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1.
Sci Rep ; 10(1): 12745, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728182

RESUMO

Compound Kushen injection (CKI), a medicine in widespread clinical use in China, has proven therapeutic effects on cancer. However, few molecular mechanism analyses have been carried out. To address this problem, bioinformatics approaches combining weighted gene co-expression network analysis with network pharmacology methods were undertaken to elucidate the underlying molecular mechanisms of CKI in the treatment of esophageal cancer (ESCA). First, the key gene modules related to the clinical traits of ESCA were analysed by WCGNA. Based on the results, the hub genes related to CKI treatment for ESCA were explored through network pharmacology. Molecular docking simulation was performed to recognize the binding activity of hub genes with CKI compounds. The results showed that the potential hub targets, including EGFR, ErbB2, CCND1 and IGF1R, are therapeutic targets of CKI for the treatment of ESCA. Moreover, these targets were significantly enriched in many pathways related to cancer and signalling pathways, such as the PI3K-Akt signalling pathway and ErbB signalling pathway. In conclusion, this research partially highlighted the molecular mechanism of CKI in the treatment of ESCA, offering great potential in the identification of the effective compounds in CKI and biomarkers for ESCA treatment.


Assuntos
Antineoplásicos/farmacologia , Biologia Computacional/métodos , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Esofágicas/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Algoritmos , Antineoplásicos/química , Ciclina D1/química , Ciclina D1/metabolismo , Bases de Dados Genéticas , Medicamentos de Ervas Chinesas/química , Receptores ErbB/química , Receptores ErbB/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Modelos Moleculares , Simulação de Acoplamento Molecular , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Análise de Sequência de RNA
2.
Oncol Rep ; 43(6): 2093-2104, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32236617

RESUMO

Human epidermal growth factor receptor 2 (HER2) is composed of an extracellular domain (ECD), a lipophilic transmembrane region and an intracellular domain (ICD). The most commonly used method to determine the status of HER2 is immunohistochemistry. However, false­negative results are sometimes given, which causes some patients to lose the opportunity for anti­HER2 therapy. We found that calpain­10 may prohibit HER2­ECD into peripheral blood resulting in a HER2­negative result by the immunohistochemical method. We enrolled 289 patients into our experiment to assess the relationship between sHER2­ECD and calpain­10. The results showed that there was a positive correlation between sHER2­ECD and calpain­10. Moreover, we also investigated the prognostic values of sHER2­ECD and calpain­10 in breast cancer patients. According to the follow­up results, positive sHER2­ECD and tissue calpain­10 were indicative of a poor prognosis in breast cancer patients. Subsequently, we further validated the relationship between the two molecules in in vitro experiments. In the in vitro experiments, the level of HER2­ECD in the culture medium was increased or decreased with a decrease or increase in calpain­10 by transfection technology, showing an inverse association. The results indicated that sHER2­ECD and tissue calpain­10 levels were powerful factors to assess the status of HER2. In combination with tissue HER2 detection, the occurrence of false­negative HER2 was reduced, providing patients with additional treatment opportunities. In conclusion, sHER2­ECD and tissue calpain­10 may be used as new prognostic indices for breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Calpaína/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Calpaína/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Domínios Proteicos , Regulação para Cima
3.
Crit Rev Oncol Hematol ; 149: 102927, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32172224

RESUMO

Anti-HER2 monoclonal antibodies (mAbs) such as trastuzumab are effective for all stages of HER2-positive breast cancer (BC). However, intrinsic or acquired resistance to these drugs may occur in a significant number of patients (pts) and, except for HER2 status, no validated predictive factors of response/resistance have been identified to date. This lack is in part due to the not yet fully elucidated mechanism of action of mAbs in vivo. Increasing evidence suggests a significant contribution of both innate and adaptive immunity to the antitumor effects of mAbs. The aim of this review was to describe the role of innate and adaptive immunity in the efficacy of anti-HER2 mAbs and to report known and novel strategies to be used for optimizing immune effects of anti-HER2 therapies for HER2-positive BC.


Assuntos
Imunidade Adaptativa , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/uso terapêutico , Anticorpos Monoclonais/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Humanos , Receptor ErbB-2/química
4.
J Med Chem ; 63(10): 5074-5088, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32027502

RESUMO

Membrane-bound mucins belong to a heterogeneous family of large O-glycoproteins involved in numerous cancers and inflammatory diseases of the epithelium. Some of them are also involved in protein-protein interactions, with receptor tyrosine kinase ErbB2, and fundamental and clinical data showed that these complexes have a detrimental impact on cancer outcome, thus raising interest in therapeutic targeting. This paper aims to demonstrate that MUC3, MUC4, MUC12, MUC13, and MUC17 have a common evolutionary origin and share a common structural organization with EGF-like and SEA domains. Theoretical structure-function relationship analysis of the conserved domains indicated that the studied membrane-bound mucins share common biological properties along with potential specific functions. Finally, the potential druggability of these complexes is discussed, revealing ErbB2-related pathways of cell signaling to be targeted.


Assuntos
Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Fator de Crescimento Epidérmico/metabolismo , Mucinas/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Membrana Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/química , Humanos , Mucinas/antagonistas & inibidores , Mucinas/química , Estrutura Secundária de Proteína , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/química , Transdução de Sinais/fisiologia
5.
BMC Cancer ; 20(1): 114, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046665

RESUMO

BACKGROUND: The HER2 extracellular domain shed in blood (HER2ECD) is reported to rise and fall in parallel with HER2+ breast cancer behavior. In this study, we evaluated the clinical relevance of plasma HER2ECD values in patients with metastatic breast cancer treated in the SAKK22/99 trial comparing trastuzumab monotherapy followed by trastuzumab-chemotherapy combination at progression versus upfront combination therapy. METHODS: Quantitative assessment of plasma HER2ECD was performed in 133 patients at baseline; after 2-24 h; at 3 weeks; at first response evaluation (8-9 weeks); and at tumor progression. Associations with tumor characteristics, disease course and trial treatment were evaluated. RESULTS: Baseline HER2ECD levels were stable within 24 h after the first trastuzumab injection. These plasma values correlated positively with the HER2 gene ratio (rs = 0.39, P < 0.001) and HER2 protein expression levels (rs = 0.36, P < 0.001) but not with ER/PR status of the primary tumor. HER2ECD baseline levels were positively associated with the presence of visceral disease (P = 0.05) and poor patients' outcome (Cox-regression: P = 0.009). Patients with high baseline levels (> 35 ng/ml) had the worst overall survival (P = 0.03) if treated with upfront combination therapy. Conversely, patients with low HER2ECD baseline values (< 15 ng/ml) had longer time to progression on combined trastuzumab-chemotherapy when first treated with trastuzumab monotherapy (P = 0.02). Monitoring HER2ECD levels during the course of the trial revealed significant time (P = 0.001) and time-treatment arm interactions (P = 0.0007). Under upfront trastuzumab alone, the HER2ECD levels remained stable until just before disease progression. In patients responding to combination treatment HER2ECD levels decreased to > 20%. CONCLUSIONS: Plasma HER2ECD levels in patients with metastatic breast cancer reflect HER2 disease status. This robust biomarker might help identifying patients without visceral disease profiting from a sequential treatment's modality. Monitoring HER2ECD levels during trastuzumab monotherapy could help defining the optimal time to introduce chemotherapy. TRIAL REGISTRATION: Registration Number by ClinicalTrials.gov: NCT00004935, Trial number: SAKK22/99. Registered on 27 January 2003.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/sangue , Neoplasias da Mama/mortalidade , Domínios Proteicos , Receptor ErbB-2/sangue , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Prognóstico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/química , Resultado do Tratamento
6.
J Biol Chem ; 295(11): 3563-3575, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32024694

RESUMO

The Src homology phosphatase 2 (SHP2) is a cytoplasmic enzyme that mediates signaling induced by multiple receptor tyrosine kinases, including signaling by the epidermal growth factor receptor (EGFR) family (EGFR1-4 or the human homologs HER1-4). In EGFR (HER1) and EGFR2 (HER2) signaling, SHP2 increases the half-life of activated Ras by blocking recruitment of Ras GTPase-activating protein (RasGAP) to the plasma membrane through dephosphorylation of docking sites on the receptors. However, it is unclear how SHP2 selectively recognizes RasGAP-binding sites on EGFR and HER2. In this report, we show that SHP2-targeted pTyr residues exist in a specific amino acid context that allows selective binding. More specifically, we show that acidic residues N-terminal to the substrate pTyr in EGFR and HER2 mediate specific binding by the SHP2 active site, leading to blockade of RasGAP binding and optimal signaling by the two receptors. Molecular modeling studies revealed that a peptide derived from the region of pTyr992-EGFR packs well and makes stronger interactions with the SHP2 active site than with the SHP1 active site, suggesting a built-in mechanism that enables selective substrate recognition by SHP2. A phosphorylated form of this peptide inhibits SHP2 activity in vitro and EGFR and HER2 signaling in cells, suggesting inhibition of SHP2 protein tyrosine phosphatase activity by this peptide. Although we do not expect this peptide to be a strong inhibitor by itself, we foresee that the insights into SHP2 selectivity described here will be useful in future development of active-site small molecule-based inhibitors.


Assuntos
Aminoácidos/química , Domínio Catalítico , Receptores ErbB/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptor ErbB-2/química , Sequência de Aminoácidos , Animais , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Humanos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genética , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Receptor ErbB-2/metabolismo , Transdução de Sinais , Especificidade por Substrato
7.
Breast Cancer ; 27(2): 213-224, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31559601

RESUMO

BACKGROUND: Breast cancer is one of the most lethal types of cancer in women worldwide. The human epidermal growth factor receptor 2 (HER2) is considered as a validated target in breast cancer therapy. Previously, we have used quantitative structure activity relationship QSAR equations and their associated pharmacophore models to screen for new promising HER2 structurally diverse inhibitory leads which were tested against HER2-overexpressing SKOV3 ovarian cancer cell line. OBJECTIVE: In this study, we sought to explore the effect of most active ligands against different normal and breast cancer cell lines that represent different breast cancer subtypes with distinguished expression levels in HER2 and HER1. METHODS: We have tested the promising compounds against SKBR3, MDA-MB-231, MCF7, human fibroblast, and MCF10 cell lines. To understand the inhibitory effects of the active ligands against HER2 over expressed breast cancer cell lines, all inhibitors and the control compound, lapatinib, were docked into the active site of HER2 enzyme performed using Ligand Fit docking engine and PMF scoring function. RESULTS: Five ligands exhibited promising results with relatively low IC50 values on cells that amplify HER2 and high IC50 on those that do not express such a receptor. The most potent compound (compound 13) showed an IC50 of 0.046 µM. To test their toxicity against normal cells, the active compounds were tested against both normal fibroblast and normal breast cancer cell MCF-10 and relatively high IC50 values were scored. The IC50 values on HER2 over-expressed breast cancer and normal fibroblast cells provided a promising safety index. Docking results showed the highest similarity in the binding site between the most active ligand and the lapatinib. CONCLUSION: Our pharmacophore model resulted in a high potent ligand that shows high potency against HER2 positive breast cancer and relatively low toxicity towards the normal human cells.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Lapatinib/química , Lapatinib/farmacologia , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/química , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo
8.
Protein Expr Purif ; 166: 105505, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31563543

RESUMO

Recombinant antibodies can be expressed as fusion constructs in combination with tags which simplify their engineering into reliable and homogeneous immunoreagents by allowing site-specific, 1:1 functionalization. Several tags and corresponding reagents for recombinant protein derivatization have been proposed but benchmarking surveys for the evaluation of their effect on the characteristics of recombinant antibodies have not been reported. In this work we evaluated the impact on expression yields, shelf-stability, thermostability and binding affinity of a set of C-terminal tags fused to the same anti-Her2 nanobody. Furthermore, we assessed the efficiency of the derivatization process. The constructs always bore a 6xHis tag plus either the controls (EGFP and C-tag) or CLIP, HALO, AviTag, the LEPTG sequence recognized by Sortase A (Sortase tag), or a free cysteine. The advantages and drawbacks of the different systems were analyzed and discussed.


Assuntos
Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/genética , Ligação Competitiva , Cisteína/metabolismo , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Oxirredutases/química , Oxirredutases/genética , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Estabilidade Proteica , Receptor ErbB-2/química , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusão/química , Anticorpos de Domínio Único/química
9.
J Med Chem ; 63(1): 40-51, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31414802

RESUMO

The ErbB receptor tyrosine kinase family members EGFR (epidermal growth factor receptor) and Her2 are among the prominent mutated oncogenic drivers of non-small cell lung cancer (NSCLC). Their importance in proliferation, apoptosis, and cell death ultimately renders them hot targets in cancer therapy. Small-molecule tyrosine kinase inhibitors seem well suited to be tailor-made therapeutics for EGFR mutant NSCLC; however, drug resistance mutations limit their success. Against this background, the elucidation and visualization of the three-dimensional structure of cancer-related kinases provide valuable insights into their molecular functions. This field has undergone a revolution because X-ray crystal structure determinations aided structure-based drug design approaches and clarified the effect of activating and resistance-conferring mutations. Here, we present an overview of important mutations affecting EGFR and Her2 and highlight their influence on the kinase domain conformations and active site accessibility.


Assuntos
Receptores ErbB/química , Receptor ErbB-2/química , Carcinoma Pulmonar de Células não Pequenas/genética , Domínio Catalítico/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação Puntual , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
10.
J Pept Sci ; 26(2): e3231, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31749266

RESUMO

The binding process of A9 peptide toward HER2-DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2-DIVMP, multiple ligand-based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.


Assuntos
Peptídeos/química , Peptídeos/farmacologia , Receptor ErbB-2/metabolismo , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Receptor ErbB-2/química , Ressonância de Plasmônio de Superfície
11.
Pharm Res ; 36(12): 177, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31696314

RESUMO

PURPOSE: The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation. METHODS: This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters. RESULTS: The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action. CONCLUSIONS: These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.


Assuntos
Antineoplásicos/química , Medicamentos Biossimilares/química , Trastuzumab/química , Animais , Ligação Competitiva , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Cinética , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais , Ligação Proteica , Receptor ErbB-2/química , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Relação Estrutura-Atividade
12.
Mol Biol Rep ; 46(6): 6361-6370, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31583572

RESUMO

In spite of several studies that have shown the cytotoxic effects of amygdalin on the different cancer cell lines, however, the chemopreventive potential of amygdalin on the breast cancer cell line is not completely understood. We investigated the effect of amygdalin on the cell death and the level of pro-apoptotic Bax protein and anti-apoptotic Bcl-2 protein in SK-BR-3 human breast cancer cell line. The cell viability of SK-BR-3 cells was evaluated by MTT assay in different concentration of amygdalin. The level of Bax and Bcl-2 in SK-BR-3 cells were measured by western blot analysis. For statistical analysis, One-way ANOVA was used for the comparison of Bax and Bcl-2 protein level and percent of cell viability between groups. The molecular docking studies of amygdalin within the Bcl-2 (PDB ID: 4LVT) and HER2 (PDB ID: 3RCD) active site, were performed using AutoDock 4.2.5. Amygdalin induced a significant reduction of cell viability in SK-BR-3 after 24-h treatment in a dose-dependent manner. Also, amygdalin causes an increase in pro-apoptotic Bax protein and a decrease in anti-apoptotic Bcl-2 protein expression in the SK-BR-3 cells. Molecular docking studies showed that amygdalin interacts with the active site amino acids of Bcl-2 and HER2 through hydrogen bonding and some hydrophobic interactions. Amygdalin can induce apoptotic death in SK-BR-3 cells by increasing pro-apoptotic Bax protein and decreasing anti-apoptotic Bcl-2 protein expression. The results suggest that amygdalin may be a valuable candidate for the treatment of breast cancer, especially in HER2 positive cells.


Assuntos
Amigdalina/farmacologia , Neoplasias da Mama/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor ErbB-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Amigdalina/química , Neoplasias da Mama/tratamento farmacológico , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas c-bcl-2/química , Receptor ErbB-2/química , Transdução de Sinais/efeitos dos fármacos
13.
Chem Asian J ; 14(23): 4268-4273, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31591824

RESUMO

The 9-mer peptide MFCH401 (N: 165-173: DTILWKDIF), which is located in the extracellular domain of HER2, has been predicted to be a novel epitope. Self-adjuvanting anti-HER2 vaccine constructs were designed and synthesized via covalently attaching MFCH401 or its linear tandem repeats (2×MFCH401, 3×MFCH401) to a lipopeptide Pam3 CSK4 via iterative condensation reaction. The in vivo results showed the Pam3 CSK4 -MFCH401 vaccine construct can induce higher antibody titers of IgG and IgM than those of other conjugates, and the analysis of changes in plasma cytokines level indicate the activation of Th1 cells and NK cells. In addition, the Pam3 CSK4 -MFCH401 vaccine conjugate induced a specific immune response to HER2-overexpressing human BT474 cells. Our data clearly indicated that MFCH401 is a promising epitope; moreover, its linear tandem repeats were unsuitable for anticancer vaccine design when conjugating with Pam3 CSK4 , which provided useful evidence for developing further anti-HER2 cancer vaccines.


Assuntos
Vacinas Anticâncer/imunologia , Peptídeos/imunologia , Animais , Anticorpos/sangue , Anticorpos/imunologia , Anticorpos/metabolismo , Vacinas Anticâncer/química , Linhagem Celular Tumoral , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Peptídeos/síntese química , Peptídeos/química , Receptor ErbB-2/química , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo
14.
J Recept Signal Transduct Res ; 39(3): 243-252, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31538848

RESUMO

Simultaneous inhibition of EGFR and HER2 by dual-targeting inhibitors is an established anti-cancer strategy. Therefore, a recent trend in drug discovery involves understanding the features of such dual inhibitors. In this study, three different G-QSAR models were developed corresponding to individual EGFR, HER2 and the dual-model for both receptors. The dual-model provided site-specific information wherein (i) increasing electronegative character and higher index of saturated carbon at R4 position; (ii) presence of chlorine atom at R2 position; (iii) decreasing alpha modified shape index at R1 and R3 positions; and (iv) less electronegativity at R2 position; were found important for enhancing the dual activity. Also, comparison of dual-model with the EGFR/HER2 individual models revealed that it incorporates the properties of both models and, thus, represents a combination of EGFR/HER2. Further, fragment analysis revealed that R2 and R4 are important for imparting high potency while specificity is decided by R1/R3 fragment. We also checked the predictive ability of the dual-model by determining applicability domain using William's plot. Also, analysis of active molecules showed they show favorable substitutions that agree with the constructed dual-model. Thus, we have been successful in developing a single dual-response QSAR model to get an insight into various structural features influencing EGFR/HER2 activity.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Receptores ErbB/química , Relação Quantitativa Estrutura-Atividade , Receptor ErbB-2/química , Humanos , Modelos Moleculares
15.
Mikrochim Acta ; 186(8): 495, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270702

RESUMO

A method is presented for electrochemical determination of the breast cancer biomarker HER2. A glassy carbon electrode (GCE) was modified with densely packed gold nanoparticles placed on a composite consisting of electrochemically reduced graphene oxide and single walled carbon nanotubes (ErGO-SWCNTs). An aptamer directed against HER2 was then immobilized ono the GCE. The modified GCE was characterized by cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. The immobilized aptamer selectively recognizes HER2 on the electrode interface, and this leads to an increased charge transfer resistance (Rct) of the electrode when using ferri/ferro-cyanide as the electrochemical probe. The method has a low limit of detection (50 fg·mL-1) and a wide analytical range (0.1 pg·mL-1 to 1 ng·mL-1). The assay is highly reproducible and specific. Clinical application was demonstrated by analysis of the HER2 levels in serum samples, and sera of breast cancer patients were successfully discriminated from sera of healthy persons. Graphical abstract An electrochemical aptasensor for HER2 is described that is based on the immobilization of anti-HER2 aptamer on a glassy carbon electrode modified with a nanocomposite prepred fromreduced graphene oxide, carbon nanotubes and gold nanoparticles.


Assuntos
Aptâmeros de Nucleotídeos/química , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Técnicas Eletroquímicas , Receptor ErbB-2/sangue , Biomarcadores Tumorais/química , Carbono/química , Eletrodos , Feminino , Ouro/química , Grafite/química , Humanos , Nanopartículas Metálicas/química , Nanotubos de Carbono/química , Receptor ErbB-2/química
16.
J Pharm Biomed Anal ; 174: 608-617, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31265987

RESUMO

A facile electrochemical sandwich immunosensor for the detection of a breast cancer biomarker, the human epidermal growth factor receptor 2 (HER2), was designed, using lead sulfide quantum dots-conjugated secondary HER2 antibody (Ab2-PbS QDs) as a label. Using Ab2-PbS QDs in the development of electrochemical immunoassays leads to many advantages such as straightforward synthesis and well-defined stripping signal of Pb(II) through acid dissolution, which in turn yields better sensing performance for the sandwiched immunosensor. In the bioconjugation of PbS QDs, the available amine and hydroxyl groups from secondary anti-HER2 and capped PbS QDs were bound covalently together via carbonyldiimidazole (CDI) acting as a linker. In order to quantify the biomarker, SWV signal was obtained, where the Pb2+ ions after acid dissolution in HCl was detected. The plated mercury film SPCE was also detected in situ. Under optimal conditions, HER2 was detected in a linear range from 1-100 ng/mL with a limit of detection of 0.28 ng/mL. The measures of satisfactory recoveries were 91.3% to 104.3% for the spiked samples, displaying high selectivity. Therefore, this method can be applied to determine HER2 in human serum.


Assuntos
Anticorpos/química , Técnicas Eletroquímicas , Chumbo/química , Pontos Quânticos , Receptor ErbB-2/química , Sulfetos/química , Algoritmos , Biomarcadores Tumorais/sangue , Técnicas Biossensoriais , Neoplasias da Mama/sangue , Carbono/química , Feminino , Humanos , Imidazóis/química , Imunoensaio , Limite de Detecção , Modelos Lineares , Mercúrio , Nanopartículas Metálicas , Nanomedicina , Nanoestruturas , Reprodutibilidade dos Testes
17.
Br J Cancer ; 121(3): 237-248, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209328

RESUMO

BACKGROUND: Despite overexpression of the ErbB (EGFR/HER2/ErbB3/ErbB4) family in castration-resistant prostate cancer (CRPC), some inhibitors of this family, including the dual EGFR/HER2 inhibitor lapatinib, failed in Phase II clinical trials. Hence, we investigated mechanisms of lapatinib resistance to determine whether alternate ErbB inhibitors can succeed. METHODS: The CWR22 human tumour xenograft and its CRPC subline 22Rv1 and sera from lapatinib-treated CRPC patients from a previously reported Phase II trial were used to study lapatinib resistance. Mechanistic studies were conducted in LNCaP, C4-2 and 22Rv1 cell lines. RESULTS: Lapatinib increased intratumoral HER2 protein, which encouraged resistance to this treatment in mouse models. Sera from CRPC patients following lapatinib treatment demonstrated increased HER2 levels. Investigation of the mechanism of lapatinib-induced HER2 increase revealed that lapatinib promotes HER2 protein stability, leading to membrane localisation, EGFR/HER2 heterodimerisation and signalling, elevating cell viability. Knockdown of HER2 and ErbB3, but not EGFR, sensitised CRPC cells to lapatinib. At equimolar concentrations, the recently FDA-approved pan-ErbB inhibitor dacomitinib decreased HER2 protein stability, prevented ErbB membrane localisation (despite continued membrane integrity) and EGFR/HER2 heterodimerisation, thereby decreasing downstream signalling and increasing apoptosis. CONCLUSIONS: Targeting the EGFR axis using the irreversible pan-ErbB inhibitor dacomitinib is a viable therapeutic option for CRPC.


Assuntos
Lapatinib/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Quinazolinonas/uso terapêutico , Receptor ErbB-2/biossíntese , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Receptores ErbB/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias de Próstata Resistentes à Castração/metabolismo , Multimerização Proteica , Receptor ErbB-2/sangue , Receptor ErbB-2/química
18.
J Pharm Biomed Anal ; 174: 441-449, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31220702

RESUMO

XMT-1522, an antibody-drug conjugate (ADC) currently in Phase I clinical development, represents the first Dolaflexin®-based, cleavable ADC with a high drug-antibody ratio (DAR). In this work, a novel immunocapture LC-MS/MS method was successfully developed for the simultaneous quantification of both total antibody and cleavable antibody-conjugated drug auristatin F-hydroxypropylamide (AF-HPA) in human plasma. This method utilized microwave-assisted enzymatic digestion for the total antibody and chemical release of the drug from ADC on a 96-well based immunocapture sample preparation platform. The total antibody and the conjugated drug AF-HPA were separated and subsequently quantified concurrently by LC-MS/MS. The linear range of the standard curve for total antibody was from 50 to 5000 ng/mL and for AF-HPA was from 3.3 to 330 ng/mL. The linearities showed R2 ≥ 0.993 for total antibody and R2 ≥ 0.996 for AF-HPA, respectively. The intra- and inter-day precision and accuracy were well within 15%. The validated method, with the characteristics of high efficiency, great selectivity, free of carryover, short LC-MS/MS time (˜3.5 min) and low sample volume (20 µl), was successfully applied for analyzing Phase 1 cancer patient samples.


Assuntos
Anticorpos/sangue , Cromatografia Líquida , Imunoconjugados/sangue , Espectrometria de Massas , Anticorpos/química , Calibragem , Hemólise , Humanos , Hidrólise , Imunoconjugados/química , Modelos Lineares , Micro-Ondas , Peptídeos/química , Controle de Qualidade , Receptor ErbB-2/química , Reprodutibilidade dos Testes , Tripsina/química
19.
J Nucl Med ; 60(11): 1569-1578, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31171598

RESUMO

Human epidermal growth factor receptor 2 (HER2) is used as a tumor biomarker and therapeutic target. Pertuzumab is an anti-HER2 antibody, and its binding to tumor cells requires HER2 to be present at the cell membrane. However, the cellular distribution of HER2 protein in gastric tumors is dynamic, and HER2 internalization decreases antibody binding to tumor cells. These features preclude the use of pretargeted strategies for molecular imaging and therapy. We explored the pharmacological modulation of HER2 endocytosis as a strategy to improve pertuzumab uptake in HER2-positive gastric tumors and allow the use of a pretargeted imaging approach. Methods: We conducted in vitro and in vivo studies with NCI-N87 gastric cancer cells to determine how HER2 endocytosis affects pertuzumab binding to tumor cells. Lovastatin, a clinically approved cholesterol-lowering drug, was used to modulate caveolae-mediated HER2 endocytosis. Results: Administration of lovastatin to NCI-N87 cancer cells resulted in significant accumulation of non-activated HER2 dimers at the cell surface. Pretreatment of NCI-N87 cells with lovastatin increased in vitro specific accumulation of membrane-bound 89Zr-labeled pertuzumab. Lovastatin-enhanced pertuzumab tumor uptake was also observed in NCI-N87 gastric cancer xenografts, allowing tumor detection as early as 4 h and high-contrast images at 48 h after tracer administration via PET. Temporal enhancement of HER2 membrane availability by lovastatin allowed imaging of cell surface HER2 with transcyclooctene-conjugated antibodies and 18F-labeled tetrazine. Conclusion: Temporal pharmacological modulation of membrane HER2 may be clinically relevant and exploitable for pretargeted molecular imaging and therapy in gastric tumors.


Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Membrana Celular/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Humanos , Lovastatina/farmacologia , Terapia de Alvo Molecular , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Transporte Proteico/efeitos dos fármacos , Receptor ErbB-2/química , Trastuzumab/farmacologia
20.
Mikrochim Acta ; 186(7): 439, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197538

RESUMO

Convenient and sensitive detection of human epidermal growth factor receptor 2 (HER2) dimerization is highly desirable for molecule subtyping and guiding personalized HER2 targeted therapy of breast cancer. A colocalization-triggered DNA nanoassembly (CtDNA) strategy was developed for amplified imaging of HER2 dimerization. It exploits (a) the advantage of the specificity of aptamer proximity hybridization, and (b) the high sensitivity of hairpin-free nonlinear HCR. The mechanism of step-by-step hairpin-free nonlinear HCR for DNA dendritic nanoassembly was studied by native polyacrylamide gel electrophoresis, atomic force microscopy and fluorometry. The results revealed a high specificity, sensitivity, and excellent controllability of the DNA dendritic nanoassembly. The method was used to identify HER2 homodimers and HER2/HER3 heterodimers in various breast cancer cell lines using fluorescence microscopy. It was then extended to image and quantitatively evaluate HER2 homodimers in clinical formalin-fixed paraffin-embedded breast cancer tissue specimens. This revealed its remarkable accuracy and practicality for clinical diagnostics. Graphical abstract Schematic presentation of amplified imaging of human epidermal growth factor receptor 2 (HER2) dimerization on cancer cell surfaces by using a co-localization triggered DNA nanoassembly (CtDNA).


Assuntos
DNA/química , Nanoestruturas/química , Multimerização Proteica , Receptor ErbB-2/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dendrímeros/química , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Estudo de Prova de Conceito , Receptor ErbB-2/química
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