RESUMO
5-trifluoromethanesulfonyl-uracil (OTfU), a recently proposed radiosensitizer, is decomposed in the gas-phase by attachment of low-energy electrons. OTfU is a derivative of uracil with a triflate (OTf) group at the C5-position, which substantially increases its ability to undergo effective electron-induced dissociation. We report a rich assortment of fragments formed upon dissociative electron attachment (DEA), mostly by simple bond cleavages (e.g., dehydrogenation or formation of OTf-). The most favorable DEA channel corresponds to the formation of the triflate anion alongside with the reactive uracil-5-yl radical through the cleavage of the O-C5 bond, particularly at about 0 eV. Unlike for halouracils, the parent anion was not detected in our experiments. The experimental findings are accounted by a comprehensive theoretical study carried out at the M06-2X/aug-cc-pVTZ level. The latter comprises the thermodynamic thresholds for the formation of the observed anions calculated under the experimental conditions (383.15 K and 3 × 10-11 atm). The energy-resolved ion yield of the dehydrogenated parent anion, (OTfU-H)-, is discussed in terms of vibrational Feshbach resonances arising from the coupling between the dipole bound state and vibrational levels of the transient negative ion. We also report the mass spectrum of the cations obtained through ionization of OTfU by electrons with a kinetic energy of 70 eV. The current study endorses OTfU as a potential radiosensitizer agent with possible applications in radio-chemotherapy.
Assuntos
Elétrons , Radiossensibilizantes/química , Uracila/química , Estrutura Molecular , Termodinâmica , Uracila/metabolismoRESUMO
The TYT and TXT trimeric oligonucleotides, where X stands for a native nucleobase, T (thymine), C (cytosine), A (adenine), or G (guanine), and Y indicates a brominated analogue of the former, were irradiated with ionizing radiation generated by a (60)Co source in aqueous solutions containing Tris as a hydroxyl radical scavenger. In the past, these oligomers were bombarded with low energy electrons under an ultra-high vacuum and significant damage to TXT trimers was observed. However, in aqueous solution, hydrated electrons do not produce serious damage to TXT trimers although the employed radiation dose exceeded many times the doses used in radiotherapy. Thus, our studies demonstrate unequivocally that hydrated electrons, which are the major form of electrons generated during radiotherapy, are a negligible factor in damage to native DNA. It was also demonstrated that all the studied brominated nucleobases have a potential to sensitize DNA under hypoxic conditions. Strand breaks, abasic sites and the products of hydroxyl radical attachment to nucleobases have been identified by HPLC and LC-MS methods. Although all the bromonucleobases lead to DNA damage under the experimental conditions of the present work, bromopyrimidines seem to be the radiosensitizers of choice since they lead to more strand breaks than bromopurines.
Assuntos
Dano ao DNA , DNA/química , Elétrons , Água/química , Conformação de Ácido Nucleico , Solubilidade , SoluçõesRESUMO
The critical role of the vasculature in cancer progression is predominantly studied at the capillary level, and often equated with angiogenesis. However, the mechanisms that ensure the supply of increasing blood volume to the expanding tumor microcirculation remain presently unclear. Here we used established mouse tumor models to document the enlargement (arteriogenesis), of macroscopic feeding vessels at considerable distances upstream from the malignant lesion, but not contralaterally. These changes are not affected by the procoagulant host tissue factor (TF), but are modulated by vascular ageing and atherosclerosis in ApoE-/- mice. Moreover, arteriogenic growth involves infiltration of bone marrow derived (YFP-labeled) cells and changes the gene expression profiles in the vessel wall. Thus, our observations suggest that in addition to local angiogenesis tumors influence distant remodeling of regional macroscopic blood vessels in a manner that is modulated by certain vascular comorbidities.
RESUMO
A low proliferating fraction in solid tumors limits the effectiveness of cell cycle-dependent chemotherapeutic agents. To understand the molecular basis of such "kinetic" resistance we cultured tumor cells as multicellular spheroids and examined levels of p27Kip1, a cyclin-dependent kinase inhibitor known to be upregulated by intercellular contact in normal cells. When transferred from monolayer to three-dimensional culture, a consistent upregulation (up to 15-fold) of p27 protein was observed in a panel of mouse and human carcinoma cell lines. Antisense-oligonucleotide-mediated downregulation of p27 in EMT-6 mammary tumor cell spheroids reduced intercellular adhesion, increased cell proliferation, sensitized tumor cells to 4-hydroperoxycyclophosphamide, and restored drug- or radiation-induced cell-cycle perturbations repressed in spheroid culture. Our results implicate p27 as a regulator of drug resistance in solid tumors and suggest that tumor-targeted p27 antagonists may be useful chemosensitizers in conjunction with conventional anticancer therapy.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Supressoras de Tumor , Animais , Adesão Celular , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Camundongos , Esferoides Celulares/metabolismo , Células Tumorais CultivadasRESUMO
We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.
Assuntos
Cromossomos Humanos Par 6/imunologia , Genes MHC da Classe II , Antígenos HLA-D/genética , Mutagênese , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/química , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular Transformada , Membrana Celular/química , Membrana Celular/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Genes MHC da Classe II/imunologia , Teste de Complementação Genética , Antígenos HLA-D/biossíntese , Antígenos HLA-D/metabolismo , Antígeno HLA-DR3/imunologia , Herpesvirus Humano 4 , Antígenos de Histocompatibilidade Classe II/metabolismo , Homozigoto , Humanos , Fenótipo , Conformação Proteica , RNA Mensageiro/biossíntese , Dodecilsulfato de Sódio , Coloração e RotulagemRESUMO
OBJECTIVE: To identify new IgG autoantibodies in sera from patients with rheumatoid arthritis (RA). METHODS: We tested serum samples from 19 patients with RA with given human leukocyte antigen (HLA)-DR genotypes, from 7 patients with spondylarthropathy, 2 patients with lupus, 4 patients with systemic sclerosis and 10 healthy individuals on 8268 human protein arrays. RESULTS: We identified four antigens (peptidyl arginine deiminase 4 (PAD4), protein kinase Cbeta1 (PKCbeta1), phosphatylinositol 4 phosphate 5 kinase type II gamma (PIP4K2C) and v raf murine sarcoma viral oncogene homologue B1 catalytic domain (BRAF)) that were recognised almost uniquely by sera from patients with RA on protein arrays. Using purified proteins, we confirmed that PAD4 and BRAF are recognised almost uniquely by patients with RA. CONCLUSION: We identified PAD4 and BRAF as RA specific autoantigens.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Imunoglobulina G/imunologia , Análise Serial de Proteínas , Autoanticorpos/imunologia , Western Blotting/métodos , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Hidrolases/sangue , Hidrolases/imunologia , Proteína Quinase C/sangue , Proteína Quinase C/imunologia , Proteína Quinase C beta , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteínas Proto-Oncogênicas B-raf/sangue , Proteínas Proto-Oncogênicas B-raf/imunologiaRESUMO
Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptotic member of the Bcl-2 family. Since exogenous Bak could only partially restore susceptibility to anoikis in the ras-transformed cells, the existence of at least another component of the apoptotic machinery mediating the effect of activated ras on anoikis was suggested. Indeed, here we show that, in nonmalignant rat and human intestinal epithelial cells, detachment from the ECM or disruption of the cytoskeleton results in a significant downregulation of the antiapoptotic effector Bcl-X(L), and that activated H- or K-ras oncogenes completely abrogate this downregulation. In addition, we found that enforced downregulation of Bcl-X(L) in the ras-transformed cells promotes anoikis and significantly inhibits tumorigenicity, indicating that disruption of the adhesion-dependent regulation of Bcl-X(L) is an essential part of the molecular changes associated with transformation by ras. While the ras-induced downregulation of Bak could be reversed by pharmacological inhibition of phosphatidylinositol 3 kinase (PI 3-kinase), the effect of ras on Bcl-X(L) was PI 3-kinase- and mitogen-activated protein kinase (MAP kinase)-independent. We conclude that ras-induced resistance to anoikis in intestinal epithelial cells is mediated by at least two distinct mechanisms: one that triggers downregulation of Bak and another that stabilizes Bcl-X(L) expression in the absence of the ECM.
Assuntos
Apoptose/fisiologia , Transformação Celular Neoplásica , Matriz Extracelular/fisiologia , Genes ras , Mucosa Intestinal/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Adesão Celular , Linhagem Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Transfecção , Proteína bcl-XRESUMO
Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Apoptose/fisiologia , Intestinos/citologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Adesão Celular/fisiologia , Células Epiteliais , Epitélio/fisiologia , Regulação da Expressão Gênica/fisiologia , Mutação/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Ratos , Esferoides Celulares/citologia , Fatores de Tempo , Células Tumorais Cultivadas/citologiaRESUMO
Recent studies have demonstrated the importance of E-cadherin, a homophilic cell-cell adhesion molecule, in contact inhibition of growth of normal epithelial cells. Many tumor cells also maintain strong intercellular adhesion, and are growth-inhibited by cell- cell contact, especially when grown in three-dimensional culture. To determine if E-cadherin could mediate contact-dependent growth inhibition of nonadherent EMT/6 mouse mammary carcinoma cells that lack E-cadherin, we transfected these cells with an exogenous E-cadherin expression vector. E-cadherin expression in EMT/6 cells resulted in tighter adhesion of multicellular spheroids and a reduced proliferative fraction in three-dimensional culture. In addition to increased cell-cell adhesion, E-cadherin expression also resulted in dephosphorylation of the retinoblastoma protein, an increase in the level of the cyclin-dependent kinase inhibitor p27(kip1) and a late reduction in cyclin D1 protein. Tightly adherent spheroids also showed increased levels of p27 bound to the cyclin E-cdk2 complex, and a reduction in cyclin E-cdk2 activity. Exposure to E-cadherin-neutralizing antibodies in three-dimensional culture simultaneously prevented adhesion and stimulated proliferation of E-cadherin transfectants as well as a panel of human colon, breast, and lung carcinoma cell lines that express functional E-cadherin. To test the importance of p27 in E-cadherin-dependent growth inhibition, we engineered E-cadherin-positive cells to express inducible p27. By forcing expression of p27 levels similar to those observed in aggregated cells, the stimulatory effect of E-cadherin-neutralizing antibodies on proliferation could be inhibited. This study demonstrates that E-cadherin, classically described as an invasion suppressor, is also a major growth suppressor, and its ability to inhibit proliferation involves upregulation of the cyclin-dependent kinase inhibitor p27.
Assuntos
Caderinas/fisiologia , Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Supressoras de Tumor , Animais , Caderinas/genética , Caderinas/imunologia , Adesão Celular/fisiologia , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27 , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Testes de Neutralização , Fosforilação , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: 5-methoxy-N,N-dimethyltryptamine (hereinafter referred to as 5-MeO-DMT) is a psychedelic substance found in the secretion from the parotoid glands of the Bufo alvarius toad. Inhalation of vapor from toad secretion containing 5-MeO-DMT has become popular in naturalistic settings as a treatment of mental health problems or as a means for spiritual exploration. However, knowledge of the effects of 5-MeO-DMT in humans is limited. AIMS: The first objective of this study was to assess sub-acute and long-term effects of inhaling vapor from dried toad secretion containing 5-MeO-DMT on affect and cognition. The second objective was to assess whether any changes were associated with the psychedelic experience. METHODS: Assessments at baseline, within 24 h and 4 weeks following intake, were made in 42 individuals who inhaled vapor from dried toad secretion at several European locations. RESULTS: Relative to baseline, ratings of satisfaction with life and convergent thinking significantly increased right after intake and were maintained at follow-up 4 weeks later. Ratings of mindfulness also increased over time and reached statistical significance at 4 weeks. Ratings of depression, anxiety, and stress decreased after the session, and reached significance at 4 weeks. Participants that experienced high levels of ego dissolution or oceanic boundlessness during the session displayed higher ratings of satisfaction with life and lower ratings of depression and stress. CONCLUSION: A single inhalation of vapor from dried toad secretion containing 5-MeO-DMT produces sub-acute and long-term changes in affect and cognition in volunteers. These results warrant exploratory research into therapeutic applications of 5-MeO-DMT.
Assuntos
Alucinógenos/administração & dosagem , Transtornos Mentais/psicologia , Metoxidimetiltriptaminas/administração & dosagem , Atenção Plena/métodos , Satisfação Pessoal , Vaping/psicologia , Administração por Inalação , Adulto , Animais , Bufonidae , Cognição/efeitos dos fármacos , Cognição/fisiologia , Feminino , Seguimentos , Humanos , Masculino , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/epidemiologiaRESUMO
OBJECTIVE: To test whether the presence of RA associated HLA-DRB1*0101, HLA-DRB1*0401 and HLA-DRB1*0404 alleles individually influences anti-cyclic citrullinated peptide antibodies (anti-CCP) production. METHODS: The frequency of anti-CCP antibodies was calculated in the sera of 260 RA patients expressing either two (double dose genotypes SE+/SE+), one (single dose genotypes SE+/SE-) or no RA associated HLA-DR alleles (SE-/SE-). Anti-CCP antibodies titers were also determined. RESULTS: RA associated HLA-DR alleles are not mandatory for production of anti-CCP. We found that 68% of SE-/SE- patients were anti-CCP positive. There was no significant difference in anti-CCP between SE negative patient (SE-/SE-) and patients expressing at least one SE (SE+/SE+ and SE+/SE-) (p=0.140). We observed no statistical difference in anti-CCP between RA patients expressing one or two SE (82% vs. 77%, p=0.577). Among SE+/SE-patients, HLA-DRB1*0404 was associated with anti-CCP with a statistically significant difference compared with SE negative patients (90% anti-CCP positive, p=0.02). HLA-DRB1*0404 was also associated with high titers of anti CCP with a statistically significant difference compared with HLA-DRB1*0401 and HLA-DRB1*0101 patients (p=0.025). CONCLUSIONS: The RA-associated HLA-DRB1*0404 allele was the most strongly associated with the presence of anti-CCP in RA sera. Moreover, HLA-DRB1*0404 patients had higher titers of anti CCP than patients with other RA associated HLA-DR alleles.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Antígenos HLA-DR/genética , Peptídeos Cíclicos/imunologia , Artrite Reumatoide/sangue , Autoanticorpos/genética , Autoanticorpos/imunologia , Estudos de Casos e Controles , Feminino , Antígenos HLA-DR/imunologia , Cadeias HLA-DRB1 , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/imunologiaRESUMO
We have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV-mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV-related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum-induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells. Abbreviations: CryoTEM: cryo-transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC-5i: human brain endothelial cells; MES: mesenchymal cells; MTS - [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMT1: proneural-to-mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting.
RESUMO
Essentials Tumor-bearing mice were employed to follow oncogenic HRAS sequences in plasma, and blood cells. Cancer DNA accumulated in leukocytes above levels detected in exosomes, platelets and plasma. Extracellular vesicles and nucleosomes are required for uptake of tumor DNA by leukocytes. Uptake of tumor-derived extracellular vesicles by leukocytes triggers coagulant phenotype. SUMMARY: Background Tumor-derived extracellular vesicles (EVs) and free nucleosomes (NSs) carry into the circulation a wealth of cancer-specific, bioactive and poorly understood molecular cargoes, including genomic DNA (gDNA). Objective Here we investigated the distribution of extracellular oncogenic gDNA sequences (HRAS and HER2) in the circulation of tumor-bearing mice. Methods and Results Surprisingly, circulating leukocytes (WBCs), especially neutrophils, contained the highest levels of mutant gDNA, which exceeded the amount of this material recovered from soluble fractions of plasma, circulating EVs, platelets, red blood cells (RBCs) and peripheral organs, as quantified by digital droplet PCR (ddPCR). Tumor excision resulted in disappearance of the WBC-associated gDNA signal within 2-9 days, which is in line with the expected half-life of these cells. EVs and nucleosomes were essential for the uptake of tumor-derived extracellular DNA by neutrophil-like cells and impacted their phenotype. Indeed, the exposure of granulocytic HL-60 cells to EVs from HRAS-driven cancer cells resulted in a selective increase in tissue factor (TF) procoagulant activity and interleukin 8 (IL-8) production. The levels of circulating thrombin-antithrombin complexes (TAT) were markedly elevated in mice harboring HRAS-driven xenografts. Conclusions Myeloid cells may represent a hitherto unrecognized reservoir of cancer-derived, EV/NS-associated oncogenic gDNA in the circulation, and a possible novel platform for liquid biopsy in cancer. In addition, uptake of this material alters the phenotype of myeloid cells, induces procoagulant and proinflammatory activity and may contribute to systemic effects associated with cancer.
Assuntos
DNA de Neoplasias/sangue , Vesículas Extracelulares/química , Genes erbB-2 , Genes ras , Células Mieloides/química , Neutrófilos/química , Animais , Antitrombina III , Plaquetas/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica , DNA de Neoplasias/farmacocinética , Exossomos/química , Feminino , Células HL-60 , Xenoenxertos , Humanos , Interleucina-8/biossíntese , Camundongos , Camundongos SCID , Células Mieloides/metabolismo , Transplante de Neoplasias , Neutrófilos/metabolismo , Nucleossomos/química , Peptídeo Hidrolases/sangue , Plasma/química , Ratos , Células THP-1 , Tromboplastina/biossíntese , Carga TumoralRESUMO
Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.
Assuntos
Apoptose/genética , Apoptose/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Genes ras , Mucosa Intestinal/metabolismo , Intestinos/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Animais , Cromonas/farmacologia , Células Clonais , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Humanos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Transfecção , Proteína Killer-Antagonista Homóloga a bcl-2RESUMO
Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Neuroblastoma/tratamento farmacológico , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Vimblastina/uso terapêutico , Animais , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos Fitogênicos/efeitos adversos , Células Cultivadas , Terapia Combinada , Relação Dose-Resposta a Droga , Fluorescência , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neovascularização Patológica , Neuroblastoma/irrigação sanguínea , Neuroblastoma/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Células Tumorais Cultivadas , Vimblastina/efeitos adversosAssuntos
Proteínas de Transporte/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica/fisiopatologia , Animais , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/fisiologia , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Linfocinas/biossíntese , Linfocinas/fisiologia , Camundongos , Camundongos Nus , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The possible role of statins in cancer is controversial. Indeed, among the multiplicity of biological effects ascribed to these widely used cholesterol lowering agents some could, at least in theory, inhibit tumor growth (e.g. by inhibiting Ras oncoproteins), while other actions are inert, or may even stimulate cancer aggressiveness (e.g. through promoting neovascularization). In order to address some of these controversies, we set out to compare the effects of statins on growth of cancer and endothelial cells in vitro, to the impact of these drugs on angiogenesis-dependent expansion of the corresponding tumors in vivo. Water-soluble fluvastatin was used at concentrations (0-800 ng/ml) against human umbilical vein endothelial cells (HUVEC), and several well-characterized cancer cell lines in culture, including: carcinoma (LLC), melanoma (B16F1) and fibrosarcoma driven by mutant H-ras (528ras1). Endpoints were based on 3H-thymidine incorporation assay, cell morphology and tumorigenicity in mice. The growth inhibitory effects of fluvastatin varied among cancer cell lines (LLC>B16F1>528ras1), irrespectively of their mutant H-ras status. Fluvastatin also blocked the action of angiogenic factors on cultured endothelial cells, but was relatively ineffective against highly angiogenic and aggressive tumors both in young mice (6-8 weeks), and in less aggressively growing tumors in aged (80-90 weeks) mice. Thus, antitumor and antiangiogenic activity of fluvastatin in vitro is not recapitulated in vivo. Tumors may display a form of resistance to statins through a mechanism operative only in vivo.
Assuntos
Células Endoteliais/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Indóis/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica , Animais , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fluvastatina , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: De novo or acquired resistance to chemotherapeutic drugs continues to be one of the most important obstacles hindering the successful treatment of cancer patients. Consequently, enhancing the efficacy of conventional chemotherapeutic drugs has become an important research goal. Our previous studies using the mouse EMT-6 mammary carcinoma selected for resistance to various alkylating agents in vivo demonstrated that such acquired drug resistance may be manifested in vitro only in cells growing in a three-dimensional configuration but not in conventional monolayer culture. We also found that this phenomenon, which we refer to as "acquired multicellular resistance," is associated with an increase in intercellular adhesion or compaction of the alkylating agent-resistant cell lines grown as aggregates in three-dimensional culture. PURPOSE: The present study further investigates the impact of three-dimensional architecture on acquired multicellular drug resistance and its influence on cell cycle kinetics, cell cycle arrest, and cell survival. METHODS: To test the hypothesis that an increase in three-dimensional compaction is related to the drug resistance properties of the cells, we did the following: 1) selected clones of the EMT-6 cell line that spontaneously formed tightly or loosely adherent aggregates and assessed their respective drug resistance properties in vitro; 2) assayed tumorigenic potential of the tight and loose clones after exposure to defined concentrations of the activated form of cyclophosphamide, 4-hydroperoxycyclophosphamide (4-HC) in vitro; and 3) treated the tight clones with hyaluronidase, an agent capable of disrupting EMT-6 spheroids, and assayed what effect this treatment had on chemosensitivity. We used fluorescence-activated cell sorter analysis to monitor any potential alterations in cell cycle kinetics. RESULTS: The increase in compaction in three-dimensional culture was sufficient to confer resistance to 4-HC. This increase in intercellular adhesion was also associated with a lower proliferating fraction of tumor cells and with an almost completely diminished ability of the cells to arrest in the G2/M phase of the cell cycle after drug exposure. Furthermore, these changes were detectable only in three-dimensional culture, not in conventional monolayer culture. In conventional monolayer culture, all cell types consistently showed a high level of proliferation and arrested in G2/M after exposure to 4-HC. Moreover, hyaluronidase was able to disrupt intercellular adhesion and chemosensitize tumor cells both in vitro and in vivo in an ascites model. CONCLUSION: Earlier studies have demonstrated that hyaluronidase is able to sensitize tumor cells to various anticancer agents. Our studies now demonstrate that this sensitization can occur by a mechanism independent of increased drug penetration. This mechanism is likely to be related to the "anti-adhesive" effect of hyaluronidase, which overrides cell contact-dependent growth inhibition, recruits cells into the cycling pool, and renders tumor cells more sensitive to cytotoxic agents that preferentially kill rapidly dividing cells. IMPLICATIONS: Other tumor-specific "anti-adhesives" should be explored that can be effective chemosensitizers when used in combination with cell cycle-specific drugs for the treatment of small, solid tumors.
Assuntos
Antineoplásicos/farmacologia , Moléculas de Adesão Celular/metabolismo , Ciclofosfamida/análogos & derivados , Hialuronoglucosaminidase/fisiologia , Neoplasias Mamárias Experimentais/fisiopatologia , Animais , Bromodesoxiuridina/metabolismo , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/fisiologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclofosfamida/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Human melanomas can become progressively resistant to the growth-inhibitory effects of a broad family of structurally diverse cytokines which includes interleukin 6 (IL-6). Uncovering this multicytokine resistance was made possible by the availability of cell lines established from early-stage radial growth phase or vertical growth phase primary melanomas as well as more advanced primary lesions and distant metastases. Because Oncostatin M (OSM) is also a member of the IL-6 family we evaluated the effects of this cytokine on the growth of human melanoma cell lines obtained from different stages of disease progression. The results showed that three different cell lines derived from early-stage melanomas were strongly growth inhibited by OSM, as they are by IL-6. Three cell lines, established from advanced-stage melanomas, were growth inhibited by OSM, but much higher concentrations (in the range of 10-fold) were required to obtain 50% growth inhibition; these cell lines were not inhibited by IL-6. Three other cell lines that were IL-6 resistant (two of which were advanced stage) were also found to be OSM resistant. Only one advanced-stage IL-6-resistant cell line was found to be highly sensitive to OSM-mediated growth inhibition. In addition, we found that variants isolated from early-stage WM35 melanoma cells that possess a much more aggressive tumorigenic phenotype in nude mice were significantly more resistant to both OSM- and IL-6-mediated growth inhibition. The results demonstrate that OSM can function as a growth inhibitor of human melanoma cells but that its ability to do so is progressively diminished or lost with disease progression. This finding is consistent with the concept of acquired "multicytokine resistance" during melanoma progression.
Assuntos
Melanoma/patologia , Peptídeos/farmacologia , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-6/farmacologia , Fator Inibidor de Leucemia , Linfocinas/farmacologia , Estadiamento de Neoplasias , Oncostatina M , Células Tumorais CultivadasRESUMO
The growth of solid tumors in vivo beyond 1-2 mm in diameter requires induction and maintenance of an angiogenic response. This can occur through the release of various angiogenic growth factors from tumor cells. One such factor is vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), a secreted and specific mitogen for vascular endothelial cells. We show that one of the most commonly encountered genetic changes detected in human cancer, i.e., expression of mutant ras oncogenes, is associated with marked up-regulation of VEGF/VPF in transformed epithelial cells. Thus, elevation of the levels of both VEGF/VPF mRNA and secreted functional protein were detected in human and rodent tumor cell lines expressing mutant K-ras or H-ras oncogenes, respectively. Genetic disruption of the mutant K-ras allele in human colon carcinoma cells was associated with a reduction in VEGF/VPF activity. Furthermore, pharmacological disruption of mutant RAS protein function in H-ras transformed rat intestinal epithelial cells by treatment with L-739,749 (a protein farnesyltransferase inhibitor) caused a significant suppression of VEGF/VPF. The results suggest that dominantly acting ras oncogenes may contribute to the growth of solid tumors in vivo not only by a direct effect on tumor cell proliferation but also indirectly, i.e., by facilitating tumor angiogenesis. Hence, pharmacologically targeting mutant ras oncogenes could conceivably suppress solid tumor growth in vivo, in part, by inhibiting tumor-induced angiogenesis.