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1.
Medicine (Baltimore) ; 99(36): e22128, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899099

RESUMO

RATIONALE: Brain metastasis (BM) is a serious complication in non-small cell lung cancer (NSCLC) patients. Pemetrexed is one of the preferred agents in nonsquamous NSCLC with BM; however, the traditional chemotherapy demonstrated limited efficacy partly due to drug resistance and the blood-brain barrier. PATIENT CONCERNS: A 52-year-old male non-smoker was admitted for irritating cough, chest distress, and back pain. DIAGNOSES: Epidermal growth factor receptor wild-type, anaplastic lymphoma kinase-negative primary lung adenocarcinoma with an asymptomatic solitary BM (cTxNxM1b, IVA). INTERVENTIONS: Pemetrexed (500 mg/m of body surface area) and carboplatin (area under the curve of 5) were firstly administered every 3 weeks for 3 cycles, followed by pemetrexed/carboplatin plus anlotinib (12 mg daily; 2 weeks on and 1 week off) for another 3 cycles. Then maintenance anlotinib monotherapy was continued for a year, without unacceptable adverse events. OUTCOMES: The BM was slightly enlarged after 3 cycles of pemetrexed/carboplatin; however, a complete remission was achieved after the combination therapy. His intracranial progression-free survival was more than 2 years. LESSONS: Pemetrexed/carboplatin plus anlotinib could be considered for the treatment of epidermal growth factor receptor wild-type, anaplastic lymphoma kinase-negative lung adenocarcinoma with BM. Further well-designed trials are warranted to verify this occasional finding.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Quinase do Linfoma Anaplásico/metabolismo , Carboplatina/uso terapêutico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Indóis/uso terapêutico , Masculino , Pessoa de Meia-Idade , Pemetrexede/uso terapêutico , Quinolinas/uso terapêutico
2.
Eur Rev Med Pharmacol Sci ; 24(16): 8606-8620, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32894568

RESUMO

OBJECTIVE: COVID-19 immune syndrome is a multi-systemic disorder induced by the COVID-19 infection. Pathobiological transitions and clinical stages of the COVID-19 syndrome following the attack of SARS-CoV-2 on the human body have not been fully explored. The aim of this review is to outline the three critical prominent phase regarding the clinicogenomics course of the COVID-19 immune syndrome. MATERIALS AND METHODS: In the clinical setting, the COVID-19 process presents as "asymptomatic/pre-symptomatic phase", "respiratory phase with mild/moderate/severe symptoms" and "multi-systemic clinical syndrome with impaired/disproportionate and/or defective immunity". The corresponding three genomic phases include the "ACE2, ANPEP transcripts in the initial phase", "EGFR and IGF2R transcripts in the propagating phase" and the "immune system related critical gene involvements of the complicating phase". RESULTS: The separation of the phases is important since the genomic features of each phase are different from each other and these different mechanisms lead to distinct clinical multi-systemic features. Comprehensive genomic profiling with next generation sequencing may play an important role in defining and clarifying these three unique separate phases for COVID-19. From our point of view, it is important to understand these unique phases of the syndrome in order to approach a COVID-19 patient bedside. CONCLUSIONS: This three-phase approach may be useful for future studies which will focus on the clinical management and development of the vaccines and/or specific drugs targeting the COVID-19 processes. ANPEP gene pathway may have a potential for the vaccine development. Regarding the specific disease treatments, MAS agonists, TXA127, Angiotensin (1-7) and soluble ACE2 could have therapeutic potential for the COVID-19 course. Moreover, future CRISPR technology can be utilized for the genomic editing and future management of the clinical course of the syndrome.


Assuntos
Doenças Assintomáticas , Infecções por Coronavirus/patologia , Sistema Imunitário/metabolismo , Pneumonia Viral/patologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/complicações , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Humanos , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/patologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Prognóstico , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Sepse/complicações , Sepse/patologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
PLoS One ; 15(8): e0237098, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745124

RESUMO

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 µg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 µg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.


Assuntos
Caspase 3/metabolismo , Receptores ErbB/genética , MicroRNAs/genética , Neoplasias Experimentais/terapia , Terapêutica com RNAi/métodos , Adenoviridae/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células , Cetuximab/administração & dosagem , Cetuximab/uso terapêutico , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo
4.
Life Sci ; 258: 118158, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32750435

RESUMO

AIMS: Glioblastoma multiforme (GBM) is characterized by aggressive infiltration and terrible lethality. The overwhelming majority of chemotherapeutic drugs fail to exhibit the desired treatment effects. Polydatin (PD), which was initially extracted from Polygonum cuspidatum, is distinguished for its outstanding cardioprotective, hepatoprotective, and renal protective effects, as well as significant anticancer activities. However, the anti-GBM effect of PD is unclear. MATERIALS AND METHODS: Cell proliferation and apoptosis after PD intervention were estimated using MTT, colony formation and flow cytometry assays in vitro, while wound-healing and Transwell assays were applied to assess cell migration and invasion. In addition, the anti-GBM effects of PD in vivo were detected in the subcutaneous tumor model of nude mice. Moreover, Western blot, immunofluorescence and immunohistochemical staining assays were employed to elaborate the relevant molecular mechanisms. KEY FINDINGS: The present study demonstrated that PD repressed cell proliferation, migration, invasion and stemness and promoted apoptosis in GBM cells. Moreover, by correlating the molecular characteristics of cancer cells with different sensitivities to PD and employing diverse analytical methods, we ultimately verified that the cytotoxicity of PD was related to EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway inhibition, in which multiple components were vital therapeutic targets of GBM. SIGNIFICANCE: This work demonstrated that PD could inhibit proliferation, migration, invasion and stemness and induce apoptosis by restraining multiple components of the EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway in GBM cells.


Assuntos
Antineoplásicos/uso terapêutico , Glioblastoma/tratamento farmacológico , Glucosídeos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Estilbenos/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Glucosídeos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Estilbenos/farmacologia
5.
PLoS Biol ; 18(7): e3000778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678845

RESUMO

The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/metabolismo , Endocitose , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Oncogenes , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular Tumoral , Progressão da Doença , Endossomos/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/enzimologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores da Transferrina/metabolismo , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
6.
Toxicon ; 185: 129-146, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32682827

RESUMO

The search for novel and relevant cancer therapeutics is continuous and ongoing. Cancer adaptations, resulting in therapeutic treatment failures, fuel this continuous necessity for new drugs to novel targets. Recently, researchers have started to investigate the effect of venoms and venom components on different types of cancer, investigating their mechanisms of action. Receptor tyrosine kinases (RTKs) comprise a family of highly conserved and functionally important druggable targets for cancer therapy. This research exploits the novelty of complex venom mixtures to affect phosphorylation of the epidermal growth factor receptor (EGFR) and related RTK family members, dually identifying new activities and unexplored avenues for future cancer and venom research. Six whole venoms from diverse species taxa, were evaluated for their ability to illicit changes in the phosphorylated expression of a panel of 49 commonly expressed RTKs. The triple negative breast cancer cell line MDA-MB-468 was treated with optimised venom doses, pre-determined by SDS PAGE and Western blot analysis. The phosphorylated expression levels of 49 RTKs in response to the venoms were assessed with the use of Human Phospho-RTK Arrays and analysed using ImageLab 5.2.1 analysis software (BioRad). Inhibition of EGFR phosphorylation occurred with treatment of venom from Acanthoscurria geniculata (Theraphosidae), Heterometrus swammerdami (Scorpionidae), Crotalus durissus vegrandis (Crotalidae) and Naja naja (Elapidae). Western green mamba Dendroaspis viridis venom increased EGFR phosphorylation. Eph, HGFR and HER were the most affected receptor families by venoms. Whilst the importance of these changes in terms of effect on MDA-MB-468 cells' long-term viability and functionality are still unclear, the findings present exciting opportunities for further investigation as potential drug targets in cancer and as tools to understand better how these pathways interact.


Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Venenos de Serpentes/farmacologia , Animais , Antivenenos , Crotalus , Venenos Elapídicos , Elapidae , Humanos , Fosforilação
7.
Nat Commun ; 11(1): 3660, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694521

RESUMO

High expression or aberrant activation of epidermal growth factor receptor (EGFR) is related to tumor progression and therapy resistance across cancer types, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) are first-line therapy for NSCLC. However, patients eventually deteriorate after inevitable acquisition of EGFR TKI-resistant mutations, highlighting the need for therapeutics with alternative mechanisms of action. Here, we report that the elevated tribbles pseudokinase 3 (TRIB3) is positively associated with EGFR stability and NSCLC progression. TRIB3 interacts with EGFR and recruits PKCα to induce a Thr654 phosphorylation and WWP1-induced Lys689 ubiquitination in the EGFR juxtamembrane region, which enhances EGFR recycling, stability, downstream activity, and NSCLC stemness. Disturbing the TRIB3-EGFR interaction with a stapled peptide attenuates NSCLC progression by accelerating EGFR degradation and sensitizes NSCLC cells to chemotherapeutic agents. These findings indicate that targeting EGFR degradation is a previously unappreciated therapeutic option in EGFR-related NSCLC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Adulto , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Fosforilação/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , Taxa de Sobrevida , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
9.
Proc Natl Acad Sci U S A ; 117(32): 19507-19516, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723814

RESUMO

Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.


Assuntos
Núcleo Celular/metabolismo , Citomegalovirus/fisiologia , Monócitos/virologia , Proteínas do Envelope Viral/metabolismo , Núcleo Celular/virologia , Células Cultivadas , DNA Viral/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Monócitos/metabolismo , Ligação Proteica , Transdução de Sinais , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia
10.
Anticancer Res ; 40(8): 4675-4680, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727791

RESUMO

BACKGROUND: From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. MATERIALS AND METHODS: The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. RESULTS: R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC50 value of TX-2036 was 1.8 µM. CONCLUSION: R-configured TXs interacted with Lys721 and Thr766 of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile765 and Thr766 (TX-2044), Ser696, Thr766, and Thr830 (TX-2031), Gly772, Cys773, and Thr830 (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.


Assuntos
Domínios Proteicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Isomerismo , Radiossensibilizantes/farmacologia , Estereoisomerismo
11.
Int J Nanomedicine ; 15: 4691-4703, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636625

RESUMO

Purpose: Gd-encapsulated carbonaceous dots (Gd@C-dots) have excellent stability and magnetic properties without free Gd leakage, therefore they can be considered as a safe alternative T1 contrast agent to commonly used Gd complexes. To improve their potential for cancer diagnosis and treatment, affibody-modified Gd@C-dots targeting non-small-cell lung cancer (NSCLC) EGFR-positive tumors with enhanced renal clearance were developed and synthesized. Materials and Methods: Gd@C-dots were developed and modified with Ac-Cys-ZEGFR:1907 through EDC/NHS. The size, morphology, and optical properties of the Gd@C-dots and Gd@C-dots-Cys-ZEGFR:1907 were characterized. Targeting ability was evaluated by in vitro and in vivo experiments, respectively. Residual gadolinium concentration in major organs was detected with confocal imaging and inductively coupled plasma mass spectrometry (ICP-MS) ex vivo. H&E staining was used to assess the morphology of these organs. Results: Gd@C-dots with nearly 20 nm in diameter were developed and modified with Ac-Cys-ZEGFR:1907. EGFR expression in HCC827 cells was higher than NCI-H520. In cell uptake assays, EGFR-expressing HCC827 cells exhibited significant MR T1WI signal enhancement when compared to NCI-H520 cells. Cellular uptake of Gd@C-dots-Cys-ZEGFR:1907 was reduced, when Ac-Cys-ZEGFR:1907 was added. In vivo targeting experiments showed that the probe signal was significantly higher in HCC827 than NCI-H520 xenografts at 1 h after injection. In contrast to Gd@C-dots, Gd@C-dots-Cys-ZEGFR:1907 nanoparticles can be efficiently excreted through renal clearance. No morphological changes were observed by H&E staining in the major organs after injection of Gd@C-dots-Cys-ZEGFR:1907. Conclusion: Gd@C-dots-Cys-ZEGFR:1907 is a high-affinity EGFR-targeting probe with efficient renal clearance and is therefore a promising contrast agent for clinical applications such as diagnosis and treatment of NSCLC EGFR-positive malignant tumors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Meios de Contraste/farmacocinética , Neoplasias Pulmonares/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Pontos Quânticos/química , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Meios de Contraste/química , Receptores ErbB/metabolismo , Feminino , Gadolínio/química , Gadolínio/farmacocinética , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Nus , Nanopartículas/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
12.
PLoS Pathog ; 16(7): e1008656, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32639985

RESUMO

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by IAV and transmit cell entry signals. However, how IAV's binding to sialic acid leads to engagement and activation of EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV's AFs and its functional receptor EGFR at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AFs and EGFR are organized in partially overlapping submicrometer clusters in the plasma membrane of A549 cells. Within AF domains, the local AF concentration reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a flat membrane. The results predict that the local AF concentration strongly influences the distinct mobility pattern of IAVs, in a manner consistent with live-cell single-virus tracking data. In contrast to AFs, EGFR resides in smaller clusters. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, indicating the activation of pre-formed clusters, which we show are long-lived. Taken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, thus linking their spatial organization to their functional role during virus-cell binding and receptor activation.


Assuntos
Vírus da Influenza A/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Células A549 , Receptores ErbB/metabolismo , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/metabolismo , Internalização do Vírus
13.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 40(7): 981-987, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32701235

RESUMO

OBJECTIVE: To investigate the inhibitory effect of epidermal growth factor receptor tyrosine kinase inhibitor (EGFRTKI) HS-10296 on the proliferation of triple-negative breast cancer (TNBC) MDA-MB-231 cells and explore the possible molecular mechanism. METHODS: MDA-MB-231 cells were treated with HS-10296 for 24, 48, or 72 h, and CCK-8 assay was used to assess the changes in the cell viability. The inhibitory effect of HS-10296 on cell proliferation was determined by clonogenic assay. JC-1 and flow cytometry were employed for analyzing the cell apoptosis, and the ultrastructure of the cells was observed under electron microscope. After pretreatment with autophagy inhibitor chloroquine (CQ), MDA-MB-231 cells were divided into control group, CQ treatment group, HS-10296 (4 and 6 µmol/L) treatment groups and combined treatment groups, and the sensitivity of the treated cells to HS-10296 was determined using CCK-8 assay. The effects of HS-10296 on EGFR pathway and apoptosis- and autophagy-related proteins in MDA-MB-231 cells were investigated using Western blotting. RESULTS: HS-10296 significantly inhibited the proliferation of MDA-MB-231 cells with IC50 values at 24, 48 and 72 h of 8.393, 2.777 and 2.016 µmol/L, respectively. JC-1 and flow cytometry showed that HS-10296 induced obvious apoptosis of MDA-MB-231 cells, which showed an apoptosis rate of (21.63 ± 2.97)% following treatment with 8 µmol/L HS-10296. Autophagy vesicles were observed in the cells treated with HS-10296 under electron microscope. In MDA-MB-231 cells pretreated with CQ, inhibition of autophagy significantly enhanced HS-10296-induced cell death. Western blotting showed that the apoptosis-related protein caspase-3 was activated after HS-10296 treatment to cut its substrate PARP. The expression of autophagy-related protein light chain 3B (LC3B) was significantly enhanced after HS-10296 treatment (P < 0.01), which also resulted in inhibited phosphorylation of EGFR and AKT proteins in the cells. CONCLUSIONS: HS-10296 can inhibit the proliferation and induce autophagy and apoptosis of MDA-MB-231 cells by inhibiting the EGFR/PI3K/AKT signaling pathway.


Assuntos
Apoptose , Autofagia , Inibidores de Proteínas Quinases , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos
14.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G63-G73, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538139

RESUMO

Hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously demonstrated that both CD44 and TLR4, but predominately TLR4, mediated HA stimulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal mice. Here we address the questions of which cell type expresses the relevant TLR4 in driving intestinal growth and what are the downstream events from TLR4 activation. Studies were done in 14-day-old mice: wild type (WT), mice deficient in cyclooxygenase 2 (COX2), mice deficient in myeloid cell TLR4, and mice deficient in epithelial cell epidermal growth factor receptor (EGFR). Biological end points included crypt fission and Lgr5 cell proliferation. In WT mice, treatment with NS-398 (a COX2 inhibitor), clodronate (a macrophage-depleting agent), or tyrphostin (an EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with control mice. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5+ stem cell proliferation compared with WT mice. Administration of dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5+ stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS-398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. We conclude that, in postnatal mice, ~30% of intestinal growth as manifested by crypt fission and Lgr5+ stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages, inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5+ epithelial stem cells, resulting in Lgr5+ stem cell proliferation and crypt fission.NEW & NOTEWORTHY This study, in newborn mice, describes a novel molecular pathway regulating Lgr5+ epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5+ stem cells resulting in proliferation. Lgr5+ stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.


Assuntos
Dinoprostona/metabolismo , Receptores ErbB/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Receptor 4 Toll-Like/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Receptores ErbB/metabolismo , Ácido Hialurônico/antagonistas & inibidores , Intestinos/efeitos dos fármacos , Camundongos Knockout , Receptor 4 Toll-Like/metabolismo , Ativação Transcricional/efeitos dos fármacos
15.
PLoS One ; 15(6): e0234719, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555629

RESUMO

BACKGROUND: Colostrum, the milk produced during first few days after birth, is rich in immunoglobulins, antimicrobial peptides & growth factors. Multiple clinical trials using bovine colostrum are ongoing but with no assessment of test product bioactivity. OBJECTIVES: To examine variability of bioactivity between 20 commercial colostrum products, contribution of TGFß and EGFR in mediating effects, heat sensitivity of bioactivity and changes in bioactivity of colostrum milkings in the days following calving. DESIGN: In vitro bioactivity used AGS, RIE-1 and Caco-2 cell proliferation (Alamar blue) and migration (wounded monolayers) assays. Changes in colostrum bioactivity determined following addition of TGFß-neutralising antibody, EGFR blocker (Typhostin) and after heating (40-60°C, 60 min). In vivo bioassay assessed ability of colostrum gavage (2ml, 7mg/ml) to reduce gastric damage (NSAID + restraint) in rats. Milkings from 6 cows, days 0-3 post calving were assessed for bioactivity and growth factor concentrations. RESULT: Six-fold differences in pro-proliferative and migratory activity were seen comparing commercial products. Comparison of most- and least-active samples from in vitro studies showed two- to three-fold differences in ability to reduce gastric injury (86% reduction using most-active vs 48% using least-active, p<0.01). Tyrphostin reduced pro-migratory and proliferative activity by 23% and 55%. TGFß neutralisation reduced migratory activity by 83% but did not affect proliferation Heating colostrum powder to 50°C did not affect immunoactivity of haptoglobin, EGF, TGFß, IgG, IGF-1 or betacellulin but decreased bioactivity by >40%. Milking studies showed high bioactivity during first and second milkings on day 0 but 77% reduction by day 3. Changes in total protein, haptoglobin, EGF, TGFß, IgG and IGF-1 paralleled falls in bioactivity. CONCLUSION: Commercial colostrum products possess widely different bioactivity. Variation in heat exposure and/or proportion of day 0 colostrum content may contribute to this. Assessment of colostrum bioactivity has advantages to growth factor quantitation for quality control.


Assuntos
Ensaios Clínicos como Assunto , Colostro/metabolismo , Idoso , Animais , Células CACO-2 , Bovinos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Temperatura Alta , Humanos , Masculino , Gravidez , Fator de Crescimento Transformador beta/metabolismo
16.
Environ Toxicol ; 35(10): 1058-1069, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32485087

RESUMO

Glioblastoma is the most common primary brain tumor with poor survival rate and without effective treatment strategy. Notably, amplification and active mutation of epidermal growth factor receptor (EGFR) occur frequently in glioblastoma patient that may be a potential treatment target. Several studies indicated that various type of herbal compounds not only regulate anti-depressant effect but also shown capacity to suppress glioblastoma growth via inducing apoptosis and inhibiting oncogene signaling transduction. Hyperforin, an herb compound derived from St. John's wort was used to treat depressive disorder by inhibiting neuronal reuptake of several neurotransmitters. Although hyperforin can reduce matrix metallopeptidases-2 (MMPs) and -9-mediated metastasis of glioblastoma, the detail mechanism of hyperforin on glioblastoma is remaining unclear. Here, we suggested that hyperforin may induce extrinsic/intrinsic apoptosis and suppress anti-apoptotic related proteins expression of glioblastoma. We also indicated that hyperforin-mediated anti-apoptotic potential of glioblastoma was correlated to inactivation of EGFR/extracellular signal-regulated kinases (ERK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Floroglucinol/análogos & derivados , Terpenos/farmacologia , Fator de Transcrição RelA/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Hypericum/química , Floroglucinol/isolamento & purificação , Floroglucinol/farmacologia , Transdução de Sinais , Terpenos/isolamento & purificação , Fator de Transcrição RelA/genética
17.
Nature ; 583(7815): 265-270, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581361

RESUMO

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.


Assuntos
Segregação de Cromossomos/genética , Evolução Molecular , Genoma/genética , Neoplasias/genética , Alelos , Animais , Reparo do DNA , Replicação do DNA , Receptores ErbB/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Mutação , Neoplasias/patologia , Seleção Genética , Transdução de Sinais , Troca de Cromátide Irmã , Transcrição Genética , Quinases raf/metabolismo , Proteínas ras/metabolismo
18.
Cancer Sci ; 111(9): 3268-3278, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32533590

RESUMO

Fibroblast growth factor receptor 4 (FGFR4) is known to induce cancer cell proliferation, invasion, and antiapoptosis through activation of RAS/RAF/ERK and PI3K/AKT pathways, which are also known as major molecular bases of colon cancer carcinogenesis related with epidermal growth factor receptor (EGFR) signaling. However, the interaction between FGFR4 and EGFR signaling in regard to colon cancer progression is unclear. Here, we investigated a potential cross-talk between FGFR4 and EGFR, and the effect of anti-EGFR therapy in colon cancer treatment. To explore the biological roles of FGFR4 in cancer progression, RNA sequencing was carried out using FGFR4 transfected colon cell lines. Gene ontology data showed the upregulation of genes related to EGFR signaling, and we identified that FGFR4 overexpression secretes EGFR ligands such as amphiregulin (AREG) with consequent activation of EGFR and ErbB3. This result was also shown in in vivo study and the cooperative interaction between EGFR and FGFR4 promoted tumor growth. In addition, FGFR4 overexpression reduced cetuximab-induced cytotoxicity and the combination of FGFR4 inhibitor (BLU9931) and cetuximab showed profound antitumor effect compared to cetuximab alone. Clinically, we found the positive correlation between FGFR4 and AREG expression in tumor tissue, but not in normal tissue, from colon cancer patients and these expressions were significantly correlated with poor overall survival in patients treated with cetuximab. Therefore, our results provide the novel mechanism of FGFR4 in connection with EGFR activation and the combination of FGFR4 inhibitor and cetuximab could be a promising therapeutic option to achieve the optimal response to anti-EGFR therapy in colon cancer.


Assuntos
Anfirregulina/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Linhagem Celular Tumoral , Cetuximab/farmacologia , Neoplasias do Colo/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
19.
J Toxicol Sci ; 45(6): 349-363, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32493877

RESUMO

9,10-Phenanthrenequinone (9,10-PQ) is a polycyclic aromatic hydrocarbon quinone contaminated in diesel exhaust particles and particulate matter 2.5. It is an efficient electron acceptor that induces redox cycling with electron donors, resulting in excessive reactive oxygen species and oxidized protein production in cells. The current study examined whether 9,10-PQ could activate epidermal growth factor receptor (EGFR) signaling in A431 cells through S-oxidation of its negative regulators such as protein tyrosine phosphatase (PTP) 1B. 9,10-PQ oxidized recombinant human PTP1B at Cys215 and inhibited its catalytic activity, an effect that was blocked by catalase (CAT), whereas cis-9,10-dihydroxy-9,10-dihydrophenanthrene (DDP), which lacks redox cycling activity, had no effect on PTP1B activity. Exposure of A431 cells to 9,10-PQ, but not DDP, activated signaling through EGFR and its downstream extracellular signal-regulated kinase 1/2 (ERK1/2), coupled with a decrease of cellular PTP activity. Immunoprecipitation and UPLC-MSE revealed that PTP1B easily undergoes oxidation during exposure of A431 cells to 9,10-PQ. Pretreatment with polyethylene glycol conjugated with CAT (PEG-CAT) abolished 9,10-PQ-generated H2O2 production and significantly blocked the activation of EGFR-ERK1/2 signaling by 9,10-PQ, indicating the involvement of H2O2 in the activation because scavenging agents for hydroxyl radicals had no effect on the redox signal activation. These results suggest that such an air pollutant producing H2O2, activates EGFR-ERK1/2 signaling, presumably through the S-oxidation of PTPs such as PTP1B, and activation of the signal cascade may contribute, at least in part, to cellular responses in A431 cells.


Assuntos
Receptores ErbB/metabolismo , Fenantrenos/efeitos adversos , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Cultivadas , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
20.
J Vis Exp ; (159)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32510498

RESUMO

Numerous somatic mutations occurring in the epidermal growth factor receptor (EGFR) family (ErbB) of receptor tyrosine kinases (RTK) have been reported from cancer patients, although relatively few have been tested and shown to cause functional changes in ErbBs. The ErbB receptors are dimerized and activated upon ligand binding, and dynamic conformational changes of the receptors are inherent for induction of downstream signaling. For two mutations shown experimentally to alter EGFR function, A702V and the Δ746ELREA750 deletion mutation, we illustrate in the following protocol how molecular dynamics (MD) simulations can probe the (1) conformational stability of the mutant tyrosine kinase structure in comparison with wild-type EGFR; (2) structural consequences and conformational transitions and their relationship to observed functional changes; (3) effects of mutations on the strength of binding ATP as well as for binding between the kinase domains in the activated asymmetric dimer; and (4) effects of the mutations on key interactions within the EGFR binding site associated with the activated enzyme. The protocol provides a detailed step-by-step procedure as well as guidance that can be more generally useful for investigation of protein structures using MD simulations as a means to probe structural dynamics and the relationship to biological function.


Assuntos
Simulação de Dinâmica Molecular , Mutação , Trifosfato de Adenosina/metabolismo , Estabilidade Enzimática , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Ligação Proteica , Conformação Proteica , Transdução de Sinais
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