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1.
Immunity ; 53(4): 805-823.e15, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33053330

RESUMO

The activating receptor CD226 is expressed on lymphocytes, monocytes, and platelets and promotes anti-tumor immunity in pre-clinical models. Here, we examined the role of CD226 in the function of tumor-infiltrating lymphocytes (TILs) and resistance to immunotherapy. In murine tumors, a large proportion of CD8+ TILs had decreased surface expression of CD226 and exhibited features of dysfunction, whereas CD226hi TILs were highly functional. This correlation was seen also in TILs isolated from HNSCC patients. Mutation of CD226 at tyrosine 319 (Y319) led to increased CD226 surface expression, enhanced anti-tumor immunity and improved efficacy of immune checkpoint blockade (ICB). Mechanistically, tumor-derived CD155, the ligand for CD226, initiated phosphorylation of Y319 by Src kinases, thereby enabling ubiquitination of CD226 by CBL-B, internalization, and proteasomal degradation. In pre-treatment samples from melanoma patients, CD226+CD8+ T cells correlated with improved progression-free survival following ICB. Our findings argue for the development of therapies aimed at maintaining the expression of CD226.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptores Virais/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Inibidores de Checkpoint Imunológico/imunologia , Imunoterapia/métodos , Células Jurkat , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL
2.
Am J Hum Genet ; 107(4): 778-787, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871102

RESUMO

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Proteínas de Neoplasias/genética , Netrinas/genética , Alelos , Animais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica/métodos , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Netrinas/metabolismo , Fenótipo , Locos de Características Quantitativas , Risco
3.
J Biol Chem ; 294(47): 17951-17961, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31597697

RESUMO

Naïve CD4+ T cells in the periphery differentiate into regulatory T cells (Tregs) in which Foxp3 is expressed for their suppressive function. NLRP3, a pro-inflammatory molecule, is known to be involved in inflammasome activation associated with several diseases. Recently, the expression of NLRP3 in CD4+ T cells, as well as in myeloid cells, has been described; however, a role of T cell-intrinsic NLRP3 in Treg differentiation remains unknown. Here, we report that NLRP3 impeded the expression of Foxp3 independent of inflammasome activation in Tregs. NLRP3-deficient mice elevate Treg generation in various organs in the de novo pathway. NLRP3 deficiency increased the amount and suppressive activity of Treg populations, whereas NLRP3 overexpression reduced Foxp3 expression and Treg abundance. Importantly, NLRP3 interacted with Kpna2 and translocated to the nucleus from the cytoplasm under Treg-polarizing conditions. Taken together, our results identify a novel role for NLRP3 as a new negative regulator of Treg differentiation, mediated via its interaction with Kpna2 for nuclear translocation.


Assuntos
Diferenciação Celular , Núcleo Celular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , alfa Carioferinas/metabolismo , Animais , Feminino , Fatores de Transcrição Forkhead/metabolismo , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Transporte Proteico
4.
Int J Cancer ; 141(3): 614-620, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28445609

RESUMO

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Exossomos/patologia , Neoplasias Pulmonares/patologia , Mesoderma/patologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Células Tumorais Cultivadas
5.
BMC Complement Altern Med ; 16: 42, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26832364

RESUMO

BACKGROUND: Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. METHODS: A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. RESULTS: A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. CONCLUSIONS: Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.


Assuntos
Papillomavirus Humano 16/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Infecções por Papillomavirus/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Plantas Medicinais/química
6.
Mol Biol Rep ; 40(7): 4507-19, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23649764

RESUMO

It has been reported that extracts from Asian traditional/medical herbs possess therapeutic agents against cancers, metabolic diseases, inflammatory diseases, and other intractable diseases. In this study, we assessed the molecular mechanisms involved in the anticancer effects of A1E, the extract of Korean medicinal herbs. We examined the role of the cytotoxic and apoptotic pathways in the cancer chemopreventive activity in non-small-cell lung cancer (NSCLC) cell lines NCI-H460 and NCI-H1299. A1E inhibited the proliferation of NCI-H460 more efficiently than NCI-H1299 (p53(-/-)) cells. The apoptosis was detected by nuclear morphological changes, annexin V-FITC/PI staining, cell cycle analysis, western blot, RT-PCR, and measurement of mitochondrial membrane potential. A1E induced cellular morphological changes and nuclear condensation at 24 h in a dose-dependent manner. A1E also perturbed cell cycle progression at the sub-G1 stage and altered cell cycle regulatory factors in NCI-H460 cells. Furthermore, A1E inhibited the PI3K/Akt and NF-κB survival pathways, and it activated apoptotic intrinsic and extrinsic pathways. A1E increased the expression levels of members of the extrinsic death receptor complex FasL and FADD. In addition, A1E treatment induced cleavage of caspase-8, caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), whereas the expression levels of Bcl-2 and Bcl-xl were downregulated. A1E induced mitochondrial membrane potential collapse and cytochrome C release. Our results suggest that A1E induces apoptosis via activation of both extrinsic and intrinsic pathways and inhibition of PI3K/Akt survival signaling pathways in NCI-H460 cells. In conclusion, these data demonstrate the potential of A1E as a novel chemotherapeutic agent in NSCLC.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/toxicidade , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/química , Extratos Vegetais/toxicidade , Plantas Medicinais , Proteínas Proto-Oncogênicas c-akt/metabolismo
7.
J Extracell Vesicles ; 11(9): e12266, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36124834

RESUMO

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.


Assuntos
Vesículas Extracelulares , Animais , Cromatografia em Gel , Vesículas Extracelulares/química , Filtração/métodos , Humanos , Camundongos , Reprodutibilidade dos Testes , Ultracentrifugação/métodos
8.
Nat Commun ; 12(1): 3543, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112803

RESUMO

Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.


Assuntos
Neoplasias da Mama/sangue , Quimiocina CCL2/metabolismo , Exossomos/metabolismo , Receptores CCR2/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/patologia , Citocinas/metabolismo , Exossomos/imunologia , Exossomos/patologia , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteoglicanas/metabolismo , Receptores de Citocinas/metabolismo , Baço/imunologia , Baço/metabolismo , Baço/patologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia
9.
Front Immunol ; 11: 1308, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32655574

RESUMO

Within the tumor microenvironment, there is an intricate communication happening between tumor and stromal cells. This information exchange, in the form of cytokines, growth factors, extracellular vesicles, danger molecules, cell debris, and other factors, is capable of modulating the function of immune cells. The triggering of specific responses, including phenotypic alterations, can ultimately result in either immune surveillance or tumor cell survival. Macrophages are a well-studied cell lineage illustrating the different cellular phenotypes possible, depending on the tumor microenvironmental context. While our understanding of macrophage responses is well documented in vitro, surprisingly, little work has been done to confirm these observations in the cancer microenvironment. In fact, there are examples of opposing reactions of macrophages to cytokines in cell culture and in vivo tumor settings. Additionally, it seems that different macrophage lineages, for example tissue-resident and monocyte-derived macrophages, respond differently to cytokines and other cancer-derived signals. In this review article, we will describe and discuss the diverging reports on how cancer cells influence monocyte-derived and tissue-resident macrophage traits in vivo.


Assuntos
Macrófagos/imunologia , Microambiente Tumoral/imunologia , Animais , Citocinas/imunologia , Vesículas Extracelulares/imunologia , Humanos , Fenótipo
10.
Genome Biol ; 21(1): 8, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910858

RESUMO

BACKGROUND: Genome-wide association studies have identified 196 high confidence independent signals associated with breast cancer susceptibility. Variants within these signals frequently fall in distal regulatory DNA elements that control gene expression. RESULTS: We designed a Capture Hi-C array to enrich for chromatin interactions between the credible causal variants and target genes in six human mammary epithelial and breast cancer cell lines. We show that interacting regions are enriched for open chromatin, histone marks for active enhancers, and transcription factors relevant to breast biology. We exploit this comprehensive resource to identify candidate target genes at 139 independent breast cancer risk signals and explore the functional mechanism underlying altered risk at the 12q24 risk region. CONCLUSIONS: Our results demonstrate the power of combining genetics, computational genomics, and molecular studies to rationalize the identification of key variants and candidate target genes at breast cancer GWAS signals.


Assuntos
Neoplasias da Mama/genética , Cromatina/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos
11.
BioDrugs ; 33(4): 411-422, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31190280

RESUMO

BACKGROUND: SB3 has been developed as a trastuzumab biosimilar, a therapeutic monoclonal antibody targeted to human epidermal growth factor receptor 2 (HER2), and approved by the European Commission and United States (US) Food and Drug Administration (FDA). During the developmental period of a biosimilar, setting an appropriate quality target is critical for assessing the similarity of the biosimilar product to the reference product. A stepwise approach should be taken to assessing similarity, beginning with extensive characterization of the reference product to establish the quality target. OBJECTIVE: In this study, we evaluated the similarity of SB3 to the reference product and the impact of changes in the biological profile of the reference product on similarity assessment. METHODS: Analytical similarity was assessed with defined test procedures in terms of critical quality attributes (CQAs) that could affect efficacy, potency, and safety, as well as for the non-CQAs that are related to process consistency. The quality target was established using up to 154 lots of European Union (EU)- and US-sourced Herceptin® (reference product), analyzed during the developmental period of SB3. RESULTS: Trends of the EU- and US-sourced reference product showed that the biological profile exhibited two marked changes for Fc-related attributes, and then recovered to pre-change quality level. Since the similarity range set by pre-change lots was considered most relevant, the changed lots were excluded from establishing the similarity range, which resulted in tightened acceptance criteria. As shown in the results of similarity assessment using the stringent quality target ranges, SB3 exhibits highly similar functional activities compared to the reference product in terms of both CQAs and non-CQAs. CONCLUSION: SB3 has been developed as a trastuzumab biosimilar approved in the EU and USA, and its manufacturing process is deemed to be robust and well-controlled within stringent quality target ranges.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Medicamentos Biossimilares/farmacologia , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Bioensaio/normas , Medicamentos Biossimilares/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/normas , Humanos , Receptor ErbB-2/metabolismo , Padrões de Referência , Trastuzumab/uso terapêutico
12.
Mol Oncol ; 13(4): 725-737, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30478887

RESUMO

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrPC expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrPC or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Krüppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy.


Assuntos
Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Platina/uso terapêutico , Proteínas Priônicas/metabolismo , Transdução de Sinais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Progressão da Doença , Receptores ErbB/metabolismo , Humanos , Platina/farmacologia , Resultado do Tratamento , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
JCI Insight ; 52019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30830863

RESUMO

Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.


Assuntos
Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Priônicas/metabolismo , Adulto , Antraciclinas/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Epirubicina/farmacologia , Epirubicina/uso terapêutico , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Seleção de Pacientes , Proteínas Priônicas/sangue , Proteínas Priônicas/genética , Prognóstico , Ligação Proteica , Isoformas de Proteínas/sangue , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo
14.
Anal Chem ; 80(17): 6724-30, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18672891

RESUMO

Bismuth telluride (Bi 2Te 3 ) is a benchmark material for thermoelectric power generation and cooling applications. Electrodeposition is a versatile technique for preparing thin films of this material; however, it affords films of variable composition depending on the preparation history. A simple and rapid assay of electrodeposited films, therefore, has both fundamental and practical importance. In this study, a new protocol for the electroanalysis of Bi 2Te 3 thin films is presented by combining the two powerful and complementary techniques of electrochemical quartz crystal microgravimetry (EQCM) and stripping voltammetry. First, any free (and excess) tellurium in the electrodeposited film was reduced to soluble Te ( 2- ) species by scanning to negative potentials in a 0.1 M Na 2SO 4 electrolyte, and the accompanying frequency increase (mass loss) was used to determine the content of free tellurium. The film was again subjected to cathodic stripping in the same medium (to generate Bi (0) and soluble Te (2-) from the Bi 2 Te 3 film component of interest), and the EQCM frequency change was used to determine the content of chemically bound Te in the Bi 2Te 3 thin film and thereby the compound stoichiometry. Finally, the EQCM frequency change during Bi oxidation to Bi (3+) and the difference between total Bi and Bi in Bi 2Te 3 resulted in the assay of free (excess) Bi in the electrodeposited film. Problems associated with the chemical/electrochemical stability of the free Bi species were circumvented by a flow electroanalysis approach. Data are also presented on the sensitivity of electrodeposited Bi 2Te 3 film composition to the electrodeposition potential. This newly developed method can be used for the compositional analysis of other thermoelectric thin-film material candidates in general.

15.
Mol Oncol ; 12(10): 1811-1826, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30171795

RESUMO

Transforming growth factor ß (TGFß) is overexpressed in advanced cancers and promotes tumorigenesis by inducing epithelial-mesenchymal transition (EMT), which enhances invasiveness and metastasis. Although we previously reported that EMT could be induced by increasing CK2 activity alone, it is not known whether CK2 also plays an essential role in TGFß-induced EMT. Therefore, in the present study, we investigated whether TGFß signaling could activate CK2 and, if so, whether such activation is required for TGFß-induced EMT. We found that CK2 is activated by TGFß treatment, and that activity peaks at 48 h after treatment. CK2 activation is dependent on TGFß receptor (TGFBR) I kinase activity, but independent of SMAD4. Inhibition of CK2 activation through the use of either a CK2 inhibitor or shRNA against CSNK2A1 inhibited TGFß-induced EMT. TGFß signaling decreased CK2ß but did not affect CK2α protein levels, resulting in a quantitative imbalance between the catalytic α and regulatory ß subunits, thereby increasing CK2 activity. The decrease in CK2ß expression was dependent on TGFBRI kinase activity and the ubiquitin-proteasome pathway. The E3 ubiquitin ligases responsible for TGFß-induced CK2ß degradation were found to be CHIP and WWP1. Okadaic acid (OA) pretreatment protected CK2ß from TGFß-induced degradation, suggesting that dephosphorylation of CK2ß by an OA-sensitive phosphatase might be required for CK2 activation in TGFß-induced EMT. Collectively, our results suggest CK2 as a therapeutic target for the prevention of EMT and metastasis of cancers.


Assuntos
Caseína Quinase II/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática , Células HEK293 , Humanos , Fosforilação , Proteólise , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4 , Ubiquitina-Proteína Ligases/metabolismo
16.
Oncogene ; 37(31): 4214-4225, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29713057

RESUMO

Hypoxia arises frequently in solid tumors and is a poor prognostic factor as it promotes tumor cell proliferation, invasion, angiogenesis, therapy resistance, and metastasis. Notably, there are two described forms of hypoxia present in a growing tumor: chronic hypoxia, caused by abnormal tumor vasculature, and intermittent hypoxia, caused by transient perfusion facilitated by tumor-supplying blood vessels. Here, we demonstrate that intermittent hypoxia, but not chronic hypoxia, endows breast cancer cells with greater metastatic potential. Using an immunocompetent and syngeneic murine model of breast cancer, we show that intermittent hypoxia enhances metastatic seeding and outgrowth in lungs in vivo. Furthermore, exposing mammary tumor cells to intermittent hypoxia promoted clonal diversity, upregulated metastasis-associated gene expression, induced a pro-tumorigenic secretory profile, increased stem-like cell marker expression, and gave rise to tumor-initiating cells at a relatively higher frequency. This work demonstrates that intermittent hypoxia, but not chronic hypoxia, induces a number of genetic, molecular, biochemical, and cellular changes that facilitate tumor cell survival, colonization, and the creation of a permissive microenvironment and thus enhances metastatic growth.


Assuntos
Neoplasias da Mama/patologia , Hipóxia/patologia , Metástase Neoplásica/patologia , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/patologia , Fenótipo
17.
Front Immunol ; 9: 871, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867925

RESUMO

Tumor-derived exosomes are being recognized as essential mediators of intercellular communication between cancer and immune cells. It is well established that bone marrow-derived macrophages (BMDMs) take up tumor-derived exosomes. However, the functional impact of these exosomes on macrophage phenotypes is controversial and not well studied. Here, we show that breast cancer-derived exosomes alter the phenotype of macrophages through the interleukin-6 (IL-6) receptor beta (glycoprotein 130, gp130)-STAT3 signaling pathway. Addition of breast cancer-derived exosomes to macrophages results in the activation of the IL-6 response pathway, including phosphorylation of the key downstream transcription factor STAT3. Exosomal gp130, which is highly enriched in cancer exosomes, triggers the secretion of IL-6 from BMDMs. Moreover, the exposure of BMDMs to cancer-derived exosomes triggers changes from a conventional toward a polarized phenotype often observed in tumor-associated macrophages. All of these effects can be inhibited through the addition of a gp130 inhibitor to cancer-derived exosomes or by blocking BMDMs exosome uptake. Collectively, this work demonstrates that breast cancer-derived exosomes are capable of inducing IL-6 secretion and a pro-survival phenotype in macrophages, partially via gp130/STAT3 signaling.


Assuntos
Exossomos/imunologia , Macrófagos/imunologia , Neoplasias Mamárias Experimentais/imunologia , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/imunologia , Receptor gp130 de Citocina/metabolismo , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Hidrazinas/farmacologia , Interleucina-6/imunologia , Interleucina-6/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Quinoxalinas/farmacologia , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
18.
MAbs ; 9(4): 704-714, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28296619

RESUMO

A biosimilar product needs to demonstrate biosimilarity to the originator reference product, and the quality profile of the latter should be monitored throughout the period of the biosimilar's development to match the quality attributes of the 2 products that relate to efficacy and safety. For the development of a biosimilar version of trastuzumab, the reference product, Herceptin®, was extensively characterized for the main physicochemical and biologic properties by standard or state-of-the-art analytical methods, using multiple lots expiring between March 2015 and December 2019. For lots with expiry dates up to July 2018, a high degree of consistency was observed for all the tested properties. However, among the lots expiring in August 2018 or later, a downward drift was observed in %afucose (G0+G1+G2). Furthermore, the upward drift of %high mannose (M5+M6) was observed in the lots with expiry dates from June 2019 to December 2019. As a result, the combination of %afucose and %high mannose showed 2 marked drifts in the lots with expiry dates from August 2018 to December 2019, which was supported by the similar trend of biologic data, such as FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Considering that ADCC is one of the clinically relevant mechanisms of action for trastuzumab, the levels of %afucose and %high mannose should be tightly monitored as critical quality attributes for biosimilar development of trastuzumab.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Medicamentos Biossimilares , Trastuzumab , Medicamentos Biossimilares/química , Medicamentos Biossimilares/farmacologia , Linhagem Celular , Humanos , Controle de Qualidade , Trastuzumab/química , Trastuzumab/farmacologia
19.
J Environ Pathol Toxicol Oncol ; 33(3): 219-31, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25272060

RESUMO

Luteolin is a common flavonoid that exists in medicinal herbs, fruits, and vegetables. Luteolin has biochemical functions including anti-allergy, anti-inflammation, and anti-cancer functions. However, its efficacy and precise mode of action against breast cancer are still under study. To elucidate whether luteolin exhibits an anticancer effect in breast cancer, MCF-7 breast cancer cells were incubated with luteolin, and apoptosis was assessed by observing nuclear morphological changes and by performing cell viability assay, cell cycle analysis, annexin V-FITC/PI double staining, western blotting, RT-PCR, and mitochondrial membrane potential measurements. Luteolin inhibited growth through perturbation of cell cycle progression at the sub-G1 and G1 phases in MCF-7 cells. Furthermore, luteolin enhanced the expression of death receptors, such as DR5, and activated caspase cascades. It enhanced the activities of caspase-8/-9/-3 in a dose-dependent manner, followed by inactivation of PARP. Activation of caspase-8 and caspase-9 induced caspase-3 activity, respectively, in apoptosis of extrinsic and intrinsic pathways. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and increased Bax expression by inhibiting expression of Bcl-2. Taken together, these results suggest that luteolin provokes cell cycle arrest and induces apoptosis by activating the extrinsic and intrinsic pathways.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Luteolina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Anexinas/química , Western Blotting , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Fluoresceína-5-Isotiocianato/química , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Oncol Rep ; 31(6): 2683-91, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24789165

RESUMO

Luteolin, a flavonoid extracted from a number of plants with recognized anticancer, anti-inflammatory and anti-oxidative activities, inhibits angiogenic processes and modulates multidrug resistance. However, the efficacy and mechanisms of action of this flavonoid agent are still undergoing study. In order to elucidate whether luteolin exhibits an anticancer effect in cervical cancer cells, HeLa cells were incubated with luteolin and apoptosis was assessed by observing nuclear morphological changes, and performing Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Cell cycle analysis, western blotting, RT-PCR and mitochondrial membrane potential measurements were also carried out. Luteolin showed a significant dose-dependent cytotoxic effect only in human papillomavirus (HPV)-positive cervical cancer cells, when compared to its effect on HPV-negative cervical cancer C33A cells. Expression levels of human papilloma virus E6 and E7 oncogenes were suppressed, those of related factors pRb and p53 were recovered and E2F5 was increased by luteolin treatment. Furthermore, luteolin enhanced the expression of death receptors and death receptor downstream factors such as Fas/FasL, DR5/TRAIL and FADD in HeLa cells, and activated caspase cascades. In particular, luteolin enhanced the activity of caspase-3 and -8 in a dose-dependent manner. Activation of caspase-3 induced caspase-8 activity and vice versa. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited Bcl-2 and Bcl-xL expression. In conclusion, luteolin exerts anticarcinogenic activity through inhibition of E6 and E7 expression and cross-activation of caspase-3 and -8. Taken together, these results suggest that luteolin induces inactivation of HPV-18 oncogene expression and apoptosis by activating the intrinsic and extrinsic pathways.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/biossíntese , Neoplasias do Colo do Útero/genética , Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HeLa , Papillomavirus Humano 18/patogenicidade , Humanos , Luteolina , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
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