RESUMO
Tumor-associated macrophages (TAMs) are integral components of the tumor microenvironment. They are involved in various aspects of tumor cell biology, driving pathological processes such as tumor cell proliferation, metastasis, immunosuppression, and resistance to therapy. TAMs exert their tumorigenic effects by secreting growth factors, cytokines/chemokines, metabolites, and other soluble bioactive molecules. These mediators directly promote tumor cell proliferation and modulate interactions with immune and stromal cells, facilitating further tumor growth. As research into therapies targeting TAMs intensifies, there is a growing need for reliable methods to comprehend the impact of TAMs on cancer progression and to validate novel therapeutics directed at TAMs. The traditional "M1-M2" macrophage classification based on transcriptional profiles of TAMs is not only too simplistic to describe their physiological roles, it also does not explain differences observed between mouse and human macrophages. In this context, methods that assess how TAMs influence tumor or immune cells, either through direct contact or the release of soluble factors, offer a more promising approach. We describe here comprehensive protocols for in vitro functional assays to study TAMs, specifically regarding their impact on the growth of lung cancer cells. We have applied these methods to both mouse and human macrophages, achieving similar outcomes in promoting the proliferation of cancer cells. This methodology can serve as a standardized approach for testing novel therapeutic approaches, targeting TAMs with novel immunotherapeutic compounds, or utilizing gene-editing techniques. Taken together, the described methodology may contribute to our understanding of complex macrophage-tumor interactions and support the development of innovative therapeutic strategies.
Assuntos
Microambiente Tumoral , Macrófagos Associados a Tumor , Humanos , Animais , Camundongos , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Proliferação de Células , Macrófagos/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Linhagem Celular Tumoral , Neoplasias/patologia , Neoplasias/metabolismoRESUMO
OBJECTIVE: Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN: We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS: We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION: In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.
Assuntos
Carcinoma Ductal Pancreático , Quinases Dyrk , Macrófagos , Neoplasias Pancreáticas , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Microambiente Tumoral , Animais , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Camundongos , Humanos , Macrófagos/metabolismo , Modelos Animais de Doenças , Linhagem Celular Tumoral , FagocitoseRESUMO
Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV-focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.
Assuntos
Autofagia/fisiologia , Matriz Extracelular/patologia , Animais , Linhagem Celular , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Adesões Focais/metabolismo , Adesões Focais/patologia , Integrina alfaV/metabolismo , Camundongos , Células NIH 3T3 , Neoplasias/metabolismo , Neoplasias/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Células Estromais/metabolismoRESUMO
Expansion of neoplastic lesions generates the initial signal that instigates the creation of a tumor niche. Nontransformed cell types within the microenvironment continuously coevolve with tumor cells to promote tumorigenesis. Here, we identify p38MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. We found that lung cancer growth depends on short-distance cues produced by the cancer niche in a p38-dependent manner. We identified fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to carcinoma-associated fibroblasts (CAFs), and cancer cell proliferation. Systemic down-regulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in fibroblasts has a significant tumor-suppressive effect on K-ras lung tumorigenesis. Furthermore, both Kras-driven mouse lung tumors and orthotopically grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-the-counter inhibitor of hyaluronan synthesis. We propose that p38MAPK-hyaluronan-dependent reprogramming of the tumor microenvironment plays a critical role in driving lung tumorigenesis, while blocking this process could have far-reaching therapeutic implications.
Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Ácido Hialurônico/metabolismo , Neoplasias Pulmonares/fisiopatologia , Microambiente Tumoral/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antineoplásicos/farmacologia , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Reprogramação Celular/genética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Fibroblastos , Regulação Neoplásica da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The metabolism of glioma cells exhibits significant heterogeneity and is partially responsible for treatment outcomes. Given this variability, we hypothesized that the effectiveness of treatments targeting various metabolic pathways depends on the bioenergetic profiles and mitochondrial status of glioma cells. To this end, we analyzed mitochondrial biomass, mitochondrial protein density, oxidative phosphorylation (OXPHOS), and glycolysis in a panel of eight glioma cell lines. Our findings revealed considerable variability: mitochondrial biomass varied by up to 3.2-fold, the density of mitochondrial proteins by up to 2.1-fold, and OXPHOS levels by up to 7.3-fold across the cell lines. Subsequently, we stratified glioma cell lines based on their mitochondrial status, OXPHOS, and bioenergetic fitness. Following this stratification, we utilized 16 compounds targeting key bioenergetic, mitochondrial, and related pathways to analyze the associations between induced changes in cell numbers, proliferation, and apoptosis with respect to their steady-state mitochondrial and bioenergetic metrics. Remarkably, a significant fraction of the treatments showed strong correlations with mitochondrial biomass and the density of mitochondrial proteins, suggesting that mitochondrial status may reflect glioma cell sensitivity to specific treatments. Overall, our results indicate that mitochondrial status and bioenergetics are linked to the efficacy of treatments targeting metabolic pathways in glioma.
Assuntos
Biomassa , Metabolismo Energético , Glioma , Mitocôndrias , Proteínas Mitocondriais , Fosforilação Oxidativa , Glioma/metabolismo , Glioma/patologia , Humanos , Linhagem Celular Tumoral , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proliferação de Células , Glicólise , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/tratamento farmacológico , ApoptoseRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Linfócitos T CD8-Positivos , Fibroblastos Associados a Câncer/metabolismo , Interleucina-17/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Proteínas de Homeodomínio , Neoplasias PancreáticasRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor in children. Transcriptional profiling has so far delineated four major MB subgroups of which one is driven by uncontrolled Hedgehog (Hh) signaling (SHH-MB). This pathway is amenable to drug targeting, yet clinically approved compounds exclusively target the transmembrane component Smoothened (SMO). Unfortunately, drug resistance against SMO inhibitors is encountered frequently, making the identification of novel Hh pathway components mandatory, which could serve as novel drug targets in the future. Here, we have used MB as a tool to delineate novel modulators of Hh signaling and have identified the Acidic Nuclear Phosphoprotein 32 (ANP32) family of proteins as novel regulators. The expression of all three family members (ANP32A, ANP32B, ANP32E) is increased in Hh-induced MB and their expression level is negatively associated with overall survival in SHH-MB patients. Mechanistically, we could find that ANP32 proteins function as positive modulators of mammalian Hh signaling upstream of GLI transcription factors. These findings add hitherto unknown regulators to the mammalian Hh signaling cascade and might spur future translational efforts to combat Hh-driven malignancies.
Assuntos
Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Fibroblastos , Humanos , Meduloblastoma/genética , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Neuroblastoma (NB), a pediatric cancer of the peripheral sympathetic nervous system, represents the most frequent solid malignancy in infants. Treatment of high-risk patients is still challenging and, depending on the genetic make-up and involved risk factors, the 5-year survival rate can drop to only 30%. Here, we found that the expression of the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 3 (DYRK3) is increased in NB and is associated with decreased survival in NB patients. We further identified DYRK3 as a cytoplasmic kinase in NB cells and found that its levels are increased by hypoxic conditions. Further mechanistic studies revealed that DYRK3 acts as a negative regulator of HIF-driven transcriptional responses, suggesting that it functions in a negative feedback loop controlling the hypoxic response. Moreover, DYRK3 negatively impacted on NB cell differentiation, proposing an oncogenic role of this kinase in the etiology of NB. In summary, we describe novel functions of the DYRK3 kinase in NB, which will help to further improve the understanding of this disease eventually leading to the design of improved therapeutic concepts.
Assuntos
Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/análise , Proteínas Tirosina Quinases/análise , Hipóxia TumoralRESUMO
Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.
Assuntos
Carcinoma Ductal Pancreático , Progressão da Doença , Vesículas Extracelulares , Interleucina-33 , Mastócitos , Neoplasias Pancreáticas , Microambiente Tumoral , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Proliferação de Células , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Interleucina-33/genética , Mastócitos/metabolismo , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/imunologiaRESUMO
Pancreatic cancer represents one of the most desmoplastic malignancies and is characterized by an extensive deposition of extracellular matrix. The latter is provided by activated cancer-associated fibroblasts (CAFs), which are abundant cells in the pancreatic tumor microenvironment. Many recent studies have made it clear that CAFs are not a singular cellular entity but represent a multitude of potentially dynamic subgroups that affect tumor biology at several levels. As mentioned before, CAFs significantly contribute to the fibrotic reaction and the biomechanical properties of the tumor, but they can also modulate the local immune environment and the response to targeted, chemo or radiotherapy. As the number of known and emerging CAF subgroups is steadily increasing, it is becoming increasingly difficult to keep up with these developments and to clearly discriminate the cellular subsets identified so far. This review aims to provide a helpful overview that enables readers to quickly familiarize themselves with field of CAF heterogeneity and to grasp the phenotypic, functional and therapeutic distinctions of the various stromal subpopulations.
RESUMO
Transcriptional cancer subtypes which correlate with traits such as tumor growth, drug sensitivity or the chances of relapse and metastasis, have been described for several malignancies. The core regulatory circuits (CRCs) defining these subtypes are established by chromatin super enhancers (SEs) driving key transcription factors (TFs) specific for the particular cell state. In neuroblastoma (NB), one of the most frequent solid pediatric cancer entities, two major SE-directed molecular subtypes have been described: A more lineage-committed adrenergic (ADRN) and a mesenchymal (MES) subtype. Here, we found that a small isoxazole molecule (ISX), a frequently used pro-neural drug, reprogrammed SE activity and switched NB cells from an ADRN subtype towards a growth-retarded MES-like state. The MES-like state shared strong transcriptional overlap with ganglioneuroma (GN), a benign and highly differentiated tumor of the neural crest. Mechanistically, ISX suppressed chromatin binding of N-MYC, a CRC-amplifying transcription factor, resulting in loss of key ADRN subtype-enriched components such as N-MYC itself, PHOX2B and ALK, while concomitently, MES subtype markers were induced. Globally, ISX treatment installed a chromatin accessibility landscape typically associated with low risk NB. In summary, we provide evidence that CRCs and cancer subtype reprogramming might be amenable to future therapeutic targeting.
RESUMO
The abnormal tumor microenvironment (TME) often dictates the therapeutic response of cancer to chemo- and immuno-therapy. Aberrant expression of pericentromeric satellite repeats has been reported for epithelial cancers, including lung cancer. However, the transcription of tandemly repetitive elements in stromal cells of the TME has been unappreciated, limiting the optimal use of satellite transcripts as biomarkers or anti-cancer targets. We found that transcription of pericentromeric satellite DNA (satDNA) in mouse and human lung adenocarcinoma was observed in cancer-associated fibroblasts (CAFs). In vivo, lung fibroblasts expressed pericentromeric satellite repeats HS2/HS3 specifically in tumors. In vitro, transcription of satDNA was induced in lung fibroblasts in response to TGFß, IL1α, matrix stiffness, direct contact with tumor cells and treatment with chemotherapeutic drugs. Single-cell transcriptome analysis of human lung adenocarcinoma confirmed that CAFs were the cell type with the highest number of satellite transcripts. Human HS2/HS3 pericentromeric transcripts were detected in the nucleus, cytoplasm, extracellularly and co-localized with extracellular vesicles in situ in human biopsies and activated fibroblasts in vitro. The transcripts were transmitted into recipient cells and entered their nuclei. Knock-down of satellite transcripts in human lung fibroblasts attenuated cellular senescence and blocked the formation of an inflammatory CAFs phenotype which resulted in the inhibition of their pro-tumorigenic functions. In sum, our data suggest that satellite long non-coding (lnc) RNAs are induced in CAFs, regulate expression of inflammatory genes and can be secreted from the cells, which potentially might present a new element of cell-cell communication in the TME.
Assuntos
Adenocarcinoma , Fibroblastos Associados a Câncer , Neoplasias Pulmonares , RNA Longo não Codificante , Humanos , Animais , Camundongos , Fibroblastos Associados a Câncer/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fibroblastos/metabolismo , DNA Satélite , Neoplasias Pulmonares/patologia , Adenocarcinoma/genética , Pulmão , Carcinogênese/genética , Microambiente Tumoral/genéticaRESUMO
Although being rare in absolute numbers, neuroblastoma (NB) represents the most frequent solid tumor in infants and young children. Therapy options and prognosis are comparably good for NB patients except for the high risk stage 4 class. Particularly in adolescent patients with certain genetic alterations, 5-year survival rates can drop below 30%, necessitating the development of novel therapy approaches. The developmentally important Hedgehog (Hh) pathway is involved in neural crest differentiation, the cell type being causal in the etiology of NB. However, and in contrast to its function in some other cancer types, Hedgehog signaling and its transcription factor GLI1 exert tumor-suppressive functions in NB, rendering GLI1 an interesting new candidate for anti-NB therapy. Unfortunately, the therapeutic concept of pharmacological Hh/GLI1 pathway activation is difficult to implement as NB cells have lost primary cilia, essential organelles for Hh perception and activation. In order to bypass this bottleneck, we have identified a GLI1-activating small molecule which stimulates endogenous GLI1 production without the need for upstream Hh pathway elements such as Smoothened or primary cilia. This isoxazole compound potently abrogates NB cell proliferation and might serve as a starting point for the development of a novel class of NB-suppressive molecules.
RESUMO
Formulated forms of cancer therapeutics enhance the efficacy of treatment by more precise targeting, increased bioavailability of drugs, and an aptitude of some delivery systems to overcome multiple drug resistance of tumors. Drug carriers acquire importance for anti-cancer interventions via targeting tumor-associated macrophages with active molecules capable to either eliminate them or change their polarity. Although several packaged drug forms have reached the market, there is still a high demand for novel carrier systems to hurdle limitations of existing drugs on active molecules, toxicity, bioeffect, and stability. Here, we report a facile assembly and delivery methodology for biodegradable polymeric multilayer capsules (PMC) with the purpose of further use in injectable drug formulations for lung cancer therapy via direct erosion of tumors and suppression of the tumor-promoting function of macrophages in the tumor microenvironment. We demonstrate delivery of low-molecular-weight drug molecules to lung cancer cells and macrophages and provide details on in vivo distribution, cellular uptake, and disintegration of the developed PMC. Poly-l-arginine and dextran sulfate alternately adsorb on a â¼500 nm CaCO3 sacrificial template followed by removal of the inorganic core to obtain hollow capsules for consequent loading with drug molecules, gemcitabine or clodronate. The capsules further compacted upon loading down to â¼250 nm in diameter via heat treatment. A comparative study of the capsule internalization rate in vitro and in vivo reveals the benefits of a diminished carrier size. We show that macrophages and epithelial cells of the lungs and liver internalize capsules with efficacy higher than 75%. Using an in vivo mouse model of lung cancer, we also confirm that tumor lungs better retain smaller capsules than the healthy lung tissue. The pronounced cytotoxic effect of the encapsulated gemcitabine on lung cancer cells and the ability of the encapsulated clodronate to block the tumor-promoting function of macrophages prove the efficacy of the developed capsule loading method in vitro. Our study taken as a whole demonstrates the great potential of the developed PMC for in vivo treatment of cancer via transporting active molecules, including those that are water-soluble with low molecular weight, to both cancer cells and macrophages through the bloodstream.
Assuntos
Antineoplásicos , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Pulmonares/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Cápsulas , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacocinética , Desoxicitidina/farmacologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polímeros/química , Polímeros/metabolismo , Distribuição Tecidual , GencitabinaRESUMO
With the purpose to replace expensive and significantly cytotoxic positively charged polypeptides in biodegradable capsules formed via Layer-by-Layer (LbL) assembly, multilayers of bovine serum albumin (BSA) and tannic acid (TA) are obtained and employed for encapsulation and release of model drugs with different solubility in water: hydrophilic-tetramethylrhodamine-isothiocyanate-labeled BSA (TRITC-BSA) and hydrophobic 3,4,9,10-tetra-(hectoxy-carbonyl)-perylene (THCP). Hydrogen bonding is proposed to be predominant within thus formed BSA/TA films. The TRITC-BSA-loaded capsules comprising 6 bilayers of the protein and polyphenol are benchmarked against the shells composed of dextran sulfate (DS) and poly-l-arginine (PARG) on degradability by two proteolytic enzymes with different cleavage site specificity (i.e., α-chymotrypsin and trypsin) and toxicity for murine RAW264.7 macrophage cells. Capsules of both types possess low cytotoxicity taken at concentrations equal or below 50 capsules per cell, and evident susceptibility to α-chymotrypsin resulted in release of TRITC-BSA. While the BSA/TA-based capsules clearly display resistance to treatment with trypsin, the assemblies of DS/PARG extensively degrade. Successful encapsulation of THCP in the TRITC-BSA/TA/BSA multilayer is confirmed, and the release of the model drug is observed in response to treatment with α-chymotrypsin. The thickness, surface morphology, and enzyme-catalyzed degradation process of the BSA/TA-based films are investigated on a planar multilayer comprising 40 bilayers of the protein and polyphenol deposited on a silicon wafer. The developed BSA/TA-based capsules with a protease-specific degradation mechanism are proposed to find applications in personal care, pharmacology, and the development of drug delivery systems including those intravenous injectable and having site-specific release capability.
Assuntos
Preparações de Ação Retardada , Sistemas de Liberação de Medicamentos , Soroalbumina Bovina/administração & dosagem , Taninos/administração & dosagem , Animais , Arginina/química , Plásticos Biodegradáveis/química , Plásticos Biodegradáveis/farmacologia , Cápsulas/administração & dosagem , Cápsulas/química , Bovinos , Quimotripsina/administração & dosagem , Humanos , Ligação de Hidrogênio , Camundongos , Soroalbumina Bovina/química , Taninos/químicaRESUMO
The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Transformação Celular Viral/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Animais , Linhagem Celular , Ativação Enzimática/fisiologia , Receptor alfa de Estrogênio/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Células NIH 3T3 , Proteínas de Fusão Oncogênica/metabolismo , RNA Interferente Pequeno , TransfecçãoAssuntos
Neoplasias/enzimologia , Neoplasias/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antineoplásicos/farmacologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Inflamação , Pulmão/metabolismo , Camundongos , Ratos-Toupeira , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/citologia , Ratos , Microambiente TumoralRESUMO
Atherosclerosis commonly causes coronary and cerebrovascular diseases, which are major morbidities worldwide. Controlling these conditions remains a challenge owing to an incomplete understanding of underlying molecular mechanisms. We have recently shown that PPM1D/WIP1 phosphatase plays a crucial role in regulating atherosclerosis in mice. Deletion of Ppm1d results in the suppression of lipid droplet accumulation in macrophages, which prevents the formation of foam cells, and ultimately the development of atherosclerotic plaques. This process is controlled by the ATM-MTOR pathway and depends on the activation of selective autophagy to regulate cholesterol efflux from macrophage foam cells. Our data suggest that modulating autophagy through the PPM1D-ATM-MTOR pathway may be beneficial at both early and advanced stages of atherosclerosis.