RESUMO
Predicting a response of osteosarcoma patients to chemotherapy, such as doxorubicin or high-dose methotrexate cocktail, remains a challenge in the clinic. Moreover, the prognostic value of currently used necrosis analysis is debatable. New markers of the therapeutic response or the prognostic response are urgently needed. The microenvironment plays a key role in the vascularization of highly heterogeneous tumors. Using the syngeneic MOS-J mouse model of osteosarcoma, we focused our study on the immunohistochemistry of tumor vascularization in order to identify new vessel markers, and to search for potential markers of the therapeutic response. Endomucin+, CD31+, and α-SMA+-positive elements were quantified in control (n=6) and doxorubicin-treated (n=6) mice in three different intra-tumor locations. We also used co-labeling to assess CD31+/Endomucin+ and CD31+/α-SMA+ co-expression. We identified a central tumor zone with a low vascularization profile for all of these markers. We identified two distinct types of vessels: CD31+/Endomucin+ vessels with a sprouting, neo-angiogenic, interlaced appearance, and CD31+/α-SMA+ vessel with a well-defined, mature structure. Doxorubicin appeared to reduce CD31+ expression in the tumor invasion front. In the doxorubicin-sensitive model, there were four times more CD31+/α-SMA+ elements than in the poorly responsive model. Therefore, we propose a methodology based on immunohistochemistry and multiplexed immunofluorescence to use endomucin as a promising new vascular marker in the osteosarcoma model. Moreover, our results suggest that CD31+/α-SMA+ vessels could be considered to be indicators of vasculature normalization and they may be used as specific markers of a good therapeutic response.
RESUMO
Tumor microenvironment plays an important role in melanoma progression. Recent studies reported endothelial cells (EC) are involved in endothelial-to-mesenchymal transition (EndMT). During this phenotypic switch, EC progressively lose their endothelial markers and acquire mesenchymal properties. Depending on their concentration, reactive oxygen species (ROS) can control tumor growth. In EC, ROS are mainly produced by NAPDH oxidases (NOX) such as NOX1 and NOX2. The aim of the present study was to determine the role of these enzymes in EndMT induced by conditioned media (CM) from SK-MEL 28 melanoma cells. The capacity of CM to induce EndMT in HUVEC after 24 h, 48 h or 72 h has been evaluated by following endothelial HUVECs proliferation, migration and their capacity to form capillary on ECMgel®. Furthermore, EndMT was confirmed by western blot and flow cytometry. To determine the role of NOX in EndMT, specific NOX1 and/or NOX2 inhibitors has been tested. TGF-ß2 + /- IL-1ß was used as positive control. ROS production was determined through DCFDA assay. An altered endothelial phenotype was found in CM-treated HUVECs. This phenotypic modification was correlated with a decrease in both capillary formation on ECMgel® and cell proliferation and an increase in cell migration. Exposure to CM for 48 h significantly enhanced intracellular HUVECs ROS production and this increase was prevented by the dual pharmacological inhibition of NOX1 and NOX2. Furthermore, inhibition of NOX1/2 also leads to a partial reversion of CM-induced EndMT. These data confirmed the role of NOX1 and NOX2 in EndMT induced by melanoma cancer cell secretome.
Assuntos
Células Endoteliais , Melanoma , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Transição Epitelial-Mesenquimal , Humanos , Espécies Reativas de Oxigênio , Microambiente TumoralRESUMO
Knowledge about the hematopoietic niche has evolved considerably in recent years, in particular through in vitro analyzes, mouse models and the use of xenografts. Its complexity in the human bone marrow, in particular in a context of hematological malignancy, is more difficult to decipher by these strategies and could benefit from the knowledge acquired on the niches of solid tumors. Indeed, some common features can be suspected, since the bone marrow is a frequent site of solid tumor metastases. Recent research on solid tumors has provided very interesting information on the interactions between tumoral cells and their microenvironment, composed notably of mesenchymal, endothelial and immune cells. This review thus focuses on recent discoveries on tumor niches that could help in understanding hematopoietic niches, with special attention to 4 particular points: i) the heterogeneity of carcinoma/cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs), ii) niche cytokines and chemokines, iii) the energy/oxidative metabolism and communication, especially mitochondrial transfer, and iv) the vascular niche through angiogenesis and endothelial plasticity. This review highlights actors and/or pathways of the microenvironment broadly involved in cancer processes. This opens avenues for innovative therapeutic opportunities targeting not only cancer stem cells but also their regulatory tumor niche(s), in order to improve current antitumor therapies.
Assuntos
Microambiente Tumoral , Animais , Fibroblastos Associados a Câncer , Comunicação Celular , Citocinas , Endotélio Vascular , Humanos , Células-Tronco Mesenquimais , Neoplasias , Neovascularização PatológicaRESUMO
BACKGROUND: The poor survival rate of patients with osteosarcoma (OS), specifically with metastases at diagnosis, undergoes the urgency to develop new therapeutic strategies. Although we recently demonstrated the key role of YAP/TEAD signaling in the growth of OS primary tumor, the molecular mechanisms by which YAP regulates metastases development remain poorly understood. METHODS: The molecular mechanisms by which YAP regulates metastases development were studied using an overexpression of mutated forms of YAP able or not able to interact with TEAD. Molecular signatures were identified using RNA-sequencing analysis and gene set enrichment. Interactions between YAP and Smad3 were studied using proximity ligation assay (PLA), immunoprecipitation, and promoter/specific gene assays. The involvement of the TGF-ß pathway in the ability of YAP to stimulate metastatic development in vivo was studied using an inhibitor of the TGF-ß cascade in a preclinical model of OS and in vitro on the ability of OS cells to migrate and invade. RESULTS: Our work shows that a high YAP expression is associated with the presence of lung metastases which predicts a poor prognosis. Molecular analysis indicates that TGF-ß signaling is involved in YAP-driven osteosarcoma cell pro-migratory phenotype, epithelial mesenchymal transition, cell migration, and in vivo lung metastasis development. Regardless of its ability to bind to TEAD, YAP interacts with Smad3 and stimulates the transcriptional activity of TGF-ß/Smad3, thereby enhancing the ability of TGF-ß to stimulate lung metastasis development. CONCLUSIONS: We demonstrated the crucial involvement of the TGF-ß/Smad3 signaling pathway in YAP-driven lung metastasis development in OS.
RESUMO
Osteosarcoma (OS) and Ewing's sarcoma (ES) are the most common malignant bone tumors in children and adolescents. In many cases, the prognosis remains very poor. The Sonic hedgehog (SHH) signaling pathway, strongly involved in the development of many cancers, regulate transcription via the transcriptional factors Gli1-3. In this context, RNAseq analysis of OS and ES cell lines reveals an increase of some major compounds of the SHH signaling cascade in ES cells, such as the transcriptional factor Gli1. This increase leads to an augmentation of the transcriptional response of Gli1 in ES cell lines, demonstrating a dysregulation of Gli1 signaling in ES cells and thus the rationale for targeting Gli1 in ES. The use of a preclinical model of ES demonstrates that GANT61, an inhibitor of the transcriptional factor Gli1, reduces ES primary tumor growth. In vitro experiments show that GANT61 decreases the viability of ES cell, mainly through its ability to induce caspase-3/7-dependent cell apoptosis. Taken together, these results demonstrates that GANT61 may be a promising therapeutic strategy for inhibiting the progression of primary ES tumors.
RESUMO
Cancer is one of the most important causes of morbidity and mortality worldwide. Tumor cells grow in a complex microenvironment constituted of immune, stromal, and vascular cells that supports growth, angiogenesis, and metastasis. Endothelial cells (ECs) are major components of the vascular microenvironment. These cells have been described for their plasticity and potential to transdifferentiate into mesenchymal cells through a process known as endothelial-to-mesenchymal transition (EndMT). This complex process is controlled by various factors, by which ECs convert into a phenotype characterized by mesenchymal protein expression and motile, contractile morphology. Initially described in normal heart development, EndMT is now identified in several pathologies, and especially in cancer. In this review, we highlight the process of EndMT in the context of cancer and we discuss it as an important adaptive process of the tumor microenvironment that favors tumor growth and dissemination but also resistance to treatment. Thus, we underline targeting of EndMT as a potential therapeutic strategy.
RESUMO
Osteosarcomas are the most frequent primary bone sarcomas, affecting mainly children, adolescents, and young adults, and with a second peak of incidence in elderly individuals. The current therapeutic management, a combined regimen of poly-chemotherapy and surgery, still remains largely insufficient, as patient survival has not improved in recent decades. Osteosarcomas are very heterogeneous tumors, both at the intra- and inter-tumor level, with no identified driver mutation. Consequently, efforts to improve treatments using targeted therapies have faced this lack of specific osteosarcoma targets. Nevertheless, these tumors are inextricably linked to their local microenvironment, composed of bone, stromal, vascular and immune cells and the osteosarcoma microenvironment is now considered to be essential and supportive for growth and dissemination. This review describes the different actors of the osteosarcoma microenvironment and gives an overview of the past, current, and future strategies of therapy targeting this complex ecosystem, with a focus on the role of extracellular vesicles and on the emergence of multi-kinase inhibitors.
Assuntos
Terapia de Alvo Molecular , Osteossarcoma/patologia , Osteossarcoma/terapia , Microambiente Tumoral , Animais , Remodelação Óssea , Humanos , Sistema Imunitário/patologia , Células-Tronco Mesenquimais/patologia , Osteossarcoma/imunologia , Osteossarcoma/fisiopatologiaRESUMO
Ceramide-induced endothelial cell apoptosis boosts intestinal stem cell radiosensitivity. However, the molecular connection between these two cellular compartments has not been clearly elucidated. Here we report that ceramide and its related enzyme acid sphingomyelinase (ASM) are secreted by irradiated endothelial cells and act as bystander factors to enhance the radiotoxicity of intestinal epithelium. Ceramide and the two isoforms of ASM were acutely secreted in the blood serum of wild-type mice after 15 Gy radiation dose, inducing a gastrointestinal syndrome. Interestingly, serum ceramide was not enhanced in irradiated ASMKO mice, which are unable to develop intestinal failure injury. Because ASM/ceramide were secreted by primary endothelial cells, their contribution was studied in intestinal epithelium dysfunction using coculture of primary endothelial cells and intestinal T84 cells. Adding exogenous ASM or ceramide enhanced epithelial cell growth arrest and death. Conversely, blocking their secretion by endothelial cells using genetic, pharmacologic, or immunologic approaches abolished intestinal T84 cell radiosensitivity. Use of enteroid models revealed ASM and ceramide-mediated deleterious mode-of-action: when ceramide reduced the number of intestinal crypt-forming enteroids without affecting their structure, ASM induced a significant decrease of enteroid growth without affecting their number. Identification of specific and different roles for ceramide and ASM secreted by irradiated endothelial cells opens new perspectives in the understanding of intestinal epithelial dysfunction after radiation and defines a new class of potential therapeutic radiomitigators. SIGNIFICANCE: This study identifies secreted ASM and ceramide as paracrine factors enhancing intestinal epithelial dysfunction, revealing a previously unknown class of mediators of radiosensitivity.
Assuntos
Ceramidas/metabolismo , Células Endoteliais/metabolismo , Mucosa Intestinal/patologia , Lesões por Radiação/patologia , Esfingomielina Fosfodiesterase/metabolismo , Animais , Efeito Espectador/efeitos da radiação , Células Cultivadas , Ceramidas/sangue , Técnicas de Cocultura , Desipramina/farmacologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/efeitos da radiação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Masculino , Camundongos , Camundongos Knockout , Comunicação Parácrina/genética , Comunicação Parácrina/efeitos da radiação , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Lesões por Radiação/sangue , Lesões por Radiação/etiologia , Lesões por Radiação/prevenção & controle , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/sangue , Esfingomielina Fosfodiesterase/genéticaRESUMO
Primary bone tumors can be divided into two classes, benign and malignant. Among the latter group, osteosarcoma and Ewing sarcoma are the most prevalent malignant primary bone tumors in children and adolescents. Despite intensive efforts to improve treatments, almost 40% of patients succumb to the disease. Specifically, the clinical outcome for metastatic osteosarcoma or Ewing sarcoma remains poor; less than 30% of patients who present metastases will survive 5 years after initial diagnosis. One common and specific point of these bone tumors is their ability to deregulate bone homeostasis and remodeling and divert them to their benefit. Over the past years, considerable interest in the Sonic Hedgehog (SHH) pathway has taken place within the cancer research community. The activation of this SHH cascade can be done through different ways and, schematically, two pathways can be described, the canonical and the non-canonical. This review discusses the current knowledge about the involvement of the SHH signaling pathway in skeletal development, pediatric bone sarcoma progression and the related therapeutic options that may be possible for these tumors.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Progressão da Doença , Proteínas Hedgehog/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Transdução de Sinais , Desenvolvimento Ósseo , Neoplasias Ósseas/terapia , Criança , Humanos , Osteossarcoma/terapiaRESUMO
Cancer cells evolve in a very complex tumor microenvironment, composed of several cell types, among which the endothelial cells are the major actors of the tumor angiogenesis. Today, these cells are also characterized for their plasticity, as endothelial cells have demonstrated their potential to modify their phenotype to differentiate into mesenchymal cells through the endothelial-to-mesenchymal transition (EndoMT). This cellular plasticity is mediated by various stimuli including transforming growth factor-ß (TGF-ß) and is modulated dependently of experimental conditions. Recently, emerging evidences have shown that EndoMT is involved in the development and dissemination of cancer and also in cancer cell to escape from therapeutic treatment. In this review, we summarize current updates on EndoMT and its main induction pathways. In addition, we discuss the role of EndoMT in tumorigenesis, metastasis, and its potential implication in cancer therapy resistance.
RESUMO
By gating the traffic of molecules and cells across the vessel wall, endothelial cells play a central role in regulating cardiovascular functions and systemic homeostasis and in modulating pathophysiological processes such as inflammation and immunity. Accordingly, the loss of endothelial cell integrity is associated with pathological disorders that include atherosclerosis and cancer. The p38 mitogen-activated protein kinase (MAPK) cascades are major signaling pathways that regulate several functions of endothelial cells in response to exogenous and endogenous stimuli including growth factors, stress and cytokines. The p38 MAPK family contains four isoforms p38α, p38ß, p38γ and p38δ that are encoded by four different genes. They are all widely expressed although to different levels in almost all human tissues. p38α/MAPK14, that is ubiquitously expressed is the prototype member of the family and is referred here as p38. It regulates the production of inflammatory mediators, and controls cell proliferation, differentiation, migration and survival. Its activation in endothelial cells leads to actin remodeling, angiogenesis, DNA damage response and thereby has major impact on cardiovascular homeostasis, and on cancer progression. In this manuscript, we review the biology of p38 in regulating endothelial functions especially in response to oxidative stress and during the metastatic process.
RESUMO
Ionizing radiation causes oxidative stress, leading to acute and late cellular responses. We previously demonstrated that irradiation of non-proliferating endothelial cells, as observed in normal tissues, induces early apoptosis, which can be inhibited by pretreatment with Sphingosine-1-Phosphate. We now propose to better characterize the long-term radiation response of endothelial cells by studying the molecular pathways associated with senescence and its link with acute apoptosis. First, senescence was validated in irradiated quiescent microvascular HMVEC-L in a dose- and time-dependent manner by SA ß-galactosidase staining, p16Ink4a and p21Waf1 expression, pro-inflammatory IL-8 secretion and DNA damage response activation. This premature aging was induced independently of Sphingosine 1-Phosphate treatment, supporting its non-connection with acute IR-induced apoptosis. Then, senescence under these conditions showed persistent activation of p53 pathway and mitochondrial dysfunctions, characterized by O2·- generation, inhibition of respiratory complex II activity and over-expression of SOD2 and GPX1 detoxification enzymes. Senescence was significantly inhibited by treatment with pifithrin-α, a p53 inhibitor, or by MnTBAP, a superoxide dismutase mimetic, validating those molecular actors in IR-induced endothelial cell aging. However, MnTBAP, but not pifithrin-α, was able to limit superoxide generation and to rescue the respiratory complex II activity. Furthermore, MnTBAP was not modulating p53 up-regulation, suggesting that IR-induced senescence in quiescent endothelial cells is provided by at least 2 different pathways dependent of the mitochondrial oxidative stress response and the p53 activation. Further characterization of the actors involved in the respiratory complex II dysfunction will open new pharmacological strategies to modulate late radiation toxicity.
Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Células Endoteliais/fisiologia , Microvasos/patologia , Mitocôndrias/metabolismo , Superóxidos/metabolismo , Apoptose , Linhagem Celular , Senescência Celular , Células Endoteliais/efeitos da radiação , Humanos , Interleucina-8/metabolismo , Lisofosfolipídeos/metabolismo , Estresse Oxidativo , Radiação Ionizante , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Superóxido Dismutase/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The p38 MAPK signaling pathway is essential in the cellular response to stress stimuli, in particular in the endothelial cells that are major target of external stress. The importance of the bioactive sphingolipid ceramide generated by acid sphingomyelinase is also firmly established in stress-induced endothelial apoptotic cell death. Despite a suggested link between the p38 MAPK and ceramide pathways, the exact molecular events of this connection remain elusive. In the present study, by using two different activators of p38 MAPK, namely anisomycin and ionizing radiation, we depicted how ceramide generated by acid sphingomyelinase was involved in p38 MAPK-dependent apoptosis of endothelial cells. We first proved that both anisomycin and ionizing radiation conducted to apoptosis through activation of p38 MAPK in human microvascular endothelial cells HMEC-1. We then found that both treatments induced activation of acid sphingomyelinase and the generation of ceramide. This step was required for p38 MAPK activation and apoptosis. We finally showed that irradiation, as well as treatment with exogenous C16-ceramide or bacterial sphingomyelinase, induced in endothelial cells a deep reorganization of the plasma membrane with formation of large lipid platforms at the cell surface, leading to p38 MAPK activation and apoptosis in endothelial cells. Altogether, our results proved that the plasma membrane reorganization leading to ceramide production is essential for stress-induced activation of p38 MAPK and apoptosis in endothelial cells and established the link between the acid sphingomyelinase/ceramide and p38 MAPK pathways.
Assuntos
Apoptose , Membrana Celular/metabolismo , Ceramidas/metabolismo , Sistema de Sinalização das MAP Quinases , Esfingomielina Fosfodiesterase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Células Endoteliais/metabolismo , Ativação Enzimática , Humanos , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Estresse FisiológicoRESUMO
Oxidative stress is a leading cause of endothelial dysfunction. The p38 MAPK pathway plays a determinant role in allowing cells to cope with oxidative stress and is tightly regulated by a balanced interaction between p38 protein and its interacting partners. By using a proteomic approach, we identified nucleophosmin (NPM) as a new partner of p38 in HUVECs. Coimmunoprecipitation and microscopic analyses confirmed the existence of a cytosolic nucleophosmin (NPM)/p38 interaction in basal condition. Oxidative stress, which was generated by exposure to 500 µM H2O2, induces a rapid dephosphorylation of NPM at T199 that depends on phosphatase PP2A, another partner of the NPM/p38 complex. Blocking PP2A activity leads to accumulation of NPM-pT199 and to an increased association of NPM with p38. Concomitantly to its dephosphorylation, oxidative stress promotes translocation of NPM to the nucleus to affect the DNA damage response. Dephosphorylated NPM impairs the signaling of oxidative stress-induced DNA damage via inhibition of the phosphorylation of ataxia-telangiectasia mutated and DNA-dependent protein kinase catalytic subunit. Overall, these results suggest that the p38/NPM/PP2A complex acts as a dynamic sensor, allowing endothelial cells to react rapidly to acute oxidative stress.-Guillonneau, M., Paris, F., Dutoit, S., Estephan, H., Bénéteau, E., Huot, J., Corre, I. Oxidative stress disassembles the p38/NPM/PP2A complex, which leads to modulation of nucleophosmin-mediated signaling to DNA damage response.
Assuntos
Células Endoteliais/fisiologia , Proteínas Nucleares/metabolismo , Estresse Oxidativo/fisiologia , Proteína Fosfatase 2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Dano ao DNA , Regulação Enzimológica da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Nucleofosmina , Fosforilação , Proteína Fosfatase 2/genética , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Tumor areas can now be very precisely delimited thanks to technical progress in imaging and ballistics. This has also led to the development of novel radiotherapy protocols, delivering higher doses of ionizing radiation directly to cancer cells. Despite this, radiation toxicity in healthy tissue remains a major issue, particularly with dose-escalation in these new protocols. Acute and late tissue damage following irradiation have both been linked to the endothelium irrigating normal tissues. The molecular mechanisms involved in the endothelial response to high doses of radiation are associated with signaling from the plasma membrane, mainly via the acid sphingomyelinase/ceramide pathway. This review describes this signaling pathway and discusses the relevance of targeting endothelial signaling to protect healthy tissues from the deleterious effects of high doses of radiation.
Assuntos
Membranas/efeitos da radiação , Neoplasias/radioterapia , Tolerância a Radiação/genética , Transdução de Sinais/genética , Apoptose/efeitos da radiação , Células Endoteliais/patologia , Humanos , Neoplasias/patologia , Doses de Radiação , Radiação IonizanteRESUMO
Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.
Assuntos
Movimento Celular/efeitos da radiação , Endotélio Vascular/efeitos da radiação , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Amidas/farmacologia , Apoptose/efeitos da radiação , Adesão Celular/efeitos da radiação , Linhagem Celular , Citoesqueleto/metabolismo , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Fibronectinas/metabolismo , Humanos , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
A previous in vitro study showed that sphingosine-1-phosphate (S1P), a ceramide antagonist, preserved endothelial cells in culture from radiation-induced apoptosis. We proposed to validate the role of S1P in tissue radioprotection by inhibiting acute gastrointestinal (GI) syndrome induced by endothelial cell apoptosis after high dose of radiation. Retro-orbital S1P was injected in mice exposed to 15 Gy, a dose-inducing GI syndrome within 10 days. Overall survival and apoptosis on intestines sections were studied. Intestinal cell type targeted by S1P and early molecular survival pathways were researched using irradiated in vitro cell models and in vivo mouse models. We showed that retro-orbital S1P injection before irradiation prevented GI syndrome by inhibiting endothelium collapse. We defined endothelium as a specific therapeutic target because only these cells and not intestinal epithelial cells, or B and T lymphocytes, were protected. Pharmacologic approaches using AKT inhibitor and pertussis toxin established that S1P affords endothelial cell protection in vitro and in vivo through a mechanism involving AKT and 7-pass transmembrane receptors coupled to Gi proteins. Our results provide strong pharmacologic and mechanistic proofs that S1P protects endothelial cells against acute radiation enteropathy.
Assuntos
Apoptose/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Apoptose/efeitos da radiação , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Medula Óssea/efeitos da radiação , Linhagem Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/patologia , Trato Gastrointestinal/efeitos da radiação , Humanos , Imuno-Histoquímica , Intestino Delgado/citologia , Intestino Delgado/efeitos da radiação , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/patologia , Tecido Linfoide/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Lesões Experimentais por Radiação/tratamento farmacológico , Esfingosina/farmacologia , SíndromeRESUMO
Despite significant expression level in cancer cells, the role of the angiotensin II Type 2 receptor (AT2R) in cancer progression remains poorly understood. We aimed to investigate the involvement of AT2R in tumorigenesis, hypothesizing a role in tumor cell proliferation and/or tumor angiogenesis. Two animal tumor models were used: fibrosarcoma induced by 3-methylcholanthrene (3-MCA) in FVB/N mice invalidated for AT2R (AT2R-KO) and carcinoma LL/2 cells injected in C57BL/6N mice treated with AT2R antagonist PD123,319. Tumor growth was monitored, microvascular density (MVD) evaluated by CD31 staining. Proliferation index of LL/2 and 3-MCA tumor cells was evaluated by expression of Ki-67. Angiogenesis was assessed by aorta ring assay and angiogenic mediators' expression by real-time RT-PCR. Tumor induction by 3-MCA was significantly delayed in AT2R-KO compared to wild-type mice (56 days vs. 28 days). Tumorigenesis following LL/2 cell injection in mice was also significantly reduced by early administration of the antagonist PD123,319. In vitro, inactivation or invalidation of AT2R inhibited proliferation of LL/2 and 3-MCA tumor cells, respectively. Tumor MVD was reduced in mice treated early with PD123,319. Ex vivo experiments revealed a significant decrease in angiogenesis after PD123,319 treatment or in AT2R-KO mice. Finally, we identified vascular endothelial growth factor (VEGF) as a soluble proangiogenic factor produced by LL/2 cells and we showed that in LL/2 and 3-MCA tumor cells, inhibition or deficiency of AT2R was associated with impaired production of proangiogenic factors included VEGF. This study uncovered novel mechanisms by which AT2R would promote tumor development, favoring both malignant cell proliferation and tumor angiogenesis.
Assuntos
Bloqueadores do Receptor Tipo 2 de Angiotensina II , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Fibrossarcoma/irrigação sanguínea , Fibrossarcoma/metabolismo , Receptor Tipo 2 de Angiotensina/deficiência , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Fibrossarcoma/patologia , Deleção de Genes , Imidazóis/farmacologia , Metilcolantreno , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Piridinas/farmacologia , Receptor Tipo 2 de Angiotensina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
For decades, DNA has been considered as the main cellular target of deleterious effects of ionizing radiation (IR). Nevertheless, molecular signals initiated at cellular membranes are now identified as critical events in a large spectrum of radiation-induced cellular processes. If IR provokes DNA damage directly by energy deposit on the DNA double helix and indirectly by reactive species, origin of IR-induced molecular events initiated at the plasma membrane remains more obscure. Generation of reactive oxygen/nitrogen species (ROS/RNS) inducing proteins and lipids modifications seems to be the prevalent hypothesis. However, spatial and temporal relocalization of proteins and/or lipids represents also potential mechanisms of cell signaling generation. In the context of an oxidative stress such as IR, the best example is the translocation of the enzyme acid sphingomyelinase (ASMase) from lysosomes to the outer layer of cell membrane, which then induces sphingomyelin hydrolysis and ceramide formation. Ceramide coalescence with cholesterol forms lipids microdomains in the plasma membrane, enhancing clustering of signaling receptors (death receptors like FAS, TNF, CD40, TRAIL or G protein-coupled receptors). In this manuscript, we propose to overview the different key molecular mechanisms induced at the plasma membrane after IR in perspective with their linked molecular actors.