RESUMO
Acute radiation syndrome (ARS) is a major cause of lethality following radiation disasters. A TLR5 agonist, entolimod, is among the most powerful experimental radiation countermeasures and shows efficacy in rodents and non-human primates as a prophylactic (radioprotection) and treatment (radiomitigation) modality. While the prophylactic activity of entolimod has been connected to the suppression of radiation-induced apoptosis, the mechanism by which entolimod functions as a radiomitigator remains poorly understood. Uncovering this mechanism has significant and broad-reaching implications for the clinical development and improvement of TLR5 agonists for use as an effective radiation countermeasure in scenarios of mass casualty resulting from accidental exposure to ionizing radiation. Here, we demonstrate that in contrast to radioprotection, neutrophils are essential for the radiomitigative activity of entolimod in a mouse model of lethal ARS. Neutrophils express functional TLR5 and rapidly exit the bone marrow (BM), accumulate in solid tissues, and release MMP-9 following TLR5 stimulation which is accompanied by an increase in the number of active hematopoietic pluripotent precursors (HPPs) in the BM. Importantly, recombinant MMP-9 by itself has radiomitigative activity and, in the absence of neutrophils, accelerates the recovery of the hematopoietic system. Unveiling this novel TLR5-neutrophil-MMP-9 axis of radiomitigation opens new opportunities for the development of efficacious radiation countermeasures to treat ARS following accidental radiation disasters.
RESUMO
The Toll-like receptor 5 (TLR5) agonist entolimod, a derivative of Salmonella flagellin, has therapeutic potential for several indications including radioprotection and cancer immunotherapy. However, in Phase 1 human studies, entolimod induced a rapid neutralizing immune response, presumably due to immune memory from prior exposure to flagellated enterobacteria. To enable multi-dose applications, we used structure-guided reengineering to develop a next-generation, substantially deimmunized entolimod variant, GP532. GP532 induces TLR5-dependent NF-κB activation like entolimod but is smaller and has mutations eliminating an inflammasome-activating domain and key B- and T-cell epitopes. GP532 is resistant to human entolimod-neutralizing antibodies and shows reduced de novo immunogenicity. GP532 also has improved bioavailability, a stronger effect on key cytokine biomarkers, and a longer-lasting effect on NF-κB. Like entolimod, GP532 demonstrated potent prophylactic and therapeutic efficacy in mouse models of radiation-induced death and tissue damage. These results establish GP532 as an optimized TLR5 agonist suitable for multi-dose therapies and for patients with high titers of preexisting flagellin-neutralizing antibodies.
Assuntos
Peptídeos/farmacologia , Transdução de Sinais , Receptor 5 Toll-Like/agonistas , Linhagem Celular Tumoral , Células HEK293 , HumanosRESUMO
Curaxins are small molecules that bind genomic DNA and interfere with DNA-histone interactions leading to the loss of histones and decondensation of chromatin. We named this phenomenon 'chromatin damage'. Curaxins demonstrated anti-cancer activity in multiple pre-clinical tumor models. Here, we present data which reveals, for the first time, a role for the immune system in the anti-cancer effects of curaxins. Using the lead curaxin, CBL0137, we observed elevated expression of several group of genes in CBL0137-treated tumor cells including interferon sensitive genes, MHC molecules, some embryo-specific antigens suggesting that CBL0137 increases tumor cell immunogenicity and improves recognition of tumor cells by the immune system. In support of this, we found that the anti-tumor activity of CBL0137 was reduced in immune deficient SCID mice when compared to immune competent mice. Anti-tumor activity of CBL0137 was abrogated in CD8+ T cell depleted mice but only partially lost when natural killer or CD4+ T cells were depleted. Further support for a key role for the immune system in the anti-tumor activity of CBL0137 is evidenced by an increased antigen-specific effector CD8+ T cell and NK cell response, and an increased ratio of effector T cells to Tregs in the tumor and spleen. CBL0137 also elevated the number of CXCR3-expressing CTLs in the tumor and the level of interferon-γ-inducible protein 10 (IP-10) in serum, suggesting IP-10/CXCR3 controls CBL0137-elicited recruitment of effector CTLs to tumors. Our collective data underscores a previously unrecognized role for both innate and adaptive immunity in the anti-tumor activity of curaxins.
Assuntos
Carbazóis/farmacologia , Cromatina/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Imunidade/imunologia , Animais , Apoptose , Proliferação de Células , Quimiocinas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Citocinas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor necrosis factor alpha (TNF) is capable of inducing regression of solid tumors. However, TNF released in response to Toll-like receptor 4 (TLR4) activation by bacterial lipopolysaccharide (LPS) is the key mediator of cytokine storm and septic shock that can cause severe tissue damage limiting anticancer applications of this cytokine. In our previous studies, we demonstrated that activation of another Toll-like receptor, TLR5, could protect from tissue damage caused by a variety of stresses including radiation, chemotherapy, Fas-activating antibody and ischemia-reperfusion. In this study, we tested whether entolimod could counteract TNF-induced toxicity in mouse models. We found that entolimod pretreatment effectively protects livers and lungs from LPS- and TNF-induced toxicity and prevents mortality caused by combining either of these agents with the sensitizer, D-galactosamine. While LPS and TNF induced significant activation of apoptotic caspase 3/7, lipid tissue peroxidation and serum ALT accumulation in mice without entolimod treatment, these indicators of toxicity were reduced by entolimod pretreatment to the levels of untreated control mice. Entolimod was effective when injected 0.5-48 hours prior to, but not when injected simultaneously or after LPS or TNF. Using chimeric mice with hematopoiesis differing in its TLR5 status from the rest of tissues, we showed that this protective activity was dependent on TLR5 expression by non-hematopoietic cells. Gene expression analysis identified multiple genes upregulated by entolimod in the liver and cultured hepatocytes as possible mediators of its protective activity. Entolimod did not interfere with the antitumor activity of TNF in mouse hepatocellular and colorectal tumor models. These results support further development of TLR5 agonists to increase tissue resistance to cytotoxic cytokines, reduce the risk of septic shock and enable safe systemic application of TNF as an anticancer therapy.
Assuntos
Antineoplásicos/farmacologia , Peptídeos/farmacologia , Receptor 5 Toll-Like/agonistas , Fator de Necrose Tumoral alfa/toxicidade , Animais , Linhagem Celular Tumoral , Células Cultivadas , Galactosamina , Hematopoese/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Biológicos , NF-kappa B/metabolismo , Substâncias Protetoras/farmacologia , Análise de Sobrevida , Receptor 5 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/sangue , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Radiation treatment of head and neck cancer frequently causes severe collateral damage to normal tissues including mouth mucosa, salivary glands and skin. This toxicity limits the radiation dose that can be delivered and affects the patient's quality of life. Previous studies in mice and nonhuman primates showed that entolimod, a toll-like receptor 5 (TLR5) agonist derived from bacterial flagellin, effectively reduced radiation damage to hematopoietic and gastrointestinal tissues in both total-body and local irradiation scenarios, with no protection of tumors. Here, using a mouse model, we analyzed the efficacy of entolimod administered before or after irradiation in reducing damage to normal tissues. Animals received local fractionated radiation to the head and neck area, thus modeling radiotherapy of head and neck cancer. Tissue damage was evaluated through histomorphological examination of samples collected at different time points up to four weeks, mice were exposed locally to five daily fractions of 5, 6 or 7 Gy. A semiquantitative scoring system was used to assess the severity of observed pathomorphological changes. In this model, radiation damage was most severe in the lips, tongue and skin, moderate in the upper esophagus and minor in salivary glands. The kinetics of injury appearance and recovery of normal morphology varied among tissues, with maximal damage to the tongue, esophagus and salivary glands developing at earlier times (days 8-11 postirradiation) relative to that of lip and skin mucosa (days 11-15 postirradiation). While both tested regimens of entolimod significantly reduced the extent of radiation damage and accelerated restoration of normal structure in all tissues analyzed, administration of entolimod 1 h after each irradiation was more effective than treatment 30 min before irradiation. These results support the potential clinical use of entolimod as an adjuvant for improving the therapeutic index of head and neck cancer radiotherapy by reducing the radiation toxicity in normal tissues.
Assuntos
Epitélio/lesões , Epitélio/patologia , Neoplasias de Cabeça e Pescoço/radioterapia , Peptídeos/administração & dosagem , Lesões por Radiação/patologia , Lesões por Radiação/prevenção & controle , Animais , Fracionamento da Dose de Radiação , Relação Dose-Resposta a Droga , Epitélio/efeitos da radiação , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Camundongos , Protetores contra Radiação/administração & dosagem , Receptor 5 Toll-Like/antagonistas & inibidores , Resultado do TratamentoRESUMO
Activation of an anticancer innate immune response is highly desirable because of its inherent ability to generate an adaptive antitumor T-cell response. However, insufficient safety of innate immune modulators limits clinical use to topical applications. Toll-like receptor 5 (TLR5) agonists are favorably positioned as potential systemic immunotherapeutic agents because of unusual tissue specificity of expression, uniquely safe profile of induced cytokines, and antitumor efficacy demonstrated in a number of animal models. Here, we decipher the molecular and cellular events underlying the metastasis suppressive activity of entolimod, a clinical stage TLR5 agonist that activates NF-κB-, AP-1-, and STAT3-driven immunomodulatory signaling pathways specifically within the liver. Used as a single agent in murine colon and mammary metastatic cancer models, entolimod rapidly induces CXCL9 and -10 that support homing of blood-borne CXCR3-expressing NK cells to the liver predominantly through an IFN-γ signaling independent mechanism. NK cell-dependent activation of dendritic cells is followed by stimulation of a CD8(+) T-cell response, which exert both antimetastatic effect of entolimod and establishment of tumor-specific and durable immune memory. These results define systemically administered TLR5 agonists as organ-specific immunoadjuvants, enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Peptídeos/farmacologia , Receptor 5 Toll-Like/agonistas , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Células Matadoras Naturais/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Uveal melanoma (UM) is the most common primary cancer of the eye in adults and progresses to metastatic disease predominantly of the liver in ~50% of patients. In these cases, life expectancy averages just 9 months due to the lack of effective treatment options. The Toll-like receptor 5 (TLR5) agonist entolimod (former name CBLB502) rapidly activates TLR5-NF-κB signaling in hepatocytes and suppresses growth of both TLR5-expressing and non-expressing tumors in the liver through mobilization and activation of innate and adaptive immune mechanisms. The goal of this study was to explore the potential of entolimod as an immunotherapeutic agent against hepatic metastasis of UM using the TLR5-positive B16LS9 mouse model of ocular melanoma. Mice were given seven subcutaneous injections of vehicle or entolimod given 72 h apart started one day before, on the same day or three days after intraocular injection of B16LS9 cells. All tested regimens of entolimod treatment resulted in significantly reduced B16LS9 metastasis to the liver. Entolimod induced mobilization of natural killer (NK) cells to the liver and stimulated their maturation, differentiation and activation. Antibody-mediated depletion of NK cells from mice abrogated entolimod's antimetastatic activity in the liver and eliminated the entolimod-elicited in vitro cytotoxic activity of hepatic lymphocytes against B16LS9 cells. These results provide pre-clinical evidence of entolimod's efficacy against hepatometastasis of UM and support its further development as an anticancer immunotherapeutic drug.
Assuntos
Antineoplásicos/uso terapêutico , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas , Melanoma Experimental/patologia , Melanoma/patologia , Peptídeos/uso terapêutico , Neoplasias Uveais/patologia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Hepatócitos/metabolismo , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Imunoterapia/métodos , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos C57BL , Receptor 5 Toll-Like/agonistas , Fator de Transcrição RelA/metabolismoRESUMO
Myelosuppression and gastrointestinal damage are common side effects of cancer treatment limiting efficacy of DNA-damaging chemotherapeutic drugs. The Toll-like receptor 5 (TLR5) agonist Entolimod has demonstrated efficacy in mitigating damage to hematopoietic and gastrointestinal tissues caused by radiation. Here, using 5-Fluorouracil (5-FU) treated mice as a model of chemotherapy-induced side effects, we demonstrated significant reduction in the severity of 5-FU-induced morbidity and increased survival accompanied by the improved integrity of intestinal tissue and stimulated the restoration of hematopoiesis. Entolimod-stimulated IL-6 production was essential for Entolimod's ability to rescue mice from death caused by doses of 5-FU associated with hematopoietic failure. In contrast, IL-6 induction was not necessary for protection and restoration of drug-damaged gastrointestinal tissue by Entolimod. In a syngeneic mouse CT26 colon adenocarcinoma model, Entolimod reduced the systemic toxicity of 5-FU, but did not reduce its antitumor efficacy indicating that the protective effect of Entolimod was selective for normal, non-tumor, tissues. These results suggest that Entolimod has clinical potential to broaden the therapeutic window of genotoxic anticancer drugs by reducing their associated hematopoietic and gastrointestinal toxicities.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/tratamento farmacológico , Fluoruracila/farmacologia , Peptídeos/farmacologia , Receptor 5 Toll-Like/agonistas , Adenocarcinoma/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Sinergismo Farmacológico , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição AleatóriaRESUMO
Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. The "danger context" elicited by TLR agonists allows an initially non-immunogenic antigen to become immunogenic. This ability to alter environment is highly relevant in tumor immunity, since it is inherently difficult for the immune system to recognize host-derived tumors as immunogenic. However, immune cells may have encountered certain TLR ligands associated with tumor development, yet the endogenous stimulation is typically not sufficient to induce spontaneous tumor rejection. Of special interest are TLR5 agonists, because there are no endogenous ligands that bind TLR5. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. We examined the effect of CBLB502 on tumor immunity using two syngeneic lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T-cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell- and perforin-dependent. In addition, CBLB502 stimulates clearance of the B-cell lymphoma A20 in BALB/c mice in a CD8(+) T cell-dependent fashion. Analysis on the cellular level via ImageStream flow cytometry reveals that CD11b(+) and CD11c(+) cells, but neither NK nor T cells, directly respond to CBLB502 as determined by NFκB nuclear translocation. Our findings demonstrate that CBLB502 stimulates a robust antitumor response by directly activating TLR5-expressing accessory immune cells, which in turn activate cytotoxic lymphocytes.
Assuntos
Adjuvantes Imunológicos/farmacologia , Flagelina/química , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfoma de Células T/imunologia , Peptídeos/farmacologia , Receptor 5 Toll-Like/agonistas , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/uso terapêutico , Animais , Linhagem Celular Tumoral , Citocinas/sangue , Imunidade Inata/efeitos dos fármacos , Linfoma de Células T/sangue , Linfoma de Células T/tratamento farmacológico , Camundongos , Peptídeos/química , Peptídeos/uso terapêutico , Perforina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Pathogen recognition receptors (PRRs) are essential components of host innate immune systems that detect specific conserved pathogen-associated molecular patterns (PAMPs) presented by microorganisms. Members of two families of PRRs, transmembrane Toll-like receptors (TLRs 1, 2, 4, 5, and 6) and cytosolic NOD receptors (NOD1 and NOD2), are stimulated upon recognition of various bacterial PAMPs. Such stimulation leads to induction of a number of immune defense reactions, mainly triggered via activation of the transcription factor NF-κB. While coordination of responses initiated via different PRRs sensing multiple PAMPS present during an infection makes clear biological sense for the host, such interactions have not been fully characterized. Here, we demonstrate that combined stimulation of NOD1 and TLR5 (as well as other NOD and TLR family members) strongly potentiates activity of NF-κB and induces enhanced levels of innate immune reactions (e.g., cytokine production) both in vitro and in vivo. Moreover, we show that an increased level of NF-κB activity plays a critical role in formation of downstream responses. In live mice, synergy between these receptors resulting in potentiation of NF-κB activity was organ specific, being most prominent in the gastrointestinal tract. Coordinated activity of NOD1 and TLR5 significantly increased protection of mice against enteroinvasive Salmonella infection. Obtained results suggest that cooperation of NOD and TLR receptors is important for effective responses to microbial infection in vivo.
Assuntos
Imunidade Inata/fisiologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Salmonella typhimurium , Receptor 5 Toll-Like/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , Proteína Adaptadora de Sinalização NOD1/genética , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Receptor 5 Toll-Like/genéticaRESUMO
Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-κB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-κB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1-) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.
Assuntos
Fígado/metabolismo , Peptídeos/farmacologia , Fator de Transcrição STAT3/metabolismo , Receptor 5 Toll-Like/agonistas , Animais , Anticarcinógenos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Feminino , Citometria de Fluxo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Transplante de Neoplasias , Neutrófilos/metabolismo , Protetores contra Radiação/farmacologia , Transdução de Sinais , Receptor fas/metabolismoRESUMO
Allogeneic hematopoietic cell transplantation is an established treatment for hematologic and nonhematologic malignancies. Donor-derived immune cells can identify and attack host tumor cells, producing a graft-versus-tumor (GVT) effect that is crucial to the effectiveness of the transplantation therapy. CBLB502 is a novel agonist for TLR5 derived from Salmonella flagellin. On the basis of TLR5-mediated immunomodulatory function, we examined the effect of CBLB502 on GVT activity. Using two tumor models that do not express TLR5, and thereby do not directly respond to CBLB502, we found that CBLB502 treatment significantly enhanced allogeneic CD8(+) T cell-mediated GVT activity, which was evidenced by decreased tumor burden and improved host survival. Importantly, histopathologic analyses showed that CBLB502 treatment did not exacerbate the moderate graft-versus-host disease condition caused by the allogeneic CD8(+) T cells. Moreover, mechanistic analyses showed that CBLB502 stimulates CD8(+) T cell proliferation and enhances their tumor killing activity mainly indirectly through a mechanism that involves the IL-12 signaling pathway and the CD11c(+) and CD11b(+) populations in the bone marrow cells. This study demonstrates a new beneficial effect of CBLB502, and suggests that TLR5-mediated immune modulation may be a promising approach to improve GVT immunity without exacerbating graft-versus-host disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Flagelina/farmacologia , Efeito Enxerto vs Tumor/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Imunidade Celular/efeitos dos fármacos , Neoplasias Experimentais/terapia , Salmonella/química , Receptor 5 Toll-Like/agonistas , Animais , Linfócitos T CD8-Positivos/patologia , Proliferação de Células/efeitos dos fármacos , Flagelina/química , Flagelina/imunologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Tumor/imunologia , Imunidade Celular/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Receptor 5 Toll-Like/imunologia , Transplante HomólogoRESUMO
Unlike reversible quiescence, cellular senescence is characterized by a large flat cell morphology, ß-gal staining and irreversible loss of regenerative (i.e., replicative) potential. Conversion from proliferative arrest to irreversible senescence, a process named geroconversion, is driven in part by growth-promoting pathways such as mammalian target of rapamycin (mTOR). During cell cycle arrest, mTOR converts reversible arrest into senescence. Inhibitors of mTOR can suppress geroconversion, maintaining quiescence instead. It was shown that hypoxia inhibits mTOR. Therefore, we suggest that hypoxia may suppress geroconversion. Here we tested this hypothesis. In HT-p21-9 cells, expression of inducible p21 caused cell cycle arrest without inhibiting mTOR, leading to senescence. Hypoxia did not prevent p21 induction and proliferative arrest, but instead inhibited the mTOR pathway and geroconversion. Exposure to hypoxia during p21 induction prevented senescent morphology and loss of regenerative potential, thus maintaining reversible quiescence so cells could restart proliferation after switching p21 off. Suppression of geroconversion was p53- and HIF-1-independent, as hypoxia also suppressed geroconversion in cells lacking functional p53 and HIF-1α. Also, in normal fibroblasts and retinal cells, hypoxia inhibited the mTOR pathway and suppressed senescence caused by etoposide without affecting DNA damage response, p53/p21 induction and cell cycle arrest. Also hypoxia suppressed geroconversion in cells treated with nutlin-3a, a nongenotoxic inducer of p53, in cell lines susceptible to nutlin-3a-induced senescence (MEL-10, A172, and NKE). Thus, in normal and cancer cell lines, hypoxia suppresses geroconversion caused by diverse stimuli. Physiological and clinical implications of the present findings are discussed.
Assuntos
Senescência Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Etoposídeo/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Development of mucositis is a frequent side effect of radiotherapy of patients with head-and-neck cancer. We have recently reported that bacterial flagellin, an agonist of Toll-like receptor 5 (TLR5), can protect rodents and primates from acute radiation syndrome caused by total body irradiation. Here we analyzed the radioprotective efficacy of TLR5 agonist under conditions of local, single dose or fractionated radiation treatment. METHODS AND MATERIALS: Mice received either single-dose (10, 15, 20, or 25 Gy) or fractioned irradiation (cumulative dose up to 30 Gy) of the head-and-neck area with or without subcutaneous injection of pharmacologically optimized flagellin, CBLB502, 30 min before irradiation. RESULTS: CBLB502 significantly reduced the severity of dermatitis and mucositis, accelerated tissue recovery, and reduced the extent of radiation induced weight loss in mice after a single dose of 15 or 20 Gy but not 25 Gy of radiation. CBLB502 was also protective from cumulative doses of 25 and 30 Gy delivered in two (10 + 15 Gy) or three (3 × 10 Gy) fractions, respectively. While providing protection to normal epithelia, CBLB502 did not affect the radiosensitivity of syngeneic squamous carcinoma SCCVII grown orthotopically in mice. Use of CBLB502 also elicited a radiation independent growth inhibitory effect upon TLR5-expressing tumors demonstrated in the mouse xenograft model of human lung adenocarcinoma A549. CONCLUSION: CBLB502 combines properties of supportive care (radiotherapy adjuvant) and anticancer agent, both mediated via activation of TLR5 signaling in the normal tissues or the tumor, respectively.
Assuntos
Neoplasias de Cabeça e Pescoço/radioterapia , Peptídeos/uso terapêutico , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Radiodermite/prevenção & controle , Estomatite/prevenção & controle , Receptor 5 Toll-Like/agonistas , Adenocarcinoma/metabolismo , Animais , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Mucosa Bucal/efeitos da radiação , Tolerância a Radiação/efeitos dos fármacos , Estomatite/etiologia , Receptor 5 Toll-Like/metabolismo , Redução de Peso/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The toxicity of ionizing radiation is associated with massive apoptosis in radiosensitive organs. Here, we investigate whether a drug that activates a signaling mechanism used by tumor cells to suppress apoptosis can protect healthy cells from the harmful effects of radiation. We studied CBLB502, a polypeptide drug derived from Salmonella flagellin that binds to Toll-like receptor 5 (TLR5) and activates nuclear factor-kappaB signaling. A single injection of CBLB502 before lethal total-body irradiation protected mice from both gastrointestinal and hematopoietic acute radiation syndromes and resulted in improved survival. CBLB502 injected after irradiation also enhanced survival, but at lower radiation doses. It is noteworthy that the drug did not decrease tumor radiosensitivity in mouse models. CBLB502 also showed radioprotective activity in lethally irradiated rhesus monkeys. Thus, TLR5 agonists could potentially improve the therapeutic index of cancer radiotherapy and serve as biological protectants in radiation emergencies.
Assuntos
NF-kappa B/metabolismo , Peptídeos/farmacologia , Lesões Experimentais por Radiação/prevenção & controle , Tolerância a Radiação/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Receptor 5 Toll-Like/agonistas , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Quimioterapia Adjuvante , Flagelina/química , Flagelina/farmacologia , Raios gama , Sistema Hematopoético/efeitos dos fármacos , Sistema Hematopoético/efeitos da radiação , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/efeitos da radiação , Macaca mulatta , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/radioterapia , Peptídeos/administração & dosagem , Peptídeos/química , Peptídeos/toxicidade , Doses de Radiação , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/química , Protetores contra Radiação/toxicidade , Salmonella enterica , Transdução de Sinais , Receptor 5 Toll-Like/metabolismo , Irradiação Corporal TotalRESUMO
Inactivation of p53 function, which frequently occurs in tumors, can significantly modulate tumor cell sensitivity to radiation and chemotherapeutic drugs. However, in addition to acting on malignant cells, anticancer agents act on the cells of tumor stroma, causing activation of a p53 response. The effect of this response on treatment outcome has been the subject of the present study. Tumors with p53-deficient stroma were generated using mouse tumorigenic packaging cells that produce a p53 inhibitory retrovirus, encoding a dominant-negative p53 mutant. Tumors maintaining wild-type p53 in their stroma were formed by cells of similar origin but deficient in retroviral production due to the deletion of the packaging signal in the retroviral vector. Comparison of these tumor models, differing only in p53 status of their stromas, showed that tumors with p53-deficient stroma were significantly more sensitive to experimental chemotherapy and radiotherapy. A similar effect was achieved when anticancer treatment was combined with pharmacologic suppression of p53 by the cyclic form of pifithrin alpha, a small-molecule inhibitor of p53. Potentiation of the anticancer effect of chemotherapy and radiotherapy by p53 suppression in the tumor stroma is likely to be due to the increased sensitivity of p53-deficient endothelium to genotoxic stress as shown both in cell culture and in experimental tumors. Thus, reversible pharmacologic suppression of p53 may be a viable approach to improving anticancer treatment via an enhanced antiangiogenic effect of chemotherapy and radiotherapy.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Benzotiazóis/uso terapêutico , Carcinoma Pulmonar de Lewis/terapia , Ciclofosfamida/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/uso terapêutico , Radiossensibilizantes/uso terapêutico , Tiazóis/uso terapêutico , Tolueno/análogos & derivados , Proteína Supressora de Tumor p53/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Benzotiazóis/farmacologia , Benzotiazóis/toxicidade , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/radioterapia , Linhagem Celular Transformada , Ciclofosfamida/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais/efeitos da radiação , Fibroblastos , Imidazóis/farmacologia , Imidazóis/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Conformação Proteica , Radiossensibilizantes/farmacologia , Radiossensibilizantes/toxicidade , Ratos , Células Estromais/efeitos dos fármacos , Células Estromais/efeitos da radiação , Tiazóis/farmacologia , Tiazóis/toxicidade , Tolueno/farmacologia , Tolueno/uso terapêutico , Tolueno/toxicidade , Transdução Genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
p53-dependent apoptosis contributes to the side effects of cancer treatment, and genetic or pharmacological inhibition of p53 function can increase normal tissue resistance to genotoxic stress. It has recently been shown that p53 can induce apoptosis through a mechanism that does not depend on transactivation but instead involves translocation of p53 to mitochondria. To determine the impact of this p53 activity on normal tissue radiosensitivity, we isolated a small molecule named pifithrin-mu (PFTmu, 1) that inhibits p53 binding to mitochondria by reducing its affinity to antiapoptotic proteins Bcl-xL and Bcl-2 but has no effect on p53-dependent transactivation. PFTmu has a high specificity for p53 and does not protect cells from apoptosis induced by overexpression of proapoptotic protein Bax or by treatment with dexamethasone (2). PFTmu rescues primary mouse thymocytes from p53-mediated apoptosis caused by radiation and protects mice from doses of radiation that cause lethal hematopoietic syndrome. These results indicate that selective inhibition of the mitochondrial branch of the p53 pathway is sufficient for radioprotection in vivo.
Assuntos
Raios gama/efeitos adversos , Mitocôndrias/metabolismo , Lesões Experimentais por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Tiazóis/uso terapêutico , Tolueno/análogos & derivados , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Benzotiazóis , Linhagem Celular , Dexametasona/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Transporte Proteico , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Protetores contra Radiação/química , Protetores contra Radiação/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Timo/efeitos dos fármacos , Timo/metabolismo , Timo/patologia , Tolueno/química , Tolueno/farmacologia , Tolueno/uso terapêutico , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
Renal cell carcinomas (RCC) commonly retain wild-type but functionally inactive p53, which is repressed by an unknown dominant mechanism. To help reveal this mechanism, we screened a diverse chemical library for small molecules capable of restoring p53-dependent transactivation in RCC cells carrying a p53-responsive reporter. Among the compounds isolated were derivatives of 9-aminoacridine (9AA), including the antimalaria drug quinacrine, which strongly induced p53 function in RCC and other types of cancer cells. Induction of p53 by these compounds does not involve genotoxic stress and is mediated by suppression of NF-kappaB activity. In contrast to agents that target IkappaB kinase 2, 9AA and quinacrine can effectively suppress both basal and inducible activities of NF-kappaB, representing inhibitors of a previously undescribed type that convert NF-kappaB from a transactivator into a transrepressor, leading to accumulation of inactive nuclear complexes with unphosphorylated Ser-536 in the p65/RelA subunit. p53 function in RCC can be restored by ectopic expression of a superrepressor of IkappaB as effectively as by 9AA-derived compounds. These findings suggest that the complete or partial repression of p53 observed in many tumors can be the result of constitutive activation of NF-kappaB. The results demonstrate, in principle, the possibility to kill cancer cells selectively through simultaneous inhibition of NF-kappaB and activation of p53 by a single small molecule and suggest anticancer applications for the well known antimalaria drug quinacrine.
Assuntos
Aminacrina/farmacologia , Carcinoma de Células Renais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , NF-kappa B/antagonistas & inibidores , Quinacrina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Colorimetria , Humanos , Relação Estrutura-Atividade , beta-Galactosidase/metabolismoRESUMO
The basis of constitutive activation of NF-kappaB, essential for survival and resistance to apoptosis in many tumors, is not well understood. We find that transforming growth factor beta2 (TGFbeta2), predominantly in its latent form, is secreted by several different types of tumor cell lines that exhibit constitutively active NF-kappaB and that TGFbeta2 potently stimulates the activation of NF-kappaB in reporter cells. Suppression of TGFbeta2 expression by small interfering RNA kills prostate cancer PC3 cells, indicating that the TGFbeta2-NF-kappaB pathway is important for their viability. These findings identify TGFbeta2 as a potential target for therapeutic strategies to inhibit the growth of tumor cells that depend on constitutively active NF-kappaB, or to sensitize them to treatment with cytotoxic drugs.