RESUMEN
Cancer stem cells (CSCs) play an important role in cancer recurrence and metastasis. It is suggested that the CSC properties in heterogeneous cancer cells can be induced by ionizing radiation (IR). This study investigated the role of DLX2 in the radioresistance and CSC properties induced by IR in NSCLC cancer cells. Here, A549 cells were exposed to fractionated irradiation at a cumulative dose of 52 Gy (4 Gy × 13 times) for a generation of radioresistant cells. After fractionated irradiation, surviving A549 cells exhibited resistance to IR and enhanced expression of various cancer stem cell markers. They also showed upregulation of mesenchymal molecular markers and downregulation of epithelial molecular markers, correlating with an increase in the migration and invasion. Fractionated irradiation triggered the secretion of TGF-ß1 and DLX2 expression. Interestingly, the increased DLX2 following fractionated irradiation seemed to induce the expression of the gene for the EGFR-ligand betacellulin via Smad2/3 signaling. To contrast, DLX2 knockdown dramatically decreased the expression of CSC markers, migration, and proliferation. Moreover, A549 cells expressing DLX2 shRNA formed tumors with a significantly smaller volume compared to those expressing control shDNA in a mouse xenograft assay. These results suggest that DLX2 overexpression in surviving NSCLC cancer cells after fractionated IR exposure is involved in the cancer stemness, radioresistance, EMT, tumor survival, and tumorigenic capability.
Asunto(s)
Autorrenovación de las Células/efectos de la radiación , Rayos gamma , Proteínas de Homeodominio/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de la radiación , Transducción de Señal/efectos de la radiación , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células A549 , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de la radiación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Técnicas de Inactivación de Genes , Humanos , Ratones , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mouse models are widely used in the study of musculoskeletal radiobiology both in vivo and in vitro. Two of the most commonly used mouse strains are C57BL/6 and BALB/c. However, little is known about their equivalence in response to ionizing radiation. In this study we compare the responses of marrow stromal cells derived from both of these strains to X rays in vitro at passages 0 and 2. Colony-forming efficiency was significantly higher in BALB/c marrow stromal cells at passage 0. Radiation-induced decreases in colony-forming unit (CFU) formation at passage 0 were comparable across both strains at 0-2 Gy, but BALB/c stromal cells were more radiosensitive than C57BL/6 stromal cells at 3-7 Gy. Osteogenic differentiation at passage 2 was not affected by radiation for either strain. This work demonstrates that commonly used inbred mouse strains differ in their early-passage marrow stromal cell responses to X rays, including self-renewal and differentiation potential. This variability is an important point to consider when selecting an animal model for in vivo or in vitro study.
Asunto(s)
Células de la Médula Ósea/citología , Tolerancia a Radiación , Células del Estroma/citología , Células del Estroma/efectos de la radiación , Animales , Diferenciación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Ratones , Osteogénesis/efectos de la radiación , Especificidad de la EspecieRESUMEN
Glioblastoma multiforme (GBM) requires radiotherapy (RT) as its definitive management. However, GBM still has a high local recurrence rate even after RT. Cancer stem-like cells (CSCs) might enable GBM to evade irradiation damage and cause therapeutic failure. The optimal RT plan should achieve a planning target volume (PTV) coverage of more than 95% but cannot always meet the requirements. Here, we demonstrate that irradiation with different tumor coverage rates to different brain areas has similar effects on GBM. To retrospectively analyze the relationship between PTV coverage and the survival rate in 26 malignant glioblastoma patients, we established primary cell lines from patient-derived malignant glioblastoma cells with the PTV95 (PTV coverage of more than 95%) program (GBM-MG1 cells) and the Non-PTV95 (poor PTV coverage of less than 95%) program (GBM-MG2 cells). The clinical results of PTV95 and Non-PTV95 showed no difference in the overall survival (OS) rate (P = .390) between the two different levels of PTV coverage. GBM-MG1 (PTV95 program) cells exhibited higher radioresistance than GBM-MG2 (Non-PTV95 program) cells. CD44 promotes radioresistance, CSC properties, angiogenesis and cell proliferation in GBM-MG1 (PTV95 program) cells. GBM patients receiving RT with the PTV95 program exhibited higher radioresistance, CSC properties, angiogenesis and cell proliferation than GBM patients receiving RT with the Non-PTV95 program. Moreover, CD44 plays a crucial role in these properties of GBM patients with the PTV95 program.
Asunto(s)
Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Receptores de Hialuranos/metabolismo , Tolerancia a Radiación , Planificación de la Radioterapia Asistida por Computador/métodos , Adulto , Anciano , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Encéfalo/efectos de la radiación , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de la radiación , Femenino , Estudios de Seguimiento , Glioblastoma/diagnóstico , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Cultivo Primario de Células , Supervivencia sin Progresión , Criterios de Evaluación de Respuesta en Tumores Sólidos , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Photostimulation has been widely used in neuromodulation. However, existing optogenetics techniques require genetic alternation of the targeted cell or tissue. Here, we report that neural stem cells (NSCs) constitutionally express blue/red light-sensitive photoreceptors. The proliferation and regulation of NSCs to neuronal or glial cells are wavelength-specific. Our results showed a 4.3-fold increase in proliferation and 2.7-fold increase in astrocyte differentiation for cells under low-power blue monochromatic light exposure (455â¯nm, 300⯵W/cm2). The melanopsin (Opn4)/transient receptor potential channel 6 (TRPC6) non-visual opsin serves as a key photoreceptor response to blue light irradiation. Two-dimensional gel electrophoresis coupled with mass spectrometry further highlighted the Jun activation domain-binding protein 1 (Jab1) as a novel and specific modulator in phototransduction pathways induced by blue light exposure. Quiescent adult NSCs reside in specific regions of the mammalian brain. Therefore, we showed that melanopsin/TRPC6 expressed in these regions and blue light stimulation through optical fibers could directly stimulate the NSCs in vivo. Upconversion nanoparticles (UCNPs) converted deep-penetrating near-infrared (NIR) light into specific wavelengths of visible light. Accordingly, we demonstrated that UCNP-mediated NIR light could be used to modulate in vivo NSC differentiation in a less invasive manner. In the future, this light-triggered system of NSCs will enable nongenetic and noninvasive neuromodulation with therapeutic potential for central nervous system diseases.
Asunto(s)
Diferenciación Celular , Células-Madre Neurales/citología , Neuroglía/citología , Neuronas/citología , Optogenética , Animales , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de la radiación , Rayos Infrarrojos , Fototransducción/efectos de la radiación , Ratones Endogámicos C57BL , Nanopartículas/química , Células-Madre Neurales/efectos de la radiación , Neuroglía/efectos de la radiación , Neuronas/efectos de la radiaciónAsunto(s)
ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Células de la Médula Ósea/citología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/efectos de la radiación , Células Cultivadas , Fluorouracilo/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Unión al ARN/genética , Radiación Ionizante , Transcriptoma/efectos de los fármacos , Transcriptoma/efectos de la radiaciónRESUMEN
Ovarian cancer stem cells (OCSCs) are considered the reason for ovarian cancer's emergence and recurrence. Ultrasound-targetted microbubble destruction (UTMD), a non-vial, safe, and promising delivery method for miRNA, is reported to transfect cancer stem cells (CSCs). In the present study, we investigated to transfect miR-let-7b into OCSCs using UTMD. The CD133+ OCSCs, accounted for only 0.1% of ovarian cancer cell line A2780, were separated by flow cytometry, and the CSC characteristics of CD133+ OCSCs have been proved by spheroid formation and self-renewal assay. The miR-let-7b transfection efficiency using UTMD was significantly higher than other groups except lipofectamine group through flow cytometry. The cell viability of all groups decreased after transfection, and the late apoptosis rate of CD133+ OCSCs after miR-let7b transfection induced by UTMD was 2.62%, while that of non-treated cells was 0.02% (P<0.05). Furthermore, the Western blot results demonstrated that the stem cells surface marker of CD133 expression has decreased. Therefore, our results indicated that UTMD-mediated miRNA delivery could be a promising platform for CSC therapy.
Asunto(s)
Autorrenovación de las Células/genética , MicroARNs/genética , Células Madre Neoplásicas/patología , Neoplasias Ováricas/genética , Antígeno AC133/genética , Línea Celular Tumoral , Autorrenovación de las Células/efectos de la radiación , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Microburbujas , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/patología , Transfección , Ondas UltrasónicasRESUMEN
There is a substantial unmet clinical need for new strategies to protect the hematopoietic stem cell (HSC) pool and regenerate hematopoiesis after radiation injury from either cancer therapy or accidental exposure. Increasing evidence suggests that sex hormones, beyond their role in promoting sexual dimorphism, regulate HSC self-renewal, differentiation, and proliferation. We and others have previously reported that sex-steroid ablation promotes bone marrow (BM) lymphopoiesis and HSC recovery in aged and immunodepleted mice. Here we found that a luteinizing hormone (LH)-releasing hormone antagonist (LHRH-Ant), currently in wide clinical use for sex-steroid inhibition, promoted hematopoietic recovery and mouse survival when administered 24 h after an otherwise-lethal dose of total-body irradiation (L-TBI). Unexpectedly, this protective effect was independent of sex steroids and instead relied on suppression of LH levels. Human and mouse long-term self-renewing HSCs (LT-HSCs) expressed high levels of the LH/choriogonadotropin receptor (LHCGR) and expanded ex vivo when stimulated with LH. In contrast, the suppression of LH after L-TBI inhibited entry of HSCs into the cell cycle, thus promoting HSC quiescence and protecting the cells from exhaustion. These findings reveal a role of LH in regulating HSC function and offer a new therapeutic approach for hematopoietic regeneration after hematopoietic injury.
Asunto(s)
Autorrenovación de las Células/genética , Células Madre Hematopoyéticas/metabolismo , Hormona Luteinizante/metabolismo , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/efectos de la radiación , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Hematopoyesis/efectos de la radiación , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Hormona Luteinizante/farmacología , Ratones , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Receptores de HL/genética , Regeneración/efectos de los fármacos , Regeneración/genética , Regeneración/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Irradiación Corporal TotalRESUMEN
Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering.
Asunto(s)
Proteína Morfogenética Ósea 4/administración & dosificación , Portadores de Fármacos , Terapia por Luz de Baja Intensidad/métodos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de la radiación , Poloxámero/química , Ingeniería de Tejidos/métodos , Andamios del Tejido , Adolescente , Adulto , Animales , Proteína Morfogenética Ósea 4/química , Regeneración Ósea , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Humanos , Hidrogeles , Inyecciones , Láseres de Semiconductores , Terapia por Luz de Baja Intensidad/instrumentación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Ratones Desnudos , FN-kappa B/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/efectos de la radiación , Hueso Parietal/lesiones , Hueso Parietal/patología , Hueso Parietal/cirugía , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Medulloblastoma (MB) is the most common pediatric brain tumor, comprising four distinct molecular variants, one of which characterized by activation of the Sonic Hedgehog (SHH) pathway, driving 25-30% of sporadic MB. SHH-dependent MBs arise from granule cell precursors (GCPs), are fatal in 40-70% of cases and radioresistance strongly contributes to poor prognosis and tumor recurrence. Patched1 heterozygous (Ptch1 +/-) mice, carrying a germ-line heterozygous inactivating mutation in the Ptch1 gene, the Shh receptor and negative regulator of the pathway, are uniquely susceptible to MB development after radiation damage in neonatal cerebellum. Here, we irradiated ex-vivo GCPs isolated from cerebella of neonatal WT and Ptch1 +/- mice. Our results highlight a less differentiated status of Ptch1-mutated cells after irradiation, influencing DNA damage response. Increased expression levels of pluripotency genes Nanog, Oct4 and Sal4, together with greater clonogenic potential, clearly suggest that radiation induces expansion of the stem-like cell compartment through cell-reprogramming and self-renewal maintenance, and that this mechanism is strongly dependent on Nanog. These results contribute to clarify the molecular mechanisms that control radiation-induced Shh-mediated tumorigenesis and may suggest Nanog as a potential target to inhibit for adjuvant radiotherapy in treatment of SHH-dependent MB.
Asunto(s)
Autorrenovación de las Células/efectos de la radiación , Reprogramación Celular/efectos de la radiación , Meduloblastoma/patología , Proteína Homeótica Nanog/metabolismo , Receptor Patched-1/deficiencia , Receptor Patched-1/metabolismo , Animales , Apoptosis/efectos de la radiación , Carcinogénesis/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Línea Celular Tumoral , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Técnicas de Inactivación de Genes , Ratones , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Receptor Patched-1/genéticaRESUMEN
The intestinal epithelium possesses a remarkable self-renewal ability, which is mediated by actively proliferating Lgr5+ stem cells. Bone morphogenetic protein (BMP) signalling represents one major counterforce that limits the hyperproliferation of intestinal epithelium, but the exact mechanism remains elusive. Here we demonstrate that epithelial BMP signalling plays an indispensable role in restricting Lgr5+ stem cell expansion to maintain intestinal homeostasis and prevent premalignant hyperproliferation on damage. Mechanistically, BMP inhibits stemness of Lgr5+ stem cells through Smad-mediated transcriptional repression of a large number of stem cell signature genes, including Lgr5, and this effect is independent of Wnt/ß-catenin signalling. Smad1/Smad4 recruits histone deacetylase HDAC1 to the promoters to repress transcription, and knockout of Smad4 abolishes the negative effects of BMP on stem cells. Our findings therefore demonstrate that epithelial BMP constrains the Lgr5+ stem cell self-renewal via Smad-mediated repression of stem cell signature genes to ensure proper homeostatic renewal of intestinal epithelium.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Perfilación de la Expresión Génica , Intestinos/citología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Animales , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Proliferación Celular/genética , Proliferación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de la radiación , Femenino , Regulación de la Expresión Génica/efectos de la radiación , Histona Desacetilasas/metabolismo , Homeostasis/genética , Masculino , Ratones Endogámicos C57BL , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de la radiación , Radiación Ionizante , Regeneración/efectos de la radiación , Proteínas Smad/metabolismo , Células Madre/efectos de la radiación , Transcripción Genética/efectos de la radiación , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
In this study, we examined whether the Yiqi and Yangyin Formula (YYF), used in traditional Chinese medicine, could ameliorate damage to the hematopoietic system induced by total body irradiation (TBI). Treatment with 15 g/kg of YYF increased the survival rate of Institute of Cancer Research (ICR) mice exposed to 7.5 Gy TBI. Furthermore, YYF treatment increased the white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) counts in ICR mice exposed to 2 Gy or 4 Gy TBI. Treatment with YYF also increased the number of bone marrow cells, hematopoietic progenitor cells (HPCs), hematopoietic stem cells (HSCs) and the colony-forming ability of granulocyte-macrophage cells. YYF alleviated TBI-induced suppression of the differentiation ability of HPCs and HSCs and decreased the reactive oxygen species (ROS) levels in bone marrow mononuclear cells (BMMNCs), HPCs and HSCs from mice exposed to 2 Gy or 4 Gy TBI. Overall, our data suggest that YYF can ameliorate myelosuppression by reducing the intracellular ROS levels in hematopoietic cells after TBI at doses of 2 Gy and 4 Gy.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Irradiación Corporal Total , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/efectos de la radiación , Recuento de Células , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Células Madre Hematopoyéticas/efectos de la radiación , Ratones Endogámicos ICR , Especies Reactivas de Oxígeno/metabolismo , Análisis de SupervivenciaRESUMEN
In response to muscle damage, satellite cells proliferate and undertake both differentiation and self-renewal, generating new functional muscle tissue and repopulating this new muscle with stem cells for future injury responses. For many questions relating to the physiological regulation of satellite cells, quantitative readouts of self-renewal and differentiation can be very useful. There is a particular need for a quantitative assay for satellite cell self-renewal that does not rely solely upon sectioning, staining and counting cells in sections. In this chapter, we provide detailed methods for quantifying the self-renewal and differentiation potential of a given population of satellite cells using an assay involving transplantation into injured, regenerating muscle together with specific markers for donor cell identity and state of differentiation. In particular, using the Pax7-ZsGreen transgene as a marker of satellite cell state, self-renewal can be quantified by FACS on transplanted muscle to actually count the total number of resident satellite cells at time points following transplantation.
Asunto(s)
Diferenciación Celular , Autorrenovación de las Células , Citometría de Flujo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Animales , Biomarcadores , Cardiotoxinas/efectos adversos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/efectos de la radiación , Separación Celular/métodos , Citometría de Flujo/métodos , Inmunohistoquímica , Inmunofenotipificación , Ratones , Ratones Endogámicos mdx , Desarrollo de Músculos , Regeneración , Células Satélite del Músculo Esquelético/trasplanteRESUMEN
The tumor suppressor protein, p53, plays pivotal roles in regulating apoptosis and proliferation in the embryonic and adult central nervous system (CNS) following neuronal injuries such as those induced by ionizing radiation. There is increasing evidence that p53 negatively regulates the self-renewal of neural stem cells in the adult murine brain; however, it is still unknown whether p53 is essential for self-renewal in the injured developing CNS. Previously, we demonstrated that the numbers of apoptotic cells in medaka (Oryzias latipes) embryos decreased in the absence of p53 at 12-24 h after irradiation with 10-Gy gamma rays. Here, we used histology to examine the later morphological development of the irradiated medaka brain. In p53-deficient larvae, the embryonic brain possessed similar vacuoles in the brain and retina, although the vacuoles were much smaller and fewer than those found in wild-type embryos. At the time of hatching (6 days after irradiation), no brain abnormality was observed. In contrast, severe disorganized neuronal arrangements were still present in the brain of irradiated wild-type embryos. Our present results demonstrated that self-renewal of the brain tissue completed faster in the absence of p53 than wild type at the time of hatching because p53 reduces the acute severe neural apoptosis induced by irradiation, suggesting that p53 is not essential for tissue self-renewal in developing brain.
Asunto(s)
Encéfalo/patología , Encéfalo/efectos de la radiación , Autorrenovación de las Células/efectos de la radiación , Oryzias/metabolismo , Traumatismos por Radiación/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Encéfalo/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Embrión no Mamífero/efectos de la radiación , Rayos gamma , Oryzias/embriología , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
The presence of glioma stem cells (GSCs) in tumor is relevant for glioma treatment resistance. This study assessed whether knockdown of Cathepsin L can influence GSC growth, tumor radiosensitivity, and clinical outcome. Protein levels of Cathepsin L and stem cell markers (CD133 and Nestin) were analyzed in samples from 90 gliomas of different WHO grades and 6 normal brain tissues by immunohistochemistry. Two glioma stem cell lines with overexpressed Cathepsin L were stably transfected with Cathepsin L short hairpin RNA expression vectors. The effects of Cathepsin L inhibition on radiosensitivity, self-renewal, stemness, DNA damage, and apoptosis were evaluated. In addition, an intracranial animal model and subcutaneous tumor xenografts in nude mice were used to assess tumor response to Cathepsin L inhibition in vivo. Our results proved that expressions of Cathepsin L and CD133, but not of Nestin, correlated with malignant grades of glioma tissues. GSCs with high Cathepsin L and CD133 co-expression were extraordinarily radioresistant. Cathepsin L inhibition with radiotherapy significantly reduced GSC growth, promoted apoptosis, and improved radiosensitivity. Knockdown of Cathepsin L resulted in a dramatic reduction of CD133 expression, as well as the decreased phosphorylation of DNA repair checkpoint proteins (ATM and DNA-PKcs). Furthermore, combination of Cathepsin L inhibition and radiotherapy potently blocked tumor growth and decreased blood vessel formation in vivo. Taken together, these findings suggest Cathepsin L as a promising therapeutic target for clinical therapy in GBM patients.
Asunto(s)
Neoplasias Encefálicas/radioterapia , Catepsina L/metabolismo , Técnicas de Silenciamiento del Gen , Glioma/radioterapia , Células Madre Neoplásicas/efectos de la radiación , Tolerancia a Radiación , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Catepsina L/genética , Línea Celular Tumoral , Autorrenovación de las Células/efectos de la radiación , Daño del ADN , Proteína Quinasa Activada por ADN/metabolismo , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , Glioma/genética , Glioma/patología , Glicoproteínas/metabolismo , Humanos , Ratones Desnudos , Clasificación del Tumor , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Interferencia de ARN , Factores de Tiempo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to (137)Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.