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Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2-glucosamine-based two-photon fluorescent probe, TP-ß, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that TP-ß is suitable for isolation of primary beta cells. Moreover, two-photon imaging of TP-ß-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that TP-ß has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe TP-α with TP-ß to make a "TP islet cocktail". This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies.
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Colorantes Fluorescentes/química , Glucosamina/química , Imagenología Tridimensional , Células Secretoras de Insulina/citología , Fotones , Supervivencia Celular , Células Cultivadas , Colorantes Fluorescentes/síntesis química , Glucosamina/síntesis química , Humanos , Estructura MolecularRESUMEN
Patients with IDH-wild-type glioblastomas have a poor five-year survival rate along with limited treatment efficacy due to immune cell (glioma-associated microglia and macrophages) infiltration promoting tumour growth and resistance. To enhance therapeutic options, our study investigated the unique RNA-RNA-binding protein complex LOC-DHX15. This complex plays a crucial role in driving immune cell infiltration and tumour growth by establishing a feedback loop between cancer and immune cells, intensifying cancer aggressiveness. Targeting this complex with blood-brain barrier-permeable small molecules improved treatment efficacy, disrupting cell communication and impeding cancer cell survival and stem-like properties. Focusing on RNA-RNA-binding protein interactions emerges as a promising approach not only for glioblastomas without the IDH mutation but also for potential applications beyond cancer, offering new avenues for developing therapies that address intricate cellular relationships in the body.
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Neoplasias Encefálicas , Glioblastoma , Isocitrato Deshidrogenasa , Proteínas de Unión al ARN , Microambiente Tumoral , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Humanos , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Animales , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Línea Celular Tumoral , Ratones , Mutación , Antineoplásicos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación Celular , Regulación Neoplásica de la Expresión GénicaRESUMEN
Human silencers have been shown to regulate developmental gene expression. However, the functional importance of human silencers needs to be elucidated, such as whether they can form 'super-silencers' and whether they are linked to cancer progression. Here, we show two silencer components of the FGF18 gene can cooperate through compensatory chromatin interactions to form a super-silencer. Double knockout of two silencers exhibited synergistic upregulation of FGF18 expression and changes in cell identity. To perturb the super-silencers, we applied combinational treatment of an enhancer of zeste homolog 2 inhibitor GSK343, and a repressor element 1-silencing transcription factor inhibitor, X5050 ('GR'). Interestingly, GR led to severe loss of topologically associated domains and loops, which were associated with reduced CTCF and TOP2A mRNA levels. Moreover, GR synergistically upregulated super-silencer-controlled genes related to cell cycle, apoptosis and DNA damage, leading to anticancer effects in vivo. Overall, our data demonstrated a super-silencer example and showed that GR can disrupt super-silencers, potentially leading to cancer ablation.
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Over-consumption of iron-rich red meat and hereditary or genetic iron overload are associated with increased risk of colorectal carcinogenesis, yet the mechanistic basis of how metal-mediated signaling leads to oncogenesis remains enigmatic. Using fresh colorectal cancer (CRC) samples we identify Pirin, an iron sensor, that overcomes a rate-limiting step in oncogenesis, by re-activating the dormant human-reverse-transcriptase (hTERT) subunit of telomerase holoenzyme in an iron-(Fe3+)-dependent-manner and thereby drives CRCs. Chemical genetic screens combined with isothermal-dose response fingerprinting and mass-spectrometry identified a small molecule SP2509, that specifically inhibits Pirin-mediated hTERT reactivation in CRCs by competing with iron-(Fe3+) binding. Our findings, first to document how metal ions reactivate telomerase, provide a molecular mechanism for the well-known association between red meat, and increased incidence of CRCs. Small molecules like SP2509 represent a novel modality to target telomerase that acts as driver of 90% human cancers and is yet to be targeted in clinic.
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Cancer cells undergo transcriptional reprogramming to drive tumor progression and metastasis. Using cancer cell lines and patient-derived tumor organoids, we demonstrate that loss of the negative elongation factor (NELF) complex inhibits breast cancer development through downregulating epithelial-mesenchymal transition (EMT) and stemness-associated genes. Quantitative multiplexed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (qPLEX-RIME) further reveals a significant rewiring of NELF-E-associated chromatin partners as a function of EMT and a co-option of NELF-E with the key EMT transcription factor SLUG. Accordingly, loss of NELF-E leads to impaired SLUG binding on chromatin. Through integrative transcriptomic and genomic analyses, we identify the histone acetyltransferase, KAT2B, as a key functional target of NELF-E-SLUG. Genetic and pharmacological inactivation of KAT2B ameliorate the expression of EMT markers, phenocopying NELF ablation. Elevated expression of NELF-E and KAT2B is associated with poorer prognosis in breast cancer patients, highlighting the clinical relevance of our findings. Taken together, we uncover a crucial role of the NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.
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Neoplasias de la Mama , Femenino , Humanos , Neoplasias de la Mama/patología , Carcinogénesis/genética , Línea Celular Tumoral , Cromatina , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción p300-CBP/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Recurrent cytogenetic abnormalities are the main hallmark of multiple myeloma (MM) and patients having 2 or more high-risk prognostic events are associated with extremely poor outcome. 17p13(del) and 1q21(gain) are critical and independent high-risk cytogenetic markers, however, the biological significance underlying the poor outcome in MM patients having co-occurrence of both these chromosomal aberrations has never been interrogated. Herein, we identified that patients harbouring concomitant 17p13(del) with 1q21(gain) demonstrated the worst prognosis as compared to patients with single- (either 17p13(del) or 1q21(gain)) and with no chromosomal events (WT for both chromosomal loci); and they are highly enriched for genomic instability (GI) signature. We discovered that the GI feature in the patients with concomitant 17p13(del)-1q21(gain) was recapitulating the biological properties of myeloma cells with co-existing p53-deficiency and NEIL1 mRNA-hyper-editing (associated with chromosome 17p and 1q, respectively) that have inherent DNA damage response (DDR) and persistent activation of Chk1 pathway. Importantly, this became a vulnerable point for therapeutic targeting whereby the cells with this co-abnormalities demonstrated hyper-sensitivity to siRNA- and pharmacological-mediated-Chk1 inhibition, as observed at both the in vitro and in vivo levels. Mechanistically, this was attributable to the synthetic lethal relationship between p53-NEIL1-Chk1 abnormalities. The Chk1 inhibitor (AZD7762) tested showed good synergism with standard-of-care myeloma drugs, velcade and melphalan, thus further reinforcing the translational potential of this therapeutic approach. In summary, combination of NEIL1-p53 abnormalities with an ensuing Chk1 activation could serve as an Achilles heel and predispose MM cells with co-existing 1q21(gain) and 17p13(del) to therapeutic vulnerability for Chk1 inhibition.
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Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN Glicosilasas , Mieloma Múltiple , Proteína p53 Supresora de Tumor , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Aberraciones Cromosómicas , Deleción Cromosómica , ADN Glicosilasas/genética , Inestabilidad Genómica , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mutaciones Letales Sintéticas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
What covalent modifications control the temporal ubiquitination of ERα and hence the duration of its transcriptional activity remain poorly understood. We show that GREB1, an ERα-inducible enzyme, catalyzes O-GlcNAcylation of ERα at residues T553/S554, which stabilizes ERα protein by inhibiting association with the ubiquitin ligase ZNF598. Loss of GREB1-mediated glycosylation of ERα results in reduced cellular ERα levels and insensitivity to estrogen. Higher GREB1 expression in ERα+ve breast cancer is associated with greater survival in response to tamoxifen, an ERα agonist. Mice lacking Greb1 exhibit growth and fertility defects reminiscent of phenotypes in ERα-null mice. In summary, this study identifies GREB1, a protein with an evolutionarily conserved domain related to DNA-modifying glycosyltransferases of bacteriophages and kinetoplastids, as the first inducible and the only other (apart from OGT) O-GlcNAc glycosyltransferase in mammalian cytoplasm and ERα as its first substrate.
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Neoplasias de la Mama , Receptor alfa de Estrógeno , Proteínas de la Membrana/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Glicosilación , Glicosiltransferasas/genética , Humanos , Mamíferos/metabolismo , Ratones , Proteínas de Neoplasias/metabolismoRESUMEN
The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping.
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Cromatina/metabolismo , Represión Epigenética , Histonas/genética , Neoplasias/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Línea Celular Tumoral , Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Histonas/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , RNA-Seq , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.
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Aciltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Linfoma de Células B/tratamiento farmacológico , Ácido Mirístico/metabolismo , Adenina/análogos & derivados , Aminopiridinas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dasatinib/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Ratones , Ratones SCID , Modelos Biológicos , Piperidinas , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/metabolismoRESUMEN
Endothelial cell (EC) recruitment is central to the vascularization of tumors. Although several proteoglycans have been implicated in cancer and angiogenesis, their roles in EC recruitment and vascularization during tumorigenesis remain poorly understood. Here, we reveal that Agrin, which is secreted in liver cancer, promotes angiogenesis by recruiting ECs within tumors and metastatic lesions and facilitates adhesion of cancer cells to ECs. In ECs, Agrin-induced angiogenesis and adherence to cancer cells are mediated by Integrin-ß1, Lrp4-MuSK pathways involving focal adhesion kinase. Mechanistically, we uncover that Agrin regulates VEGFR2 levels that sustain the angiogenic property of ECs and adherence to cancer cells. Agrin attributes an ECM stiffness-based stabilization of VEGFR2 by enhancing interactions with Integrin-ß1-Lrp4 and additionally stimulates endothelial nitric-oxide synthase (e-NOS) signaling. Therefore, we propose that cross-talk between Agrin-expressing cancer and ECs favor angiogenesis by sustaining the VEGFR2 pathway.
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Agrina/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Activación Enzimática , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina beta1/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Ratones , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estabilidad Proteica , Transducción de Señal , SolubilidadRESUMEN
Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess tumor-initiating (TIC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of TIC phenotypes. Many of the implicated GPCRs signal through the G protein GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in patients. GNA13 levels modulate drug resistance and TIC-like phenotypes in patient-derived head and neck squamous cell carcinoma (HNSCC) cells in vitro and in vivo. Blockade of GNA13 expression, or of select downstream pathways, using small-molecule inhibitors abrogates GNA13-induced TIC phenotypes, rendering cells vulnerable to standard-of-care cytotoxic therapies. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker for tumor progression, and that interfering with GNA13-induced signaling provides a novel strategy to block TICs and drug resistance in HNSCCs.
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Transformación Celular Neoplásica/genética , Resistencia a Antineoplásicos/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Transformación Celular Neoplásica/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Células Tumorales CultivadasRESUMEN
The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Agrina/metabolismo , Mecanotransducción Celular , Fosfoproteínas/metabolismo , Transducción de Señal , Proteínas 14-3-3/metabolismo , Animales , Carcinogénesis , Línea Celular , Línea Celular Tumoral , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Humanos , Ratones Desnudos , Unión Proteica , Estabilidad Proteica , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Transcriptional reactivation of telomerase reverse transcriptase (TERT) reconstitutes telomerase activity in the majority of human cancers. Here, we found that ectopic TERT expression increases cell proliferation, while acute reductions in TERT levels lead to a dramatic loss of proliferation without any change in telomere length, suggesting that the effects of TERT could be telomere independent. We observed that TERT determines the growth rate of cancer cells by directly regulating global protein synthesis independently of its catalytic activity. Genome-wide TERT binding across 5 cancer cell lines and 2 embryonic stem cell lines revealed that endogenous TERT, driven by mutant promoters or oncogenes, directly associates with the RNA polymerase III (pol III) subunit RPC32 and enhances its recruitment to chromatin, resulting in increased RNA pol III occupancy and tRNA expression in cancers. TERT-deficient mice displayed marked delays in polyomavirus middle T oncogene-induced (PyMT-induced) mammary tumorigenesis, increased survival, and reductions in tRNA levels. Ectopic expression of either RPC32 or TERT restored tRNA levels and proliferation defects in TERT-depleted cells. Finally, we determined that levels of TERT and tRNA correlated in breast and liver cancer samples. Together, these data suggest the existence of a unifying mechanism by which TERT enhances translation in cells to regulate cancer cell proliferation.
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Proliferación Celular , Regulación Neoplásica de la Expresión Génica , ARN de Transferencia/genética , Telomerasa/fisiología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Femenino , Células HEK293 , Humanos , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa III/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Transcriptional reactivation of TERT, the catalytic subunit of telomerase, is necessary for cancer progression in about 90% of human cancers. The recent discovery of two prevalent somatic mutations-C250T and C228T-in the TERT promoter in various cancers has provided insight into a plausible mechanism of TERT reactivation. Although the two hotspot mutations create a similar binding motif for E-twenty-six (ETS) transcription factors, we show that they are functionally distinct, in that the C250T unlike the C228T TERT promoter is driven by non-canonical NF-κB signalling. We demonstrate that binding of ETS to the mutant TERT promoter is insufficient in driving its transcription but this process requires non-canonical NF-κB signalling for stimulus responsiveness, sustained telomerase activity and hence cancer progression. Our findings highlight a previously unrecognized role of non-canonical NF-κB signalling in tumorigenesis and elucidate a fundamental mechanism for TERT reactivation in cancers, which if targeted could have immense therapeutic implications.
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Mutación Missense , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteína Proto-Oncogénica c-ets-2/metabolismo , Telomerasa/genética , Animales , Western Blotting , Línea Celular Tumoral , Citocina TWEAK , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/genética , Subunidad p52 de NF-kappa B/genética , Subunidad p52 de NF-kappa B/metabolismo , Unión Proteica , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-2/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Trasplante Heterólogo , Factores de Necrosis Tumoral/farmacologíaRESUMEN
BACKGROUND: Cerenkov luminescence imaging (CLI) is an emerging imaging technique where visible light emitted from injected beta-emitting radionuclides is detected with an optical imaging device. CLI research has mostly been focused on positive contrast imaging for ascertaining the distribution of the radiotracer in a way similar to other nuclear medicine techniques. Rather than using the conventional technique of measuring radiotracer distribution, we present a new approach of negative contrast imaging, where blood vessel attenuation of Cerenkov light emitted by [68Ga]GaCl3 is used to image vasculature. METHODS: BALB/c nude mice were injected subcutaneously in the right flank with HT-1080 fibrosarcoma cells 14 to 21 days prior to imaging. On the imaging day, [68Ga]GaCl3 was injected and the mice were imaged from 45 to 90 min after injection using an IVIS Spectrum in vivo imaging system. The mice were imaged one at a time, and manual focus was used to bring the skin into focus. The smallest view with pixel size around 83 µm was used to achieve a sufficiently high image resolution for blood vessel imaging. RESULTS: The blood vessels in the tumor were clearly visible, attenuating 7% to 18% of the light. Non-tumor side blood vessels had significantly reduced attenuation of 2% to 4%. The difference between the attenuation of light of tumor vessels (10% ± 4%) and the non-tumor vessels (3% ± 1%) was significant. Moreover, a necrotic core confirmed by histology was clearly visible in one of the tumors with a 21% reduction in radiance. CONCLUSIONS: The negative contrast CLI technique is capable of imaging vasculature using [68Ga]GaCl3. Since blood vessels smaller than 50 µm in diameter could be imaged, CLI is able to image structures that conventional nuclear medicine techniques cannot. Thus, the negative contrast imaging technique shows the feasibility of using CLI to perform angiography on superficial blood vessels, demonstrating an advantage over conventional nuclear medicine techniques.
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An intravascular MRI contrast agent Gd(DO3A-Lys), Gadolinium(III) (2,2',2â³-(10-(3-(5-benzamido-6-methoxy-6-oxohexylamino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate), has been studied for tumor angiography based on its high relaxivity and long blood half-life. The preparation procedures of the contrast agent have been modified in order to achieve higher yield and improve the synthetic reproducibility. High relaxivity of Gd(DO3A-Lys) has been confirmed by measurements at 3 T, 7 T and 9.4 T magnetic fields. The relaxivity-dependent albumin binding study indicated that Gd(DO3A-Lys) partially bound to albumin protein. In vitro cell viability in HK2 cell indicated low cytotoxicity of Gd(DO3A-Lys) up to 1.2 mM [Gd] concentration. In vivo toxicity studies demonstrated no toxicity of Gd(DO3A-Lys) on kidney tissues up to 0.2 mM [Gd]. While the toxicity on liver tissue was not observed at low dosage (1.0 mM [Gd]), Gd(DO3A-Lys) cause certain damage on hepatic tissue at high dosage (2.0 mM [Gd]). The DO3A-Lys has been labeled with (68)Ga radioisotope for biodistribution studies. (68)Ga(DO3A-Lys) has high uptake in both HT1080 and U87MG xenograft tumors, and has high accumulation in blood. Contrast-enhanced MR angiography (CE-MRA) in mice bearing U87MG xenograft tumor demonstrated that Gd(DO3A-Lys) could enhance vascular microenvironment around the tumor, and displays promising characteristics of an MRI contrast agent for tumor angiography.
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Angiografía/métodos , Medios de Contraste , Compuestos Heterocíclicos/administración & dosificación , Lisina/química , Imagen por Resonancia Magnética/métodos , Neoplasias/irrigación sanguínea , Compuestos Organometálicos/administración & dosificación , Animales , Femenino , Gadolinio/administración & dosificación , Gadolinio/química , Gadolinio/farmacocinética , Semivida , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacocinética , Humanos , Ratones , Ratones SCID , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacocinética , Distribución TisularRESUMEN
OBJECTIVES: In this study, the authors developed D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply TPGS) liposomes and further conjugated them to trastuzumab for controlled and targeted delivery of docetaxel (DTX) as a model hydrophobic drug. METHODS: DTX- or coumarin-6-loaded liposomes were prepared by solvent injection method and characterized for size and size distribution, surface charge, surface chemistry and drug encapsulation efficiency and drug release profile. SK-BR-3 cells were employed as an in vitro model for HER2-positive breast cancer and assessed for their cellular uptake and cytotoxicity of the two liposomal formulations. In vivo pharmacokinetics (PK) was investigated in Sprague-Dawley rats. RESULTS: The IC(50) value was found to be 20.23 ± 1.95, 3.74 ± 0.98, 0.08 ± 0.4 µg/ml for the marketed preparation of DTX, TPGS liposomes and trastuzumab-conjugated TPGS liposomes, respectively after 24 h incubation with SK-BR-3 cells. In vivo PK experiments showed that i.v. administration of trastuzumab-conjugated liposomes achieved 1.9 and 10 times longer half-life, respectively than PEG-coated liposomes and DTX. The area under the curve (AUC) was increased by 3.47- and 1.728-fold, respectively. CONCLUSION: The trastuzumab-conjugated vitamin E TPGS-coated liposomes showed greater potential for sustained and targeted chemotherapy in the treatment of HER2 overexpressing breast cancer.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Antineoplásicos/farmacocinética , Portadores de Fármacos , Liposomas/química , Taxoides/farmacocinética , Vitamina E/análogos & derivados , Animales , Antineoplásicos/química , Antineoplásicos/toxicidad , Área Bajo la Curva , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Rastreo Diferencial de Calorimetría , Línea Celular Tumoral , Supervivencia Celular , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Cumarinas/farmacocinética , Cumarinas/toxicidad , Docetaxel , Semivida , Humanos , Masculino , Microscopía Confocal , Tamaño de la Partícula , Polietilenglicoles/química , Ratas Sprague-Dawley , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Taxoides/química , Taxoides/toxicidad , Tiazoles/farmacocinética , Tiazoles/toxicidad , Trastuzumab , Vitamina E/químicaRESUMEN
The aim of this work was to develop a new type of D-alpha-tocopheryl polyethylene glycol 1000 succinate mono-ester (TPGS) coated multi-functional (theranostic) liposomes, which contain both docetaxel and quantum dots (QDs) for cancer imaging and therapy. Non-targeting and folate receptor targeting TPGS coated theranostic liposomes were prepared by the solvent injection method and characterized for their particle size, polydispersity, zeta potential, surface chemistry and drug encapsulation efficiency. MCF-7 breast cancer cells of folate receptor overexpression were employed as an in vitro model to assess cellular uptake and cytotoxicity of the drug and QDs loaded liposomes. The mean particle size of the non-targeting and the targeting liposomes was found to be 202 and 210 nm, respectively. High resolution field emission transmission electron microscopy (FETEM) confirmed the presence of quantum dots in the peripheral hydrophobic membranes of the liposomes. The qualitative internalization of multi-functional liposomes by MCF-7 cells was visualized by confocal laser scanning microscopy (CLSM). The IC50 value, which is the drug concentration needed to kill 50% cells in a designated time period, was found to be 9.54 ± 0.76, 1.56 ± 0.19 and 0.23 ± 0.05 µg/ml for the commercial Taxotere(®), non-targeting and targeting liposomes, respectively after 24 h culture with MCF-7 cells. The targeting multi-functional liposomes showed greater efficacy than the non-targeting liposomes and thus great potential to improve the cancer imaging and therapy.