RESUMO
Matrix metalloproteinases (MMPs) play a central role in tumor invasion and metastasis. Increased expression of MMPs occurs during development of hepatocellular carcinoma (HCC) in humans following infection with hepatitis B virus (HBV). Woodchucks are used as an animal model for hepadnavirus-induced HCC. All woodchucks infected chronically with woodchuck hepatitis virus (WHV), a virus that is closely related to HBV, develop HCC. In the present study MMPs and related molecules were investigated in woodchucks to better understand the mechanisms of extracellular matrix remodeling in HCC. Three groups of samples were studied: liver and HCC tissues from animals infected with WHV and age- and gender-matched normal liver from animals not infected with WHV. New partial gene sequences for woodchuck MMP-2, MMP-7, and MMP-9 as well as their inhibitors NGAL, TIMP-1, and TIMP-2 were identified and used for determination of expression levels in liver and HCC by qRT-PCR. Compared to liver of WHV-naïve woodchucks, high levels of MMP-1, MMP-2, MMP-7, NGAL, and TIMP-1 were detected in liver of animals infected with WHV. However, no differences were found for TIMP-2. MMP-9 expression was higher in HCC than in liver of animals not infected with WHV. Immunohistochemical staining demonstrated that MMP-9 immunoreactivity was most intense in HCC, correlating with the progression of liver disease. Upregulation of MMP-9 in HCC was confirmed by Western blotting and zymography analysis. Furthermore, the activity of woodchuck MMPs was suppressed by BiPS, a common inhibitor of mammalian MMPs. These results suggest the use of MMP inhibitors as a potential HCC treatment strategy that could be explored in woodchucks.
Assuntos
Carcinoma Hepatocelular/patologia , Expressão Gênica , Vírus da Hepatite B da Marmota/fisiologia , Interações Hospedeiro-Patógeno , Metaloproteinases da Matriz/biossíntese , Inibidores de Proteases/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Fígado/patologia , Masculino , Marmota , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus-induced hepatocellular carcinoma (HCC). Woodchucks (Marmota monax) that were chronically infected with HBV-related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery the WHV carrier woodchucks were superinfected with WHV-enveloped HDV (wHDV). Six weeks later the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real-time polymerase chain reaction assay, and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues. CONCLUSION: Cells of WHV-induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Because others previously hypothesized that hepadnavirus-induced HCCs are resistant to reinfection with a hepadnavirus in vivo, our data suggest that if such a resistance exists it likely occurs via a block at the post-entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection.
Assuntos
Carcinoma Hepatocelular/virologia , Hepadnaviridae/genética , Vírus Delta da Hepatite/genética , Neoplasias Hepáticas/virologia , Replicação Viral/genética , Animais , Biópsia por Agulha , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Coinfecção/virologia , Modelos Animais de Doenças , Hepadnaviridae/metabolismo , Vírus da Hepatite B da Marmota/genética , Vírus da Hepatite B da Marmota/metabolismo , Vírus Delta da Hepatite/metabolismo , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Marmota , RNA Viral/análise , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
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
The mechanistic target of rapamycin (mTOR) is a PI3K-related kinase that regulates cell growth, proliferation and survival in response to the availability of energy sources and growth factors. Cancer development and progression is often associated with constitutive activation of the mTOR pathway, thus justifying mTOR inhibition as a promising approach to cancer treatment and prevention. However, development of previous rapamycin analogues has been complicated by their induction of adverse side effects and variable efficacy. Since mTOR pathway regulation involves multiple feedback mechanisms that may be differentially activated depending on the degree of mTOR inhibition, we investigated whether rapamycin dosing could be adjusted to achieve chemopreventive efficacy without side effects. Thus, we tested the efficacy of two doses of a novel, highly bioavailable nanoformulation of rapamycin, Rapatar, in a mouse prostate cancer model (male mice with prostate epithelium-specific Pten-knockout). We found that the highest efficacy was achieved by the lowest dose of Rapatar used in the study. While both doses tested were equally effective in suppressing proliferation of prostate epithelial cells, higher dose resulted in activation of feedback circuits that reduced the drug's tumor preventive efficacy. These results demonstrate that low doses of highly bioavailable mTOR inhibitor, Rapatar, may provide safe and effective cancer prevention.
RESUMO
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
Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt's lymphoma in vitro and in mouse xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further expanded the compound's therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species. These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Citocinas/antagonistas & inibidores , Neoplasias Hematológicas/tratamento farmacológico , NAD/metabolismo , Niacina/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Animais , Apoptose , Proliferação de Células , Feminino , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.
Assuntos
Envelhecimento/fisiologia , Longevidade/fisiologia , Animais , Dieta Hiperlipídica , Feminino , Nível de Saúde , Masculino , Camundongos , Caracteres SexuaisRESUMO
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
There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40-60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.
Assuntos
Síndrome Aguda da Radiação/tratamento farmacológico , Peptídeos/uso terapêutico , Protetores contra Radiação/uso terapêutico , Receptor 5 Toll-Like/agonistas , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Feminino , Hematopoese , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Macaca mulatta , Masculino , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/farmacologiaRESUMO
We examined a rational approach to therapy of chronic hepatitis B virus (HBV) infection that utilized the reduction of viral load combined with appropriately timed immune modulation/stimulation. In a placebo-controlled study, chronic woodchuck hepatitis virus (WHV) carrier woodchucks received clevudine (L-FMAU), previously shown to have especially potent and sustained antiviral activity in woodchucks, for 32 weeks followed by WHV surface antigen (WHsAg) alum-adjuvanted vaccine at 32, 36, 40 and 48 weeks. Clevudine induced significant reductions in viraemia, surface antigenaemia, hepatic WHV nucleic acids, and hepatic core and surface antigens. Viral replication markers remained markedly suppressed in 75% of the clevudine-treated woodchucks following drug withdrawal, but remained at high levels in the vaccine monotherapy and placebo groups. Combination drug and vaccine therapy had benefits based on sustained reduction of viraemia, antigenaemia, and hepatic WHV DNA and RNA; inhibition of progression of chronic hepatitis; reduced frequency of chronic liver injury; and delayed onset of hepatocellular carcinoma (HCC). Combination therapy contributed to prevention of HCC in up to 38% of treated carriers, although the growth rate of established HCC was not affected. This study demonstrates enhanced benefits of combination chemo-immunotherapy against viral load and disease progression in chronic hepadnaviral infection, and provides a platform for further development of such treatment regimens.
Assuntos
Antivirais/administração & dosagem , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/administração & dosagem , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B da Marmota/imunologia , Hepatite B Crônica/prevenção & controle , Hepatite B Crônica/terapia , Animais , Antivirais/uso terapêutico , Arabinofuranosiluracila/uso terapêutico , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/prevenção & controle , Carcinoma Hepatocelular/terapia , Progressão da Doença , Feminino , Vacinas contra Hepatite B/imunologia , Vacinas contra Hepatite B/uso terapêutico , Vírus da Hepatite B da Marmota/fisiologia , Hepatite B Crônica/imunologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/prevenção & controle , Masculino , Marmota , Carga ViralRESUMO
Adult woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus (WHV) were treated orally with lamivudine (15 mg/kg per day) for 57 weeks. After 20 weeks of treatment a 2-3 log reduction in serum WHV DNA was detected. Serum titers of WHV then increased gradually, in the presence of lamivudine treatment, reaching pre-treatment values by week 40. Viral recrudescence was associated with development of mutations in the B domain of the WHV polymerase gene. Mutations observed in the highly conserved FLLA motif of the B domain were L564V, L565M, and A566T, with A566T being the most frequently observed. Beginning on week 57 of lamivudine treatment, one group (n = 3) was treated orally with adefovir dipivoxil at a dose of 15 mg/kg per day plus lamivudine, and a second group (n = 3) was treated with H2O placebo plus lamivudine. In woodchucks treated with adefovir dipivoxil, two had the A566T mutation, and one had both A566T and L565V. In the group maintained on lamivudine monotherapy, A566T alone was present in one animal, another carried both A566T and L565V, and in the third, no B-domain mutations were detected. There was a 4.5 log reduction in serum WHV DNA after 12 weeks of treatment with the adefovir/lamivudine combination, while in the lamivudine monotherapy controls, WHV DNA decreased by only 0.83 log (P > 0.001). A slight recurrence in serum titers of WHV DNA was observed one week after withdrawal of adefovir treatment but no further increase in viral load was observed during the remainder of the 12-week post-treatment follow-up period. The results demonstrate that supplemental adefovir dipivoxil treatment is effective in suppressing replication of lamivudine-resistant B-domain mutants in the woodchuck model of hepatitis B virus infection.
Assuntos
Adenina/análogos & derivados , Adenina/uso terapêutico , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Lamivudina/farmacologia , Organofosfonatos/uso terapêutico , Adenina/administração & dosagem , Adenina/farmacologia , Administração Oral , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , DNA Viral/sangue , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Hepatite B/virologia , Vírus da Hepatite B da Marmota/crescimento & desenvolvimento , Lamivudina/administração & dosagem , Marmota/virologia , Mutação de Sentido Incorreto , Organofosfonatos/administração & dosagem , Organofosfonatos/farmacologia , Estrutura Terciária de Proteína , Viremia , Replicação Viral/efeitos dos fármacosRESUMO
The circadian clock generates and regulates many daily physiological, metabolic and behavioral rhythms as well as acute responses to various types of stresses including those induced by anticancer treatment. It has been proposed that modulatory function of the clock may be used for improving the therapeutic efficacy of established anti-cancer treatments. In order to rationally exploit this mechanism, more information is needed to fully characterize the functional status of the molecular clock in tumors of different cellular origin; however, the data describing tumor clocks are still inconsistent. Here we tested the status of clock in two models of tumors derived from connective tissue: sarcomas spontaneously developed in p53-deficient mice and human fibrosarcoma cells grown as xenografts in immunocompromised severe combined immunodeficient (SCID) mice. We show that both types of tumors retain a functional clock, which is synchronized in phase with normal tissues. We also show that spontaneously developed tumors are not only oscillating in the context of an organism where they receive hormonal and metabolic signals but continue oscillating ex vivo in tissue explants demonstrating that tumors have functional clocks capable of timing all their functions. We also provide evidence that similar to liver, tumors can be synchronized by food availability independent of the central pacemaker in the suprachiasmatic nuclei (SCN). These data provide the basis for the design of anticancer therapies that take into account the circadian metabolic and physiological patterns of both the tumor and normal tissues.
Assuntos
Ritmo Circadiano , Sarcoma/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Comportamento Animal , Linhagem Celular Tumoral , Feminino , Fibrossarcoma/patologia , Humanos , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Transplante de Neoplasias , Proteínas Circadianas Period/metabolismo , Sarcoma/genética , Transdução de Sinais , Núcleo Supraquiasmático/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
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
There is a growing body of evidence that components of the circadian clock are involved in modulation of numerous signaling pathways, and that clock deregulation due to environmental or genetic factors contributes to the development of various pathologies, including cancer. Previous work performed in tissue culture and in in vivo mouse models defined mammalian PERIOD proteins as tumor suppressors, although some experimental inconsistencies (the use of mice on mixed genetic background, lack of sexual discrimination) did not allow a definitive conclusion. To address this issue in a systematic way, we performed a detailed analysis comparing the incidence of tumor development after low-dose ionizing radiation in male and female wild-type, Per1(-/-), and Per2(-/-) mice. We showed that in contrast to previous reports deficiency in either Per1 or Per2 genes by itself does not make mice more tumor-prone; moreover, some of the long-term effects of ionizing radiation in Per2-deficient mice are reminiscent more of accelerated aging rather than tumor-prone phenotype. Our histopathological analysis also revealed significant sexual dimorphism both in the rate of radiation-induced tumorigenesis and in the spectrum of tumors developed, which underscores the importance of using sex-matched experimental groups for in vivo studies. Based on our results, we suggest that the role of PER proteins as bona fide tumor suppressors needs to be reevaluated.
Assuntos
Carcinogênese/efeitos da radiação , Proteínas Circadianas Period/metabolismo , Radiação Ionizante , Envelhecimento , Alelos , Animais , Células Sanguíneas/citologia , Células Sanguíneas/patologia , Peso Corporal/efeitos da radiação , Carcinogênese/genética , Feminino , Longevidade/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/patologia , Proteínas Circadianas Period/deficiência , Proteínas Circadianas Period/genéticaRESUMO
The nutrient-sensing mTOR (mammalian Target of Rapamycin) pathway regulates cellular metabolism, growth functions, and proliferation and is involved in age-related diseases including cancer, type 2 diabetes, neurodegeneration and cardiovascular disease. The inhibition of mTOR by rapamycin, or calorie restriction, has been shown to extend lifespan and delays tumorigenesis in several experimental models suggesting that rapamycin may be used for cancer prevention. This requires continuous long-term treatment making oral formulations the preferred choice of administration route. However, rapamycin by itself has very poor water solubility and low absorption rate. Here we describe pharmacokinetic and biological properties of novel nanoformulated micelles of rapamycin, Rapatar. Micelles of Rapatar were rationally designed to increase water solubility of rapamycin to facilitate oral administration and to enhance its absorption. As a result, bioavailability of Rapatar was significantly increased (up to 12%) compared to unformulated rapamycin, which concentration in the blood following oral administration remained below level of detection. We also demonstrated that the new formulation does not induce toxicity during lifetime administration. Most importantly, Rapatar extended the mean lifespan by 30% and delayed tumor development in highly tumor-prone p53-/- mice. Our data demonstrate that water soluble Rapatar micelles represent safe, convenient and efficient form of rapamycin suitable for a long-term treatment and that Rapatar may be considered for tumor prevention.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Genes p53 , Longevidade/efeitos dos fármacos , Neoplasias/prevenção & controle , Sirolimo/administração & dosagem , Animais , Antibióticos Antineoplásicos/farmacocinética , Disponibilidade Biológica , Feminino , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Nanoestruturas , Neoplasias/genética , Sirolimo/farmacocinéticaRESUMO
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
UNLABELLED: Resolution of hepatitis B virus (HBV) infection is characterized by coordinated humoral and cellular immune responses. Immunity is durable over decades, protecting the host from reinfection and potential activation of residual HBV. Woodchucks infected at birth with woodchuck hepatitis virus (WHV) cleared viremia and developed antibodies to surface antigen (anti-WHs). Woodchucks became seronegative for anti-WHs 3-6 years later, but in some, WHV DNA was detected in serum, liver, and/or peripheral blood mononuclear cells (PBMCs). Those with WHV DNA had increased in vitro cellular immune responses to viral antigens, CD4 and CD8 markers, and Th1-type cytokines, suggesting active WHV-specific T lymphocytes. Immunosuppression for 12 weeks using cyclosporine A in such woodchucks resulted in transient reactivation of WHV replication. Serum of 1 woodchuck that became positive for WHV DNA during immunosuppression was inoculated into WHV-susceptible woodchucks, and a productive infection was demonstrated. The results indicate that after infection durable cellular immunity to WHV is essential for the long-term control of viral replication and is probably maintained by continuous priming from residual virus. CONCLUSION: These experimental observations demonstrate the potential of immunosuppression to reactivate HBV after resolution of infection.
Assuntos
Vírus da Hepatite B da Marmota/fisiologia , Hepatite B/veterinária , Terapia de Imunossupressão/métodos , Replicação Viral/fisiologia , Animais , Antígenos Virais/imunologia , Ciclosporina/farmacologia , DNA Viral/sangue , Hepatite B/imunologia , Vírus da Hepatite B da Marmota/genética , Vírus da Hepatite B da Marmota/imunologia , Imunossupressores/farmacologia , Marmota/virologia , Linfócitos T/imunologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologiaRESUMO
Integrations of woodchuck hepatitis virus (WHV) DNA and rearrangements of the N-myc 2 gene have been detected frequently in hepatocellular carcinoma (HCC) of Eastern woodchucks (Marmota monax) chronically infected with WHV. Fifty-five hepatocellular neoplasms and matched nontumor hepatic tissue specimens obtained postmortem from 13 chronic WHV carriers were analyzed and the frequency of WHV DNA integrations and of N-myc rearrangements compared in tumors of different size and histologic grade. Four small tumor nodules were classified histologically as adenomas and integrated sequences of WHV DNA were detected in two of the four tumor nodules. In one of the two nodules, there was evidence of N-myc rearrangement. Fifty-one neoplasms were classified as HCC. Seven were grade 1 HCCs. WHV DNA integrations were demonstrated in 43% but none had N-myc rearrangements. Twenty grade 2 HCCs had WHV DNA integrations in 80% and in 38% N-myc rearrangements were present. Twenty-four grade 3 HCCs had integrations of WHV DNA in 79% and N-myc rearrangements in 74%. In two other grade 3 HCCs, rearrangements of N-myc were detected in the absence of WHV DNA integrations. The 12 largest tumors in the series all were grade 2 or 3 HCCs, and in 83%, both WHV DNA integrations and N-myc rearrangements were demonstrated. In conclusion, molecular changes observed in this study suggest a progression of genetic alterations providing either a significant proliferative stimulation and/or a growth advantage in hepatocarcinogenesis of woodchucks with chronic WHV infection.
Assuntos
Carcinoma Hepatocelular/patologia , Genes myc/genética , Vírus da Hepatite B da Marmota/genética , Hepatite B Crônica/patologia , Neoplasias Hepáticas/patologia , Animais , Carcinoma Hepatocelular/virologia , Feminino , Rearranjo Gênico , Vírus da Hepatite B da Marmota/crescimento & desenvolvimento , Hepatite B Crônica/virologia , Neoplasias Hepáticas/virologia , Masculino , Marmota , Integração Viral/genética , gama-Glutamiltransferase/sangueRESUMO
The Eastern woodchuck ( Marmota monax ) harbors a DNA virus (Woodchuck hepatitis virus [WHV]) that is similar in structure and replicative life cycle to the human hepatitis B virus (HBV). Like HBV, WHV infects the liver and can cause acute and chronic hepatitis. Furthermore, chronic WHV infection in woodchucks usually leads to development of hepatocellular carcinoma (HCC) within the first 2-4 years of life. The woodchuck model has been important in the preclinical evaluation of safety and efficacy of the antiviral drugs now in use for treatment of HBV infection and continues to serve as an important, predictive model for innovative forms of therapy of hepatitis B using antiviral nucleosides and immune response modifiers alone or in combination. Almost all woodchucks that become chronic WHV carriers after experimental neonatal inoculation develop HCC with a median HCC-free survival of 24 months and a median life expectancy of 30-32 months. The woodchuck model of viral-induced HCC has been used effectively for the development of new imaging agents for enhancement of detection of hepatic neoplasms by ultrasound and magnetic resonance imaging. The chemoprevention of HCC using long-term antiviral nucleoside therapy has been shown in the woodchuck, and "proof of principal" has been established for some of the innovative, molecular methods for treatment of HCC. The model is available for fundamental investigations of the viral and molecular mechanisms responsible for hepatocarcinogenesis and should have substantial value for future development of innovative methods for chemoprevention and gene therapy of human HCC.