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1.
Molecules ; 26(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34500559

RESUMO

Endothelial dysfunction plays key roles in the pathological process of contrast media (CM)-induced acute kidney injury (CI-AKI) in patients undergoing vascular angiography or intervention treatment. Previously, we have demonstrated that an apolipoprotein A-I (apoA-I) mimetic peptide, D-4F, inhibits oxidative stress and improves endothelial dysfunction caused by CM through the AMPK/PKC pathway. However, it is unclear whether CM induce metabolic impairments in endothelial cells and whether D-4F ameliorates these metabolic impairments. In this work, we evaluated vitalities of human umbilical vein endothelial cells (HUVECs) treated with iodixanol and D-4F and performed nuclear magnetic resonance (NMR)-based metabolomic analysis to assess iodixanol-induced metabolic impairments in HUVECs, and to address the metabolic mechanisms underlying the protective effects of D-4F for ameliorating these metabolic impairments. Our results showed that iodixanol treatment distinctly impaired the vitality of HUVECs, and greatly disordered the metabolic pathways related to energy production and oxidative stress. Iodixanol activated glucose metabolism and the TCA cycle but inhibited choline metabolism and glutathione metabolism. Significantly, D-4F pretreatment could improve the iodixanol-impaired vitality of HUVECs and ameliorate the iodixanol-induced impairments in several metabolic pathways including glycolysis, TCA cycle and choline metabolism in HUVECs. Moreover, D-4F upregulated the glutathione level and hence enhanced antioxidative capacity and increased the levels of tyrosine and nicotinamide adenine dinucleotide in HUVECs. These results provided the mechanistic understanding of CM-induced endothelial impairments and the protective effects of D-4F for improving endothelial cell dysfunction. This work is beneficial to further exploring D-4F as a potential pharmacological agent for preventing CM-induced endothelial impairment and acute kidney injury.


Assuntos
Apolipoproteína A-I/metabolismo , Meios de Contraste/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Peptídeos/metabolismo , Doenças Vasculares/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Células Cultivadas , Humanos , Redes e Vias Metabólicas/fisiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
2.
mBio ; : e0209921, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34544273

RESUMO

The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence. IMPORTANCE Posttranslational modifications (PTMs) play an important role in regulating enzyme activities, protein-protein interactions, or DNA-protein recognition and, consequently, modulate many biological functions. We demonstrated that PhoP, the response regulator of PhoP/PhoQ two-component system, could be methylated on several evolutionally conserved amino acid residues. These amino acid residues were crucial for PhoP phosphorylation or dimerization, DNA-binding ability of PhoP, and Salmonella virulence. Interestingly, methylation negatively regulated the activity of PhoP. A bacterial chemotaxis methyltransferase CheR was involved in PhoP methylation. Methylation of PhoP could stabilize it in an inactive conformation. Our work provides a more informative depiction of PhoP PTM and markedly improves our understanding of the coordinate regulation of bacterial chemotaxis and virulence.

3.
Biomol NMR Assign ; 15(2): 397-402, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34247331

RESUMO

Polyketide cyclase from Mycobacterium tuberculosis (MtPC) is related to the formation of sterol derivatives, which may play a role in immune escape in the initial stage of macrophage infection by Mycobacterium tuberculosis. However, the structure and specific functions of MtPC are still unknown. Here we report the backbone and side-chain NMR resonance assignments for the MtPC. Most resonances were assigned and the secondary structure was predicted according to the assigned backbone resonances by TALOS-N and PECAN. These NMR assignments represent a first step towards researching the structure and function of MtPC.

4.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201416

RESUMO

Hypoxia is associated with clinical diseases. Extreme hypoxia leads to multiple organs failure. However, the different effects of hypoxia on brain and visceral organs still need to be clarified, and moreover, characteristics in vulnerable organs suffering from hypoxia remain elusive. In the present study, we first aimed to figure out the hypoxic sensitivity of organs. Adult male mice were exposed to 6% O2 or 8% O2 for 6 h. Control mice were raised under normoxic conditions. In vivo and in vitro imaging of anti-HIF-1α-NMs-cy5.5 nanocomposites showed that the expression level of hypoxia-inducible factor (HIF-1α) was the highest in the liver, followed by kidney and brain. HIF-1α was detected in the hepatocytes of liver, distal convoluted tubules of kidney and neurons of cerebral cortex. The liver, kidney and brain showed distinct metabolic profiles but an identical change in glutamate. Compared with kidney and brain, the liver had more characteristic metabolites and more disturbed metabolic pathways related to glutaminolysis and glycolysis. The level of O-phosphocholine, GTP, NAD and aspartate were upregulated in hypoxic mice brain, which displayed significant positive correlations with the locomotor activity in control mice, but not in hypoxic mice with impaired locomotor activities. Taken together, the liver, kidney and brain are the three main organs of the body that are strongly respond to acute hypoxia, and the liver exhibited the highest hypoxic sensitivity. The metabolic disorders appear to underlie the physiological function changes.


Assuntos
Encéfalo/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Rim/metabolismo , Fígado/metabolismo , Animais , Comportamento Animal , Western Blotting , Carbocianinas/química , Carbocianinas/farmacocinética , Hipóxia/fisiopatologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos Endogâmicos BALB C , Imagem Molecular , Nanocompostos/química
5.
Skelet Muscle ; 11(1): 17, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34229732

RESUMO

BACKGROUND: Cancer cachexia (CAC) reduces patient survival and quality of life. Developments of efficient therapeutic strategies are required for the CAC treatments. This long-term process could be shortened by the drug-repositioning approach which exploits old drugs approved for non-cachexia disease. Amiloride, a diuretic drug, is clinically used for treatments of hypertension and edema due to heart failure. Here, we explored the effects of the amiloride treatment for ameliorating muscle wasting in murine models of cancer cachexia. METHODS: The CT26 and LLC tumor cells were subcutaneously injected into mice to induce colon cancer cachexia and lung cancer cachexia, respectively. Amiloride was intraperitoneally injected daily once tumors were formed. Cachexia features of the CT26 model and the LLC model were separately characterized by phenotypic, histopathologic and biochemical analyses. Plasma exosomes and muscle atrophy-related proteins were quantitatively analyzed. Integrative NMR-based metabolomic and transcriptomic analyses were conducted to identify significantly altered metabolic pathways and distinctly changed metabolism-related biological processes in gastrocnemius. RESULTS: The CT26 and LLC cachexia models displayed prominent cachexia features including decreases in body weight, skeletal muscle, adipose tissue, and muscle strength. The amiloride treatment in tumor-bearing mice distinctly alleviated muscle atrophy and relieved cachexia-related features without affecting tumor growth. Both the CT26 and LLC cachexia mice showed increased plasma exosome densities which were largely derived from tumors. Significantly, the amiloride treatment inhibited tumor-derived exosome release, which did not obviously affect exosome secretion from non-neoplastic tissues or induce observable systemic toxicities in normal healthy mice. Integrative-omics revealed significant metabolic impairments in cachectic gastrocnemius, including promoted muscular catabolism, inhibited muscular protein synthesis, blocked glycolysis, and impeded ketone body oxidation. The amiloride treatment evidently improved the metabolic impairments in cachectic gastrocnemius. CONCLUSIONS: Amiloride ameliorates cachectic muscle wasting and alleviates cancer cachexia progression through inhibiting tumor-derived exosome release. Our results are beneficial to understanding the underlying molecular mechanisms, shedding light on the potentials of amiloride in cachexia therapy.

6.
Biomol NMR Assign ; 15(2): 373-377, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34101143

RESUMO

Tuberculosis (TB), a lethal disease caused by Mycobacterium tuberculosis (Mtb) infection, develops multidrug-resistance and needs new drugs for effective treatment. As a ribosome maturation factor protein, RimM plays an essential role in the bacterial ribosome assembly and is a potential target for antibiotics against TB. RimM is involved in the incorporation of ribosomal protein S19 into the 30 S ribosomal subunit, where the C-terminal domain of RimM is speculated to bind S19. However, the structure and dynamics features of MtbRimM remain unclear to date. Herein, we report the NMR assignments for the 1H, 13C, 15N backbone and side-chain resonances of the C-terminal domain of MtbRimM. We also provide the prediction of its secondary structure and order parameters. Our work lays the basis for solution structure, dynamics and functional studies on MtbRimM in future, and provides clues for the anti-tuberculosis drug development.

7.
Front Mol Biosci ; 8: 685362, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34124164

RESUMO

In cancer patients, chemotherapeutic medication induces aberrant ROS (reactive oxygen species) accumulation in skeletal muscles, resulting in myofiber degradation, muscle weakness, and even cachexia, which further leads to poor therapeutic outcomes. Acting as an antioxidant, taurine is extensively used to accelerate postexercise muscle recovery in athletes. The antioxidant effects of taurine have been shown in mature myotubes and myofibers but not yet in myoblasts, the myotube precursor. The proliferation and differentiation ability of myoblasts play a very important role in myofiber repair and regeneration, which is usually impaired during chemotherapeutics in cancer patients as well. Here, we explored the effects of taurine supplementation on C2C12 myoblasts exposed to cisplatin-induced ROS. We found that cisplatin treatment led to dramatically decreased cell viability; accumulated ROS level; down-regulated expressions of MyoD1 (myoblast determination protein 1), myogenin, and MHC (myosin heavy chain); and impaired myotube differentiation in myoblasts. Significantly, taurine supplementation protected myoblasts against cisplatin-induced cell viability decrease, promoted cellular ROS clearance, and, most importantly, preserved the expressions of MyoD1, myogenin, and MHC as well as myotube differentiation ability. We further conducted NMR-based metabolomic analysis to clarify the underlying molecular mechanisms. We identified 14 characteristic metabolites primarily responsible for the discrimination of metabolic profiles between cisplatin-treated cells and normal counterparts, including increased levels of BCAAs (branched-chain amino acids: leucine and isoleucine), alanine, glycine, threonine, glucose, ADP (adenosine diphosphate), phenylalanine, and PC (O-phosphocholine), and decreased levels of lysine, ß-alanine, choline, GPC (sn-glycero-3-phosphocholine), and myo-inositol. Evidently, taurine supplementation partially reversed the changing trends of several metabolites (isoleucine, threonine, glycine, PC, ß-alanine, lysine, and myo-inositol). Furthermore, taurine supplementation promoted the proliferation and myotube differentiation of myoblasts by alleviating cellular catabolism, facilitating GSH (reduced glutathione) biosynthesis, improving glucose utilization and TCA (tricarboxylic acid) cycle anaplerosis, and stabilizing cellular membranes. Our results demonstrated the protective effects of taurine on cisplatin-impaired myoblasts and elucidated the mechanistic rationale for the use of taurine to ameliorate muscle toxicity in clinical chemotherapy cancer patients.

8.
Leukemia ; 35(10): 2862-2874, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34007045

RESUMO

Herein, we screened a novel inhibitor of the Hsp70-Bim protein-protein interaction (PPI), S1g-2, from a Bcl-2 inhibitor library; this compound specifically disrupted the Hsp70-Bim PPI by direct binding to an unknown site adjacent to that of an allosteric Hsp70 inhibitor MKT-077, showing binding affinity in sub-µM concentration range. S1g-2 exhibited overall 5-10-fold higher apoptosis-inducing activity in CML cells, primary CML blasts, and BCR-ABL-transformed BaF3 cells than other cancer cells, normal lymphocytes, and BaF3 cells, illustrating Hsp70-Bim PPI driven by BCR-ABL protects CML through oncoclient proteins that enriched in three pathways: eIF2 signaling, the regulation of eIF4E and p70S6K signaling, and the mTOR signaling pathways. Moreover, S1g-2 progressively enhanced lethality along with the increase in BCR-ABL-independent TKI resistance in the K562 cell lines and is more effective in primary samples from BCR-ABL-independent TKI-resistant patients than those from TKI-sensitive patients. By comparing the underlying mechanisms of S1g-2, MKT-077, and an ATP-competitive Hsp70 inhibitor VER-155008, the Hsp70-Bim PPI was identified to be a CML-specific target to protect from TKIs through the above three oncogenic signaling pathways. The in vivo activity against CML and low toxicity endows S1g-2 a first-in-class promising drug candidate for both TKI-sensitive and resistant CML.

9.
Biomol NMR Assign ; 15(2): 311-316, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33871829

RESUMO

Human prion disease, also known as transmissible spongiform encephalopathy (TSEs), is caused by the conformational conversion of the normal cellular prion protein (PrPC) into the scrapie form (PrPSc). Pathogenic point mutations of prion proteins typically facilitate conformational conversion and lead to inherited prion diseases. A previous study has demonstrated that the pathogenic G131V mutation of human prion protein (HuPrP) brings in Gerstmann-Sträussler-Scheinker syndrome. However, the three-dimensional structure and dynamic features of the HuPrP(G131V) mutant remain unclear. It is expected that the determination of these structural bases will be beneficial to the pathogenic mechanistic understanding of G131V-related prion diseases. Here, we performed 1H, 15N, 13C backbone and side-chain resonance assignments of the G131V mutant of HuPrP(91-231) by using heteronuclear multi-dimensional NMR spectroscopy, and predicted the secondary structural elements and order parameters of the protein based on the assigned backbone chemical shifts. Our work lays the necessary foundation for further structural determination, dynamics characterization, and intermolecular interaction assay for the G131V mutant.

10.
Biomolecules ; 11(4)2021 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-33919647

RESUMO

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand ß-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD-S19 complex and indicated that the ß4-ß5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM-S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.


Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/metabolismo , Proteínas Ribossômicas/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Ribossômicas/metabolismo
11.
Molecules ; 26(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33805924

RESUMO

α-Ketoglutarate (AKG) is attracting much attention from researchers owing to its beneficial effects on anti-aging and cancer suppression, and, more recently, in nutritional supplements. Given that glucose is the main source of energy to maintain normal physiological functions of skeletal muscle, the effects of AKG supplementation for improving muscle performance are closely related to the glucose level in skeletal muscle. The differences of AKG-induced effects in skeletal muscle between two states of normal energy and energy deficiency are unclear. Furthermore, AKG-induced metabolic changes in skeletal muscles in different energy states also remain elusive. Here, we assessed the effects of AKG supplementation on mouse C2C12 myoblast cells cultured both in normal medium (Nor cells) and in low-glucose medium (Low cells), which were used to mimic two states of normal energy and energy deficiency, respectively. We further performed NMR-based metabolomic analysis to address AKG-induced metabolic changes in Nor and Low cells. AKG supplementation significantly promoted the proliferation and differentiation of cells in the two energy states through glutamine metabolism, oxidative stress, and energy metabolism. Under normal culture conditions, AKG up-regulated the intracellular glutamine level, changed the cellular energy status, and maintained the antioxidant capacity of cells. Under low-glucose culture condition, AKG served as a metabolic substrate to reduce the glutamine-dependence of cells, remarkably enhanced the antioxidant capacity of cells and significantly elevated the intracellular ATP level, thereby ensuring the normal growth and metabolism of cells in the state of energy deficiency. Our results provide a mechanistic understanding of the effects of AKG supplements on myoblasts in both normal energy and energy deficiency states. This work may be beneficial to the exploitation of AKG applications in clinical treatments and nutritional supplementations.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Espectroscopia de Ressonância Magnética , Metabolômica , Mioblastos Esqueléticos/metabolismo , Animais , Linhagem Celular , Camundongos
12.
Theranostics ; 11(12): 5926-5938, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897890

RESUMO

Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fosfofrutoquinase-1 Muscular/genética , Transcrição Genética/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
13.
Molecules ; 26(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670019

RESUMO

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Suramina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-raf/isolamento & purificação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Suramina/análogos & derivados , Suramina/química
14.
Front Oncol ; 10: 570516, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33330044

RESUMO

Metformin is a widely prescribed anti-diabetes drug with potential utilities for cancer therapies. Several studies have related metformin to the reduced risk of cholangiocarcinoma (CCA), highlighting its potentialities for the treatments of CCA. However, the underlying molecular mechanisms remain elusive. Here, we demonstrated that metformin treatment could inhibit proliferations of the human CCA cell lines Mz-ChA-1 and QBC939 in dose-dependent manners. The NMR-based metabonomic analyses showed distinct discriminations between the metformin-treated (Met) and control (Ctrl) groups of both CCA cells. Characteristic metabolites were identified by a combination of multivariate statistical analysis of 1D 1H-NMR spectral data and the pair-wise t-test of metabolite levels. We then identified four significantly altered metabolic pathways based on the characteristic metabolites, including glucose metabolism, oxidative stress-related metabolism, energy metabolism, and amino acids metabolism. Comparing CCA cells with normal human umbilical vein endothelial cells (HUVECs), we found that metformin treatment profoundly promoted glycolysis and specifically increased the levels of BCAAs and UDP-GlcNAc, implying the occurrence of autophagy and cell cycle arrest in metformin-treated CAA cells. This work provides a mechanistic understanding of the anticancer effect of metformin treatment on CAA cells, and is beneficial to further developments of metformin as an anticancer drug.

15.
Anal Methods ; 12(21): 2661-2667, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32930296

RESUMO

A constant magnetic field (CMF) was used to arrange the orientation of graphene oxide (GO) which was modified on a self-made screen-printed electrode. We evaluated the efficiency of this method for potential analytical application towards the sensing of hydrogen peroxide (H2O2). Mesoporous silica (MS)-encapsulated horseradish peroxidase (HRP) was immobilized on the electrode with vertically arranged GO to construct an H2O2 sensor (denoted as CMF/GO/HRP@MS). The linear range of the response of the CMF/GO/HRP@MS sensor to H2O2 was 0.1-235 µM, and the detection limit was as low as 0.01 µM. The results demonstrated that the vertical arrangement of GO resulting from the CMF on the electrode surface could increase the electron transfer rate. The excellent selectivity and anti-interference ability of this sensor to H2O2 in physiological samples may be attributed to the synergistic effect of mesoporous silica, GO and constant magnetic field.


Assuntos
Técnicas Biossensoriais , Peróxido de Hidrogênio , Eletrodos , Enzimas Imobilizadas , Grafite , Dióxido de Silício
16.
Nat Commun ; 11(1): 3976, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769970

RESUMO

Quintulene, a non-graphitic cycloarene with fivefold symmetry, has remained synthetically elusive due to its high molecular strain originating from its curved structure. Here we report the construction of extended quintulene, which was unambiguously characterized by mass and NMR spectroscopy. The extended quintulene represents a naturally curved nanocarbon based on its conical molecular geometry. It undergoes dimerization in solution via π-π stacking to form a metastable, but isolable bilayer complex. Thermodynamic and kinetic characterization reveals the dimerization process as entropy-driven and following second-order kinetics with a high activation energy. These findings provide a deeper understanding of the assembly of conical nanocarbons. Comparison of optical properties of monomer and dimer points toward a H-type interlayer coupling in the dimer.

17.
J Biol Chem ; 295(37): 12900-12909, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32651234

RESUMO

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.


Assuntos
Apoptose , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de Proteína , Regulação Alostérica , Proteína 11 Semelhante a Bcl-2/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Células K562 , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo
18.
Anal Chim Acta ; 1122: 31-38, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32503741

RESUMO

An upright GO (UGO) modified screen-printed electrode was prepared with the help of the external magnetic field for improving its electrochemical performance. The ratio of GO: Nafion and the magnetic field intensity on the properties of UGO were examined by scanning electron microscope, cyclic voltammetry and electrochemical impedance spectroscopy. The magnetic field intensity does not influence the electron transfer kinetics but increase the number of active sites and therefore enhance the electroactive surface area. In addition, the UGO electrode that was electrodeposited Ni nanoparticles (denotes as Ni NPs/UGO modified electrode) display excellent oxidation towards glycine using chronoamperometry. The Ni NPs/UGO modified electrode indicated an excellent performance for electrochemical COD (chemical oxide demand) analysis with a linear detection range of 0.1-400 mg/L and a lower detection limit of 0.02 mg/L. Moreover, this Ni NPs/UGO modified electrode can be applied to the rapid determination of COD in general real water samples. The results were in agreement with those obtained by using the standard method (ISO 6060).

19.
J Ethnopharmacol ; 261: 112978, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-32442586

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Huiyang Shengji formula (HSF) is a compound Chinese herbal medicine prescription, and has long been used for treating chronic non-healing wounds. AIM OF THE STUDY: The purpose of this study was to provide new insight into molecular mechanisms of healing effects of the HSF treatments. MATERIALS AND METHODS: We established a rat diabetic skin ulcer (DSU) model, and assessed healing effects of four HSF treatments on DSUs by calculating wound healing rates and immunohistochemical detection of the expressions of angiogenesis-related factors in the model rats (Mod) relative to normal rats (Nor), including Huiyang extract (HE), Shengji extract (SE), Huiyang Shengji extract (HSE) and HSE associated with acupuncture (Ac-HSE). We then performed NMR-based metabolomic analyses on skin tissues of the Nor, Mod, HSE-treated, Ac-HSE-treated rats to address metabolic mechanisms underlying these effects. RESULTS: These treatments up-regulated expressions of two angiogenesis-related factors VEGF and CD31, and improved efficacy of healing DSUs, in which HSE and Ac-HSE exhibited the most significant effects. Compared with Mod, HSE and Ac-HSE groups shared four characteristic metabolites (lactate, histidine, succinate and acetate) and four significantly altered metabolic pathways with Nor. Both HSE and Ac-HSE treatments could partly reverse the metabolically disordered pathological state of DSUs to the normal state. They might improve wound healing through promoting glucose metabolism, BCAAs metabolism, and enhancing antioxidant capacity and angiogenesis in DSU tissues. Ac-HSE significantly enhanced wound healing rates compared to HSE, potentially owing to significant capacities of enhancing anti-oxidation and angiogenesis and interfering three more metabolic pathways. CONCLUSIONS: This work provides a mechanistic understanding of the healing effects of the HSE and Ac-HSE treatments on DSUs, is of benefit to improvements of the HSF treatments for clinically healing chronic non-healing wounds.


Assuntos
Terapia por Acupuntura , Angiopatias Diabéticas/terapia , Medicamentos de Ervas Chinesas/farmacologia , Espectroscopia de Ressonância Magnética , Metabolômica , Úlcera Cutânea/terapia , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/terapia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/patologia , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais , Pele/lesões , Pele/metabolismo , Pele/patologia , Úlcera Cutânea/metabolismo , Úlcera Cutânea/patologia , Estreptozocina , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia
20.
Cancer Sci ; 111(9): 3195-3209, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32369664

RESUMO

Gastric cancer (GC) is one of the deadliest cancers worldwide, and the progression of gastric carcinogenesis (GCG) covers multiple complicated pathological stages. Molecular mechanisms of GCG are still unclear. Here, we undertook NMR-based metabolomic analysis of aqueous metabolites extracted from gastric tissues in an established rat model of GCG. We showed that the metabolic profiles were clearly distinguished among 5 histologically classified groups: control, gastritis, low-grade gastric dysplasia, high-grade gastric dysplasia (HGD), and GC. Furthermore, we carried out metabolic pathway analysis based on identified significant metabolites and revealed significantly disturbed metabolic pathways closely associated with the 4 pathological stages, including oxidation stress, choline phosphorylation, amino acid metabolism, Krebs cycle, and glycolysis. Three metabolic pathways were continually disturbed during the progression of GCG, including taurine and hypotaurine metabolism, glutamine and glutamate metabolism, alanine, aspartate, and glutamate metabolism. Both the Krebs cycle and glycine, serine, and threonine metabolism were profoundly impaired in both the HGD and GC stages, potentially due to abnormal energy supply for tumor cell proliferation and growth. Furthermore, valine, leucine, and isoleucine biosynthesis and glycolysis were significantly disturbed in the GC stage for higher energy requirement of the rapid growth of tumor cells. Additionally, we identified potential gastric tissue biomarkers for metabolically discriminating the 4 pathological stages, which also showed good discriminant capabilities for their serum counterparts. This work sheds light on the molecular mechanisms of GCG and is of benefit to the exploration of potential biomarkers for clinically diagnosing and monitoring the progression of GCG.


Assuntos
Transformação Celular Neoplásica/metabolismo , Espectroscopia de Ressonância Magnética , Metabolômica , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Metabolismo Energético , Humanos , Espectroscopia de Ressonância Magnética/métodos , Redes e Vias Metabólicas , Metaboloma , Metabolômica/métodos , Ratos , Neoplasias Gástricas/diagnóstico , Ensaios Antitumorais Modelo de Xenoenxerto
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