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The Class IA phosphoinositide-3-kinase catalytic isoforms p110α, p110ß, and p110δ have been implicated to play vital but overlapping roles in various cancers, including glioblastoma (GBM). We have previously shown that PIK3CD, encoding p110δ, is highly expressed in multiple glioma cell lines and involved in glioma cell migration and invasion. Based on the RNA sequencing data from The Cancer Genome Atlas (TCGA) database, we found the level of PIK3CD expression is significantly higher in GBM than WHO grade II and III gliomas and is closely related to poor survival. To further dissect the oncogenic roles of PIK3CD in glioma progression, we employed CRISPR/Cas9 to completely abrogate its expression in the GBM cell line U87-MG and have successfully isolated two knockout clones with different gene modifications. As expected, the knockout clones exhibited significantly lower migration and invasion capabilities when compared with their parental cells. Interestingly, knockout of PIK3CD also dramatically reduced the colony formation ability of the knockout cells. Further study revealed that PIK3CD deficiency could negate tumorigenesis in nude mice. To determine the downstream effect of PIK3CD depletion, we performed RT2 profiler PCR array of selected gene sets and found that knockout of PIK3CD impaired the activity of p-21 activated kinase 3 (PAK3) and pleckstrin 2 (PLEK2), molecules involved in cancer cell migration and proliferation. This explains why the glioma cells without the PIK3CD expression exhibited weaker oncogenic features. Further, RNAseq analysis of parent and knockout clones revealed that this interaction might happen through axonogenesis signaling pathway. Taken together, we demonstrated that PIK3CD could be a potential prognostic factor and therapeutic target for GBM patients.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Animales , Ratones , Glioblastoma/genética , Ratones Desnudos , Proteínas del Citoesqueleto/genética , Glioma/genética , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Encefálicas/genética , Quinasas p21 Activadas/genéticaRESUMEN
BACKGROUND: Gliomas represent the largest class of primary central nervous system neoplasms, many subtypes of which exhibit poor prognoses. Surgery followed by radiotherapy and chemotherapy has been used as a standard strategy but yielded unsatisfactory improvements in patient survival outcomes. The S-phase kinase protein 2 (Skp2), a critical component of the E3-ligase SCF complex, has been documented in tumorigenesis in various cancer types but its role in glioma has yet to be fully clarified. In this study, we investigated the function of Skp2 in the proliferation, stem cell maintenance, and drug sensitivity to temozolomide (TMZ) of glioma. METHODS: To investigate the role of Skp2 in the prognosis of patients with glioma, we first analyzed data in databases TCGA and GTEx. To further clarify the effect of Skp2 on glioma cell proliferation, we suppressed its level in glioblastoma (GBM) cell lines through knockdown and small molecule inhibitors (lovastatin and SZL-P1-41). We then detected cell growth, colony formation, sphere formation, drug sensitivity, and in vivo tumor formation in xenograft mice model. RESULTS: Skp2 mRNA level was higher in both low-grade glioma and GBM than normal brain tissues. The knockdown of Skp2 increased cell sensitivity to TMZ, decreased cell proliferation and tumorigenesis. In addition, Skp2 level was found increased upon stem cells enriching, while the knockdown of Skp2 led to reduced sphere numbers. Downregulation of Skp2 also induced senescence. Repurposing of lovastatin and novel compound SZL-P1-41 suppressed Skp2 effectively, and enhanced glioma cell sensitivity to TMZ in vitro and in vivo. CONCLUSION: Our data demonstrated that Skp2 modulated glioma cell proliferation in vitro and in vivo, stem cell maintenance, and cell sensitivity to TMZ, which indicated that Skp2 could be a potential target for long-term treatment.
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Lack of appropriate diagnostic/prognostic tools for glioblastoma (GB) is considered one of the major setbacks in the early diagnosis and treatment of this deadly brain tumor. The current gold standard for its diagnosis and staging still relies on invasive biopsy followed by histological examination as well as molecular profiling. Nevertheless, noninvasive approaches are being explored and one example is through the investigation of extracellular vesicles (EVs) in the biofluids of GB patients. EVs are known to carry molecular cargoes such as DNA, mRNA, miRNA, proteins and lipids in almost every type of body fluids. Thus, molecular signature of GB may be present in the EVs derived from these patients. This review focuses on the diagnostic/prognostic potential of EVs in GB, through presenting recent studies on (i) molecular components of EVs, (ii) links between EVs and GB tumor microenvironment, and (iii) clinical potential of EV biomarkers, together with the technical shortcomings researchers need to consider for future studies.
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Biomarcadores de Tumor/análisis , Vesículas Extracelulares/patología , Glioma/patología , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/patología , Vesículas Extracelulares/metabolismo , Glioblastoma/patología , Humanos , Pronóstico , Microambiente TumoralRESUMEN
Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary tumor in the central nervous system. One of the most widely used chemotherapeutic drugs for GBM is temozolomide, which is a DNA-alkylating agent and its efficacy is dependent on MGMT methylation status. Little progress in improving the prognosis of GBM patients has been made in the past ten years, urging the development of more effective molecular targeted therapies. Hyper-activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is frequently found in a variety of cancers including GBM, and it plays a central role in the regulation of tumor cell survival, growth, motility, angiogenesis and metabolism. Numerous PI3K inhibitors including pan-PI3K, isoform-selective and dual PI3K/mammalian target of rapamycin (mTOR) inhibitors have exhibited favorable preclinical results and entered clinical trials in a range of hematologic malignancies and solid tumors. Furthermore, combination of inhibitors targeting PI3K and other related pathways may exert synergism on suppressing tumor growth and improving patients' prognosis. Currently, only a handful of PI3K inhibitors are in phase I/II clinical trials for GBM treatment. In this review, we focus on the importance of PI3K/Akt pathway in GBM, and summarize the current development of PI3K inhibitors alone or in combination with other inhibitors for GBM treatment from preclinical to clinical studies.
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Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Terapia Molecular Dirigida , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Catálisis , Estudios Clínicos como Asunto , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Isoenzimas , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Resultado del TratamientoRESUMEN
This proposal seeks to study the potential therapeutic modality of chemoprevention and anticancer effects and mechanisms of the flavonoids from Cotinus coggygria Scop. on glioblastoma cancer. In the current study, the total flavonoids (TFs) isolated from Cotinus coggygria Scop. var. cinerea Engl. (Cotinus coggygria Scop.) and the major flavonoids of Cotinus coggygria Scop. (CCFs) were identified, and the inhibitory kinetics of TF and CCF on glioblastoma cell lines were calculated. We also investigated whether TF or CCF regulated the apoptotic mechanism in cellular models of glio-blastoma cells. Finally, we evaluated whether treatment with TF or CCF suppressed tumor growth and inhibited migration in orthotopic mouse models of glioblastoma in vivo. In this study, the CCFs were identified as rutin, myricetin, and fisetin. TF and CCF remarkably inhibited cell proliferation and downregulated the PI3K/Akt and ERK signaling pathway in glioblastoma cell lines. Furthermore, the mitochondrial caspase-dependent cascade was regulated by TF and myricetin. In addition, TF and myricetin exhibited significant antitumor effects on glioblastoma in vivo. Taken together, these results suggest that phytochemical and biological data provide evidence for the active components in Cotinus coggygria, and that the TFs are responsible for the anticancer effects on glioblastoma cell growth via induction of apoptosis. In addition, the representative compound myricetin could provide a clinically relevant therapeutic opportunity. Therefore, our data strongly suggest that myricetin-deprived CCF can serve as a potent chemopreventive herbal medicine.
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Anacardiaceae/química , Antineoplásicos Fitogénicos/farmacología , Flavonoides/farmacología , Glioblastoma/tratamiento farmacológico , Animales , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Flavonoides/análisis , Flavonoides/química , Humanos , Cinética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gliomas are a common adult central nervous system tumor, and glioblastoma (GBM), which has a poor prognosis, is the most lethal of all gliomas. The overall survival of GBM patients is only 12-14 months after diagnosis. With progress in the precision of personal medication, therapeutic options for various tumors have become gradually dependent on the molecular profiles of patients. GBM is one of the tumors in which treatment response relies largely on the molecular characteristics of the tumor. Therefore, awareness of the genetic background of each patient will help decision-making regarding the best treatment strategy to use. In this review, a novel molecular classification of gliomas based on recent findings of their genetic characteristics is introduced. Representative molecular markers, such as IDH1 mutation, 1p19q co-deletion, MGMT promoter methylation and EGFRvIII amplification, are described. Furthermore, the development of non-coding RNAs and omics studies of GBM are briefly discussed. Finally, a novel concept for non-invasive detection that could facilitate both diagnosis and treatment monitoring is presented. There is no doubt that the use of molecular profiling by biomarkers will indeed improve the overall survival and quality of life of GBM patients.
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Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Receptores ErbB/genética , Glioma/genética , Isocitrato Deshidrogenasa/genética , Proteínas Supresoras de Tumor/genética , Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patología , Deleción Cromosómica , Cromosomas Humanos Par 1 , Metilación de ADN , Expresión Génica , Glioma/clasificación , Glioma/diagnóstico , Glioma/patología , Humanos , Medicina de Precisión , Pronóstico , Regiones Promotoras GenéticasRESUMEN
MicroRNAs (miRNAs) are often deregulated in glioblastoma multiforme (GBM). Downregulation of microRNA-21 (miR-21), especially in GBM, is responsible for increased apoptosis, decreased cell proliferation and invasion, increased G0/G1 cell cycle arrest, and reduced chemotherapeutic resistance to doxorubicin. Furthermore, it is a critical regulator of multiple downstream genes and signaling pathways involved in gliomagenesis. Programmed cell death 4 (PDCD4) is critical in mediating apoptosis in GBM, and is downregulated by miR-21, which may mediate the resistance of glioblastoma cells against chemotherapy or radiation via its target genes PDCD4. Evidence is mounting that how alterations of these miRNAs transcription factors provide initiation, maintenance, or progression of tumors. This review will focus on the roles of miRNAs family members (particularly miR-21 and its target gene PDCD4) in tumors like glioblastoma and new targeting strategies, as examples some new targeting therapeutic methods and molecular mechanisms of signal pathways in glioblastoma therapeutics, to give the reader the current trends of approach to target regulation of these miRNA and genes for future glioma therapies.
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Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , MicroARNs/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Ciclo Celular/genética , Línea Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/terapia , Humanos , MicroARNs/metabolismo , Terapia Molecular Dirigida , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
Despite decades of research, glioblastoma (GBM) remains invariably fatal among all forms of cancers. The high level of inter- and intratumoral heterogeneity along with its biological location, the brain, are major barriers against effective treatment. Molecular and single cell analysis identifies different molecular subtypes with varying prognosis, while multiple subtypes can reside in the same tumor. Cellular plasticity among different subtypes in response to therapies or during recurrence adds another hurdle in the treatment of GBM. This phenotypic shift is induced and sustained by activation of several pathways within the tumor itself, or microenvironmental factors. In this review, the dynamic nature of cellular shifts in GBM and how the tumor (immune) microenvironment shapes this process leading to therapeutic resistance, while highlighting emerging tools and approaches to study this dynamic double-edged sword are discussed.
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BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive and malignant primary brain tumor characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma, which contribute to tumor recurrence and poor prognosis. Myricetin is a natural flavonoid with potent anti-oxidant, anti-inflammatory and anti-cancer activities, which may serve as a potential and harmless agent for GBM treatment. METHODS: To investigate the anti-glioblastoma effects of myricetin, GBM cells were treated with myricetin alone or in combination with temozolomide. Its effects on GBM cell motility and cytoskeletal structures including lamellipodia, focal adhesions and membrane ruffles were also evaluated. RESULTS: We showed that myricetin alone inhibited glioblastoma U-87 MG cell proliferation, migration and invasion, whereas combination of myricetin and temozolomide did not exhibit any synergistic effect. The inhibitory effect on GBM cell proliferation is independent of PTEN status. Moreover, myricetin showed less cytotoxicity to normal astrocytes than GBM cells. Formation of lamellipodia, focal adhesions, membrane ruffles and vasculogenic mimicry were blocked by myricetin, and phosphorylation of ROCK2, paxillin and cortactin was suppressed. In addition, myricetin could inhibit PI3K/Akt and JNK signaling, and bind to a series of kinases and scaffold proteins including PI3K catalytic isoforms (p110α, p110ß and p110δ), PDK1, JNK, c-Jun, ROCK2, paxillin, vinculin and VEcadherin. CONCLUSIONS: In conclusion, myricetin is a multi-targeted drug that has potent anti-migratory and antiinvasive effects on GBM cells, and suppresses formation of lamellipodia and focal adhesions, suggesting that it may serve as an alternative option for GBM treatment.
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Antineoplásicos/uso terapéutico , Movimiento Celular/efectos de los fármacos , Flavonoides/uso terapéutico , Adhesiones Focales/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Seudópodos/efectos de los fármacos , Astrocitos/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glioblastoma/patología , Humanos , Invasividad Neoplásica/patología , Transducción de Señal/efectos de los fármacos , Temozolomida/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Cell metabolism can be reprogrammed by tissue hypoxia leading to cell transformation and glioblastoma multiforme (GBM) progression. In response to hypoxia, GBM cells are able to express a transcription factor called hypoxia inducible factor-1 (HIF-1). HIF-1 belongs to a family of heterodimeric proteins that includes HIF-1α and HIF-1ß subunits. HIF-1α has been reported to play a pivotal role in GBM development and progression. In the present review, we discuss the role of HIF-1α in glucose uptake, cancer proliferation, cell mobility and chemoresistance in GBM. Evidence from previous studies indicates that HIF-1α regulates angiogenesis, metabolic and transcriptional signaling pathways. Examples of such are the EGFR, PI3K/Akt and MAPK/ERK pathways. It affects cell migration and invasion by regulating glucose metabolism and growth in GBM cells. The present review focuses on the strategies through which to target HIF-1α and the related downstream genes highlighting their regulatory roles in angiogenesis, apoptosis, migration and glucose metabolism for the development of future GBM therapeutics. Combined treatment with inhibitors of HIF-1α and glycolysis may enhance antitumor effects in clinical settings.
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Antineoplásicos/uso terapéutico , Glioblastoma/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Terapia Molecular Dirigida , Animales , Glioblastoma/enzimología , HumanosRESUMEN
BACKGROUND: We have previously reported the identification of divergent hepatitis E virus (HEV) isolated (G9, G20 and 93G) in Guangzhou, a city in southern China. They are now recognised as a new HEV subgenotype in the world. However, the relatedness and significance of these novel isolates in sporadic HEV infection in southern China is still unclear. OBJECTIVES: To perform phylogenetic analysis of nucleotide sequences from 41 HEV isolates in southern China from 1994 to 1998. STUDY DESIGN: The partial nucleotide sequence of the HEV isolates were determined and compared with reported sequences in the GenBank. Their relatedness was analysed using computer software. RESULTS: The majority of the HEV isolates, 39 out of 41, were found to belong to the Burmese-like isolates (genotype 1). The other two belonged to the Guangzhou-like isolates. The latter were only found in the samples collected in 1994. They, together with the G9 isolate, form a unique tree located between genotype 1 and genotype 4 (divergent HEV strains from northern China and Taiwan) on the phylogenetic tree. CONCLUSION: Our results suggest that the Burmese-like isolates are the main causative agents of sporadic HEV infection in southern China. The Guangzhou-like isolates, which appeared transiently in 1994, did not seem to adapt to the environment and have caused no sporadic infection since.
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Virus de la Hepatitis E/clasificación , Hepatitis E/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Niño , Preescolar , China , Femenino , Virus de la Hepatitis E/genética , Hospitales Municipales , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN Viral/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The invasive nature of malignant gliomas makes treatment by surgery alone extremely difficult. However, the preferential accumulation of photosensitisers in neoplastic tissues suggests photodynamic therapy (PDT) may be useful as an adjuvant therapy following tumour resection. In this study, the potential use of three different photosensitisers, namely Photofrin, 5-aminolevulinic acid (5-ALA) and calphostin C in the treatment of glioma was investigated. The uptake, cytotoxicity on U87 and GBM6840 glioma cell lines were determined by flow cytometry and MTT assay respectively. Their effect on glioma cell invasiveness was evaluated by (1) measuring the levels of matrix degradation enzymes matrix metalloproteinase (MMP)-2 and -9 using gelatin zymography, and (2) Matrigel invasion assay. The results showed that uptake of calphostin C reached saturation within 2 h, while Photofrin and 5-ALA induced protoporphyrin IX (PpIX) levels elevated steadily up to 24 h. Photocytotoxic effect on the two glioma cell lines was similar with LD50 at optimal uptake: 1 microg/mL Photofrin at 1.5 J/cm(2); 1 mM 5-ALA at 2 J/cm(2) and 100 nM calphostin C at 2 J/cm(2). The inhibition in cell proliferation after Photofrin treatment was similar for both cell lines, which correlated to more cells being arrested in the G0/G1 phase of the cell cycle (P<0.01). By contrast, U87 was more sensitive to calphostin C whereas GBM6840 was more susceptible to 5-ALA treatment. The ability of both cell lines to migrate through the Matrigel artificial basement membrane was significantly reduced after PDT (P<0.001). This might be due to a decreased production in MMP-2 and MMP-9, together with the reduction of adhesion molecule expression. Photofrin was most superior in inhibiting cell invasion and calphostin C was least effective in reducing adhesion molecule expression. Taken together, PDT could be useful in the treatment of gliomas but the choice of photosensitisers must be taken into consideration.
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Ácido Aminolevulínico/farmacología , Éter de Dihematoporfirina/farmacología , Glioma/patología , Naftalenos/farmacología , Fármacos Fotosensibilizantes/farmacología , Ácido Aminolevulínico/toxicidad , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Éter de Dihematoporfirina/toxicidad , Glioma/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Naftalenos/toxicidad , Invasividad Neoplásica/patología , Fármacos Fotosensibilizantes/toxicidadRESUMEN
This study is to prepare and evaluate Pluronics-modified mixed micelle (MM) to deliver polyphenolic myricetin (MYR) across the blood-brain barrier. MYR has been proven to be an effective anticancer agent against glioblastoma cells in our previous studies. However, the poor solubility of MYR limits its access to the brain. In this study, the feasibility of preparing lipid-based MM that combined sodium dodecyl sulphate (SDS) with Pluronic F68 (F68) and Labrasol was investigated. Furthermore, the nonionic surfactant coating technology for the protection of MYR against oxidation, and its attainment in oral bioavailability, was examined. On account of the altered biomaterial properties of F68/SDS-modified lipid-based micelles, myricetin-loaded mixed micelles (MYR-MMs) were prepared by solvent-evaporation method to self-assembly into MMs. The average size of MYR-MMs was 96.3 nm, with negatively charged potential and spherical in shape. The drug loading of MYR-MMs was high with the increased grafting ratio, the more prolonged drug release profile, and more effective killing glioblastoma cells in vitro. Moreover, MYR-MMs showed a higher preference for the brain than free MYR alone, suggesting the novel MMs loaded with MYR could promote absorption and increase relative bioavailability. Taken together, the F68/SDS-modified and Labrasol-modified lipid-based micelles may provide a promising method to deliver polyphenolic compounds across the brain to treat brain tumor.
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Antineoplásicos/administración & dosificación , Portadores de Fármacos/química , Flavonoides/administración & dosificación , Poloxámero/química , Dodecil Sulfato de Sodio/química , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Flavonoides/farmacocinética , Flavonoides/farmacología , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Micelas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Glioblastoma multiforme is the most aggressive malignant primary brain tumor, characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma. Both PI3K/Akt and JNK pathways are essential to glioblastoma cell survival, migration and invasion. Due to their hyperactivation in glioblastoma cells, PI3K and JNK are promising targets for glioblastoma treatment. METHODS: To investigate the combination effects of class IA PI3K catalytic isoforms (p110α, p110ß and p110δ) and JNK inhibition on tumor cell growth and motility, glioblastoma cells and xenografts in nude mice were treated with isoform-selective PI3K inhibitors in combination with JNK inhibitor. RESULTS: We showed that combined inhibition of these PI3K isoforms and JNK exerted divergent effects on the proliferation, migration and invasion of glioblastoma cells in vitro. Pharmacological inhibition of p110ß or p110δ, but not p110α, displayed synergistic inhibitory effect with JNK inhibition on glioblastoma cell proliferation and migration through decreasing phosphorylation of Akt, FAK and zyxin, leading to blockade of lamellipodia and membrane ruffles formation. No synergistic effect on invasion was observed in all the combination treatment. In vivo, combination of p110ß and JNK inhibitors significantly reduced xenograft tumor growth compared with single inhibitor alone. CONCLUSION: Concurrent inhibition of p110ß and JNK exhibited synergistic effects on suppressing glioblastoma cell proliferation and migration in vitro and xenograft tumor growth in vivo. Our data suggest that combined inhibition of PI3K p110ß isoform and JNK may serve as a potent and promising therapeutic approach for glioblastoma multiforme.
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Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Humanos , MAP Quinasa Quinasa 4/metabolismo , Ratones , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Transducción de SeñalRESUMEN
Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.
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Análisis Mutacional de ADN/métodos , Glioma/genética , Isocitrato Deshidrogenasa/genética , Mutación Missense , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Niño , Preescolar , Femenino , Glioma/enzimología , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Supervivencia , Adulto JovenRESUMEN
BACKGROUND: Single-stranded RNA has the potential to form secondary structures that may result in intrastrand misalignment of repeats and may be responsible for DNA mutation. Two amplicons obtained from amplification of hepatitis E virus (HEV) gene by reverse transcription and nested polymerase chain reaction (RT-nPCR) were of unexpected size and had the same misalignment. They did not contain the target region between the internal priming sites but contained two fragments flanking the target region joined by a 12-base sequence instead. OBJECTIVES: To determine whether the unexpected amplicons obtained were due to secondary structures present in the HEV RNA. STUDY DESIGN: HEV RNA sequences were obtained from the GenBank database and the software DNASIS was used to predict the presence of secondary structures within the amplification target regions. The free energy barriers of the secondary structures, which indicate their stability, were also calculated. Conventional RT-nPCR protocol was subsequently modified to eliminate RNA secondary structures. RESULTS: An extensive stem-loop structure was predicted to exist between the two internal priming sites of the HEV RNA by the DNASIS software. Its free energy barrier was found to be significant and might have resulted in the deletion of the target region located between the internal priming sites. Increased temperature and addition of dimethyl sulphoxide (DMSO) in the reverse transcription step gave the expected amplicon after the nested polymerase chain reaction. CONCLUSION: Spontaneous secondary structure formation can influence the outcome of RNA gene amplification and should be considered an important factor when designing primers and adopting protocols for RNA gene amplification.
Asunto(s)
Virus de la Hepatitis E/genética , Conformación de Ácido Nucleico , ARN Viral/genética , Secuencia de Bases , Cartilla de ADN , Amplificación de Genes , Virus de la Hepatitis E/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , ARN Viral/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Programas InformáticosRESUMEN
The transforming growth factor ß1 (TGF-ß1) belongs to a family of structurally related polypeptide factors. TGF-beta plays an important role in the pathobiology of invasion of malignant gliomas. The objective of the present study was to investigate the impact of TNF-α converting enzyme (TACE/ADAM17) signaling on the process of TGF-ß1-stimulated migration and invasion of T98G glioma cells. We found that TGF-ß1 increased migration and invasiveness in glioma cells. Addition of the TGF-ß1 receptor inhibitor, SB431542, reduced the TGF-ß1-stimulated migration and invasiveness of glioma cells. In addition, TGF-ß1-induced migration and invasiveness were also blocked by exposure to an ADAM17 inhibitor, TAPI-2. Furthermore, ADAM17 mRNA and protein expression were up-regulated by TGF-ß1. Treatment with SB431542 and TAPI-2 blocked TGF-ß1-induced ADAM17 protein expression. In summary, these results indicate that TGF-ß1 promotes cell migration and invasiveness of glioma cells through stimulation of ADAM17.
Asunto(s)
Proteínas ADAM/metabolismo , Activación Enzimática/efectos de los fármacos , Glioma/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Proteínas ADAM/genética , Proteína ADAM17 , Benzamidas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Dioxoles/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/genética , Humanos , Invasividad Neoplásica , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose-dependent manner. BrdU and tube formation assays indicated that gallic acid significantly decreased glioma cell proliferation and tube formation in mouse brain endothelial cells, respectively. In addition, gallic acid decreased U87 cell invasion in vitro. Western blot analysis showed that expression of ADAM17, p-Akt and p-Erk was suppressed by gallic acid in both U87 and U251n cell lines. These data suggest that suppression of ADAM17 and downregulation of PI3K/Akt and Ras/MAPK signaling pathways may contribute to gallic acid-induced decrease of invasiveness. Gallic acid may be a valuable candidate for treatment of brain tumor.