The effect and mechanism of Xihuang pill inhibits the formation of vasculogenic mimicry in human glioblastoma cells by down-regulating HIF-1α/VEGFA/VEGFR2 signaling pathway / 药学学报

Our studies were aimed to explore the effect and mechanism of the inhibition of the formation of vasculogenic mimicry (VM) in human glioblastoma cells by Xihuang pill (XHP) medicated serum through regulating the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. The medicated serum of XHP was prepared by gavage for 7 days to male SD rats (approval number of animal experiment ethics: 202105A051). The hypoxia model of U251 cells was established using 200 μmol·L-1 of CoCl2. After treatment with XHP-medicated serum, cell viability and proliferation of U251 cells were detected by CCK-8 and cell cloning experiment. Cell apoptosis and cell cycle of U251 cells were determined by flow cytometry. Cell migration and invasion were evaluated by wound healing and Transwell invasion assay. The formation of VM was assessed by three-dimensional cell culture of U251 cells. The protein expression levels of HIF-1α, VEGFA, VEGFR2, phosphorylated-VEGFR2 (p-VEGFR2), vascular endothelial-cadherin (VE-cadherin), Eph receptor tyrosine kinases A2 (EphA2), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 14 (MMP14) and laminin γ2 in U251 cells were detected by Western blot. The results showed that 10% XHP-medicated serum had little effect on the cell viability, proliferation, apoptosis and cell cycle of U251 cells under hypoxia. Compared with the model group, 10% XHP-medicated serum at 1.0, 1.5 and 2.0 h significantly decreased the migration rate (P < 0.01) and the number of invading U251 cells (P < 0.01). 10% XHP-medicated serum at 2.0 h significantly suppressed the formation of VM tubular structures in U251 cells under the condition of hypoxia (P < 0.01). Western blot experiment showed that 10% XHP-medicated serum significantly down-regulated the expression of HIF-1α, VEGFA, phospho-VEGFR2, VE-cadherin, EphA2 and MMP14 proteins (P < 0.05). In conclusion, XHP could inhibit the formation of VM in human glioblastoma U251 cells to suppress the angiogenesis by down-regulating the HIF-1α/VEGFA/VEGFR2 signaling pathway.

Role of Ectodermal-neural cortex 1 protein in human glioma progression, identification of a peptide internalized by human glioblastoma cells and development of an alternative method to generate growth curves of adherent cultures / Papel da proteína Ectodermal-neural cortex 1 (ENC1) na progressão de glioma humano, identificação de um peptídeo internalizado por células de glioblastoma humano e desenvolvimento de um método alternativo para gerar curvas de crescimento celular

Gliomas are the most common form of primary intracranial malignancy, among which astrocytomas are the most frequent. Ectodermal-cortex protein 1 (ENC 1), also known as Nuclear Restricted Protein/Brain (NRP/B), was first characterized as a protein which interacts with the cytoskeleton by binding to actin through Kelch-like domains, being related to neural fate specification during development of the nervous system. The first chapter of this thesis confirms ENC1 as a tumor suppression properties by a genomic edition approach, analyses ENC1 expression in a set of patient glioma samples and describes the correlation these data with patients survival and progression-free survival, concluding that ENC1 expression may constitute a biomarker for glioma aggressiveness. The second chapter refers to the identification and in vitro characterization of the LHTNELQ peptide, which was selected by the Phage Display method using human glioblastoma cells. This new peptide is able to be internalized by these cells and features as a new tool for the development of glioma therapeutics. The third chapter report an alternative method to generate growth curves of adherent cell cultures, which is based on the CFSE fluorescence decay over time. It is an alternative method to determine growth curves of cultured cells, with smaller variation among technical replicates than that of counting-based methods

Gliomas são a forma mais comum de malignidades primárias intracranianas, dentre os quais os astrocitomas são os mais frequentes. A proteína Ectodermal-neural cortex 1 (ENC1), também conhecida como Nuclear Restricted Protein/Brain (NRP/B), foi primeiramente caracterizada como uma proteína que interage com o citoesqueleto por meio de ligação à actina através de domínios Kelch-like, sendo relacionada com diferenciação neuronal durante o desenvolvimento do sistema nervoso. O primeiro capítulo desta tese descreve confirmação da capacidade supressora tumoral de ENC1 por abordagem de edição genômica, analisa a expressão de ENC1 em um conjunto de amostras de pacientes com gliomas e correlaciona esses dados com tempo de sobrevida geral e sobrevida livre de progressão tumoral nos pacientes, concluindo que a expressão de ENC1 pode ser utilizada como um biomarcador da agressividade do glioma. O segundo capítulo apresenta a identificação e caracterização in vitro do peptídeo LHTNELQ, que foi selecionado pela metodologia de Phage display utilizandose de células de glioblastoma humano. Este novo peptídeo é capaz de internalizar-se nestas células e figura como uma nova ferramenta para o desenvolvimento de estratégias terapêuticas para glioblastomas. No terceiro capítulo propõe-se um método alternativo para gerar curvas de crescimento celular de cultura aderente, o qual é baseado no decaimento da fluorescência do reagente CFSE ao longo do tempo. Tratase de um método alternativo para a determinação de curvas de crescimento de culturas aderentes, com menor variação entre as réplicas técnicas do que os métodos baseados em contagem das células

Construction of monoclonal cells derived from the human glioblastoma U87 cell line and study of their heterogeneity / 医学研究生学报

Objective The human glioblastoma (GBM) U87 cell line is employed as a model for studying the heterogeneity of GBM. This study was to examine the phenotypic profiles and genetic backgrounds of different monoclonal cells derived from the human GBM U87 cell line and explore the molecular mechanisms underlying the phenotypic difference. Methods Using the finite dilution method labeled with 5(6)-carboxyfluorescein diacetate N-hydroxy succinimidyl ester (CFSE), we constructed the monoclonal cell lines CF5 and G11 with typical morphological characteristics derived from the human GBM U87 cell line and identified them by short tandem repeat (STR). We detected the proliferation of the cells by CCK8 assay, EdU incorporation and colony-formation assay, their self-renewal capability by tumor sphere formation assay, their adhesion ability by immunofluorescence and CCK8 adhesion assay, their invasion ability with a 3D culture model, and their sensitivity to chemotherapeutic agents by Annexin V/PI double-staining flow cytometry. We performed transcriptome sequencing and bioinformatics analysis on the genetic profiles and determined the mRNA expressions of the representative differential genes in the enriched pathway by real-time quantitative PCR (qRT-PCR). Results The CF5 and G11 monoclonal cell lines morphologically typical of U87 were successfully constructed, the former small, short and thick, while the latter big, long and thin. Compared with the U87 and G11 cell lines, the CF5 cells showed a significantly higher proliferation ability (P < 0.01), though higher in the U87 than in the G11 cell line, a higher proportion of EdU-positive cells (0.35 ± 0.03 and 0.44 ± 0.03 vs 0.54 ± 0.05, P < 0.01), though higher in the U87 than in the G11 cell line, and a higher tumor-sphere formation ability (P < 0.01), though higher in the U87 than in the G11 cell line. In comparison with the U87 and CF5 cell lines, the G11 cells exhibited remarkably higher abilities of adhesion (P < 0.01) and invasion (P < 0.05), though both higher in the U87 than in the CF5 group. Totally, 159 genes were down-regulated and 303 up-regulated in the CF5 cells compared with those in the U87 and G11 cells, while 281 were down-regulated and 116 up-regulated in the G11 cells compared with those in the CF5 and U87 cells. The CF5 and G11 cells manifested the highest enrichment in the extracellular matrix-associated pathways, which were shown to be closely associated with the invasiveness and drug-resistance of the tumor. Conclusion We successfully constructed human GBM U87-derived monoclonal cell lines CF5 and G11 with different morphological features, phenotypic profiles and genetic backgrounds, which has paved the ground for further studies of the heterogeneity of GBM.

Experimental study on inhibitory effect of hawthorn leaves flavonoids on human glioblastoma U87 cells / 中国药理学通报

Aim To explore the inhibitory effect of hawthorn leaves flavonoids (HLF) on human glioblastoma U87 cells. Methods U87 cells were cultured in vitro. The concentration of HLF was 25, 50, 100 mg . L¹ as drug concentration. The effects of HLF on the proliferation, migration, invasion and adhesion of U87 cells were detected by CCK-8 assay, scratch assay, Transwell assay and adhesion assay, and clone forming ability was detected by cell clonogenic assay. Results HLF inhibited the proliferation, migration, invasion and adhesion of U87 cells in a concentration-dependent manner, and the difference was statistically significant compared with control group (P < 0. 05). When HLF reached 50 mg . L¹ the inhibition of tumor growth was the most significant. Conclusion HLF has a certain inhibitory effect on glioma U87 cells.

Effect and mechanism of Stellettin B induced human glioblastoma SF295 cells autophagy / 国际生物医学工程杂志

Objective To study the autophagy of human glioblastoma SF295 cells induced by sponge-derived triterpenoid Stellittin B (Stel B) and to discuss the related mechanism. Methods The proliferation inhibitory activity of Stel B on SF295 cells was studied by WST-8 assay. The autophagy effect induced by Stel B on SF295 cells was evaluated bya variety of experimental techniques, including MDC staining, Western Blot assay, and mRFP-GFP-LC3 plasmid transfection method.The activity of Stel B on the representative signal proteins in PI3K/Akt/mTOR pathway in SF295 cells was examined by Western Blot. The anti-tumor activity of Stel B was detected by WST-8 assay after blocking autophagy by autophagy inhibitor chloroquine. Results Stel B could significantly inhibit the proliferation of SF295 cells with IC50 value as 0.026 μmol/L. Plenty of autophagosome was found in Stel B treated A549 cells by MDC staining. The expression of autophagy marker proteins including LC3B-II increased. The confocal microscopy results showed that Stel B promoted autophagosome forming and inhibitedthe fusion of autophagosome with lysosome. The Western Blot results showed that Stel B inhibited the expression of PI3K-p110 protein in SF295 cells and decreased the phosphorylation level of Akt and mTOR.The inhibitory effects of Stel B with different concentrations on SF295 cells were all enhanced by the inhibition of autophagy with chloroquine, and the differences were statistically significant (all P<0.05). Conclusion Stel B can induce SF295 cells autophagy via blocking PI3K/Akt/mTOR pathway. This autophagy effect is beneficial to the survival of SF295 cells. The antitumor activity of Stel B can be enhanced by a combination of autophagy inhibitors.

Inhibitory mechanisms of timosaponin AⅢ on proliferation and growth of human glioblastoma cells / 中国中药杂志

To explore the inhibitory effect of timosaponin AⅢ on the proliferation of human glioblastoma cell line U87MG and investigate its related mechanism. As compared with the model group, the tumor weight was significantly reduced in timosaponin AⅢ-treated group. Timosaponin AⅢinhibited the proliferation of U87MG cell line in a dose-dependent manner. It up-regulated the gene and protein expression levels of p21, meanwhile inhibited the protein expression levels of β-Catenin, Cyclin D1 and Bcl-2. It also inhibited the translocation of β-Catenin into nucleus, suppressed the phosphorylation expression of ERK, but increased the phosphorylation expression of p38 and JNK. Combined use of JNK inhibitor SP600125 and p38 inhibitor SB203580 could decrease p21 and increase β-Catenin protein expressions. Timosaponin AⅢ inhibited the proliferation of human glioblastoma cell line U87MG partly by intervening MAPK and Wnt/β-Catenin signal pathways.

Establecimiento y caracterización de una línea celular derivada de un glioblastoma multiforme / Initiation and characterization of a glioblastoma multiforme derived cell line

Introducción: Las líneas celulares y los cultivos primarios son una excelente herramienta para el estudio de la biología, desarrollo y respuesta a la terapia en tumores cerebrales. Objetivo: Establecer y caracterizar una línea celular derivada de un glioblastoma multiforme como un modelo de estudio in vitro para la extrapolación y aplicación futura en terapia génica. Material y métodos: Se obtuvo una muestra de un paciente con diagnóstico clínico e histopatológico de glioblastoma multiforme, se caracterizó mediante inmunohistoquímica en cortes de tejido y por inmunocitoquímica sobre células cultivadas a partir del tumor desde el inicio del cultivo y durante los seis primeros pases, con dos tipos de marcadores específicos para glía: GFAP (glial fibrillary acidic protein) y S-100 (proteína de unión a calcio). Además, se evaluó la expresión de p53 y Bcl-2, como moduladores de apoptosis. Por último se hizo la caracterización citogenética. Resultados: Histopatológicamente, se confirmó el diagnóstico de glioblastoma multiforme. En los cultivos primarios se encontraron características citomorfológicas propias de un glioblastoma: células fibroblastoides planas, células con escaso citoplasma con 3 ó más procesos y por último bipolares o unipolares. Se encontró una expresión diferencial con los cuatro marcadores, con un patrón de marcaciones a nivel citoplasmático y nuclear a través de los pases estudiados. La línea celular se caracterizó por ser en su mayoría aneuploide con un número modal cromosómico entre 43 y 45, con un gran número de poliploidías (55-102 <4n>, XXYY) y endo-reduplicaciones (end 45, X, -Y). Conclusión: Se estableció una línea celular derivada de un glioblastoma multiforme con un fenotipo estable, con un notable mantenimiento del perfil glial y citogenético.

Introduction: Cell lines and primary cultures are a useful tool for studying basic biology, development and therapy responses in cancer and nervous system tumors. Aim: To establish and characterize a human glioblastoma multiforme (GBM) derived cell line as an in vitro biological model to study nervous system cancer chemotherapy and gene therapy. Materials and methods: A resected tumor piece was obtained from a patient with clinical and histopathological diagnosis of GBM. It was processed to obtain viable cells to culture and histological sections, which were immunostained to glial fibrillary acid protein (GFAP) and S-100 protein (calcium binding protein) and to evaluate expression of apoptosis related proteins p53 and Bcl-2. Finally a cytogenetic evaluation was carried out. Results: Histopathological examination confirmed classic findings of GBM. Typical cytomorphological features of GBM were found in cells of the primary cultures: bipolar or unipolar cells, flat fibroblastoid cells, process-bearing cells with scant cytoplasm and 3 or more processes. It was found a differential expression of the four markers, which had a nuclear and cytoplasmatic staining pattern throughout studied subcultures. Cell line exhibited a high level of aneuploidy with modal chromosomal number between 43-45, with presence of poliploidy (55-102 <4n>, XXYY) and endoreduplication (end 45, X, -Y). Conclusion: It was established a GBM derived cell line with a stable phenotype, maintaining morphological cell and cytogenetic characteristics.

STUDY OF WHOLE-MOUNT CYTOSKELETON OF HUMAN GLIOBLASTOMA BT_(325) CELLS BY CARBONPLATINUM COATING AND IMMUNOELECTRON MICROSCOPY / 解剖学报

Cells of BT_(325), a human glioblastoma cell line, were treated with 0.5% Triton X-100 and immunostained with a monoclonal antibody to vimentin and antiserum to glial fibrillary acidic protein (GFAP), and followed by fluorescence labeled secondary antibody. After observation with fluorescence microscope the cells were dehydrated in increasing grades of ethanol and then critical point-dried. The carbon-platinum coating replicas of cytoskeleton were observed by transmission electron microscopy. The morphology of whole cytoskeleton as well as the arrangement of microtubules, microfilaments and intermediate filaments were clearly observed by this technique. It was observed that the diameter of intermediate filaments highly increased in the cells stained with the monoclonal antibody to vimentin. In the samples stained with anti-GFAP intermediate filaments were decorated in some cells but undecorated in other cells. The results indicates that all BT_(325) cells express vimentin, but only a fraction of the cells express GFAP. It was also observed that if the cells were fixed with formaldehyde before immunostaing with the monoclonal antibody to vimentin, the diameter of the intermediate filaments only slightly increased. However, the diameter of the intermediate filaments highly increaed in the cells stained firstly with the monoclonal antibody to vimentin and followed by formaldehyde fixation. This result is in accord with that of our previous immunofluorescence study. It indicates that formaldehyde fixation may "mask" certain epitopes on vimentin molecules.