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BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.
Assuntos
Capsídeo , Dependovirus , Animais , Camundongos , Humanos , Capsídeo/química , Capsídeo/metabolismo , Sorogrupo , Dependovirus/genética , Transdução Genética , Vetores Genéticos , Fígado/metabolismo , Peptídeos/análise , Peptídeos/genética , Peptídeos/metabolismo , Terapia Genética/métodosRESUMO
CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1's pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals (mice, rats, dogs and monkeys) and patients with metastatic pancreatic cancer. To assess tissue disposition, [3H]-CEND-1 radioligand was administered intravenously to mice bearing orthotopic 4T1 mammary carcinoma, followed by tissue measurement using quantitative whole-body autoradiography or quantitative radioactivity analysis. The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratos , Camundongos , Animais , Cães , Infusões Intravenosas , Peptídeos , Microambiente TumoralRESUMO
Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time-of-day dependent changes of proliferation and DNA damage in HCC. Using transgenic c-myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ-H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev-erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ-H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time-of-day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Neoplasias Experimentais/genética , Proteínas Circadianas Period/genética , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Proliferação de Células/genética , Cloretos/administração & dosagem , Cloretos/toxicidade , Cronoterapia , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/terapia , Fotoperíodo , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Crescimento Transformador alfa/genética , Células Tumorais Cultivadas , Compostos de Zinco/administração & dosagem , Compostos de Zinco/toxicidadeRESUMO
BACKGROUND: The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport. METHODS: To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer. FINDINGS: Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer. INTERPRETATION: We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer. FUNDING: Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute.
Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular , Modelos Animais de Doenças , Neoplasias Hepáticas , Diester Fosfórico Hidrolases , Animais , Camundongos , Biomarcadores Tumorais/sangue , Diester Fosfórico Hidrolases/sangue , Diester Fosfórico Hidrolases/metabolismo , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , alfa-Fetoproteínas/metabolismo , Masculino , Humanos , Linhagem Celular Tumoral , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Oligopeptídeos/administração & dosagemRESUMO
Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.
RESUMO
The nuclear factor kappa beta (NFκB) signaling pathway plays an important role in liver homeostasis and cancer development. Tax1-binding protein 1 (Tax1BP1) is a regulator of the NFκB signaling pathway, but its role in the liver and hepatocellular carcinoma (HCC) is presently unknown. Here we investigated the role of Tax1BP1 in liver cells and murine models of HCC and liver fibrosis. We applied the diethylnitrosamine (DEN) model of experimental hepatocarcinogenesis in Tax1BP1+/+ and Tax1BP1-/- mice. The amount and subsets of non-parenchymal liver cells in in Tax1BP1+/+ and Tax1BP1-/- mice were determined and activation of NFκB and stress induced signaling pathways were assessed. Differential expression of mRNA and miRNA was determined. Tax1BP1-/- mice showed increased numbers of inflammatory cells in the liver. Furthermore, a sustained activation of the NFκB signaling pathway was found in hepatocytes as well as increased transcription of proinflammatory cytokines in isolated Kupffer cells from Tax1BP1-/- mice. Several differentially expressed mRNAs and miRNAs in livers of Tax1BP1-/- mice were found, which are regulators of inflammation or are involved in cancer development or progression. Furthermore, Tax1BP1-/- mice developed more HCCs than their Tax1BP1+/+ littermates. We conclude that Tax1BP1 protects from liver cancer development by limiting proinflammatory signaling.
Assuntos
Carcinogênese/metabolismo , Carcinoma Hepatocelular/metabolismo , Hepatite/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/patologia , Citometria de Fluxo , Hepatite/patologia , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
: Today, efficient delivery of sorafenib to hepatocellular carcinoma remains a challenge for current drug formulation strategies. Incorporating the lipophilic molecule into biocompatible and biodegradable theranostic nanocarriers has great potential for improving the efficacy and safety of cancer therapy. In the present study, three different technologies for the encapsulation of sorafenib into poly(d,l-lactide-co-glycolide) and polyethylene glycol-poly(d,l-lactide-co-glycolide) copolymers were compared. The particles ranged in size between 220 and 240 nm, with encapsulation efficiencies from 76.1 ± 1.7% to 69.1 ± 10.1%. A remarkable maximum drug load of approximately 9.0% was achieved. Finally, a gadolinium complex was covalently attached to the nanoparticle surface, transforming the nanospheres into theranostic devices, allowing their localization using magnetic resonance imaging. The manufacture of sorafenib-loaded nanoparticles alongside the functionalization of the particle surface with gadolinium complexes resulted in a highly efficacious nanodelivery system which exhibited a strong magnetic resonance imaging signal, optimal stability features, and a sustained release profile.
RESUMO
Tumor progression largely depends on the presence of alternatively polarized (M2) tumor-associated macrophages (TAMs), whereas the classical M1-polarized macrophages can promote anti-tumorigenic immune responses. Thus, selective inhibition of M2-TAMs is a desirable anti-cancer approach in highly resistant tumor entities such as hepatocellular carcinoma (HCC) or breast cancer. We here examined whether a peptide that selectively binds to and is internalized by in vitro-differentiated murine M2 macrophages as compared to M1 macrophages, termed M2pep, could be used to selectively target TAMs in HCC and breast carcinoma. We confirmed selectivity of M2pep for in vitro M2 polarized macrophages. Upon incubation of suspended mixed 4T1 tumor cells with M2pep, high amounts of the TAMs were found to be associated with M2pep, whereas in mixed tumor cell suspensions from two HCC mouse models, M2pep showed only low-degree binding to TAMs. M2pep also showed low-degree targeting of liver macrophages. This indicates that the TAMs in different tumor entities show different targeting of M2pep and that M2pep is a very promising approach to develop selective M2-TAM-targeting in tumor entities containing M2-TAMs with significant amounts of the so far elusive M2pep receptor(s).
Assuntos
Neoplasias Hepáticas Experimentais/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Genes myc , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Macrófagos/classificação , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Peptídeos/farmacocinética , Ligação Proteica , Fator de Crescimento Transformador alfa/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown.Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial-mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs.Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), HIF2α, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration.Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin Cancer Res; 23(14); 3896-905. ©2017 AACR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Hepatocelular/genética , RNA Helicases DEAD-box/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Ribonuclease III/genética , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Hipóxia Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKCι inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Regulação Alostérica , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos NusRESUMO
MicroRNAs (miRNAs) circulating extracellularly in the blood are currently intensively studied as novel disease markers. However, the preanalytical factors influencing the levels of the extracellular miRNAs are still incompletely explored. In particular, it is unknown, whether the incubation of blood samples as occurring in clinical routine can lead to a release of miRNAs from blood cells and thus alter the extracellular miRNA levels before the preparation of serum or plasma from the blood cells. Using a set of marker miRNAs and quantitative RT-PCR, we found that the levels of extracellular miRNA-1, miRNA-16, and miRNA-21 were increased in EDTA and serum collection tubes incubated for 1-3 hours at room temperature and declined thereafter; the levels of the liver-specific miRNA-122 declined monophasically. These events occurred in the absence of significant hemolysis. When the blood was supplemented with Ribonuclease A inhibitor, the levels of miRNA-1, miRNA-16, and miRNA-21 increased substantially during the initial 3 hours of incubation and those of miRNA-122 remained unchanged, indicating that the release of blood cell-derived miRNAs occurred during the initial 3 hours of incubation of the blood tubes, but not at later time points. Separation of 5-hour preincubated blood into vesicle and nonvesicle fractions revealed a selective increase in the portion of vesicle-associated miRNAs. Together, these data indicate that the release of vesicle-associated miRNAs from blood cells can occur in blood samples within the time elapsing in normal clinical practice until their processing without significant hemolysis. This becomes particularly visible on the inhibition of miRNA degradation by Ribonuclease A inhibitor.
Assuntos
Células Sanguíneas/metabolismo , MicroRNAs/sangue , HumanosRESUMO
Nanoparticle (NP)-based contrast agents that enable high resolution anatomic T1-weighted magnetic resonance imaging (MRI) offer the prospect of improving differential diagnosis of liver tumors such as hepatocellular carcinoma (HCC). In the present study, we investigated the possibility of employing novel non-toxic human serum albumin nanoparticles conjugated with Gd-DTPA and rhodamine 123 (Gd-Rho-HSA-NPs) for the detection of HCC by T1-weighted MRI. In addition, the influence of surface coating of the NPs with poloxamine 908, which alters the absorptive behavior of NPs and changes their distribution between the liver and tumor was examined. MRI of transgenic mice with endogenously formed HCCs following intravenous injection of Gd-Rho-HSA-NPs revealed a strong negative contrast of the tumors. Contrasting of the HCCs by NP-enhanced MRI required less Gd as compared to gadolinium-ethoxybenzyl-diethylenetriaminepentaacetic acid-enhanced MRI, which currently provides the most sensitive detection of HCC in patients. Immunohistochemical analyses revealed that the Gd-Rho-HSA-NPs were localized to macrophages, which were - similar to HCC in patients - fewer in number in HCC as compared to the liver tissue, which is in agreement with the negative contrasting of HCC in Gd-Rho-HSA-NP-enhanced MRI. Poloxamine-coated NPs showed lower accumulation in the tumor macrophages and caused a longer lasting enhancement of the MRI signal. These data indicate that Gd-Rho-HSA-NPs enable sensitive detection of HCC by T1-weighted MRI in mice with endogenous HCC through their uptake by macrophages. Poloxamine coating of the NPs delayed the tumor localization of the NPs.
Assuntos
Carcinoma Hepatocelular/diagnóstico , Meios de Contraste , Gadolínio DTPA , Neoplasias Hepáticas/diagnóstico , Imageamento por Ressonância Magnética/métodos , Rodamina 123 , Albumina Sérica , Animais , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Etilenodiaminas , Excipientes , Genes myc/genética , Humanos , Fígado/patologia , Neoplasias Hepáticas/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Nanopartículas , Tamanho da Partícula , Polietilenoglicóis , Distribuição Tecidual , Fator de Crescimento Transformador alfa/genéticaRESUMO
iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Oligopeptídeos/administração & dosagem , Compostos de Fenilureia/administração & dosagem , Administração Intravenosa , Motivos de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Doxorrubicina/farmacocinética , Sistemas de Liberação de Medicamentos , Azul Evans/administração & dosagem , Gadolínio DTPA , Células Hep G2/efeitos dos fármacos , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos Nus , Camundongos Transgênicos , Niacinamida/administração & dosagem , Oligopeptídeos/química , Sorafenibe , Distribuição TecidualRESUMO
Tumor visualization by magnetic resonance imaging (MRI) and nanoparticle-based contrast agents may improve the imaging of solid tumors such as hepatocellular carcinoma (HCC). In particular, human serum albumin (HSA) nanoparticles appear to be a suitable carrier due to their safety and feasibility of functionalization. In the present study HSA nanoparticles were conjugated with gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) using carbodiimide chemistry. The nanoparticles had a uniform spherical shape and a diameter of 235±19nm. For better optical visualization in vitro and in vivo, the HSA-Gd nanoparticles were additionally labeled with rhodamine 123. As shown by confocal microscopy and flow cytometry analysis, the fluorescent nanoparticles were readily taken up by Huh-7 hepatocellular carcinoma cells. After 24h incubation in blood serum, less than 5% of the Gd(III) was released from the particles, which suggests that this nanoparticulate system may be stable in vivo and, therefore, may serve as potentially safe T1 MRI contrast agent for MRI of hepatocellular carcinoma.
Assuntos
Albuminas/química , Materiais Biocompatíveis/química , Carcinoma Hepatocelular/diagnóstico , Meios de Contraste/química , Gadolínio DTPA/química , Neoplasias Hepáticas/diagnóstico , Imageamento por Ressonância Magnética , Nanopartículas/química , Albuminas/farmacocinética , Animais , Materiais Biocompatíveis/efeitos adversos , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/efeitos adversos , Gadolínio DTPA/farmacocinética , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/diagnóstico , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Nanopartículas/efeitos adversos , Tamanho da Partícula , Albumina Sérica/química , Propriedades de SuperfícieRESUMO
Iron oxide-containing magnetic nanoparticles (MNPs) have certain advantages over currently used contrast agents for tumor imaging by magnetic resonance imaging (MRI) as they offer the possibility of functionalization with ligands and tracers. Functionalized MNPs also may be used for targeted tumor therapy. In the current study nanoparticles (NPs) consisting of recombinant human serum albumin (rHSA) with incorporated hydrophilic (NH4)2Ce(IV)(NO3)6-γ-Fe2O3 particles (CAN maghemite particles) for medical imaging were produced and characterized. For this purpose CAN maghemite particles were incorporated into an rHSA matrix to yield rHSA-NPs. The resulting NPs were analyzed by transmission electron microscopy, photon correlation spectroscopy, and atomic absorption. The sizes of the manufactured NP were 170 ± 10 nm, and the zeta-potential was -50 ± 3 mV. The NPs remained stable when stored after lyophilization with sucrose 3% [w/v] as a cryoprotector. They showed pro-inflammatory properties without cell and animal toxicity in vivo and were highly contrasting in MRI. In conclusion, this report introduces novel rHSA NP with favorable properties containing iron oxide for detection by MRI.
Assuntos
Meios de Contraste , Diagnóstico por Imagem/métodos , Compostos Férricos , Nanopartículas de Magnetita , Albumina Sérica , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/administração & dosagem , Meios de Contraste/toxicidade , Estabilidade de Medicamentos , Eletroquímica , Compostos Férricos/administração & dosagem , Compostos Férricos/química , Compostos Férricos/toxicidade , Humanos , Imageamento por Ressonância Magnética , Magnetismo , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Neoplasias/diagnóstico , Tamanho da Partícula , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/toxicidade , Albumina Sérica/administração & dosagem , Albumina Sérica/toxicidadeRESUMO
BACKGROUND: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. METHODS: The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. RESULTS: The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. CONCLUSIONS: Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.
Assuntos
Biomarcadores Tumorais/sangue , Eritrócitos/metabolismo , MicroRNAs/sangue , MicroRNAs/química , Soro/metabolismo , Inibidores Enzimáticos/farmacologia , Voluntários Saudáveis , Humanos , Estabilidade de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease Pancreático/antagonistas & inibidoresRESUMO
Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.