U87MG Glioblastoma Cells Research Articles

Characterization of glioblastoma spheroid models for drug screening and phototherapy assays

Glioblastoma (GBM) spheroids present as a promising alternative testing platform for in vivo models of brain cancer, aiming at the 3Rs principle of animal use in pre-clinical trials. However, the poor characterization and irreproducibility of various GBM-based spheroids have hampered the achievement of statistically relevant data for drug screening and photodynamic therapy (PDT) assays. In a previous study (Calori et al., 2022), we defined the conditions for inducing tight compaction of U87MG, T98G, UW473, A172, and U251 GBM cell lines into reproducible 3D spheroids using type I collagen as an extracellular matrix. Herein, GBM spheroids were characterized in their growth profile, proliferation rate, cell viability, necrotic nucleus, and cell death mechanisms and applied in PDT assays. Driven by proliferation, U87MG and A172 spheroids grew with time, at least up to day 12, whereas T98G and U251 demonstrated little change. The order of cell viability of spheroids over time was U87MG > A172 > U251 > T98G. A necrotic nucleus was formed over time for all cell lines, as shown by histological assays and the inverse size-cell viability relationship. When subjected to PDT, all spheroids showed a dose-dependent response, with an excellent dose-light-cell viability correlation. The higher light dose required for PDT of GBM 3D spheroids compared with the simplest 2D monolayers and its proximity to in vivo dose response in the literature is proof of its applicability in pre-clinical tests to reduce the use of animals in research.

CBMS-3 SEARCH FOR COMPOUNDS WITH ANTI-TUMOUR EFFECTS AGAINST GLIOBLASTOMA

Abstract Purpose Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and the resistance to temozolomide, the standard treatment for GBM. Therefore, the development of new therapeutic agents for GBM is desired. We searched our compound library for compounds with anti-tumor activity against GBM and identified Curcumin (Cur) derivatives, Compound A and B. The purpose of this study was to investigate the antitumor activity of Compound A and B against GBM. Methods and Results To evaluate the antitumor activity of Compound A and B against GBM, we performed the MTT assay. Human glioblastoma cell lines, U87-MG and U251 cells, were treated with Compound A and B. After 96 hours, cell viability was evaluated using CCK-8, and the IC50 values of Compound A and B were calculated. In U87-MG, the IC50 of Cur was 9.78 μM, whereas the that of Compound A was 2.42 μM and that of Compound B was 1.28 μM. In U251 cells, the IC50 of Cur was 9.50 μM, whereas that of Compound A was 2.27 μM and that of Compound B was 0.64 μM. Next, to examine the effects of Compound A and B on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. At the concentrations at which antitumor effects were observed (Compound A; 3 μM, B; 1.5 μM), there was no reduction in cell viability in primary rat cultured astrocytes. Discussion The present study shows that Compound A and B exhibit antitumor activity against human glioblastoma cells at lower concentrations than Cur without affecting normal cells. We will examine the effects of Compound A and B in a mouse model of brain tumor transplanted with U87-RFP cells in the future, which may lead to the development of noble brain tumor therapeutics.

Ruxolitinib enhances cytotoxic and apoptotic effects of temozolomide on glioblastoma cells by regulating WNT signaling pathway-related genes.

Although temozolomide is the primary chemotherapeutic agent in glioblastoma, current studies have focused on its combinational applications to overcome resistance by targeting multiple pathways. JAK/STAT and WNT are among the essential cancer-related signaling pathways. Ruxolitinib, the first approved JAK1/2 inhibitor, has promise in glioblastoma with its blood-brain barrier (BBB) crossing ability. The mentioned study aims to evaluate the anti-cancer potential of ruxolitinib individually and in combination with temozolomide on glioblastoma cells, brain cancer stem cells (BCSCs), and BBB-forming healthy cells. It also intends to determine the effects of JAK inhibitor treatment in combination with temozolomide on WNT signaling, which is known to cross-talk with the JAK/STAT pathway. The U87MG, BCSC, and HBMEC cell lines were the in vitro models. The cytotoxic and apoptotic effects of ruxolitinib and the combination were determined by the WST-1 test and Annexin V assay, respectively. The expression level changes of WNT signaling pathway genes caused by ruxolitinib and the combination treatments were defined by the qRT-PCR method. Network analysis of significantly upregulated and downregulated genes was performed via the GO KEGG pathway enrichment module of the String V11.5 database. The IC50 value of the ruxolitinib on U87MG glioblastoma cells was determined as 94.07µM at 24th h. The combination of temozolomide and ruxolitinib had a synergistic effect on U87MG cells at 24th h. The combination index (CI) was determined as 0.796, and ED60 values of ruxolitinib and temozolomide were determined as 89.75 and 391.48µM, respectively. Ruxolitinib improves the apoptotic effect of temozolomide on glioblastoma cells and brain cancer stem cells. Ruxolitinib regulates the WNT signaling pathway both individually and in combination with temozolomide. Our study indicates the potential of ruxolitinib to increase the cytotoxic and apoptotic activity of temozolomide in glioblastoma cells, also considering CSCs and healthy BBB-forming cells. As supported by gene expression and network analyses, the BBB-crossing agent ruxolitinib promises the potential to increase the efficacy of temozolomide in glioblastoma by affecting multiple signaling pathways in both cancer cells and CSCs.

Euphol from Tapinanthus sp. Induces Apoptosis and Affects Signaling Proteins in Glioblastoma and Prostate Cancer Cells

Plants play an important role in cancer therapy. They are source of natural molecules which can induce apoptosis in cancer cells by affecting molecular mechanisms implicated in cancer progression. The MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways are two classical signaling pathways implicated in cancer progression and constitute therapeutic targets against cancer. This study aimed to evaluate the effect of euphol on MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways in glioblastoma and prostate cancer cells. Euphol is a tetracyclique triterpene alcohol isolated from Tapinanthus sp. which is a hemi parasitic plant belonging to Loranthaceae family. Plant powder was extracted by maceration and euphol was isolated and described using respectively column chromatography separation on silica gel and spectroscopic data. Cytotoxic effect of euphol was evaluated using XTT assay and its effect on MAP Kinase/ERK1/2 and PI3K/AKT protein expression was investigated by Western immunoblot analysis. Apotosis was analyzed by evaluating caspase-3/7 activity. Our investigations demonstrated that this compound has an important cytotoxic effect on C6 and U87 MG glioblastoma (GBM) cells and PC-3 prostate cancer cells. Furthermore, euphol-induced apoptosis revealed by elevated caspase 3/7 activity, was correlated with a significant inhibition of MAP kinase/Erk 1/2 and PI3K/Akt signaling pathway in glioblastoma U87 MG cells. The reverse effect was observed in C6 glioblastoma cells, where apoptosis was correlated with a long-lasting activation of Erk 1/2. In PC-3 cells, euphol had no or limited effect on Erk 1/2 and Akt activity. These results indicate that euphol induces cell death in glioblastoma and prostate cancer cells and regulates significantly Erk1/2 and Akt activity in glioblastoma cells.

Isoginkgetin—A Natural Compound to Control U87MG Glioblastoma Cell Growth and Migration Activating Apoptosis and Autophagy

Isoginkgetin (Iso) is a natural bioflavonoid isolated from the leaves of Ginkgo biloba, this natural substance exhibits many healing properties, among which the antitumor effect stands out. Here we tested the effect of Iso on the growth of U87MG glioblastoma cells. Growth curves and MTT toxicity assays showed time and dose-dependent growth inhibition of U87MG after treatment with Iso (15/25 µM) for 1, 2, and 3 days. The cell growth block of U87MG was further investigated with the colony formation test, which showed that iso treatment for 24 h reduced colony formation. The present study also aimed to evaluate the effect of Iso on U87MG glioblastoma cell migration. The FACS analysis, on the other hand, showed that treatment with Iso 15 µM determines a blockage of the cell cycle in the S1 phase. Further investigation shows that Iso treatment of U87MG altered the protein pathways of homeostasis including autophagy and apoptosis. The present study demonstrated, for the first time, that Iso could represent an excellent adjuvant drug for the treatment of glioblastoma by simultaneously activating multiple mechanisms that control the growth and migration of neoplastic cells.

Upregulated FKBP1A Suppresses Glioblastoma Cell Growth via Apoptosis Pathway

Glioblastoma (GBM), the most deadly primary brain tumor, presents a major medical difficulty. The need for better therapeutic targets in GBM is therefore urgent. A growing body of evidence suggests that the gene FKBP1A plays an important role in tumor progression and may be therapeutically useful. However, the role of FKBP1A in glioblastoma and the underlying biologic mechanism remain unclear. The purpose of this study was to identify the role of FKBP1A in GBM and its molecular mechanism. We demonstrated that FKBP1A was the hub gene in GBM via a weighted correlation network analysis (WGCNA) and differentially expressed genes (DEGs) analysis based on the bulk RNA-seq data from TCGA and GTEx. Afterwards, we proved that the upregulated FKBP1A protein could promote GBM cell death by CCK-8 assays in U87MG and t98g GBM cell lines. We further demonstrated two key pathways of FKBP1A in GBM by bioinformatics methods: 'Apoptosis' and 'mTOR signaling pathway'. Subsequently, the key pathways were verified by flow cytometry and Western blot. We identified that upregulated FKBP1A could inhibit GBM growth via the apoptosis pathway. Together, these findings may contribute to future GBM treatment.

Cannabis sativa ethanolic extract demonstrated significant anti-tumor effects associated with elevated expression of AXIN1 protein in glioblastoma U87-MG cell line

Glioblastoma as the most common and lethal primary brain malignancy in adults is extremely resistant to conventional treatments. Recent evidence indicated that Cannabis sativa inhibits cell growth and induce apoptosis in cancer cells. Therefore, this study was conducted to investigate how Cannabis sativa ethanolic extract can affect U87-MG glioblastoma cell line through expression analysis of AXIN1 protein as a key regulator of Wnt/β-catenin signaling. Human U87-MG cells were treated with different concentrations of C. sativa ethanolic extract (40, 80, 120, 160, 200, 240, and 280 μg/ml) for 24 h. The cell viability and the protein levels of AXIN1 were evaluated using MTT, western blotting, and Immunofluorescence (ICC) assays, respectively. Our findings demonstrated that administration of Cannabis sativa extract decreased the viability of U87-MG cells in a dose-dependent manner. Moreover, a significant increase in the protein levels of AXIN1 was observed in U87-MG cells treated with 4.4, and 8.8 μl/ml for 24 h in comparison to un-exposed and the cells incubated with the dose of 2.2 μl/ml (P ≤ 0.05 each). Our results indicated that decreased cell viability of glioblastoma cell line in a dose-dependent manner in response to Cannabis sativa extract is associated with increased expression of AXIN1 protein. This anti-cancer effect of Cannabis sativa could suggest a novel therapeutic approach for the treatment of glioblastoma.

ONX-0914 Induces Apoptosis and Autophagy with p53 Regulation in Human Glioblastoma Cells.

Glioblastoma is believed to be one of the most aggressive brain tumors in the world. ONX-0914 (PR957) is a selective inhibitor of proteasome subunit beta type-8 (PSMB8). Previous studies have shown that inhibiting PSMB8 expression in glioblastoma reduces tumor progression. Therefore, this study aimed to determine whether ONX-0914 has antitumor effects on human glioblastoma. The results indicated that ONX-0914 treatment inhibited survival in LN229, GBM8401, and U87MG glioblastoma cells. Cell cycle analysis showed that ONX-0914 treatment caused cell cycle arrest at the G1 phase and apoptosis in glioblastoma cells. The protein expression of BCL-2 was reduced and PARP was cleaved after ONX-0914 treatment. Furthermore, the levels of p53 and phosphorylated p53 were increased by ONX-0914 treatment in glioblastoma cells. ONX-0914 also induced autophagy in glioblastoma cells. Furthermore, the p53 inhibitor pifithrin attenuated apoptosis but enhanced autophagy caused by ONX-0914. In an orthotopic mouse model, TMZ plus ONX-0914 reduced tumor progression better than the control or TMZ alone. These data suggest that ONX-0914 is a novel therapeutic drug for glioblastoma.

EXTH-16. THE ORAL IRON-MIMETIC GALLIUM MALTOLATE SUPPRESSES TREATMENT-RESISTANT GLIOBLASTOMA – IN VIVO VALIDATION

Abstract BACKGROUND We previously reported on our in vitro and in vivo studies using the metallocompound gallium maltolate (GaM) to target glioblastoma (GBM). We demonstrated a profound cytotoxic effect in vitrov and in vivo. Additionally, our preliminary in vivo data suggested a substantial inhibitory effect on tumor growth, as well as a significant extension of disease-specific survival (OS). Here, we further validate the inhibitory effects of GaM in vivo. METHODS In vitro irradiated adult GBM U87-MG cells were stereotactically implanted into the right striatum of male and female athymic rats. Advanced MR imaging at 9.4T was carried out weekly starting two weeks after implantation. Daily oral GaM (50 mg/kg) or vehicle were provided on tumor confirmation. Longitudinal advanced MRI parameters were processed for enhancing tumor ROIs in OsiriX 8.5.1 (lite) with Imaging Biometrics Software (Imaging Biometrics LLC,). Statistical analyses included Kaplan-Meier survival plots, linear mixed model (LMM) comparisons, and t-statistic for slopes comparison (as indicator of tumor growth rate). RESULTS Median OS for the male pilot cohort (5 control, 5 GaM) was 28 and 51 days, respectively; 31 and 59 days for the male replication cohort (1 control, 3 GaM); 37 and 48 days for the female replication cohort (6 control, 7 GaM). GaM-treated xenograft tumors grew significantly slower than control tumors with sex and time dependent growth rates. No significant differences were seen between male and female cohorts in both control and treatment groups at all timepoints. Hence, subsequent analyses were performed on pooled data. Median OS was 30.5 days and 48 days for control and GaM groups, respectively (p < .001). A strong correlation between treatment and survival (Χ2 = 6.238187; p = .0125) and tumor growth suppression (LMM; p < .0001) was evident. CONCLUSION We have successfully validated the inhibitory effects of GaM on GBM growth in vivo.

ANGI-04. DEPLETION OF CHITINASE-3-LIKE PROTEIN 1 (CHI3L1) IN HUMAN U87-MG GLIOMA CELLS AFFECTS TUMOUR GROWTH AND NEOVASCULATURE IN INTRACRANIAL MOUSE MODEL

Abstract Chitinase-3-like protein 1 (CHI3L1) plays multiple roles in inflammation, tissue remodelling, and wound healing. It is a new, potentially druggable target for the treatment of cancer due to its association with both extracellular matrix (ECM) modifications and potential involvement in neoangiogenesis. Expression of CHI3L1 is upregulated in glioblastoma (GBM), the most common and aggressive primary brain tumour. The role of CHI3L1 in GBM progression is not fully understood and calls for further research. We generated a knockout (KO) of CHI3L1 in human glioblastoma U87-MG cells. To evaluate the effects of CHI3L1 KO on tumour growth, wild-type (WT) or CHI3L1 KO cells were intracranially implanted into athymic mice and tumour volume was assessed with magnetic resonance imaging. We have measured the serum level of CHI3L1 in both groups and correlated it with the tumour burden. Immunofluorescent staining against endothelium marker – von Willebrand factor (vWF), and perivascular water channel – aquaporin 4 (AQP4) were employed to evaluate the tumour vasculature in both groups. We found that CHI3L1 KO in glioblastoma cells significantly reduced volume of established tumors. Blood serum level of CHI3L1 positively correlated with tumour burden of experimental animals, which corroborates its role as a biomarker. Immunostaining for vWF revealed that in CHI3L1 KO gliomas there were more non-capillary blood vessels than in controls. We observed a higher level of AQP4 in CHI3L1 KO tumours, which suggests a difference in the structure of perivascular space, and thus vasculature functionality, in CHI3L1 depleted gliomas. In conclusion, we demonstrated the involvement of glioma-derived CHI3L1 in the growth and neovasculature of experimental human gliomas. Our findings suggest that the depletion of CHI3L1 in glioma cells results in a higher number and possibly different structure of non-capillary blood vessels in the tumour. Studies supported by the grant N 2020/39/B/NZ4/02683 from the National Science Center.

Voxtalisib and low intensity pulsed ultrasound combinatorial effect on glioblastoma multiforme cancer stem cells via PI3K/AKT/mTOR.

Glioblastoma (GBM) is the most aggressive brain tumor contributed to tumor growth by cancer stem cells (CSCs). Targeting CSCs is vital to preventing differentiation into cancer cells, their proliferation, and treatment resistance. According to research, PI3K/AKT/mTOR signaling is active in GBM and GBMCSCs. Anticancer medications combined with ultrasound application have been proposed as a strategy to increase the drug intake of cancer cells. This study aims to investigate the effects of inhibition of PI3K/ AKT/ mTOR pathway with dual inhibitor Voxtalisib (Vox; also known as XL765) and low intensity pulsed ultrasound (LIPUS) combinations in GBM and GBMCSCs in the point of cell survival. F-actin was also used to evaluate cell motility. GBMCSCs were isolated from the human glioblastoma U87 MG cell line using the fluorescence-activated cell sorting (FACS) method. Cells were exposed to various concentrations of Vox, LIPUS, and their combinations. Cell count and viability assay was used to determine drug delivery doses. F-actin and mTOR immunofluorescence staining were used to identify cytoskeletal alterations and PI3K/AKT/mTOR signal pathway suppression, respectively. Additionally, the migration capacity of cells was shown with standard wound-healing experiments. High doses of Vox+LIPUS inhibited mTOR and decreased the viability in both cell groups. Inhibiting mTOR activated autophagy, and LIPUS increased autophagy in GBM cells. However, GBMCSCs were resistant to autophagy even at high drug dosages. Both in GBM and GBMCSCs, combinations of Vox and LIPUS were observed to decrease F-actin density and cell motility. The combination of Vox+LIPUS has increased drug effectiveness in targeted GBM and GBMCSCs. Combinatory treatment with PI3K/AKT/mTOR signaling pathway and LIPUS has been thought to help develop more effective therapeutic approaches for GBM.

18F-AlF-NOTA-PCP1 PET/CT Imaging Visualize Radiation-Induced PD-L1 Expression in Glioblastoma Subcutaneous Xenograft Mouse Models

<h3>Purpose/Objective(s)</h3> Remarkable clinical benefits from radiotherapy combined with PD-1/PD-L1 immune checkpoint blockades were reported across several cancer patients. However, the optimal time window of combining treatment remains undefined, which may be correlated with the timing of upregulation of PD-L1 expression. PD-L1 antibody-based PET/CT imaging was difficult to translate into clinical settings secondary to the long interval before the scan. We therefore generated a peptide-based PET radiotracer to monitor radiation-induced PD-L1 expression in a non-invasive manner, which could be used to assess the optimal time window of combining radio- and immunotherapy. <h3>Materials/Methods</h3> Peptide targeting PD-L1 (PCP1) was conjugated with NOTA and radiolabeled with 18F ([18F]-AlF-NOTA-PCP1). The Binding assay was performed in U87MG and GL261 glioblastoma cell lines with different levels of PD-L1 expression. [18F]-AlF-NOTA-PCP1 PET/CT imaging was prepared in U87MG and GL261 xenografts and syngeneic tumor models. <i>In vivo</i> imaging data was confirmed by <i>ex vivo</i> biodistribution and immunohistochemistry (IHC). To evaluate the radiation-induced PD-L1 upregulation, [18F]-AlF-NOTA-PCP1 PET/CT scan was prepared 3 days after U87MG tumor on mice subjecting to 20 Gy × 1F external radiation therapy. <h3>Results</h3> [18F]-AlF-NOTA-PCP1 was developed with high radiochemical purity and stability. The Binding assay showed more than 20 folds [18F]-AlF-NOTA-PCP1 binding to U87MG compared with GL261 cell line (p = 0.0495). The U87MG and GL261 tumors and excretory organs were identified by [18F]-AlF-NOTA-PCP1 PET/CT imaging. [18F]-AlF-NOTA-PCP1 PET/CT data showed different %ID/cc (percentage of injected dose per cubic centimeter of tissue) values of ROIs (regions of interest) among tumor models as evaluated by ex vivo methods. Preliminary results demonstrated the increase in PD-L1 expression of irradiated tumors on [18F]-AlF-NOTA-PCP1 PET/CT, showing a %ID/cc value of nearly 4 folds development post-radiation. <h3>Conclusion</h3> PET imaging with [18F]-AlF-NOTA-PCP1 is an effective approach for specific and non-invasive visualization of tumor PD-L1 level. PD-L1 expression can be induced by radiotherapy treatments and [18F]-AlF-NOTA-PCP1 PET can quantify these changes in glioblastoma subcutaneous xenograft mouse models.

Spironolactone Induces Apoptotic Cell Death in Human Glioblastoma U87-MG Cancer Cells

Background: Spironolactone is a conventional drug widely in use for the treatment of heart failure and hypertension patients. On the other side recent studies have reported spironolactone can prevent growth and drug resistance in cancer stem cells (CSCs), by inhibiting DNA double-strand break (DSB) repair; suggesting its potential application in cancer therapy. Objectives: Our study aimed at assessing the potential cytotoxicity of spironolactone in human U87-MG glioblastoma cells. Methods: Different concentrations of spironolactone (0 - 50 μM) for 48 and 72 h were used for treatment. Cell viability assay was carried out by the 4, 5-dimethylthiazole-2-yl, 2, 5-diphenyl tetrazolium (MTT) method. Apoptosis was evaluated using annexin V/PI staining and flow cytometry and colorimetric measurement of caspase 8 and 9 activity. Results: Our findings showed a significant dose-dependent cytotoxic effect of spironolactone with maximum effect in 30 μM (P-value &lt; 0.05). Spironolactone can induce approximately 20% apoptotic cell death in U87-MG cancer cells which were mainly related to early apoptotic cells. Indeed, the activity of caspase 8 and 9 was significantly elevated in spironolactone-treated cells compared to mock control. Conclusions: Findings showed the cytotoxic effect of spironolactone in U87-MG glioblastoma cancer cells in a mechanism dependent on apoptosis cell death induction. Our findings suggest the potential application of spironolactone in the treatment of glioblastoma in vitro.

Brain Tumour Lessons From Alzheimer’s Disease: Beta-Amyloid and SorLA Expression in Glioblastoma

Abstract AIMS Exploring how two proteins strongly associated with Alzheimer’s disease are expressed in glioma. Amyloid beta (Aβ) is a peptide cleaved from amyloid precursor protein (APP) that is shuttled around the cell by sorting-related receptor with A-type repeats (SorLA). Low expression of SorLA has been linked to increased risk of Alzheimer’s disease as APP processing shifts towards production of the plaque-forming Aβ42 rather than the more benign Aβ40. METHOD Expression of Aβ40, Aβ42 and SorLA was determined in immortalised cell lines (grade 4 glioblastoma U87MG, grade 2 astrocytoma 1321N1, foetal astrocytes SVGp12), glioblastoma patient-derived cells (PD301, PD304) and normal human astrocytes through western blotting or immunocytochemistry. RESULTS SorLA was present in all cells. Relative optical density was more than 60-fold higher for U87MG glioblastoma cells and approximately 10-fold higher for 1321N1 astrocytoma cells than SVGp12 cells. Similarly, SorLA had a significantly higher expression (p&amp;lt;0.01) in patient derived cells than normal human astrocytes. High expression of SorLA was accompanied by lower Aβ42 in patient derived cells compared to normal human astrocytes. Expression of Aβ40 was similar across patient derived cells and normal human astrocytes. CONCLUSION These data suggest that SorLA is retained in glioblastoma and remains functional to drive APP processing away from Aβ42 production. Future work will determine how high SorLA expression contributes to the increased motility and invasiveness seen in glioblastoma cells by transfecting siRNA to knock down SorLA.

Inhibitor of differentiation 1 in U87MG glioblastoma cells promotes HUVEC sprouting through endothelin-1.

Anti-angiogenesis therapy, a promising remedy against tumor progression, is now widely used to treat numerous types of cancer. Since vascular endothelial growth factor (VEGF) is the most vital factor in angiogenesis, most anti-angiogenesis drugs target the VEGF-related pathway. However, in glioblastoma (GBM), the therapeutic strategy involving the inhibition of VEGF signaling is ineffective. The present study demonstrated that the potential angiogenic function of endothelin-1 (EDN1) was upregulated by inhibitor of differentiation 1 (ID1) independent of VEGF during tumor angiogenesis. Anatomic structure transcriptomes of patients with GBM revealed that the expression levels of ID1 and EDN1 were specifically upregulated in the vascular-related region. The aortic ring assay and endothelial sprouting assay demonstrated that EDN1 more potently promoted endothelial sprouting ability than VEGF. The activity of EDN1 was induced by endothelin receptor, which seemed to mediate regulation via positive feedback. Finally, in patients with GBM who did not respond to bevacizumab, a VEGF antagonist, EDN1 expression was higher than that in bevacizumab responders. Collectively, the present study demonstrated that EDN1 is a potent angiogenic factor inducing endothelial sprouting and may be a novel target for inhibiting glioma angiogenesis.

Cell death induced in glioblastoma cells by Plasma-Activated-Liquids (PAL) is primarily mediated by membrane lipid peroxidation and not ROS influx

Since first identified in 1879, plasma, the fourth state of matter, has been developed and utilised in many fields. Nonthermal atmospheric plasma, also known as cold plasma, can be applied to liquids, where plasma reactive species such as reactive Oxygen and Nitrogen species and their effects can be retained and mediated through plasma-activated liquids (PAL). In the medical field, PAL is considered promising for wound treatment, sterilisation and cancer therapy due to its rich and relatively long-lived reactive species components. This study sought to identify any potential antagonistic effect between antioxidative intracellularly accumulated platinum nanoparticles (PtNPs) and PAL. We found that PAL can significantly reduce the viability of glioblastoma U-251MG cells. This did not involve measurable ROS influx but instead lead to lipid damage on the plasma membrane of cells exposed to PAL. Although the intracellular antioxidative PtNPs showed no protective effect against PAL, this study contributes to further understanding of principle cell killing routes of PAL and discovery of potential PAL-related therapy and methods to inhibit side effects.

Chrysomycin A Inhibits the Proliferation, Migration and Invasion of U251 and U87-MG Glioblastoma Cells to Exert Its Anti-Cancer Effects.

Chrysomycin A (Chr-A), an antibiotic from Streptomyces, is reported to have anti-tumor and anti-tuberculous activities, but its anti-glioblastoma activity and possible mechanism are not clear. Therefore, the current study was to investigate the mechanism of Chr-A against glioblastoma using U251 and U87-MG human cells. CCK8 assays, EdU-DNA synthesis assays and LDH assays were carried out to detect cell viability, proliferation and cytotoxicity of U251 and U87-MG cells, respectively. Transwell assays were performed to detect the invasion and migration abilities of glioblastoma cells. Western blot was used to validate the potential proteins. Chr-A treatment significantly inhibited the growth of glioblastoma cells and weakened the ability of cell migration and invasion by down regulating the expression of slug, MMP2 and MMP9. Furthermore, Chr-A also down regulated Akt, p-Akt, GSK-3β, p-GSK-3β and their downstream proteins, such as β-catenin and c-Myc in human glioblastoma cells. In conclusion, Chr-A may inhibit the proliferation, migration and invasion of glioblastoma cells through the Akt/GSK-3β/β-catenin signaling pathway.

Locked and Loaded: β-Galactosidase Activated Photodynamic Therapy Agent Enables Selective Imaging and Targeted Treatment of Glioblastoma Multiforme Cancer Cells.

Selective detection and effective therapy of brain cancer, specifically, the very aggressive glioblastoma multiforme (GBM), remains one of the paramount challenges in clinical settings. While radiotherapy combined surgery is proposed as the main treatment course, it has several drawbacks such as complexity of the operation and common development of recurrent tumors in this course of patient care. Unique opportunities presented by photodynamic therapy (PDT) offer promising, effective, and precise therapy against GBM cells along with simultaneous imaging opportunities. However, activatable, theranostic molecular systems in PDT modality for GBM remained scarce. Specifically, even though elevated β-galactosidase (β-gal) activity in glioblastoma cells is well-documented, targeted, activatable therapeutic PDT agents have not been realized. Herein, we report a β-galactosidase (β-gal) activatable phototheranostic agent based on an iodinated resorufin core (RB-1) which was realized in only three steps with commercial reagents in 29% overall yield. RB-1 showed very high singlet oxygen (1O2) quantum yield (54%) accompanied by a remarkable turn-on response in fluorescence upon enzymatic activation. RB-1 was tested in different cell lines and revealed selective photocytotoxicity in U-87MG glioblastoma cells. Additionally, thanks to almost 7% fluorescence quantum yield (ΦF) despite extremely high 1O2 generation yield, RB-1 was also demonstrated as a successful agent for fluorescence imaging of U-87MG cells. Due to significantly lower (β-gal) activity in healthy cells (NIH/3T3), RB-1 stayed in a passive state and showed minimal photo and dark toxicity. RB-1 marks the first example of a β-gal activatable phototheranostic agent toward effective treatment of glioblastoma.

Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells.

The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial–mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells’ infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.

Antitumor activity of copper(II) complexes with Schiff bases derived from N′-tosylbenzene-1,2-diamine

The electrochemical oxidation of anodic metal copper in a solution of the ligands N-[(5-tert-butyl-2-hydroxyphenyl)methylidine]-N′-tosylbenzene-1,2-diamine [H2L1] and N-[(3,5-di-tert-butyl-2-hydroxyphenyl)methylidine]-N′-tosylbenzene-1,2-diamine, [H2L2] afforded homoleptic [CuL] compounds or solvate [CuLS] complexes. The addition to the electrochemical cell of coligands (L') such as 2,2′-bipyridine (2-bpy), 4,4′-bipyridine(4-bpy) or 1,10-phenanthroline (phen) allowed the synthesis, in one step, of heteroleptic [CuLL'] compounds, namely [CuL1(H2O)] (1), [CuL1(2,2′-bpy)]⋅CH3CN (2), [CuL1(phen)]·H2O (3), [Cu2L12(4,4′-bpy)] (4), [CuL2(CH3OH)] (5), [CuL2(2,2′-bpy)] (6), [CuL2(phen)] (7) and [Cu2L22(4,4′-bpy)] (8). The crystal structures of both ligands, H2L1, H2L2, and those of the complexes (2), (4), (5), (6) and (7) have been determined by X-ray diffraction techniques. Coordination polyhedron around metal atom is square planar for [CuL2(CH3OH)] (5) and [Cu2L12(4,4′-bpy)] (4) and square pyramid for the other complexes with additional chelating ligands. The cytotoxic activity of this new series of copper(II) complexes against the SH-SY5Y neuroblastoma cell line and U87-MG and U373-MG glioblastoma cell lines has been investigated. Most of the test compounds showed higher activity than cisplatin in the three cell lines. Among this series, compound [CuL1(phen)] (3) displayed the highest activity with IC50 equal to 1.77 μM on SH-SY5Y whereas compound [Cu2L12(4.4′-bpy)] (4) resulted the most potent compounds on U87 MG and U373 MG glioblastoma cell lines. Studies on the cytotoxic activity of these derivatives suggest that these compounds induce cell death by a mechanism other than apoptosis.