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The versatility of the Passerini three component reaction (Passerini-3CR) is herein exploited for the synthesis of an amphiphilic diblock copolymer, which self-assembles into polymersomes. Carboxy-functionalized poly(ethylene glycol) methyl ether is reacted with AB-type bifunctional monomers and tert-butyl isocyanide in a single process via Passerini-3CR. The resultant diblock copolymer (P1) is obtained in good yield and molar mass dispersity and is well tolerated in model cell lines. The Passerini-3CR versatility and reproducibility are shown by the synthesis of P2, P3, and P4 copolymers. The ability of the Passerini P1 polymersomes to incorporate hydrophilic molecules is verified by loading doxorubicin hydrochloride in P1DOX polymersomes. The flexibility of the synthesis is further demonstrated by simple post-functionalization with a dye, Cyanine-5 (Cy5). The obtained P1-Cy5 polymersomes rapidly internalize in 2D cell monolayers and penetrate deep into 3D spheroids of MDA-MB-231 triple-negative breast cancer cells. P1-Cy5 polymersomes injected systemically in healthy mice are well tolerated and no visible adverse effects are seen under the conditions tested. These data demonstrate that new, biodegradable, biocompatible polymersomes having properties suitable for future use in drug delivery can be easily synthesized by the Passerini-3CR.
Assuntos
Sistemas de Liberação de Medicamentos , Polímeros , Animais , Doxorrubicina/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Reprodutibilidade dos TestesRESUMO
Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.
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Asthma is a chronic inflammatory lung disease with considerable unmet medical needs for new and effective therapies. Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme that is ultimately responsible for the production of eicosanoids implicated in the pathogenesis of asthma. We investigated a novel cPLA(2)α inhibitor, PF-5212372, to establish the potential of this drug as a treatment for asthma. PF-5212372 was a potent inhibitor of cPLA(2)α (7 nM) and was able to inhibit prostaglandin (PG)D(2) and cysteinyl leukotriene release from anti-IgE-stimulated human lung mast cells (0.29 and 0.45 nM, respectively). In a mixed human lung cell population, PF-5212372 was able to inhibit ionomycin-stimulated release of leukotriene B(4), thromboxane A(2), and PGD(2) (2.6, 2.6, and 4.0 nM, respectively) but was significantly less effective against PGE(2) release (>301 nM; p < 0.05). In an in vitro cell retention assay, PF-5212372 retained its potency up to 24 h after being washed off. In a sheep model of allergic inflammation, inhalation of PF-5212372 significantly inhibited late-phase bronchoconstriction (78% inhibition; p < 0.001) and airway hyper-responsiveness (94% inhibition; p < 0.001), and isolated sheep lung mast cell assays confirmed species translation via effective inhibition of PGD(2) release (0.78 nM). Finally, PF-5212372 was assessed for its ability to inhibit the contraction of human bronchi induced by AMP. PF5212372 significantly inhibited AMP-induced contraction of human bronchi (81% inhibition; p < 0.001); this finding, together with the ability of this drug to be effective in a wide range of preclinical asthma models, suggests that inhibition of cPLA(2)α with PF-5212372 may represent a new therapeutic option for the treatment of asthma.
Assuntos
Asma/tratamento farmacológico , Citosol/enzimologia , Inibidores Enzimáticos/uso terapêutico , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fenilpropionatos/farmacologia , Sulfonamidas/farmacologia , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Broncoconstrição/efeitos dos fármacos , Ionóforos de Cálcio/farmacologia , Linhagem Celular , Humanos , Mastócitos/fisiologia , Prostaglandina D2/metabolismo , OvinosRESUMO
Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.
Assuntos
Neoplasias de Mama Triplo Negativas , Bicarbonatos , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Humanos , Hipóxia/genética , Sódio , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The chemotherapy resistance of esophageal adenocarcinomas (EACs) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that, by targeting the tumor-promoting functions of the predominant TME cell type, cancer-associated fibroblasts (CAFs) with phosphodiesterase type 5 inhibitors (PDE5i), we can enhance the efficacy of standard-of-care chemotherapy. In ex vivo conditions, PDE5i prevent the transdifferentiation of normal fibroblasts to CAF and abolish the tumor-promoting function of established EAC CAFs. Using shotgun proteomics and single-cell RNA-seq, we reveal PDE5i-specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAFs drive chemotherapy resistance in EACs and can be targeted by repurposing PDE5i, a safe and well-tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.
Assuntos
Adenocarcinoma , Fibroblastos Associados a Câncer , Neoplasias Esofágicas , Adenocarcinoma/tratamento farmacológico , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Humanos , Masculino , Inibidores da Fosfodiesterase 5/farmacologia , Microambiente TumoralRESUMO
BACKGROUND AND AIMS: Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines. METHODS: Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT. RESULTS: The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins. CONCLUSION: The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.
Assuntos
Infecções por Helicobacter/metabolismo , Helicobacter pylori/patogenicidade , Proteínas de Neoplasias/biossíntese , Neoplasias Gástricas/microbiologia , Animais , Transformação Celular Neoplásica/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Células Epiteliais/patologia , Gastrinas/biossíntese , Gastrinas/genética , Gastrinas/fisiologia , Regulação Neoplásica da Expressão Gênica , Infecções por Helicobacter/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/fisiologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Regulação para Cima , VirulênciaRESUMO
Glioblastoma is a malignant brain tumour with a median survival of 14.6 months from diagnosis. Despite maximal surgical resection and concurrent chemoradiotherapy, reoccurrence is inevitable. To try combating the disease at a stage of low residual tumour burden immediately post-surgery, we propose a localised drug delivery system comprising of a spray device, bioadhesive hydrogel (pectin) and drug nanocrystals coated with polylactic acid-polyethylene glycol (NCPPs), to be administered directly into brain parenchyma adjacent to the surgical cavity. We have repurposed pectin for use within the brain, showing in vitro and in vivo biocompatibility, bio-adhesion to mammalian brain and gelling at physiological brain calcium concentrations. Etoposide and olaparib NCPPs with high drug loading have shown in vitro stability and drug release over 120 h. Pluronic F127 stabilised NCPPs to ensure successful spraying, as determined by dynamic light scattering and transmission electron microscopy. Successful delivery of Cy5-labelled NCPPs was demonstrated in a large ex vivo mammalian brain, with NCPP present in the tissue surrounding the resection cavity. Our data collectively demonstrates the pre-clinical development of a novel localised delivery device based on a sprayable hydrogel containing therapeutic NCPPs, amenable for translation to intracranial surgical resection models for the treatment of malignant brain tumours.
Assuntos
Antineoplásicos/administração & dosagem , Encéfalo/metabolismo , Portadores de Fármacos , Etoposídeo/administração & dosagem , Lactatos/química , Nanopartículas , Pectinas/química , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Polietilenoglicóis/química , Adesividade , Aerossóis , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Composição de Medicamentos , Liberação Controlada de Fármacos , Etoposídeo/química , Etoposídeo/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Hidrogéis , Masculino , Camundongos Nus , Ftalazinas/química , Ftalazinas/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Ratos , Solubilidade , Distribuição TecidualRESUMO
Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-ß) signalling, and of ß-catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the ß-catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-ß and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients.
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Triple negative or basal-like breast cancer (TNBC) is characterised by aggressive progression, lack of standard therapies and poorer overall survival rates for patients. The bad prognosis, high rate of relapse and resistance against anticancer drugs have been associated with a highly abnormal loss of redox control in TNBC cells. Here, we developed docetaxel (DTX)-loaded micellar-like nanoparticles (MLNPs), designed to address the aberrant TNBC biology through the placement of redox responsive cross-links designed into a terpolymer. The MLNPs were derived from poly(ethyleneglycol)-b-poly(lactide)-co-poly(N3-α-ε-caprolactone) with a disulfide linker pendant from the caprolactone regions in order to cross-link adjacent chains. The terpolymer contained both polylactide and polycaprolactone to provide a balance of accessibility to reductive agents necessary to ensure stability in transit, but rapid micellar breakdown and concomitant drug release, when in breast cancer cells with increased levels of reducing agents. The empty MLNPs did not show any cytotoxicity in vitro in 2D monolayers of MDA-MB-231 (triple negative breast cancer), MCF7 (breast cancer) and MCF10A (normal breast epithelial cell line), whereas DTX-loaded reducible crosslinked MLNPs exhibited higher cytotoxicity against TNBC and breast cancer cells which present high intracellular levels of glutathione. Crosslinked and non-crosslinked MLNPs showed high and concentration-dependent cellular uptake in monolayers and tumour spheroids, including when assessed in co-cultures of TNBC cells and cancer-associated fibroblasts. DTX loaded crosslinked MLNPs showed the highest efficacy against 3D spheroids of TNBC, in addition the MLNPs also induced higher levels of apoptosis, as assessed by annexin V/PI assays and increased caspase 3/7 activity in MDA-MB-231 cells in comparison to cells treated with DTX-loaded un-crosslinked MLNP (used as a control) and free DTX. Taken together these data demonstrate that the terpolymer micellar-like nanoparticles with reducible crosslinks have high efficacy in both 2D and 3D in vitro cancer models by targeting the aberrant biology, i.e. loss of redox control of this type of tumour, thus may be promising and effective carrier systems for future clinical applications in TNBC.
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Antineoplásicos , Nanopartículas , Neoplasias de Mama Triplo Negativas , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Docetaxel/uso terapêutico , Humanos , Micelas , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
The spatial resolution and light detected in fluorescence imaging for small animals are limited by light scattering, absorption and autofluorescence. To address this, novel near-infrared fluorescent contrast agents and imaging configurations have been investigated. In this paper, the influence of the light wavelength and imaging configurations (full-field illumination system and scanning system) on fluorescence imaging are compared quantitatively. The surface radiance for both systems is calculated by modifying the simulation tool Near-Infrared Fluorescence and Spectral Tomography. Fluorescent targets are embedded within a scattering medium at different positions. The surface radiance and spatial resolution are obtained for emission wavelengths between 620 nm and 1000 nm. It was found that the spatial resolution of the scanning system is independent of the tissue optical properties, whereas for full-field illumination, the spatial resolution degrades at longer wavelength. The full width at half maximum obtained by the scanning system is 25% lower than that obtained by the full-field illumination system when the targets are located in the middle of the phantom. The results indicate that although imaging at near-infrared wavelength can achieve a higher surface radiance, it may produce worse spatial resolution.
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This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycine-extended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC(Min) mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin-2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC(Min) mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK-2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy.
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Células Endoteliais/efeitos dos fármacos , Fator de Crescimento Epidérmico/fisiologia , Gastrinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Embrião de Galinha , Neoplasias do Colo/sangue , Neoplasias do Colo/irrigação sanguínea , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Gastrinas/antagonistas & inibidores , Gastrinas/sangue , Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Soros Imunes , Peptídeos e Proteínas de Sinalização Intercelular , Isoenzimas/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Omeprazol/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidoresRESUMO
Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40-80%, when tested in a panel of lung epithelial cells. Considering the formulation 'developability', there were no significant changes in the complexes' size and integrity on aerosolisation by microsprayer (PenCentury™) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA.
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Quitosana/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacocinética , Água/metabolismo , Células A549 , Administração por Inalação , Animais , Quitosana/química , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , RNA Interferente Pequeno/química , Solubilidade , Água/química , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
BACKGROUND: A model to predict chemotherapy response would provide a marked clinical benefit, enabling tailored treatment of oesophageal cancer, where less than half of patients respond to the routinely administered chemotherapy. METHODS: Cancer cells were established from tumour biopsies taken from individual patients about to undergo neoadjuvant chemotherapy. A 3D-tumour growth assay (3D-TGA) was developed, in which cancer cells were grown with or without supporting mesenchymal cells, then subjected to chemo-sensitivity testing using the standard chemotherapy administered in clinic, and a novel emerging HDAC inhibitor, Panobinostat. RESULTS: Individual patient's cancer cells could be expanded and screened within a clinically applicable timescale of 3 weeks. Incorporating mesenchymal support within the 3D-TGA significantly enhanced both the growth and drug resistance profiles of the patient's cancer cells. The ex vivo drug response in the presence, but not absence, of mesenchymal cells accurately reflected clinical chemo-sensitivity, as measured by tumour regression grade. Combination with Panobinostat enhanced response and proved efficacious in otherwise chemo-resistant tumours. CONCLUSIONS: This novel method of establishing individual patient oesophageal cancers in the laboratory, from small endoscopic biopsies, enables clinically-relevant chemo-sensitivity testing, and reduces use of animals by providing more refined in vitro models for pre-screening of drugs. The 3D-TGA accurately predicted chemo-sensitivity in patients, and could be developed to guide tailored patient treatment. The incorporation of mesenchymal cells as the stromal cell component of the tumour micro-environment had a significant effect upon enhancing chemotherapy drug resistance in oesophageal cancer, and could prove a useful target for future drug development.
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Técnicas de Cultura de Células , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Medicina de Precisão , Idoso , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas/terapia , Feminino , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fenótipo , Medicina de Precisão/métodos , Resultado do Tratamento , Carga TumoralRESUMO
Gastrin is a growth factor for both gastrointestinal and non-gastrointestinal tumours. Endocytosis of gastrin has been demonstrated in tumour cell lines expressing cholecystokinin-B/gastrin receptor (CCK-BR); this has raised the possibility of receptor targeted therapy. The aim of this study was to examine endocytosis of gastrin and CCK-BR in tumour cell lines. A small gastrin analogue, RG-G7, and the anti-CCK-BR antibody, anti-GRE1, were fluorescently labelled and uptake by cancer cell lines including AR42J, HepG2, and C170HM2 as well as transfected NIH3T3 fibroblast cells was assessed using standard and confocal fluorescence microscopy. CCK-BR expression of cell lines was assayed by reverse transcription-polymerase chain reaction and Western blotting. Apoptosis was detected using a fluorescent TUNEL method. RG-G7 and anti-GRE1 antibody were specifically taken up by all cell lines expressing CCK-BR. In addition to cytoplasmic uptake with RG-G7 and anti-GRE1 the latter also showed specific uptake into the nucleus. A coincidence of anti-GRE1 and apoptosis was seen. Targeting CCK-BR by peptide or antibody may offer therapeutic opportunities for some cancers.
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Carcinoma Hepatocelular/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor de Colecistocinina B/metabolismo , Animais , Apoptose/fisiologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Endocitose , Humanos , Immunoblotting/métodos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Microscopia Confocal/métodos , Células NIH 3T3 , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Receptor de Colecistocinina B/biossíntese , Receptor de Colecistocinina B/genéticaRESUMO
Mechanisms by which premalignant Barrett's metaplasia (BM) progresses to esophageal adenocarcinoma are currently being sought. This study investigated the role played by the polypeptide hormone gastrin, specifically its antiapoptotic effects through activation of protein kinase B/Akt (PKB/Akt). In esophageal cell lines with low basal levels of activated PKB/Akt, phosphorylation could be induced by exogenous amidated gastrin. High basal levels of activated PKB/Akt were linked to endogenous gastrin expression and were reduced by treatment with a cholecystokinin-type 2 receptor (CCK-2R) antagonist. Expression of a constitutively active splice variant of the CCK-2R additionally increased basal activation of PKB/Akt. It is proposed that gastrin acting in an autocrine and endocrine manner via a CCK-2R isoform may activate PKB/Akt and that with expression of gastrin and CCK-2R isoforms increasing in BM samples, gastrin may aid progression of BM through amplification of antiapoptotic pathways. Evidence for this proposal was provided through the observed specific up-regulation of PKB/Akt in BM samples.
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Apoptose , Esôfago de Barrett/metabolismo , Gastrinas/fisiologia , Proteínas Serina-Treonina Quinases , Receptor de Colecistocinina B/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Esôfago de Barrett/patologia , Benzodiazepinas/farmacologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Esôfago/metabolismo , Esôfago/patologia , Antagonistas de Hormônios/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptor de Colecistocinina B/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
The gastrin gene is expressed widely in pancreatic adenocarcinomas and the study aimed to assess its role in both the resistance of cancer cells to apoptosis and the sensitivity of cells to chemotherapeutic agents. Two human pancreatic cell lines, PAN1 and BXPC3, expressed gastrin at both the RNA and protein levels and are shown to be representative of human pancreatic adenocarcinomas in terms of gastrin expression. Inhibition of endogenous gastrin production by tumor cells was achieved with neutralizing gastrin antiserum and transfection with a gastrin antisense plasmid. Gastrin antiserum synergized with both taxotere and gemcitabine in inhibiting the in vitro growth of the PAN1 cell line with the inhibitory effect of the antiserum increasing from 12.7% to 70.2% with taxotere (P < 0.05) and 28.6% with gemcitabine (P < 0.01) after controlling for the effects of the cytotoxics. Synergy was only achieved with taxotere in BXPC3 cells with the inhibitory effect of gastrin antiserum increasing from 22.9% to 50.0% (P < 0.005). Cells transfected with gastrin antisense had reduced in vitro growth in low serum conditions and were poorly tumorigenic in nude mice at an orthotopic site. Gastrin antisense-transfected PAN1 cells had increased sensitivity to the antiproliferative effects of both gemcitabine (IC50 of > 100 microg/ml reduced to 0.1 microg/ml) and taxotere (IC50 of 20 microg/ml reduced to < 0.01 microg/ml) when compared with vector controls. The increased sensitivity of PAN1 antisense coincided with increased caspase-3 activity and reduced protein kinase B/Akt phosphorylation in response to both gemcitabine and taxotere. Gastrin gene circumvention may be an optimal adjunct to chemotherapeutic agents, such as taxotere and gemcitabine, in pancreatic adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Desoxicitidina/análogos & derivados , Gastrinas/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/terapia , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , DNA Antissenso/genética , Desoxicitidina/farmacologia , Docetaxel , Gastrinas/biossíntese , Gastrinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Fosforilação , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Taxoides/farmacologia , Transfecção , GencitabinaRESUMO
There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Inibidores de Histona Desacetilases/uso terapêutico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Padrão de Cuidado , Células Estromais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Biomarcadores , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/cirurgia , Estadiamento de Neoplasias , Fenótipo , Células Estromais/metabolismo , Técnicas de Cultura de Tecidos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
With growing international calls for the enhanced involvement of Indigenous peoples and their biocultural knowledge in managing conservation and the sustainable use of physical environment, it is timely to review the available literature and develop cross-cultural approaches to the management of biocultural resources. Online spatial databases are becoming common tools for educating land managers about Indigenous Biocultural Knowledge (IBK), specifically to raise a broad awareness of issues, identify knowledge gaps and opportunities, and to promote collaboration. Here we describe a novel approach to the application of internet and spatial analysis tools that provide an overview of publically available documented Australian IBK (AIBK) and outline the processes used to develop the online resource. By funding an AIBK working group, the Australian Centre for Ecological Analysis and Synthesis (ACEAS) provided a unique opportunity to bring together cross-cultural, cross-disciplinary and trans-organizational contributors who developed these resources. Without such an intentionally collaborative process, this unique tool would not have been developed. The tool developed through this process is derived from a spatial and temporal literature review, case studies and a compilation of methods, as well as other relevant AIBK papers. The online resource illustrates the depth and breadth of documented IBK and identifies opportunities for further work, partnerships and investment for the benefit of not only Indigenous Australians, but all Australians. The database currently includes links to over 1500 publically available IBK documents, of which 568 are geo-referenced and were mapped. It is anticipated that as awareness of the online resource grows, more documents will be provided through the website to build the database. It is envisaged that this will become a well-used tool, integral to future natural and cultural resource management and maintenance.
Assuntos
Conservação dos Recursos Naturais/métodos , Bases de Dados Factuais , Havaiano Nativo ou Outro Ilhéu do Pacífico , Austrália , HumanosRESUMO
PURPOSE: To produce antitumor monoclonal antibodies (mAbs) targeting glycans as they are aberrantly expressed in tumors and are coaccessory molecules for key survival pathways. EXPERIMENTAL DESIGN: Two mAbs (FG88.2 and FG88.7) recognizing novel tumor-associated Lewis (Le) glycans were produced by immunizations with plasma membrane lipid extracts of the COLO205 cell line. RESULTS: Glycan array analysis showed that both mAbs bound Le(c)Le(x), di-Le(a), and Le(a)Le(x), as well as Le(a)-containing glycans. These glycans are expressed on both lipids and proteins. Both mAbs showed strong tumor reactivity, binding to 71% (147 of 208) of colorectal, 81% (155 of 192) of pancreatic, 54% (52 of 96) of gastric, 23% (62 of 274) of non-small cell lung, and 31% (66 of 217) of ovarian tumor tissue in combination with a restricted normal tissue distribution. In colorectal cancer, high FG88 glyco-epitope expression was significantly associated with poor survival. The mAbs demonstrated excellent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), in addition to direct tumor cell killing via a caspase-independent mechanism. Scanning electron microscopy revealed antibody-induced pore formation. In addition, the mAbs internalized, colocalized with lysosomes, and delivered saporin that killed cells with subnanomolar potency. In vivo, the mAbs demonstrated potent antitumor efficacy in a metastatic colorectal tumor model, leading to significant long-term survival. CONCLUSIONS: The mAbs direct and immune-assisted tumor cell killing, pan-tumor reactivity, and potent in vivo antitumor efficacy indicate their potential as therapeutic agents for the treatment of multiple solid tumors. In addition, internalization of saporin conjugates and associated tumor cell killing suggests their potential as antibody drug carriers.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Animais , Anticorpos Monoclonais Murinos/metabolismo , Antineoplásicos/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Neoplasias Hepáticas Experimentais/secundário , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Polissacarídeos/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The role of the gut microbiome in human health, and non-invasive measurement of gut dysbiosis are of increasing clinical interest. New high-throughput methods are required for the rapid measurement of gut microbial metabolites and to establish reference ranges in human populations. We used ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) -- positive and negative electrospray ionization modes, multiple reaction monitoring transitions -- to simultaneously measure three urinary metabolites (phenylacetylglutamine, 4-cresyl sulphate and hippurate) that are potential biomarkers of gut function, among multi-ethnic US men and women aged 40-59 from the INTERMAP epidemiologic study (n = 2000, two timed 24-hr urine collections/person). Metabolite concentrations were quantified via stable isotope labeled internal standards. The assay was linear in the ranges 1ng/mL (lower limit of quantification) to 1000ng/mL (phenylacetylglutamine and 4-cresyl sulfate) and 3ng/mL to 3000ng/mL (hippurate). These quantitative data provide new urinary reference ranges for population-based human samples: mean (standard deviation) 24-hr urinary excretion for phenylacetylglutamine was: 1283.0 (751.7) µmol/24-hr (men), 1145.9 (635.5) µmol/24-hr (women); for 4-cresyl sulphate, 1002.5 (737.1) µmol/24-hr (men), 1031.8 (687.9) µmol/24-hr (women); for hippurate, 6284.6 (4008.1) µmol/24-hr (men), 4793.0 (3293.3) µmol/24-hr (women). Metabolic profiling by UPLC-MS/MS in a large sample of free-living individuals has provided new data on urinary reference ranges for three urinary microbial co-metabolites, and demonstrates the applicability of this approach to epidemiological investigations.