Migration and internalization of cells and polystyrene microspheres in tumor cell spheroids

The movement and internalization of ³H-labelled cells and of inert polystyrene microspheres within multicellular spheroids has been examined through histological sectioning and autoradiography. EMT6 and RIF-1 spheroids were cultured in spinner flasks for approx. 2.5 weeks. At this time, ³H-labelled cells and/or microspheres were allowed to adhere to the spheroid surface. Microspheres, ³H-labelled RIF-1 monolayer cells and ³H-labelled EMT6 monolayer cells were observed to move centripetally as a wave into EMT6 spheroids. In contrast, ³H-labelled trypsinized RIF-1 and EMT6 spheroid cells became mixed with the other non-labelled spheroid cells in homotypic RIF-1 and EMT6 spheroids, respectively. Reduction of spheroid growth by maintaining the spheroids at room temperature and by treatment with 2500 rads irradiation did not prohibit the internalization of ³H-labelled EMT6 cells and microspheres in EMT6 spheroids.     

Transformation-specific cell killing by a cancer-associated galactosyltransferase acceptor and cellular binding

Cancer-associated galactosyltransferase acceptor (CAGA glycoprotein), a small glycoprotein purified from human malignant effusion that selectively kills transformed cells, was tritiated by reductive methylation in the presence of NaB³H₄. CAGA-glycoprotein-sensitive cells (baby-hamster kidney cells transformed by polyoma virus and chick-embryo fibroblasts infected with Ts68 temperature-sensitive mutant of Rous sarcoma virus grown at 37°C, the permissive temperature) bound 3–5-fold more ³H-labelled CAGA glycoprotein than did their CAGA-glycoprotein-resistant non-transformed counterparts. The Rous-sarcoma-virus-infected chick-embryo fibroblasts grown at non-permissive temperature (41°C) bound an intermediate amount of ³H-labelled CAGA glycoprotein; however, this intermediate amount appeared to be sufficient to induce inhibition of cell growth when the infected chick-embryo fibroblasts treated at 41°C were switched to 37°C. Binding of ³H-labelled CAGA glycoprotein was time- and temperature-dependent and was not inhibited by monosaccharide. Binding was completely inhibited by the oligosaccharide liberated by endoglucosaminidase H treatment or by exhaustive Pronase digestion of intact CAGA glycoprotein. However, the isolated oligosaccharide failed to demonstrate the growth-inhibition characteristics of the intact glycopeptide. Binding of ³H-labelled CAGA glycoprotein was unaffected by co-incubation with the peptide core released by endoglucosaminidase H treatment. ³H-labelled CAGA glycoprotein bound to intact cells could be removed by trypsin treatment up to 4h after addition of the glycoprotein but not thereafter. This time course paralleled the decreasing reversibility of growth inhibition. However, all ³H-labelled CAGA glycoprotein was found in the supernatant when cells were first disrupted by sonication followed by trypsin treatment for up to 12h. ³H-labelled CAGA glycoprotein linked to Sepharose 4B failed to cause growth inhibition in CAGA-glycoprotein-sensitive cells. These findings suggest that binding of CAGA glycoprotein occurs via its oligosaccharide moiety. Binding appears to be a necessary but not sufficient condition to induce cell killing. Growth inhibition appears to depend on internalization of the glycoprotein and the presence of a transformation-specific cell process.     

Uptake of HDL unesterified and esterified cholesterol by human endothelial cells. Modulation by HDL phospholipolysis and cell cholesterol content

Human HDL (1.070-1.210), doubly labelled with ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol were incubated for 1–5 h with monolayer cultures of human endothelial cells. HDL were preincubated for 60–120 min the presence of albumin and with/without purified phospholipase A₂ (control HDL, phospholipase A₂ HDL) before dilution in the cell culture medium. Average phosphatidyl-choline (PC) degradation was 62.10% ± 2.57% (range 45–80%). A purified lipase /phospholipase A₁ from guinea pig pancreas was used in some experiments (range of PC hydrolysis: 16–70%). (1) ³H/¹⁴C-labelled unesterified cholesterol and ³H-labelled esterified cholesterol appeared in cells during 0–5 h incubations. Trypsin treatment allowed a simple adsorption of HDL onto the cell surface to be avoided, and most of the ³H-labelled esterified cholesterol transferred to cells was hydrolysed. Cell uptake of radioactive cholesterol increased as a function of HDL concentration but no saturation was achieved at the highest lipoprotein concentration used (200 μg cholesterol/ml). Flux of ³H/¹⁴C-labelled unesterified cholesterol was related to the cell cholesterol content, suggesting that it might partly represent an exchange process. The cell cholesterol content was slightly increased after 5 h incubation with HDL (+16%). (2) Pretreatment of HDL with purified phospholipase A₂ doubled on average the amount of cell recovered ³H-labelled esterified cholesterol, while the flux of ³H/¹⁴C-labelled unesterified cholesterol was enhanced by 15–25%. Both transfer and cell hydrolysis of ³H-labelled esterified cholesterol were increased. A stimulation was also observed using purified lipase/phospholipase A₁, provided that a threshold phospholipid degradation was achieved (between 27 and 45%). (3) Endothelial cells were conditioned in different media so as to modulate their charge in cholesterol. The uptake of ³H-labelled esterified cholesterol was found to be significantly higher in cholesterol-enriched cells compared to the sterol-depleted state. Finally, movements of ³H-labelled esterified cholesterol from HDL to endothelial cells were essentially unaffected by cell density or by the presence of partially purified cholesterol ester transfer protein. The possible roles of the transfer of HDL esterified cholesterol to endothelial cells and its modulation by phospholipases are discussed.     

Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells

Background

Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells.

Methods

Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways.

Results

The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation.

Conclusions

Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.

     

Platelet-derived growth factor (PDGF) induces intranuclear protein accumulation in 3T3 fibroblasts

Quiescent 3T3 fibroblasts were grown for a short time in the presence of [3H]amino acids then treated with PDGF, and the nucleo-cytoplasmic distribution of the 3H-labelled proteins was analysed by autoradiography. There was no difference in the total amount of 3H-labelled proteins in PDGF-treated and untreated cells but PDGF induced a significant increase in intranuclear protein accumulation.     

A rapid method for the determination of CTP and phosphocholine in rat liver and baby hamster kidney 21 cells

A rapid and sensitive assay for CTP and phosphocholine was required for us to determine the concentration of these compounds in tissues and cell cultures. Such a procedure was devised with CTP:phosphocholine cytidylyltransferase, an enzyme which is highly specific for CTP and phosphocholine. The 0--22% ammonium sulfate precipitate of a cytosolic extract from rat liver was used as the source of the enzyme. The amount of CTP in an extract was estimated by the conversion of [3H]phosphocholine to 3H-labelled CDP-choline. Similarly, the concentration of phosphocholine was estimated by the formation of 3H-labelled CDP-choline from 3H-labelled CTP. The conversion of CTP and phosphocholine to CDP-choline was 90% when inorganic pyrophosphatase was added to the incubations. The formation of CDP-choline was linear between 1 and 10 nmol of CTP or phosphocholine. The concentration of CTP was determined in rat liver (62 nmol/g wet weight) and baby hamster kidney 21 (BHK-21) cells (161 nmol/g wet weight). The concentration of phosphocholine in rat liver was 1.16 mumol/g wet weight whereas in BHK-21 cells it was much less (69 nmol/g wet weight). By this procedure, it may be possible to establish the importance of CTP and phosphocholine in the control of phosphatidylcholine biosynthesis.     

Cellular reactions of O6-methylguanine, a product of some alkylating carcinogens

Cultures of a purine-requiring mutant of Chinese hamster ovary cells (CHO-104b), randomly bred hamster embryo cells, or Escherichia coli B_s−1 were treated with non-toxic doses of ³H-labelled O⁶-methylguanine. DNA and RNA were isolated and subjected to enzymic digestion to nucleosides at pH8. The products of digestion were analysed by ion-exchange chromatography on columns of Dowex 50 (NH₄⁺ form) at pH8.9. No ³H-labelled O⁶-methylguanosine was detected in nucleic acid digests. ³H-labelled O⁶-methylguanine was O-demethylated yielding [³H]guanine in CHO-104b cells. Radioactivity in nucleic acid digests was associated with thymidine, guanosine, deoxyguanosine and an unidentified early-eluting product. Reports of similar unidentified products from nucleic acids labelled with various agents are discussed.     

Antitumor compound testing in glioblastoma organotypic brain cultures

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staining, an acidic environment, and tumor-surrounding astrocyte activation. Temozolomide inhibits GBM growth in brain Hi-Spots, but it is not effective in 2D cultures. Other chemotherapeutics that have proven to be inefficient in GBM treatment display low activity against GBM-derived cells growing in brain Hi-Spots in comparison to their activity against GBM 2D cultures. These findings suggest that GBM/brain Hi-Spots represent a simple system to culture cells derived from brain tumors in an orthotopic environment in vitro and that the system is reliable to test GBM targeting compounds.     

High resolution SEM analysis of acellular glomerular basement membrane following pepsin digestion: intrinsic fibrillar structures

Microdissection of acellular rat renal cortex with pepsin was carried out to investigate the morphological substructure of glomerular basement membrane (GBM) by high resolution SEM. Renal cortical blocks (less than 5 mm3) from adult male Sprague Dawley rats were rendered acellular by sequential detergent extraction and digested up to 184 hrs with 5 mg/ml pepsin (185 U/mg) in 0.5 M acetic acid (pH 2) at 10-15 degrees C. Samples were conventionally prepared for SEM, and observed at original magnifications of 500-100,000 diameters. At low magnifications (500-5,000x), acellular GBM surfaces appeared smooth at all digestion times. At higher magnifications (50,000-100,000x), control GBM surfaces were finely granular. Granule diameter ranged from 20-80 nm, with most between 30-40 nm. Pepsin digestion did not affect average granule size. Beginning at 44 hrs of digestion, intrinsic fibrillar structures comprised of linear arrays of 20-40 nm granules were observed on/in GBM surfaces. At later incubation times, this component of GBM became more extensive. At 160 hrs, the fibrillar arrays frequently bifurcated and showed distinctive "forked" termini, some of which comprised two sides of a triangle (120-150 nm on a side). Fork "handles" (310-350 nm in length) radiated from each angle of the triangle. These sometimes terminated in large granules (approximately 100 nm in diameter), two of which appeared to connect fibrillar arrays end-to-end. Together with other arrays, the interconnected triangles appeared to comprise a three-dimensional meshwork extending into the GBM and possibly providing support for, its granular components.     

Removal of glycosaminoglycans from cultures of human skin fibroblasts.

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.     

miR-135b Contributes to the Radioresistance by Targeting GSK3β in Human Glioblastoma Multiforme Cells

Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). Recent data strongly suggests the important role of miRNAs in cancer progression and therapeutic response. Here, we have established a radioresistant human GBM cell line U87R derived from parental U87 and found miR-135b expression was upregulated in U87R cells. miR-135b knockdown reversed radioresistance of U87R cells, and miR-135b overexpression enhanced radioresistance of U87 cells. Mechanically, bioinformatics analysis combined with experimental analysis demonstrated GSK3β (Glycogen synthase kinase 3 beta) was a novel direct target of miR-135b. Moreover, GSK3β protein expression was downregulated in U87R cells and restored expression of GSK3β increased radiosensitivity of U87R cells. In addition, clinical data indicated that the expression of miR-135b or GSK3β was significantly association with IR resistance of GBM samples. Our findings suggest miR-135b is involved in the radioresistance of human GBM cells and miR-135b-GSK3β axis may be a novel candidate for developing rational therapeutic strategies for human GBM treatment.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

C3G downregulation induces the acquisition of a mesenchymal phenotype that enhances aggressiveness of glioblastoma cells

Glioblastoma (GBM) is the most aggressive tumor from the central nervous system (CNS). The current lack of efficient therapies makes essential to find new treatment strategies. C3G, a guanine nucleotide exchange factor for some Ras proteins, plays a dual role in cancer, but its function in GBM remains unknown. Database analyses revealed a reduced C3G mRNA expression in GBM patient samples. C3G protein levels were also decreased in a panel of human GBM cell lines as compared to astrocytes. Based on this, we characterized C3G function in GBM using in vitro and in vivo human GBM models. We report here that C3G downregulation promoted the acquisition of a more mesenchymal phenotype that enhanced the migratory and invasive capacity of GBM cells. This facilitates foci formation in anchorage-dependent and -independent growth assays and the generation of larger tumors in xenografts and chick chorioallantoic membrane (CAM) assays, but with a lower cell density, as proliferation was reduced. Mechanistically, C3G knock-down impairs EGFR signaling by reducing cell surface EGFR through recycling inhibition, while upregulating the activation of several other receptor tyrosine kinases (RTKs) that might promote invasion. In particular, FGF2, likely acting through FGFR1, promoted invasion of C3G-silenced GBM cells. Moreover, ERKs mediate this invasiveness, both in response to FGF2- and serum-induced chemoattraction. In conclusion, our data show the distinct dependency of GBM tumors on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibition protocols. Hence, patients with a low C3G expression might not respond to EGFR inhibitors.Subject terms: CNS cancer, Metastasis     

Brahma-Related Gene-1 (BRG1) promotes the malignant phenotype of glioblastoma cells

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumour that is resistant to existing therapeutics. Identifying signalling pathways deregulated in GBM that can be targeted therapeutically is critical to improve the present dismal prognosis for GBM patients. In this report, we have identified that the BRG1 (Brahma-Related Gene-1) catalytic subunit of the SWI/SNF chromatin remodelling complex promotes the malignant phenotype of GBM cells. We found that BRG1 is ubiquitously expressed in tumour tissue from GBM patients, and high BRG1 expression levels are localized to specific brain tumour regions. Knockout (KO) of BRG1 by CRISPR-Cas9 gene editing had minimal effects on GBM cell proliferation, but significantly inhibited GBM cell migration and invasion. BRG1-KO also sensitized GBM cells to the anti-proliferative effects of the anti-cancer agent temozolomide (TMZ), which is used to treat GBM patients in the clinic, and selectively altered STAT3 tyrosine phosphorylation and gene expression. These results demonstrate that BRG-1 promotes invasion and migration, and decreases chemotherapy sensitivity, indicating that it functions in an oncogenic manner in GBM cells. Taken together, our findings suggest that targeting BRG1 in GBM may have therapeutic benefit in the treatment of this deadly form of brain cancer.     

Danthron Induced Apoptosis Through Mitochondria- and Caspase-3-Dependent Pathways in Human Brain Glioblastoma Multiforms GBM 8401 Cells

Danthron (1,8-dihydroxyanthraquinone), is one of component from Rheum palmatum L. (Polygonaceae), has been shown several biological activities but did not show to induce apoptosis in human brain tumor cells. The aim of this study is to investigate the mechanisms by danthron for the induction of apoptotic potential on human brain glioblastoma multiforms GBM 8401 cell line. Danthron showed a marked concentration- and time-dependent inhibition of GBM 8401 cell viability and induced apoptosis in a dose-and time-dependent manner. There was an attenuation of mitochondrial membrane potential (ΔΨ _m ) with the alterations of Bcl-2/Bax protein ratio in GBM 8401 cells, indicating the participation of a mitochondria-related mechanism. Pretreatment of a caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK) and caspase-3 inhibitor (Z-DEVE-FMK) significantly increased the viable of GBM 8401 cells implied that the participations of caspases. Western blotting analysis also showed the activation of initiator caspase-8 and caspase-9, and executor caspase-3 in GBM 8401 cells. Meanwhile, danthron also promoted the generation of reactive oxygen species (ROS) and cytosolic Ca²⁺ in GBM 8401 cells. Taken together, our data showed that danthron induced apoptosis in GBM 8401 cells through mitochondria-related and caspase-related pathways, and it may be further evaluated as a chemotherapeutic agent for human brain cancer.     

Zerumbone suppresses IKKα, Akt,and FOXO1 activation,resulting in apoptosis of GBM 8401 cells

Background

Zerumbone, a sesquiterpene compound isolated from subtropical ginger, Zingiber zerumbet Smith, has been documented to exert antitumoral and anti- inflammatory activities. In this study, we demonstrate that zerumbone induces apoptosis in human glioblastoma multiforme (GBM8401) cells and investigate the apoptotic mechanism.

Methods

We added a caspase inhibitor and transfected wild-type (WT) IKK and Akt into GBM 8401 cells, and measured cell viability and apoptosis by MTT assay and flow cytometry. By western blotting, we evaluated activation of caspase-3, dephosphorylation of IKK, Akt, FOXO1 with time, and change of IKK, Akt, and FOXO1 phosphorylation after transfection of WT IKK and Akt.

Results

Zerumbone (10∽50 μM) induced death of GBM8401 cells in a dose-dependent manner. Flow cytometry studies showed that zerumbone increased the percentage of apoptotic GBM cells. Zerumbone also caused caspase-3 activation and poly (ADP-ribose) polymerase (PARP) production. N-benzyloxycarbonyl -Val-Ala-Asp- fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, hindered zerumbone-induced cell death. Transfection of GBM 8401 cells with WT IKKα inhibited zerumbone-induced apoptosis, and zerumbone significantly decreased IKKα phosphorylation levels in a time-dependent manner. Similarly, transfection of GBM8401 cells with Akt suppressed zerumbone-induced apoptosis, and zerumbone also diminished Akt phosphorylation levels remarkably and time-dependently. Moreover, transfection of GBM8401 cells with WT IKKα reduced the zerumbone-induced decrease in Akt and FOXO1 phosphorylation. However, transfection with WT Akt decreased FOXO1, but not IKKα, phosphorylation.

Conclusion

The results suggest that inactivation of IKKα, followed by Akt and FOXO1 phosphorylation and caspase-3 activation, contributes to zerumbone-induced GBM cell apoptosis.     

HOXB13 promotes proliferation,migration, and invasion of glioblastoma through transcriptional upregulation of lncRNA HOXC-AS3

HOXB13 exerts a close relation in several human cancers. This study explored the role of HOXB13 in glioblastoma (GBM), a brain tissue with the highest aggressive rate and mortality in adults. Through microarray and immunohistochemistry analyses, HOXB13 was highly expressed in GBM tissues. Furthermore, we showed that high-level expression of HOXB13 in GBM was associated with worse survival, suggesting that HOXB13 could be a prognostic marker for patients with GBM. GBM cells U87 and U251 overexpressing HOXB13 showed enhanced proliferation, migration, and invasion relative to the control cells, while knockdown of HOXB13 led to decreased cell proliferation, migration, and invasion abilities. In addition, dual-luciferase report assay, chromatin immunoprecipitation assay, and quantitative real-time polymerase chain reaction data showed that HOXB13 directly bound to HOXC-AS3 promoter. HOXC-AS3 was involved in HOXB13-induced proliferation, migration, and invasion of GBM cells. In summary, this study revealed the prognostic potential of HOXB13 in GBM. We believed that HOXB13/HOXC-AS3 signaling axis can be served as therapeutic targets for this highly aggressive cancer.