B (NF-ĸB)/Signal Transducer and Activator of Transcription 3 (STAT3) inflammatory pathways. Using gene-reporter assays, we demonstrated that EPO markedly inhibits both NF-ĸB and STAT3 activity in 4T1 cells, whereas ERG had limited effect. Consistent with their effects on NF-ĸB and STAT3 activity, EPO, but not ERG, exerted anti-proliferative effects on 4T1 cells. Furthermore, EPO significant inhibited both the migration and invasion of 4T1 cells in vitro, and pre-treatment of 4T1 cells with EPO significantly inhibited the formation of experimental lung metastases in vivo. Collectively, we demonstrated that ERG and EPO can be isolated from O. gracilioides mycelia, and further identified EPO as an active constituent of its anti-metastatic effects through the inhibition of NF-ĸB and STAT3 inflammatory pathways in 4T1 breast cancer cells.

11.
BMC Complement Med Ther ; 21(1): 115, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33836731

RESUMO

BACKGROUND: Morus alba L. bark has been widely used in traditional medicine for treating several inflammatory diseases, such as hypertension, diabetes mellitus and coughing; however, the molecular mechanisms underlying its anti-inflammatory effects are not well understood. METHODS: We examined the effects of an extract of Morus alba L. bark (MabE) on Toll-like receptor (TLR) ligand-induced activation of RAW264.7 macrophages using a luciferase reporter assay and immunoassays. For the in vivo experiment, we used an imiquimod-induced ear edema model to examine the anti-inflammatory effects of MabE. RESULTS: MabE inhibited the TLR ligand-induced activation of NF-κB in RAW264.7 cells without affecting their viability. Consistent with the inhibition of NF-κB activation, MabE also inhibited the production of IL-6 and IL-1ß from TLR ligand-treated RAW264.7 cells. In vivo MabE treatment inhibited the ear swelling of IMQ-treated mice, in addition to the mRNA expression of IL-17A, IL-1ß and COX-2. The increases in splenic γδT cells in IMQ-treated mice and the production of IL-17A from splenocytes were significantly inhibited by MabE treatment. CONCLUSION: Our study suggests that the anti-inflammatory effects of MabE on the activation of the macrophage cell line RAW246.7 by TLRs and IMQ-induced ear edema are through the inhibition of NF-κB activation and IL-17A-producing γδT cells, respectively.

12.
Sci Rep ; 11(1): 6618, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758209

RESUMO

Cancer metastasis is a major cause of the high mortality rate in lung cancer patients. The cytoskeletal rearrangement and degradation of extracellular matrix are required to facilitate cell migration and invasion and the suppression of these behaviors is an intriguing approach to minimize cancer metastasis. Even though Erianthridin (ETD), a phenolic compound isolated from the Thai orchid Dendrobium formosum exhibits various biological activities, the molecular mechanism of ETD for anti-cancer activity is unclear. In this study, we found that noncytotoxic concentrations of ETD (≤ 50 µM) were able to significantly inhibit cell migration and invasion via disruption of actin stress fibers and lamellipodia formation. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was markedly downregulated in a dose-dependent manner after ETD treatment. Mechanistic studies revealed that protein kinase B (Akt) and its downstream effectors mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K) were strongly attenuated. An in silico study further demonstrated that ETD binds to the protein kinase domain of Akt with both hydrogen bonding and van der Waals interactions. In addition, an in vivo tail vein injection metastasis study demonstrated a significant effect of ETD on the suppression of lung cancer cell metastasis. This study provides preclinical information regarding ETD, which exhibits promising antimetastatic activity against non-small-cell lung cancer through Akt/mTOR/p70S6K-induced actin reorganization and MMPs expression.


Assuntos
Antineoplásicos/farmacologia , Fenantrenos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Actinas/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Humanos , Metaloproteinases da Matriz/metabolismo , Modelos Moleculares , Fenantrenos/química , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/química
13.
J Immunol Methods ; 491: 112993, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33609531

RESUMO

Although the importance of NK cells as immune effector cells in controlling growth and metastatic dissemination of tumor cells has been widely recognized, it is unclear whether NK cells in different organs similarly control tumor cell growth and metastasis. In the present study, we established a bioluminescent imaging model of mouse T cell lymphoma cells, which are highly susceptive to NK cell-dependent immune-surveillance, to monitor the dissemination of lymphoma cells using an in vivo imaging system. The use of this model is expected to be a highly sensitive method to examine the role of NK cells in controlling lymphoma dissemination in a variety of tissues.


Assuntos
Vigilância Imunológica , Células Matadoras Naturais/imunologia , Medições Luminescentes/métodos , Linfoma de Células T/imunologia , Animais , Linfoma de Células T/patologia , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos CBA
14.
Biomed Chromatogr ; 35(7): e5094, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33599311

RESUMO

Clozapine (CLZ) is a key drug in treatment-resistant schizophrenia. Therapeutic drug monitoring (TDM) of CLZ and its metabolites, N-desmethylclozapine and clozapine N-oxide, is required to monitor and manage the risks of side effects. Although quantification methods for TDM have been developed for CLZ and its metabolites, they were not sufficiently accurate for the quantification of CLZ owing to the upper limits of the calibration curves. An analytical method using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous measurement of CLZ and its metabolites in human plasma. To expand the concentration range of the calibration curves, we used a linear range shift technique using in-source collision-induced dissociation (CID). Using our approach, the linearity and quantitative range were improved compared to those reported by previous studies, and were sufficient for TDM in clinical practice. The intra- and inter-assay accuracy was 84.6%-114.8%, and the intra- and inter-assay precisions were ≤9.1% and ≤9.9%, respectively. Moreover, all samples from patients with treatment-resistant schizophrenia were successfully quantified. Therefore, our novel analytical method using in-source CID had the appropriate performance to measure the plasma concentrations of CLZ and its metabolites for TDM in clinical practice.


Assuntos
Antipsicóticos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Clozapina/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Antipsicóticos/metabolismo , Antipsicóticos/uso terapêutico , Clozapina/metabolismo , Clozapina/uso terapêutico , Monitoramento de Medicamentos , Feminino , Humanos , Masculino , Esquizofrenia/tratamento farmacológico , Espectrometria de Massas em Tandem/métodos
15.
Cancer Sci ; 112(4): 1633-1643, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33565179

RESUMO

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal-regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell-mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response-related genes, including interferon-gamma (IFNγ). We also revealed that NK cells are required for these anti-metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell-dependent anti-tumor immunity and that ASK1-targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.


Assuntos
Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MAP Quinase Quinase Quinase 5/metabolismo , Metástase Neoplásica/patologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunoterapia/métodos , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/imunologia , Células RAW 264.7
16.
Biomed Chromatogr ; 35(6): e5067, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33450064

RESUMO

Several drugs are administered to lung-transplanted patients, which are monitored using therapeutic drug monitoring (TDM). Therefore, we developed and validated a liquid chromatography-tandem mass spectrometry method to simultaneously analyze immunosuppressive drugs such as mycophenolic acid, antifungal drugs such as voriconazole and itraconazole, and its metabolite hydroxyitraconazole. Chromatographic separation was achieved using a C18 column and gradient flow of mobile phase comprising 20 mM aqueous ammonium formate and 20 mM ammonium formate-methanol solution. A simple protein precipitation treatment was performed using acetonitrile/methanol and mycophenolic acid-2 H3 , voriconazole-2 H3 , itraconazole-2 H4 , and hydroxyitraconazole-2 H4 as internal standards. The linearity ranges of mycophenolic acid, voriconazole, itraconazole, and hydroxyitraconazole were 100-20,000, 50-10,000, 5-1000, and 5-1000 ng/mL, respectively. The retention time of each target was less than 2 min. The relative errors in intra- and inter-day were within ±7.6%, the coefficient of variation was 8.9% or less for quality control low, medium, and high, and it was 15.8% or less for lower limit of quantitation. Moreover, the patient samples were successfully quantified, and they were within the linear range of measurements. Therefore, our new method may be useful for TDM in lung-transplanted patients.


Assuntos
Cromatografia Líquida/métodos , Monitoramento de Medicamentos/métodos , Transplante de Pulmão , Espectrometria de Massas em Tandem/métodos , Adulto , Antifúngicos/sangue , Antifúngicos/farmacocinética , Antifúngicos/uso terapêutico , Feminino , Humanos , Imunossupressores/sangue , Imunossupressores/farmacocinética , Imunossupressores/uso terapêutico , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Biochem J ; 478(1): 41-59, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33196080

RESUMO

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.


Assuntos
Membrana Celular/metabolismo , Parede Celular/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Proteínas Quinases/metabolismo , Tolerância ao Sal/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Citosol/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Floculação , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/genética , Peptidoglicano Glicosiltransferase/genética , Mutação Puntual , Proteínas Quinases/genética , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética
18.
Talanta ; 222: 121625, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33167273

RESUMO

Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.


Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Kluyveromyces , Reprodutibilidade dos Testes
19.
Biol Pharm Bull ; 43(12): 1899-1905, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33268707

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is considered a potential target for cancer treatment because of its relationship with cellular transformation and tumor initiation and progression. In this study, we aimed to identify a new anti-cancer drug candidate from natural products by targeting STAT3 activity. Using STAT3-luciferase reporter cell line, we screened the chemical library of natural products and found that baicalein, a flavone isolated from the roots of Scutelleria baicalensis, strongly suppressed STAT3 activity in breast cancer cells. Baicalein inhibited STAT3 transcriptional activity and its phosphorylation, and further exhibited anti-proliferative effects in breast cancer cells. Moreover, baicalein suppressed the production of interleukin (IL)-6 and the metastatic potential of breast cancer cells both in vitro and in vivo. Collectively, our study suggests baicalein as an attractive phytochemical compound for reducing metastatic potential of breast cancer cells by regulating STAT3 activity.


Assuntos
Neoplasias da Mama/metabolismo , Flavanonas/farmacologia , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Feminino , Flavanonas/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C
20.
Sci Rep ; 10(1): 19214, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154524

RESUMO

Although melanoma therapy is improved by novel molecular targeted reagents, including vemurafenib, aberrant proliferation and early metastasis remain obstacles for melanoma; therefore, novel target molecules for melanoma need to be identified. In this study, we focused on deubiquitinating enzymes, which regulate protein stability through ubiquitin-proteasome systems, and identified 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) as a molecule related to melanoma growth using siRNA library screening. Similar to a previous report, PSMD14 knockdown strongly induced p21 expression and inhibited RB phosphorylation in melanoma. After in silico analysis, TGF-ß signaling was identified as a negatively correlated gene set with PSMD14 expression. Although TGF-ß signaling is also related to the invasive phenotype of melanoma, PSMD14 knockdown suppressed melanoma migration and reduced SLUG expression, suggesting that targeting PSMD14 suppresses both growth and migration. Furthermore, SMAD3 expression increased in nucleus and SMAD3 degradation was delayed after PSMD14 knockdown. Thus, our present study suggests that targeting PSMD14 can inhibit melanoma growth and migration through either SMAD3 accumulation or SLUG reduction, respectively.


Assuntos
Proliferação de Células/fisiologia , Melanoma/genética , Complexo de Endopeptidases do Proteassoma/genética , Neoplasias Cutâneas/genética , Proteína Smad3/genética , Transativadores/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Melanoma/metabolismo , Melanoma/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Smad3/metabolismo , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo
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