RESUMO
OBJECTIVE: To investigate the effects and mechanisms of equol and its enantiomers on urethane-induced lung cancer in mice. METHODS: A total of 120 5-week-old male C57BL/6 mice were randomly divided into 8 groups: lung cancer tumor control group (CG), genistein control group (GCG), low dose racemic equol group (LEG), high dose racemic equol group (HEG), low dose R-equol group (LRE), high dose R-equol group (HRE), low dose S-equol group (LSE) and high dose S-equol group (HSE). Urethane was injected subcutaneously twice a week for 4 weeks to induce lung cancer and then the mice were fed for 4 months. The body weight and food intake of each group were measured and recorded weekly. After the mice were sacrificed, the blood, livers and lungs of the mice were collected. The incidence of lung cancer in each group was recorded. The concentration of serum superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxydeoxygunosine (8-OHdG) were detected by the corresponding kits. Western blotting was used to detect the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the livers. Between-group differences in body weight and food intake of the mice were compared using repeated measures ANOVA, and ANOVA for the differences between non-repeated measurements, with post hoc analysis using Tukey's method if there were between-group differences. Comparisons of categorical data were performed by chi-square test, and if there were differences between the groups, the Bonferroni method was used for pairwise comparison. RESULTS: A total of 49 in the 120 mice developed lung cancer. The overall incidence of lung cancer was 40.8%. Compared with the control group, the incidence of lung cancers in each experimental group was lower, and the difference was statistically significant. The incidence of lung cancer in the high-dose experimental group was significantly lower than that in the low-dose experimental group. However, the incidence of lung cancer was similar in the three equol groups and the genistein group at the same dose. Compared with the control group, the high-dose experimental group had higher serum SOD concentration, lower MDA and 8-OHdG concentrations, and the differences were statistically significant. Western blotting analysis showed that the expression levels of Nrf2 protein in the experimental groups were higher than those in the control group except the low-dose racemic equol group, and the Nrf2 protein expression level in the high-dose equol groups was higher than that in the low-dose equol groups. CONCLUSION: Racemic equol and its enantiomers mayinhibit lung carcinogenesis through antioxidant effects.
Assuntos
Equol , Neoplasias Pulmonares , Animais , Peso Corporal , Genisteína , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Superóxido Dismutase , Uretana/toxicidadeRESUMO
The carcinogen urethane induces pulmonary tumors in mice initiated by an incredibly specific Q61L/R oncogenic mutation in the proto-oncogene Kras. Previous Whole-Exome Sequencing of urethane-induced tumors revealed a bias towards AâT/G and GâA substitutions. Subsequent ultra-sensitive Maximum-Depth Sequencing of Kras shortly after urethane exposure suggest a further refinement to CAâCT/G substitutions. As C182AAâC182T/GA substitutions in Kras result in Q61L/R mutations, the extreme bias of urethane towards these genomic driver mutations can be ascribed to the specificity of the carcinogen for CAâCT/G substitutions. However, we previously found that changing rare codons to common in the Kras gene to increase protein expression shifted mutations in urethane-induced tumors away from Kras, or when detected in Kras, to G12D mutations that are usually rarely detected in such tumors. Moreover, the loss of p53 partially reversed this effect, generating tumors with either Q61L/R or G12D oncogenic Kras mutations, or no Kras mutations, presumably due to other genomic driver mutations. Determining the origin of these G12D and other unknown non-canonical genomic driver mutations would provide critical insight into the extreme bias of carcinogens for specific genomic driver mutations. We thus compared the types of Single Nucleotide Variations detected by previously performed Maximum-Depth Sequencing immediately after urethane exposure to the mutation signatures derived from Whole Exome Sequencing of urethane-induced tumors. This identified two types of non-canonical mutations. First, a V637E oncogenic mutation in the proto-oncogene Braf that conforms to the mutation signature of urethane, suggesting that the mutational bias of the carcinogen may account for this non-canonical mutation, similar to that for canonical Q61L/R mutations in Kras. Second, G12D and Q61H mutations in Kras that did not fit this mutation signature, and instead shared similarity with Single Nucleotide Variations detected by Maximum-Depth Sequencing from normal cells, suggesting that perhaps these mutations were pre-existing. We thus posit that when canonical Kras mutations are selected against that the carcinogen may instead promote the expansion of pre-existing genomic driver mutations, although admittedly we cannot rule out other mechanisms. Interrogating the mutation signatures of human lung cancers similarly identified KRAS genomic driver mutations that failed to match the mutation signature of the tumor. Thus, we also speculate that the selection for non-canonical genomic driver mutations during urethane carcinogenesis may reflect the process by which discordance between genomic driver mutations and mutational signatures arises in human cancers.
Assuntos
Neoplasias Pulmonares , Uretana , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Carcinógenos/toxicidade , Genômica , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Nucleotídeos , Proteínas Proto-Oncogênicas p21(ras)/genética , Uretana/toxicidadeRESUMO
Ethyl carbamate (EC, urethane), which is used as an anesthetic especially by veterinarians due to its very long duration of action, is also a naturally occurring compound in all fermented foods and beverages. Although the health problem of EC is related to its carcinogenic potential, the scarcity of current studies that can be used in the evaluation of usage limits encouraged us to do this study. In this context, zebrafish embryos were exposed to serial doses of EC. According to the results, it was observed that EC exposure caused a significant decrease in survival and hatching rates as well as significant body malformations. Whole-mount staining results showed that EC caused dose-dependent increased apoptosis. Oxidative stress caused by EC exposure was demonstrated by whole-mount staining, transcriptional and immunohistochemically. Furthermore, it has been shown histochemically that EC exposure causes necrosis and degeneration in the brain. In behavioral tests, it was observed that EC caused hyperactivity associated with these neuronal degenerations. In addition, a dramatic decrease in blood flow was detected in association with pericardial edema. In the light of the current results, it should be carefully considered that EC can be found naturally in many human diets, especially fermented foods.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Uretana/toxicidade , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal , Relação Dose-Resposta a Droga , Embrião não Mamífero/anormalidades , Imuno-Histoquímica , Larva/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica , Uretana/administração & dosagem , Peixe-ZebraRESUMO
This study aimed to assess the suitability of hyperpolarized 129Xe (HPXe) MRI for noninvasive longitudinal evaluation of pulmonary function in preclinical lung cancer models. A mouse model of lung cancer (LC) was induced in 5 mice by intraperitoneal injection of urethane, while a negative-control (NC) mice (N = 5) was prepared by injection of saline solution. Longitudinal HPXe MRI was performed over a 5-month period to monitor lung ventilation and gas exchange. The treatment efficacy of ethyl pyruvate (EP), an anti-inflammatory drug, to the mouse LC model was monitored using HPXe MRI by commencing administration of EP pre (early-phase) and 1-month post (late-phase) injection of urethane (N = 5 mice for each group). Gas-exchange function in LC mice was significantly reduced at 1-month after urethane injection compared with NC mice administered with saline (P < 0.01). Thereafter, it remained consistently lower than that of the NC group for the full 5-month measurement period. In contrast, the ventilation function of the LC model mice was not significantly different to that of the NC mice. Histological analysis revealed alveolar epithelial hyperplasia in LC mice alveoli at 1 month after urethane injection, and adenoma was confirmed 3 months after the injection. The early- and late-phase EP interventions were found to improve HPXe MRI metrics (reduced at 1 month postinjection of urethane) and significantly inhibit tumor growth. These results suggest that HPXe MRI gas-exchange metrics can be used to quantitatively assess changes in the precancerous lesion microenvironment and to evaluate therapeutic efficacy in cancer. Thus, HPXe MRI can be utilized to noninvasively monitor pulmonary pathology during LC progression and can visualize functional changes during therapy.
Assuntos
Inflamação/tratamento farmacológico , Neoplasias Pulmonares/patologia , Imageamento por Ressonância Magnética/métodos , Piruvatos/farmacologia , Uretana/toxicidade , Xenônio/química , Animais , Carcinógenos/toxicidade , Inflamação/etiologia , Inflamação/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/complicações , Masculino , CamundongosRESUMO
In spite of the overwhelming epidemiological evidence for cigarette smoke (CS) carcinogenicity, less attention has been paid to the effects of CS as a complex mixture. As assessed in a series of experiments in murine models, the whole-body exposure to mainstream CS induced significant increases of micronucleated cells in the respiratory tract, bone marrow and peripheral blood of adult mice as well as in the liver and peripheral blood of foetuses whose mothers had been exposed throughout pregnancy. Urethane was potently clastogenic in the same cells when injected intraperitoneally. The daily administration of extra-virgin olive oil by gavage produced evident and consistent protective effects in all monitored experimental systems. In contrast, sunflower oil exhibited some adverse effects. Curcumin did not produce any significant effect in the bone marrow of both CS-exposed adults and foetuses but it elicited a dose-dependent protective effect traceable in blood erythrocytes. However, the higher curcumin dose further increased the frequency of micronucleated pulmonary alveolar macrophages. The apparent protective effects produced by lycopene and by a carotenoid mix were overwhelmed by those produced by olive oil, and lycopene even exhibited a worsening effect on the frequency of micronucleated erythroblasts in the bone marrow of urethane-treated adult mice.
Assuntos
Mutagênicos/toxicidade , Substâncias Protetoras/farmacologia , Poluição por Fumaça de Tabaco/efeitos adversos , Uretana/toxicidade , Animais , Curcumina/farmacologia , Eritrócitos/efeitos dos fármacos , Feminino , Feto/efeitos dos fármacos , Licopeno/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Azeite de Oliva/farmacologia , GravidezRESUMO
RAS genes are commonly mutated in human cancer. Despite many possible mutations, individual cancer types often have a 'tropism' towards a specific subset of RAS mutations. As driver mutations, these patterns ostensibly originate from normal cells. High oncogenic RAS activity causes oncogenic stress and different oncogenic mutations can impart different levels of activity, suggesting a relationship between oncoprotein activity and RAS mutation tropism. Here, we show that changing rare codons to common in the murine Kras gene to increase protein expression shifts tumors induced by the carcinogen urethane from arising from canonical Q61 to biochemically less active G12Kras driver mutations, despite the carcinogen still being biased towards generating Q61 mutations. Conversely, inactivating the tumor suppressor p53 to blunt oncogenic stress partially reversed this effect, restoring Q61 mutations. One interpretation of these findings is that the RAS mutation tropism of urethane arises from selection in normal cells for specific mutations that impart a narrow window of signaling that promotes proliferation without causing oncogenic stress.
Assuntos
Genes ras/genética , Neoplasias Pulmonares/genética , Mutação/genética , Uretana/toxicidade , Animais , Carcinogênese , Transformação Celular Neoplásica/genética , Neoplasias Pulmonares/induzido quimicamente , Camundongos , Camundongos da Linhagem 129RESUMO
Short-term (26 weeks) Tg.rasH2 mouse carcinogenicity studies have been conducted as an alternative model to the conventional 2-year mouse carcinogenicity studies, using urethane as a positive control material. In these studies, urethane was used at a dose of 1,000 mg/kg/dose, administered intraperitoneally on days 1, 3, and 5. Urethane consistently produces lung adenomas and carcinomas and hemangiosarcomas of the spleen, proving validity of the assay. We conducted 3 pilot studies at 3 different sites of Charles River Laboratories using a lower dose of urethane (500 mg/kg/dose), administered on days 1, 3, and 5, followed by a 12-week observation period. Our results demonstrate that a lower dose can be used successfully with fewer number of animals per sex to prove the validity of the assay. However, based on our cumulative experience with this model, we propose to eliminate positive control dose groups in future Tg.rasH2 carcinogenicity studies.
Assuntos
Experimentação Animal , Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Esplênicas/induzido quimicamente , Uretana/toxicidade , Animais , Feminino , Neoplasias Pulmonares/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Esplênicas/fisiopatologiaRESUMO
Chemical induced carcinogenesis together with genetically engineered mouse models represent important approaches for the study of the complex mechanisms involving genotype and environmental factors in cancer development, including lung cancer. The induction of lung tumor in mice with urethane (ethyl carbamate) is considered a valuable model of Kras-driven lung cancer. However, inbred mouse strains show variable susceptibility to lung tumor formation, with C57BL/6 background, widely used to study many transgenic and null mutations, highly resistant to lung carcinogenesis. Here is described a protocol of urethane-induced lung cancer effective in lung tumor induction in C57BL/6J strain. Multiple urethane injections are needed to overcome genetic resistance and induce in a reproducible manner lung carcinogenesis in C57BL/6J background mice.
Assuntos
Neoplasias Pulmonares , Uretana , Animais , Carcinogênese/genética , Pulmão , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos C57BL , Uretana/toxicidadeRESUMO
Time-to-event data often violate the proportional hazards assumption inherent in the popular Cox regression model. Such violations are especially common in the sphere of biological and medical data where latent heterogeneity due to unmeasured covariates or time varying effects are common. A variety of parametric survival models have been proposed in the literature which make more appropriate assumptions on the hazard function, at least for certain applications. One such model is derived from the First Hitting Time (FHT) paradigm which assumes that a subject's event time is determined by a latent stochastic process reaching a threshold value. Several random effects specifications of the FHT model have also been proposed which allow for better modeling of data with unmeasured covariates. While often appropriate, these methods often display limited flexibility due to their inability to model a wide range of heterogeneities. To address this issue, we propose a Bayesian model which loosens assumptions on the mixing distribution inherent in the random effects FHT models currently in use. We demonstrate via simulation study that the proposed model greatly improves both survival and parameter estimation in the presence of latent heterogeneity. We also apply the proposed methodology to data from a toxicology/carcinogenicity study which exhibits nonproportional hazards and contrast the results with both the Cox model and two popular FHT models.
Assuntos
Teorema de Bayes , Análise de Sobrevida , Algoritmos , Animais , Camundongos , Modelos de Riscos Proporcionais , Processos Estocásticos , Uretana/toxicidadeRESUMO
In Tg-rasH2 carcinogenicity mouse models, a positive control group is treated with a carcinogen such as urethane or N-nitroso-N-methylurea to test study validity based on the presence of the expected proliferative lesions in the transgenic mice. We hypothesized that artificial intelligence-based deep learning (DL) could provide decision support for the toxicologic pathologist by screening for the proliferative changes, verifying the expected pattern for the positive control groups. Whole slide images (WSIs) of the lungs, thymus, and stomach from positive control groups were used for supervised training of a convolutional neural network (CNN). A single pathologist annotated WSIs of normal and abnormal tissue regions for training the CNN-based supervised classifier using INHAND criteria. The algorithm was evaluated using a subset of tissue regions that were not used for training and then additional tissues were evaluated blindly by 2 independent pathologists. A binary output (proliferative classes present or not) from the pathologists was compared to that of the CNN classifier. The CNN model grouped proliferative lesion positive and negative animals at high concordance with the pathologists. This process simulated a workflow for review of these studies, whereby a DL algorithm could provide decision support for the pathologists in a nonclinical study.
Assuntos
Aprendizado Profundo , Uretana , Algoritmos , Animais , Inteligência Artificial , Carcinógenos/toxicidade , Compostos de Metilureia , Camundongos , Camundongos Transgênicos , Uretana/toxicidadeRESUMO
BACKGROUND: Rubus chingii Hu is a widely cultivated fruit in China and has declared multiple bioactivities including antioxidative activity. Ethyl carbamate (EC), mostly found in fermented food and alcoholic beverages, is a recognized human carcinogen, and researchers have proposed the correlation between oxidative stress and its toxicity. This study acquired the polysaccharide from R. chingii (RP) and explored its effect on EC-induced cytotoxicity using Caco-2 cells as the cell model. RESULTS: Results showed that RP exhibited protection against EC-induced toxicity by repairing redox imbalance as indicative of mitigated mitochondrial membrane potential collapse, attenuated reactive oxygen species overproduction, and impeded glutathione depletion. Moreover, the structural features of RP were characterized and revealed that it was mainly constituted by galacturonic acid and arabinose, with an average molecular weight of 7.039 × 105 g mol-1 . CONCLUSION: Overall, our results provided a new approach dealing with the toxicity caused by EC from the perspective of oxidative stress and described a new potential healthy value of R. chingii Hu, which could contribute to the development of a promising dietary supplement and functional food. © 2020 Society of Chemical Industry.
Assuntos
Extratos Vegetais/farmacologia , Polissacarídeos/farmacologia , Substâncias Protetoras/farmacologia , Rubus/química , Uretana/toxicidade , Antioxidantes , Células CACO-2/efeitos dos fármacos , Células CACO-2/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Polissacarídeos/isolamento & purificação , Substâncias Protetoras/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lung cancer remains incurable; therefore, novel therapeutical approaches are of great demand. This study was designed to investigate the effectiveness of cisplatin nanoparticles combined with vitamin-D3 on urethane-induced early lung cancer in rats and to clarify the underlying signaling mechanisms. Early lung cancer was induced in male Wistar rats by urethane. Rats were divided into six groups: I-control, II-cancer untreated, III-cancer + free cisplatin, IV-cancer + cisplatin nanoparticles, V-cancer + free cisplatin + vitamin-D3 , VI-cancer + cisplatin nanoparticles + vitamin-D3 . Inflammation, proliferation, and apoptosis were evaluated together with the levels of tumor marker CK-19 along with histological assessment. Treatment of lung cancer with either free or nanoparticles of cisplatin alone demonstrated significant suppression in the expression of inflammatory, anti-apoptotic and tumor markers compared to rats with lung cancer. Moreover, vitamin-D3 supplementation with either cisplatin forms lead to a further decrease of all markers, markedly with cisplatin nanoparticles. The present study shows the synergistic effect of cisplatin-nanoparticles combined with vitamin-D3 as a new therapy regimen against lung cancer. Further studies with larger sample sizes and longer duration are needed to confirm these results.
Assuntos
Colecalciferol/farmacologia , Cisplatino/farmacologia , Modelos Animais de Doenças , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/administração & dosagem , Uretana/toxicidade , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose , Carcinógenos/toxicidade , Colecalciferol/administração & dosagem , Cisplatino/administração & dosagem , Quimioterapia Combinada , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Masculino , Nanopartículas/química , Ratos , Ratos Wistar , Transdução de Sinais , Vitaminas/administração & dosagem , Vitaminas/farmacologiaRESUMO
Ethyl-4-bromophenyl-carbamate (LQM 919) and Ethyl-4-chlorophenyl-carbamate (LQM 996) are compounds that inhibit egg-laying and hatching of tick larvae that are resistant to conventional ixodicides. The structure-activity relationship (SAR) to get the endpoint predictions of mutagenicity and carcinogenicity of the LQM 919 and LQM 996 was performed and the absence of mutagenicity was confirmed by Ames test. SAR analysis show no structural alerts indicating the ability of ethyl-carbamates to bind biomolecules or estrogen receptors. Endpoint of mutagenicity with and without metabolic activation showed that the ethyl-carbamates were negative (p <0.05) for mutagenicity induction in strains TA97, TA98, TA102, TA1535, TA1537 and TA1538 of Salmonella typhimurium. Pre-incubation with different ethyl-carbamate concentrations did not increase the number of spontaneously reverting colonies; moreover, the compounds did not induce a concentration-dependent increase in the number of reverting colonies in any of the strains used. This confirmed the absence of mutagenic activity in this test system. Exogenous metabolic activation did not modify these observations; suggesting that no metabolites with mutagenic activity were present. The endpoint of carcinogenicity in rats were negative for LQM 919 (p <0.05,) and LQM 996 (p <0.001). The results of the present study strongly suggest that ethyl-carbamates do not represent a risk for cancer in mammals.
Assuntos
Carcinógenos/química , Carcinógenos/toxicidade , Ixodidae/efeitos dos fármacos , Mutagênicos/química , Mutagênicos/toxicidade , Uretana/química , Uretana/toxicidade , Animais , Salmonella typhimurium/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Ethyl carbamate (EC) is a carcinogen toxicant, commonly found in fermented foods and beverages. The carcinogenic and toxic possibility of EC is thought to be related to its metabolite vinyl carbamate (VC). However, we found interesting mechanisms underlying VC-induced toxicity in this study, which were greatly different from EC. We first conducted a simple synthesis procedure for VC and found that VC possessed higher toxicity but failed to regulate levels of reactive oxygen species, glutathione, and autophagy. Notably, VC treatment resulted in upregulation of lysosomal pH, which was responsible for its cytotoxicity. Cyclic adenosine monophosphate (cAMP) pretreatment could enhance restoration of lysosomal acidity and ameliorate VC-induced damage. Inhibition of protein kinase A and cystic fibrosis transmembrane conductance regulator can block cAMP-induced cytoprotection. Together, our results provided the evidence for novel mechanisms of toxicity and possible protection method under VC exposure, which might give new perspectives on the study of EC-induced toxicity.
Assuntos
Carcinógenos/toxicidade , Lisossomos/química , Lisossomos/efeitos dos fármacos , Uretana/análogos & derivados , Ácidos/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Glutationa/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Uretana/toxicidadeRESUMO
Manganese superoxide dismutase (SOD-2), an important primary antioxidant enzyme located in mitochondria, plays a critical role in tumor progression. Reportedly, the proinflammatory cytokine, tumor necrosis factor (TNF)-α, can increase SOD-2 expression in a human lung adenocarcinoma cell line in vitro, indicating that TNF-α-mediated inflammation may regulate SOD-2 expression, which may be related to cancer promotion. Using a urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mice model, we investigated whether and how TNF-α-mediated inflammation upregulated SOD-2 expression in lung adenocarcinoma. Our results showed that SOD-2 was mostly expressed on surfactant protein-C+ AT-II cells (alveolar type II cell) and tumor cells in IDLA mice, which were surrounded by CD68+ macrophages. Blocking TNF-α-dependent inflammation downregulated SOD-2 expression in inflamed lung tissues at the protumor stage and also inhibited SOD-2 expression in tumor cells in the IDLA model. In human lung adenocarcinoma, both the number of infiltrating CD68+ macrophages and TNF-α expression correlated positively with SOD-2 expression, which is related to lymph node metastasis and TNM stage. We collected the conditioned medium from lipopolysaccharide-activated phorbol myristate acetate-induced THP1 (M1) cells to stimulate A549 and H1299 cells and observed that THP1-M1 upregulated SOD-2 by secreting TNF-α. Blocking SOD-2 expression significantly inhibited TNF-α-induced cell proliferation in A549 and H1299 cells in vitro. Thus, TNF-α-mediated lung inflammation can upregulate SOD-2 expression in lung adenocarcinoma, and macrophages contribute to SOD-2 upregulation by secreting TNF-α.
Assuntos
Adenocarcinoma de Pulmão/patologia , Proliferação de Células , Neoplasias Pulmonares/patologia , Pneumonia/complicações , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Uretana/toxicidade , Adenocarcinoma de Pulmão/etiologia , Adenocarcinoma de Pulmão/metabolismo , Animais , Apoptose , Carcinógenos/toxicidade , Citocinas , Feminino , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy fails in the majority of patients with cancer. Oncolytic viruses represent a new class of therapeutic agents, yet the therapeutic efficacy is still disappointing. Moreover, intratumoral injection of viruses is the main approach and preclinical studies mainly employ syngeneic or xenograft models. METHODS: Use an endogenous mouse lung cancer model that faithfully recapitulates human lung cancer, and various in vivo, ex vivo and in vitro assays, to investigate the efficacy, mechanism of action and resistance of systemically administered oncolytic vaccinia virus (oVV), immunotherapy and their combination, to find an effective therapy for refractory lung cancer. RESULTS: Resembling human lung cancers, the majority of which are largely resistant to PD-1/PD-L1 blockade and with decreased PD-L1 expression and T-cell activation by our analysis, urethane-induced endogenous lung tumors in mice show reduced PD-L1 expression, low tumor-infiltrating lymphocytes and innate resistance to PD-1/PD-L1 blockade. Intravenous administration of oVV has efficacy and synergizes with simultaneous but not single blockade of PD-1 and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) in this cancer model. Besides direct tumor cell killing, oVV induces T-cell lung recruitment, tumor infiltration, along with expression of PD-1 and TIM-3 on T cells and PD-1 and TIM-3 ligands on tumor cells and tumor-associated immune cells. Blockade of PD-1 or TIM-3 also causes their mutual induction on T cells. CONCLUSIONS: While systemic administration of oVV shows efficacy in lung cancer by killing tumor cells directly and recruiting and activating T cells for indirect tumor killing, its induction of PD-1 and TIM-3 on T cells and PD-1 and TIM-3 ligands on tumors and tumor-associated immune cells as well as mutual induction of PD-1 or TIM-3 on T cells by their blockade restricts the efficacy of oVV or its combination with single PD-1 or TIM-3 blockade. The triple combination therapy is more effective for refractory lung cancer, and possibly other cold cancers as well.
Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Imunoterapia/métodos , Neoplasias Pulmonares/terapia , Neoplasias Experimentais/terapia , Terapia Viral Oncolítica/métodos , Animais , Linhagem Celular Tumoral/transplante , Terapia Combinada/métodos , Feminino , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Injeções Intralesionais , Injeções Intravenosas , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/imunologia , Ativação Linfocitária , Camundongos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/imunologia , Vírus Oncolíticos/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Uretana/administração & dosagem , Uretana/toxicidadeRESUMO
Betulinic acid (BA) is a pentacyclic triterpenoid found in several plant species. Urethane (URE) is a known promutagen. Here, we examine the genotoxicity and mutagenicity of BA alone or in combination with URE using the bone marrow micronucleus assay in mice bone marrow cells and the Somatic Mutation and Recombination Test in Drosophila melanogaster. Findings revealed that BA alone was not genotoxic, but reduced the frequency of micronucleus when compared to the positive control. No significant differences were observed in the cytotoxicity. Biochemical analyzes showed no significant differences for liver (AST and ALT) or renal (creatinine and urea) function parameters, indicating the absence of hepatotoxic and nephrotoxic effects. BA alone did not increase the frequency of mutant spots, but reduced the total frequency of mutant spots when co-administered with URE in both ST and HB crosses. In addition, BA reduced the recombinogenic effect of URE at the highest concentrations of both crosses. In conclusion, under experimental conditions, BA has modulatory effects on the genotoxicity induced by URE in mice, as well as in somatic cells of D. melanogaster. We suggest that the modulatory effects of BA may be mainly due to its antioxidant and apoptotic properties.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Mutagênese/efeitos dos fármacos , Triterpenos/farmacologia , Uretana/toxicidade , Animais , Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Medula Óssea/efeitos dos fármacos , Carcinógenos/farmacologia , Drosophila melanogaster/genética , Feminino , Cabelo/efeitos dos fármacos , Masculino , Camundongos , Testes de Mutagenicidade , Triterpenos Pentacíclicos , Taxa de Sobrevida , Tricomas/efeitos dos fármacos , Triterpenos/química , Asas de Animais/efeitos dos fármacos , Ácido BetulínicoRESUMO
BACKGROUND: Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. Inhibition of Notch receptor activation (e.g. Notch3) with γ-secretase inhibitor is a potential new therapeutic approach for the targeted therapy of non-small cell lung cancer (NSCLC). However, only a few safe and effective γ-secretase inhibitors have been discovered. Evodiamine (EVO), a compound derived from Euodiae Fructus (Chinese name, Wu-Zhu-Yu), exhibits remarkable anti-NSCLC activities. However, the underlying mechanisms of action have yet to be fully elucidated. PURPOSE: We sought to determine the involvement of Notch3 signaling in the anti-NSCLC effects of EVO, and to explore whether EVO suppressed Notch3 signaling by inhibiting γ-secretase in cultured A549 and H1299 NSCLC cells and in urethane-induced lung cancer FVB mouse model. METHODS: Cell viability, migration, stemness and cell cycle distribution of EVO were examined by the MTT assay, wound healing assay, soft agar colony assay and flow cytometry analysis, respectively. The binding affinity of EVO and γ-secretase complex was analyzed by molecular docking. Cellular thermal shift assay (CETSA) was performed to study the drug-target interactions in NSCLC cells. Protein levels were determined by Western blotting. RESULTS: EVO dramatically inhibited cell viability, induced G2/M cell cycle arrest, suppressed cell migration, and reduced stemness in NSCLC cells. Mechanistic studies indicated that EVO prevented the γ-secretase cleavage of Notch3 at the cell surface and hence inhibited Notch3 activation. Moreover, EVO notably reduced tumor growth in the mouse model and inhibited Notch3 activity in the tumors. CONCLUSION: This study provides new insights into the anti-NSCLC action of EVO, and suggests that suppressing Notch3 signaling by inhibiting γ-secretase is a mechanism of action underlying the anti-NSCLC effect of EVO.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/farmacologia , Receptor Notch3/metabolismo , Células A549 , Secretases da Proteína Precursora do Amiloide/química , Animais , Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Evodia/química , Feminino , Humanos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos , Simulação de Acoplamento Molecular , Transdução de Sinais/efeitos dos fármacos , Uretana/toxicidadeRESUMO
Apolipoprotein E (ApoE) is known to regulate lipid homeostasis and associated with atherosclerogenesis. Eventhough atherosclerogenesis is associated with tumor development, the role of ApoE in lung tumorigenesis and metastasis is not clear. Thus, the tumor growth and metastasis were compared in WT and ApoE knockout (KO) mice. Urethane-induced lung tumor incidence and B16F10 lung metastasis in ApoE knockout (KO) mice were significantly reduced in comparison to that in WT mice. Knockdown of ApoE expression in lung cancer cells and B16F10 cells also decreased cancer cell growth and metastasis. The inhibitory effect of ApoE KO on tumor development and metastasis was associated with increase of infiltration of NK cells. NK cells derived from ApoE KO mice showed much greater cytotoxicity than those from WT mice. These cytotoxic effect of NK cells derived from ApoE KO mice was associated with higher expression of Granzyme B, Fas Ligand, IFN-γ, TNF-α, NKG2D, NKp46, and DNAM-1 expression. Triggering receptor expressed on myeloid cell (TREM)-1 is a proinflammatory mediator expressed on NK cells, and is known to be associated with NK cell cytotoxicity. Thus, we investigated the role of TREM-1 on ApoE KO mice originated NK cell mediated cytotoxicity for cancer cells. Blockade of TREM-1 expression with a TREM-1 antagonist prevented NK cell-mediated cytotoxicity. TREM-1 antibody recovered cytotoxic effect of NK cells derived from KO mice of T-bet, which upregulating gene for TREM-1. These data indicate that ApoE KO suppressed lung tumor development and metastasis via increase of TREM-1-dependent anti-tumor activity of NK cells.
Assuntos
Apolipoproteínas E/genética , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Proteínas com Domínio T/genética , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Humanos , Neoplasias Pulmonares/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Células Progenitoras Mieloides/metabolismo , Metástase Neoplásica/genética , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Uretana/toxicidadeRESUMO
Lung adenocarcinoma (LADC) is the leading cause of cancer death worldwide. Nevertheless, syngeneic mouse models of the disease are sparse, and cell lines suitable for transplantable and immunocompetent mouse models of LADC remain unmet needs. We established multiple mouse LADC cell lines by repeatedly exposing two mouse strains (FVB, Balb/c) to the tobacco carcinogens urethane or diethylnitrosamine and by culturing out the resulting lung tumours for prolonged periods of time. Characterization of the resulting cell lines (n = 7) showed that they were immortal and phenotypically stable in vitro, and oncogenic, metastatic and lethal in vivo. The primary tumours that gave rise to the cell lines, as well as secondary tumours generated by transplantation of the cell lines, displayed typical LADC features, such as glandular architecture and mucin and thyroid transcription factor 1 expression. Moreover, these cells exhibited marked molecular similarity with human smokers' LADC, including carcinogen-specific Kras point mutations (KrasQ61R in urethane- and KrasQ61H in diethylnitrosamine-triggered cell lines) and Trp53 deletions and displayed stemness features. Interestingly, all cell lines overexpressed proliferin, a murine prolactin orthologue, which functioned as a lung tumour promoter. Furthermore, prolactin was overexpressed and portended poor prognosis in human LADC. In conclusion, we report the first LADC cell lines derived from mice exposed to tobacco carcinogens. These cells closely resemble human LADC and provide a valuable tool for the functional investigation of the pathobiology of the disease.