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DNA double-strand breaks (DSBs) seriously damage DNA and promote genomic instability that can lead to cell death. They are the source of conditions such as carcinogenesis and aging, but also have important applications in cancer therapy. Therefore, rapid detection and quantification of DSBs in cells are necessary for identifying carcinogenic and anticancer factors. In this study, we detected DSBs using a flow cytometry-based high-throughput method to analyze γH2AX intensity. We screened a chemical library containing 9600 compounds and detected multiple DNA-damaging compounds, although we could not identify mechanisms of action through this procedure. Thus, we also profiled a representative compound with the highest DSB potential, DNA-damaging agent-1 (DDA-1), using a bioinformatics-based method we termed "molecular profiling." Prediction and verification analysis revealed DDA-1 as a potential inhibitor of topoisomerase IIα, different from known inhibitors such as etoposide and doxorubicin. Additional investigation of DDA-1 analogs and xenograft models suggested that DDA-1 is a potential anticancer drug. In conclusion, our findings established that combining high-throughput DSB detection and molecular profiling to undertake phenotypic analysis is a viable method for efficient identification of novel DNA-damaging compounds for clinical applications.
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Antineoplásicos , Quebras de DNA de Cadeia Dupla , Humanos , Histonas/metabolismo , Etoposídeo/farmacologia , Reparo do DNA , Dano ao DNA , DNARESUMO
BACKGROUND: Low-invasive stereotactic body radiation therapy is a novel anti-arrhythmic strategy. The mechanisms underlying its effects against ventricular tachycardia/fibrillation (VT/VF) are gradually becoming clear, whereas those underlying atrial tachycardia/fibrillation (AT/AF) remain unknown. This study investigated the effects of carbon ion beam on gap junction expression and sympathetic innervation.MethodsâandâResults: Atrial and ventricular tachyarrhythmia models was established in 26 hypercholesterolemic (HC) 3-year-old New Zealand white rabbits; 12 rabbits were irradiated with a single 15-Gy carbon ion beam (targeted heavy ion irradiation [THIR]) and 14 were not (HC group). Eight 3-month-old rabbits (Young) were used as a reference group. In vivo induction frequencies in the Young, HC, and HC+THIR groups were 0%, 9.9%, and 1.2%, respectively, for AT/AF and 0%, 7.8%, and 1.2%, respectively, for VT/VF (P<0.01). The conduction velocity of the atria and ventricles on optical mapping was significantly reduced in the HC group; this was reversed in the HC+THIR group. Connexin-40 immunolabelling in the atria was 66.1-78.7% lower in the HC than Young group; this downregulation was less pronounced in the HC+THIR group (by 23.1-44.4%; P<0.01). Similar results were obtained for ventricular connexin-43. Sympathetic nerve densities in the atria and ventricles increased by 41.9-65.3% in the HC vs. Young group; this increase was reversed in the HC+THIR group. CONCLUSIONS: Heavy ion radiation reduced vulnerability to AT/AF and VT/VF in HC elderly rabbits and improved cardiac conductivity. The results suggest involvement of connexin-40/43 upregulation and suppression of sympathetic nerve sprouting.
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Fibrilação Atrial , Íons Pesados , Taquicardia Ventricular , Animais , Coelhos , Átrios do Coração , Fibrilação Ventricular , Junções Comunicantes , Conexinas , CarbonoRESUMO
Relatively young (4-week-old) selenium deficient (SeD) mice, which lack the activity of selenium-dependent glutathione peroxidase (GSH-Px) isomers, were prepared using torula yeast-based SeD diet. Mice were fed the torula yeast-based SeD diet and ultra-pure water. Several different timings for starting the SeD diet were assessed. The weekly time course of liver comprehensive GSH-Px activity after weaning was monitored. Protein expression levels of GPx1 and 4 in the liver were measured by Western blot analysis. Gene expression levels of GPx1, 2, 3, 4, and 7 in the liver were measured by quantitative real-time PCR. Apoptotic activity of thymocytes after hydrogen peroxide (H2O2) exposure was compared. Thirty-day survival rates after whole-body X-ray irradiation were estimated. Pre-birth or right-after-birth starting of the SeD diet in dams was unable to lead to creation of SeD mice due to neonatal death. This suggests that Se is necessary for normal birth and healthy growing of mouse pups. Starting the mother on the SeD diet from 2 weeks after giving birth (SeD-trial-2w group) resulted in a usable SeD mouse model. The liver GSH-Px activity of the SeD-trial-2w group was almost none from 4 week olds, but the mice survived for more than 63 weeks. Protein and gene expression of GPx1 was suppressed in the SeD-trial-2w group, but that of GPx4 was not. The thymocytes of the SeD-trial-2w group were sensitive to H2O2-induced apoptosis. The SeD-trial-2w group was sensitive to whole-body X-ray irradiation compared with control mice. The SeD-trial-2w model may be a useful animal model for H2O2/hydroperoxide-induced oxidative stress.
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The establishment of cancer cell lines, which have different metastatic abilities compared with the parental cell, is considered as an effective approach to investigate mechanisms of metastasis. A highly metastatic potential mouse colon cancer cell subline, Colon-26MGS, was derived from the parental cell line Colon-26 by in vivo selection using continuous subcutaneous implanting to immunocompetent mice. To clarify the mechanisms involved in the enhancement of metastasis, morphological characteristics, cell proliferation, and gene expression profiles were compared between Colon-26MGS and the parental cell. Colon-26MGS showed over 10 times higher metastatic ability compared with the parental cell, but there were no differences in morphological characteristics and in vitro proliferation rates. In addition, the Colon-26MGS-bearing mice exhibited no marked change of splenocyte population and lung pre-metastatic niche with tumor-free mice, but there were significant differences compared to Colon-26-bearing mice. RNA-seq analyses indicated that immune costimulatory molecules were significantly up-regulated in Colon-26MGS. These results suggest that Colon-26MGS showed not only higher metastatic activity, but also less induction property of host immune response compared to parental Colon-26. Colon-26MGS has proven to be a novel useful tool for studying multiple mechanisms involving metastasis enhancement.
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Carcinoma/metabolismo , Carcinoma/secundário , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Carcinoma/genética , Carcinoma/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , RNA-SeqRESUMO
The quantitative evaluation of changes in the redox state induced by low linear energy transfer (LET) radiations such as the plateau region of heavy-ion beams via formation of reactive oxygen species is of considerable importance to eliminate the adverse effects of radiation therapy on normal tissues adjacent to a tumour. In this study, a 2,2-diphenyl-1-picrylhydrazyl radical (DPPHË) was used as a redox probe to estimate the redox states of protic and aprotic solutions irradiated by low LET carbon-ion (C-ion) beams. The dose dependence of the decrease in the absorption band due to DPPHË (which was solubilised by ß-cyclodextrin (ß-CD) in water) after irradiation with low LET C-ion beams (13 keV µm-1) was similar to that after X-irradiation. Similar results were obtained when H2O was replaced with methanol or acetonitrile although the slope values of the plots of the absorbance changes vs. radiation doses were twice larger as compared to the case in ß-CD-containing H2O. Moreover, DPPHË was more susceptible to the C-ion beam than to X-rays in isopropyl myristate (IPM), which is one of the saturated fatty acid esters.
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Our aim was to evaluate whether repetition of C-ion (carbon ion beam) irradiation induces radioresistance as well as repeated X-ray irradiation in cancer cell lines, and to find the key molecular pathway for radioresistance by comparing radioresistant cancer cells with their parental cells. A mouse squamous cell carcinoma cell line, NR-S1, and radioresistant cancer cells, NR-S1-C30 (C30) and NR-S1-X60 (X60), established by repetition of C-ion and X-ray irradiation, respectively, were used. X-ray and C-ion sensitivity, changes in lysosome, mitochondria, intracellular ATP and reactive oxygen species (ROS) level, and mechanistic target of rapamycin (mTOR) signaling were evaluated. Moreover, the effect of rapamycin on radioresistance was also assessed. X-ray and C-ion resistance of C30 cells was moderate, and the resistance of X60 cells was the highest in this study. In X60 cells, the amount of lysosome, mitochondria, intracellular ATP and ROS level were significantly increased, and mTOR and p70S6K (ribosomal protein S6 kinase p70) phosphorylation were enhanced compared with C30 and NR-S1 cells. The inhibition of mTOR signaling was effective for X-ray and C-ion radiosensitization in both cell lines, especially in X60 cells in which X-ray and C-ion resistance was decreased to the same level as that in NR-S1 cells. Our results indicated that the contribution to generate X-ray and C-ion resistance was less for repeated C-ion irradiations compared with repeated X-ray irradiation. Moreover, we found that activated mTOR signaling contributes to X-ray and C-ion resistance in the X60 cancer cells.
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Carcinoma de Células Escamosas/metabolismo , Tolerância a Radiação , Espécies Reativas de Oxigênio/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Radioterapia com Íons Pesados , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Fosforilação/efeitos da radiação , Transdução de Sinais , Terapia por Raios X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Given that the original tumor microenvironment of oral cancer cannot be reproduced, predicting the therapeutic effects of irradiation using monolayer cultures and animal models of ectopic tumors is challenging. Unique properties of carbon-ion irradiation (CIR) characterized by the Bragg peak exert therapeutic effects on tumors and prevent adverse events in surrounding normal tissues. However, the underlying mechanism remains unclear. The biological effects of CIR were evaluated on three-dimensional (3D) in vitro models of normal oral mucosa (NOMM) and oral cancer (OCM3 and OCM4) consisting of HSC-3 and HSC-4 cells. A single 10- or 20-Gy dose of CIR was delivered to NOMM, OCM3, and OCM4 models. Histopathological and histomorphometric analyses and labeling indices for Ki-67, γH2AX, and TUNEL were examined after CIR. The concentrations of high mobility group box 1 (HMGB1) were measured. NOMM exhibited epithelial thinning after CIR, which could be caused by the decreased presence of Ki-67-labeled basal cells. The relative proportion of the thickness of cancer cells to the underlying stroma in cancer models decreased after CIR. This finding appeared to be supported by changes in the three labeling indices, indicating CIR-induced cancer cell death, mostly via apoptosis. Furthermore, the three indices and the HMGB1 release levels significantly differed among the OCM4 that received different doses and with different incubation times after CIR while those of the OCM3 models did not, suggesting more radiosensitivity in the OCM4. The three 3D in vitro models can be a feasible and novel tool to elucidate radiation biology.
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Hedgehog (HH) signaling is an important regulator of embryogenesis that has been associated with the development of several types of cancer. HH signaling is characterized by Smoothened (SMO)-dependent activation of the GLI transcription factors, which regulate the expression of critical developmental genes. Neuroblastoma, an embryonal tumor of the sympathetic nervous system, was recently shown to express high levels of key molecules in this signaling cascade. Using compounds blocking SMO (cyclopamine and SANT1) or GLI1/GLI2 (GANT61) activity revealed that inhibition of HH signaling at the level of GLI was most effective in reducing neuroblastoma growth. GANT61 sensitivity positively correlated to GLI1 and negatively to MYCN expression in the neuroblastoma cell lines tested. GANT61 downregulated GLI1, c-MYC, MYCN and Cyclin D1 expression and induced apoptosis of neuroblastoma cells. The effects produced by GANT61 were mimicked by GLI knockdown but not by SMO knockdown. Furthermore, GANT61 enhanced the effects of chemotherapeutic drugs used in the treatment of neuroblastoma in an additive or synergistic manner and reduced the growth of established neuroblastoma xenografts in nude mice. Taken together this study suggests that inhibition of HH signaling is a highly relevant therapeutic target for high-risk neuroblastoma lacking MYCN amplification and should be considered for clinical testing.
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Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Neuroblastoma/prevenção & controle , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Feminino , Amplificação de Genes , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Técnicas In Vitro , Luciferases/metabolismo , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Smoothened , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
We invented a high-throughput screening method for the examination of radioprotective activity of chemical compounds using rat thymocytes. X-irradiation of the rat thymocytes induced apoptosis, leading to a significant cell shrinkage, which could be easily detected and directly quantified by the flow cytometry analysis. The protective effect of some natural antioxidants against radiation induced apoptosis in the rat thymocytes, as well as their toxicities without X-irradiation, was successfully evaluated using this method. This method provides a powerful tool to develop novel radioprotectors without toxicity and can also be widely used to estimate other oxidative stress except for radiation.
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Ensaios de Triagem em Larga Escala/métodos , Protetores contra Radiação/farmacologia , Timócitos/efeitos dos fármacos , Animais , Citometria de Fluxo , Masculino , Ratos , Ratos Wistar , Timócitos/efeitos da radiação , Raios XRESUMO
The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway.
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Adenosina Desaminase/metabolismo , Proteínas Hedgehog/metabolismo , Edição de RNA , Transdução de Sinais , Fatores de Transcrição/metabolismo , Adenosina Desaminase/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Ativação Transcricional , Proteína GLI1 em Dedos de Zinco , Quinases DyrkRESUMO
Metabotropic glutamate receptor 1 (mGluR1), a key mediator of glutamatergic signaling, is frequently overexpressed in tumor cells and is an attractive drug target for most cancers. Here, we present a targeted radiopharmaceutical therapy strategy that antagonistically recognizes mGluR1 and eradicates mGluR1+ human tumors by harnessing a small-molecule alpha (α)-emitting radiopharmaceutical, 211At-AITM. A single dose of 211At-AITM (2.96 MBq) in mGluR1+ cancers exhibits long-lasting in vivo antitumor efficacy across seven subtypes of four of the most common tumors, namely, breast cancer, pancreatic cancer, melanoma, and colon cancers, with little toxicity. Moreover, complete regression of mGluR1+ breast cancer and pancreatic cancer is observed in approximate 50% of tumor-bearing mice. Mechanistically, the functions of 211At-AITM are uncovered in downregulating mGluR1 oncoprotein and inducing senescence of tumor cells with a reprogrammed senescence-associated secretory phenotype. Our findings suggest α-radiopharmaceutical therapy with 211At-AITM can be a useful strategy for mGluR1+ pan-cancers, regardless of their tissue of origin.
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Neoplasias da Mama , Melanoma , Receptores de Glutamato Metabotrópico , Camundongos , Humanos , Animais , Feminino , Compostos Radiofarmacêuticos/uso terapêutico , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/uso terapêutico , Neoplasias da Mama/genéticaRESUMO
Cellobiose and xylobiose are disaccharides composed of two glucose or xylose units with ß-1,4 linkages. This study aimed to isolate a Trichoderma reesei mutant that lacks ß-glucosidase and ß-xylosidase activities for the simultaneous production of these disaccharides. Mutagenesis using Fe-ion beam resulted in a mutant strain, T. reesei T1640; the cellulase production in this strain was as high as that in the parent strain. Genomic analysis revealed that T1640 lost both the ß-glucosidase and ß-xylosidase activities owing to the translocation of the responsible genes. Hydrolysis of alkali-treated bagasse using the enzymes from T1640 leads to high yields (365 mg/g-biomass) and ratios (72.7% of the total sugars) of cellobiose and xylobiose.
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Celobiose , Celulase , Celulase/genética , ÁlcalisRESUMO
PURPOSE: The goal of this study is to clarify the underlying mechanisms of metastasis suppression by carbon-ion radiotherapy combined with immature dendritic cell immunotherapy (CiDC), which was shown previously to suppress pulmonary metastasis in an NR-S1-bearing C3H/He mouse model. METHODS AND MATERIALS: Mouse carcinoma cell lines (LLC, LM8, Colon-26, and Colon-26MGS) were grafted into the right hind paw of syngeneic mice (C57BL/6J, C3H/He, and BALB/c). Seven days later, the tumors on the mice were locally irradiated with carbon ions (290 MeV/n, 6 cm spread-out Bragg peak, 1 or 2 Gy). At 1.5 days after irradiation, bone marrow-derived immature dendritic cells (iDCs) were administrated intravenously into a subset of the mice. The number of lung metastases was evaluated within 3 weeks after irradiation. In vitro-cultured cancer cells were irradiated with carbon ions (290 MeV/n, mono-energy, LET approximately 70-80 keV/µm), and then cocultured with iDCs for 3 days to determine the DC maturation. RESULTS: CiDC effectively repressed distant lung metastases in cancer cell (LLC and LM8)-bearing C57BL/6J and C3H/He mouse models. However, Colon-26- and Colon-26MGS-bearing BALB/c models did not show enhancement of metastasis suppression by combination treatment. This result was evaluated further by comparing LM8-bearing C3H/He and LLC-bearing C57BL/6J models with a Colon-26-bearing BALB/c model. In vitro coculture assays demonstrated that all irradiated cell lines were able to activate C3H/He- or C57BL/6J-derived iDCs into mature DCs, but not BALB/c-derived iDCs. CONCLUSIONS: The genetic background of the host could have a strong effect on the potency of combination therapy. Future animal and clinical testing should evaluate host genetic factors when evaluating treatment efficacy.
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Imunoterapia , Neoplasias Pulmonares , Animais , Carbono , Células Dendríticas , Patrimônio Genético , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
Kindlin-2 is a recently identified FERM and PH domain containing integrin interacting protein. Kindlin-2 is ubiquitously expressed in normal tissues. So far, much effort has been spent exploring the functional aspects of Kindlin-2. However, the transcriptional regulation of Kindlin-2 has not yet been investigated. In this study we identified and functionally characterized the promoter of the human Kindlin-2 gene. We show that the core promoter of Kindlin-2 is a 39 base pair long GC rich fragment located -122/-83 upstream of the Kindlin-2 transcription start site. Functional characterization of this core promoter region by both in silico as well as in vitro/in vivo analysis shows that the transcription factor SP1 plays an important role in regulation of Kindlin-2 expression.
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Regulação da Expressão Gênica , Proteínas de Membrana , Proteínas de Neoplasias , Proteínas Recombinantes , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Chlorocebus aethiops , Bases de Dados Genéticas , Regulação da Expressão Gênica/fisiologia , Genes Reporter , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp1/genética , Sítio de Iniciação de Transcrição , Transcrição Gênica/fisiologia , TransfecçãoRESUMO
Gerbera in vitro shoots were irradiated using three types of ion beams with different line energy transfers (LETs) to investigate the effective LET and absorbed doses for mutagenesis. Furthermore, genomic mutation analyses were conducted on the obtained mutants. Survival rate analysis showed a lower lethal dose 50% (LD50) with ion beams with higher LETs. Trait/morphological mutations exhibited changes in the color and shape of petals and male sterility. Irradiation conditions with the highest growth change and trait/morphological mutation rates in each ion were C irradiation at 10 Gy, Ar irradiation at 5 Gy, and Fe irradiation at 5 Gy, with a range of absorbed dose of around LD50 to about 10 Gy lower. The highest trait/morphological mutation rate was 14.1% with Ar irradiation at 5 Gy, which was one of the criteria for ion beam irradiation of gerbera in vitro shoots. Furthermore, the genomic mutation in the flower color, petal shape, and male sterile mutants were confirmed by genotype analysis using Genotyping by Random Amplicon Sequencing-Direct technology. This is the first study to report the efficient production of gerbera mutants that could be analyzed. Our findings may lead to more efficient gerbera mutant production and analysis technology.
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The effects of Carbon ion radiation (C-ion) alone or in combination with fused toes homolog (FTS) silencing on Notch signaling were investigated in uterine cervical cancer cell lines (ME180 and CaSki). In both cell lines, upon irradiation with C-ion, the expression of Notch signaling molecules (Notch1, 2, 3 and cleaved Notch1), γ-secretase complex molecules and FTS was upregulated dose-dependently (1, 2 and 4 Gy) except Notch1 in ME180 cells where the change in expression was not significant. However, overexpression of these molecules was attenuated upon silencing of FTS. The spheroid formation, expression of stem cell markers (OCT4A, Sox2 and Nanog) and clonogenic cell survival were reduced by the combination as compared to FTS silencing or C-ion irradiation alone. Additionally, immunoprecipitation and immunofluorescence assay revealed interaction and co-localization of FTS with Notch signaling molecules. In conclusion, FTS silencing enhances the radio-sensitivity of the cervical cancer cells to C-ion by downregulating Notch signaling molecules and decreasing the survival of cancer stem cells.
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PURPOSE: The combination of radiation therapy and immunotherapy is recognized as a very promising strategy for metastatic cancer treatment. The purpose of this work is to compare the effectiveness of x-ray and high-energy carbon ion therapy in combination with checkpoint inhibitors in a murine model. METHODS AND MATERIALS: We used an osteosarcoma mouse model irradiated with either carbon ions or x-rays in combination with 2 immune checkpoint inhibitors (anti-PD-1 and anti-CTLA-4). LM8 osteosarcoma cells were injected in both hind limbs of female C3H/He mice 7 days before exposure to carbon ions or x-rays. In experimental groups receiving irradiation, only the tumor on the left limb was exposed, whereas the tumor on the right limb served as an abscopal mimic. Checkpoint inhibitors were injected intraperitoneally 1 day before exposure as well as concomitant to and after exposure. Tumor growth was measured regularly up to day 21 after exposure, when mice were sacrificed. Both tumors as well as lungs were extracted. RESULTS: A reduced growth of the abscopal tumor was most pronounced after the combined protocol of carbon ions and the immune checkpoint inhibitors administered sequentially. Radiation or checkpoint inhibitors alone were not sufficient to reduce the growth of the abscopal tumors. Carbon ions alone reduced the number of lung metastases more efficiently than x-rays, and in combination with immunotherapy both radiation types essentially suppressed the metastasis, with carbon ions being again more efficient. Investigation of the infiltration of immune cells in the abscopal tumors of animals treated with combination revealed an increase in CD8+ cells. CONCLUSIONS: Combination of checkpoint inhibitors with high-energy carbon ion radiation therapy can be an effective strategy for the treatment of advanced tumors.
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Radioterapia com Íons Pesados , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Osteossarcoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Terapia Combinada , Modelos Animais de Doenças , Feminino , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/imunologia , Camundongos , Fatores de TempoRESUMO
BACKGROUND: Indoleamine-2,3-dioxygenase 1 (IDO1) has been intensively pursued as a therapeutic target to reverse the immunosuppressive cancer-immune milieu and promote tumor elimination. However, recent failures of phase III clinical trials with IDO1 inhibitors involved in cancer immunotherapies highlight the urgent need to develop appropriate methods for tracking IDO1 when the cancer-immune milieu is therapeutically modified. METHODS: We utilized a small-molecule radiotracer, 11C-l-1MTrp, to quantitatively and longitudinally visualize whole-body IDO1 dynamics. Specifically, we first assessed 11C-l-1MTrp in mice-bearing contralateral human tumors with distinct IDO1 expression patterns. Then, we applied 11C-l-1MTrp to longitudinally monitor whole-body IDO1 variations in immunocompetent melanoma-bearing mice treated with 1-methyl-l-tryptophan plus either chemotherapeutic drugs or antibodies targeting programmedcell death 1 and cytotoxic T-lymphocyte-associated protein 4. RESULTS: 11C-l-1MTrp positron emission tomography (PET) imaging accurately delineated IDO1 expression in xenograft mouse models. Moreover, we were able to visualize dynamic IDO1 regulation in the mesenteric lymph nodes (MLNs), an off-tumor IDO1 target, where the percentage uptake of 11C-l-1MTrp accurately annotated the therapeutic efficacy of multiple combination immunotherapies in preclinical models. Remarkably, 11C-l-1MTrp signal intensity in the MLNs was inversely related to the specific growth rates of treated tumors, suggesting that IDO1 expression in the MLNs can serve as a new biomarker of the cancer-immune set point. CONCLUSIONS: PET imaging of IDO1 with 11C-l-1MTrp is a robust method to assess the therapeutic efficacy of multiple combinatorial immunotherapies, improving our understanding of the merit and challenges of IDO1 regimens. Further validation of this animal data in humans is ongoing. We envision that our results will provide a potential precision medicine paradigm for noninvasive visualizing each patient's individual response in combinatorial cancer immunotherapy, and tailoring optimal personalized combination strategies.
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Imunomodulação/imunologia , Imunoterapia/métodos , Indolamina-Pirrol 2,3,-Dioxigenase/uso terapêutico , Animais , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/farmacologia , Camundongos , Camundongos NusRESUMO
(1) Background: among all types of radiation, very heavy ions, such as Neon (Ne) or Argon (Ar), are the optimum candidates for hypoxic tumor treatments due to their reduced oxygen enhancement effect. However, their pioneering clinical use in the 1970s was halted due to severe side effects. The aim of this work was to provide a first proof that the combination of very heavy ions with minibeam radiation therapy leads to a minimization of toxicities and, thus, opening the door for a renewed use of heavy ions for therapy; (2) Methods: mouse legs were irradiated with either Ne MBRT or Ne broad beams at the same average dose. Skin toxicity was scored for a period of four weeks. Histopathology evaluations were carried out at the end of the study; (3) Results: a significant difference in toxicity was observed between the two irradiated groups. While severe da-mage, including necrosis, was observed in the broad beam group, only light to mild erythema was present in the MBRT group; (4) Conclusion: Ne MBRT is significantly better tolerated than conventional broad beam irradiations.
RESUMO
BACKGROUND: Alternative splicing is one of the key mechanisms that generate biological diversity. Even though alternative splicing also occurs in the 5' and 3' untranslated regions (UTRs) of mRNAs, the understanding of the significance and the regulation of these variations is rather limited. RESULTS: We investigated 5' UTR mRNA variants of the mouse Gli1 oncogene, which is the terminal transcriptional effector of the Hedgehog (HH) signaling pathway. In addition to identifying novel transcription start sites, we demonstrated that the expression ratio of the Gli1 splice variants in the 5' UTR is regulated by the genotype of the mouse strain analyzed. The GT allele, which contains the consensus intronic dinucleotides at the 5' splice site of intron 1B, favors exon 1B inclusion, while the GC allele, having a weaker 5' splice site sequence, promotes exon 1B skipping. Moreover, the alternative Gli1 5' UTRs had an impact on translational capacity, with the shorter and the exon 1B-skipped mRNA variants being most effective. CONCLUSIONS: Our findings implicate novel, genome-based mechanisms as regulators of the terminal events in the mouse HH signaling cascade.