U87MG Glioma Cells Research Articles

Directed cell migration towards softer environments.

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.

Hispidulin Enhances Temozolomide (TMZ)-Induced Cytotoxicity against Malignant Glioma Cells In Vitro by Inhibiting Autophagy.

Temozolomide (TMZ), an oral alkylating agent, is the widely used first-line chemotherapeutic reagent for glioma in clinical practice. However, TMZ-induced autophagy is another cellular process favoring glioma cell survival. This study aimed to explore whether hispidulin can facilitate TMZ-induced cell death of glioma. The MTT assay showed that coadministration with hispidulin and TMZ could significantly decrease the viability of glioma U87MG cells. Meanwhile, hispidulin administration was also observed to promote TMZ-induced apoptosis. Furthermore, additional hispidulin treatment further elevated TMZ-induced expression of Bax, cleaved-caspase-9, and cleaved-caspase-3 protein but decreased Bcl-2 protein expression in U87MG cells. We also observed that hispidulin suppressed TMZ-induced autophagy to promote apoptosis, as showed by decreased AVOs and LC3B-I/II protein expression. These results collectively suggested that the combination of hispidulin and TMZ could improve the antitumor efficiency of TMZ against malignant gliomas.

Bridging Size and Charge Effects of Mesoporous Silica Nanoparticles for Crossing the Blood-Brain Barrier.

The blood–brain barrier (BBB) is a highly selective cellular barrier that tightly controls the microenvironment of the central nervous system to restrict the passage of substances, which is a primary challenge in delivering therapeutic drugs to treat brain diseases. This study aimed to develop simple surface modifications of mesoporous silica nanoparticles (MSNs) without external stimuli or receptor protein conjugation, which exhibited a critical surface charge and size allowing them to cross the BBB. A series of MSNs with various charges and two different sizes of 50 and 200 nm were synthesized, which showed a uniform mesoporous structure with various surface zeta potentials ranging from +42.3 to −51.6 mV. Confocal microscopic results showed that 50 nm of strongly negatively charged N4-RMSN50@PEG/THPMP (∼−40 mV) could be significantly observed outside blood vessels of the brain in Tg(zfli1:EGFP) transgenic zebrafish embryos superior to the other negatively charged MSNs. However, very few positively charged MSNs were found in the brain, indicating that negatively charged MSNs could successfully penetrate the BBB. The data were confirmed by high-resolution images of 3D deconvoluted confocal microscopy and two-photon microscopy and zebrafish brain tissue sections. In addition, while increasing the size to 200 nm but maintaining the similar negative charge (∼40 mV), MSNs could not be detected in the brain of zebrafish, suggesting that transport across the BBB based on MSNs occurred in charge- and size-dependent manners. No obvious cytotoxicity was observed in the CTX-TNA2 astrocyte cell line and U87-MG glioma cell line treated with MSNs. After doxorubicin (Dox) loading, N4-RMSN50@PEG/THPMP/Dox enabled drug delivery and pH-responsive release. The toxicity assay showed that N4-RMSN50@PEG/THPMP could reduce Dox release, resulting in the increase of the survival rate in zebrafish. Flow cytometry demonstrated N4-RMSN50@PEG/THPMP had few cellular uptakes. Protein corona analysis revealed three transporter proteins, such as afamin, apolipoprotein E, and basigin, could contribute to BBB penetration, validating the possible mechanism of N4-RMSN50@PEG/THPMP crossing the BBB. With this simple approach, MSNs with critical negative charge and size could overcome the BBB-limiting characteristics of therapeutic drug molecules; furthermore, their use may also cause drug sustained-release in the brain, decreasing peripheral toxicity.

New metabolites (±)-bacillipyrrole A and bacillipyrazine A from the Mariana Trench-associated bacterium Bacillus subtilis SY2101

The new (±)-bacillipyrrole A (1) and bacillipyrazine A (2), together with seven known compounds, were isolated from a culture of the Mariana Trench sediment-derived bacterium Bacillus subtilis SY2101 in rice medium. Structures of the new compounds were elucidated by their HRESIMS data and NMR spectroscopic analyses. The known compound 3,5,6-trimethylpyrazine-2-methyl acetate (4) showed antiproliferative activity against human glioma U87MG cells with an IC50 value of 22.6 ± 2.8 μM.

The Protein L-Isoaspartyl (D-Aspartyl) Methyltransferase Regulates Glial-to-Mesenchymal Transition and Migration Induced by TGF-β1 in Human U-87 MG Glioma Cells.

The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-β1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-β1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-β1. In contrast, overexpressed wild-type PIMT and TGF-β1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-β1, while Snail stimulation by TGF-β1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-β1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-β1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT.

Using ELP Repeats as a Scaffold for De Novo Construction of Gadolinium-Binding Domains within Multifunctional Recombinant Proteins for Targeted Delivery of Gadolinium to Tumour Cells.

Three artificial proteins that bind the gadolinium ion (Gd3+) with tumour-specific ligands were de novo engineered and tested as candidate drugs for binary radiotherapy (BRT) and contrast agents for magnetic resonance imaging (MRI). Gd3+-binding modules were derived from calmodulin. They were joined with elastin-like polypeptide (ELP) repeats from human elastin to form the four-centre Gd3+-binding domain (4MBS-domain) that further was combined with F3 peptide (a ligand of nucleolin, a tumour marker) to form the F3-W4 block. The F3-W4 block was taken alone (E2-13W4 protein), as two repeats (E1-W8) and as three repeats (E1-W12). Each protein was supplemented with three copies of the RGD motif (a ligand of integrin αvβ3) and green fluorescent protein (GFP). In contrast to Magnevist (a Gd-containing contrast agent), the proteins exhibited three to four times higher accumulation in U87MG glioma and A375 melanoma cell lines than in normal fibroblasts. The proteins remained for >24 h in tumours induced by Ca755 adenocarcinoma in C57BL/6 mice. They exhibited stability towards blood proteases and only accumulated in the liver and kidney. The technological advantages of using the engineered proteins as a basis for developing efficient and non-toxic agents for early diagnosis of tumours by MRI as well as part of BRT were demonstrated.

Gallic Acid Enhances the Anti-Cancer Effect of Temozolomide in Human Glioma Cell Line via Inhibition of Akt and p38-MAPK Pathway

(1) Background: Temozolomide (TMZ), an oral alkylating agent, is used to treat malignant gliomas and other difficult-to-treat tumors. TMZ can enter the cerebrospinal fluid p.o. (per os) and does not need hepatic metabolism for activation of its use as a standard chemotherapeutic regimen after surgical resection of malignant glioma of the brain. However, the prognosis remains poor for most patients, and the survival rate is still unsatisfactory. Gallic acid (Ga) is a secondary metabolite existent in numerous plants. Ga shows various bioactivities, including antioxidant, anti-inflammatory, anticancer and antimicrobial effects. In this study, the latent enhanced anti-cancer efficacy of Ga in TMZ-treated U87MG cells (a human glioma line) was evaluated. (2) Methods: The U87MG cell line was cultured for 24 h. The cells were incubated with Ga alone, TMZ alone, or their combination for various time points. Cell viability and the drug combination index were evaluated by an XTT-based analysis and isobologram analysis, respectively. DNA destruction and intracellular reactive oxygen species (ROS) generation were analyzed by flow cytometer. The expression of various proteins was assessed via Western blotting. (3) Results: Compared with the action of TMZ alone or Ga alone, TMZ/Ga combination augmented the inhibition of cellular viability and apoptotic level in the U87MG glioma cell line. This enhanced anti-cancer effect correlated with the decreased expression of Bcl-2 and p-Akt, and corresponded with the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. In addition, Ga suppressed the TMZ-promoted ROS generation. (4) Conclusions: Ga can augment the anti-cancer effect of TMZ via the repression of Bcl-2 expression and Akt activation and the enhancement of the p38 MAPK pathway. Our results offer a novel probable approach for the medical treatment of malignant glioma.

Effects of endoplasmic reticulum stress-mediated CREB3L1 on apoptosis of glioma cells.

The association between endoplasmic reticulum stress (ERS) and apoptosis has been extensively studied. Cyclic adenosine monophosphate responsive element binding protein 3 like 1 (CREB3L1) has an important role in the development of glioma. In the present study, the potential association between ERS-induced apoptosis and CREB3L1 and its clinical implications were investigated. From a total of 30 cases, brain gliomas with different pathological grades surgically resected at the Department of Neurosurgery of the Affiliated Hospital of Guizhou Medical University (Guiyang, China) between January 2018 and January 2019 were collected. The expression of CREB3L1 and ERS-related proteins in gliomas with different degrees of malignancy was detected by immunohistochemistry. Furthermore, U87-MG glioma cells were cultured in vitro and treated with different concentrations of ERS inducer thapsigargin (TG). The Cell Counting Kit-8 (CCK-8) assay was performed to detect changes in cell activity at different incubation times and drug concentrations. Cell apoptosis was detected by Annexin Ⅴ-FITC/propidium iodide double staining and the protein expression levels of CREB3L1 and ERS were detected by western blot analysis. Immunohistochemical analysis suggested that the expression levels of CREB3L1, glucose-regulated protein, 78 kDa (GRP78) and C/EBP-homologous protein (CHOP) in World Health Organization (WHO) grade I glioma were higher than those in WHO grade Ⅱ-Ⅳ (all P<0.01). The results of the CCK-8 assay suggested that the activity of U87-MG glioma cells was significantly decreased after treatment with TG (all P<0.05), and this effect was time- and drug concentration-dependent. Flow cytometric analysis indicated that the apoptotic rate of the cells was increased, which was significant when the concentration of TG was 0.1 µmol/l (P<0.01). Furthermore, the protein expression of CREB3L1, GRP78 and CHOP in glioma cells treated with TG was increased (P<0.05). However, the expression level of Bcl-2 decreased (P<0.05). In conclusion, ERS may reduce the cell proliferative activity and promote apoptosis through mediating CREB3L1 expression. CREB3L1 may be a novel potential target for glioma therapy.

NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM.

Simple SummaryGlioblastoma multiforme (GBM) is the most aggressive type of glioma and exhibits extensive heterogeneity and poor prognosis with a high recurrence rate. Among the genetic alterations in GBM with different phenotypic states, a mesenchymal subtype has been associated with a worse outcome in patients with GBM. The mechanisms for the gain of the mesenchymal subtype in GBM remain unclear. Our aim was to investigate whether NOX2-induced high glycolytic activity could contribute to the gain of the mesenchymal phenotype in GBM. We revealed that NOX2-induced high glycolytic activity can induce the gain of the COL5A1-mediated mesenchymal phenotype in GBM. Our findings will provide the molecular mechanism by which NOX2 contributes to the gain of mesenchymal phenotype in GBM.The alteration of the cellular metabolism is a hallmark of glioma. The high glycolytic phenotype is a critical factor in the pathogenesis of high-grade glioma, including glioblastoma multiforme (GBM). GBM has been stratified into three subtypes as the proneural, mesenchymal, and classical subtypes. High glycolytic activity was found in mesenchymal GBM relative to proneural GBM. NADPH oxidase 2 (NOX2) has been linked to cellular metabolism and epithelial-mesenchymal transition (EMT) in tumors. The role of NOX2 in the regulation of the high glycolytic phenotype and the gain of the mesenchymal subtype in glioma remain unclear. Here, our results show that the levels of NOX2 were elevated in patients with GBM. NOX2 induces hexokinase 2 (HK2)-dependent high glycolytic activity in U87MG glioma cells. High levels of NOX2 are correlated with high levels of HK2 and glucose uptake in patients with GBM relative to benign glioma. Moreover, NOX2 increases the expression of mesenchymal-subtype-related genes, including COL5A1 and FN1 in U87MG glioma cells. High levels of NOX2 are correlated with high levels of COL5A1 and the accumulation of extracellular matrix (ECM) in patients with GBM relative to benign glioma. Furthermore, high levels of HK2 are correlated with high levels of COL5A1 in patients with GBM relative to benign glioma. Our results suggest that NOX2-induced high glycolytic activity contributes to the gain of the COL5A1-mediated mesenchymal phenotype in GBM.

PKM2 Is Overexpressed in Glioma Tissues, and Its Inhibition Highly Increases Late Apoptosis in U87MG Cells With Low-density Specificity.

Pyruvate kinase M2 (PKM2) functions as an important rate-limiting enzyme in aerobic glycolysis and is involved in tumor initiation and progression. However, there are few studies on the correlation between PKM2 expression and its role in glioma. PKM2 expression was immunohistochemically examined in human brain tumor samples. Furthermore, we studied the effects of two PKM2 inhibitors (shikonin and compound 3K) on the U87MG glioma cell line. PKM2 was overexpressed in most glioma tissues when compared to controls. Interestingly, glioma-adjacent tissues from showed slight PKM2 overexpression. This suggests that PKM2 overexpression maybe an important trigger factor for glioma tumorigenesis. We found that the PKM2 inhibitor shikonin was effective against U87MG cells at a relatively low dose and was largely dependent on low cellular density compared to the effects of the anticancer drug vincristine. Shikonin highly increased late-apoptosis of U87MG cells. We also demonstrated that autophagy was involved in the increase in late-apoptosis levels caused by shikonin. Although vincristine treatment led to a high level of G2-phase arrest in U87MG cells, shikonin did not increase G2 arrest. Co-treatment with two PKM2 inhibitors, shikonin and compound 3K, increased the inhibitory effects. Combination therapy with PKM2 inhibitors together might be more effective than combination therapy with anticancer drugs. Our findings encourage the application of PKM2-targeting in gliomas, and lay the foundation for the development of PKM2 inhibitors as promising antitumor agents for glioma.

Synergism of Temozolomide, Metformin, and Epigallocatechin Gallate Promotes Oxidative Stress-Induced Apoptosis in Glioma Cells

Aim: This study aimed to study the synergistic anti-glioma efficacies of Temozolomide, Metformin, and Epigallocatechin Gallate in U87MG and C6 glioma cells. Background: Glioblastoma (GBM) is the most malignant and invasive tumor of the central nervous system. The current standard treatment comprises surgical resection, followed by adjuvant radiotherapy and chemotherapy employing temozolomide (TMZ). Yet the survival rates for GBM patients are very low. Hence there is a need for new treatment regimes. Objective: This study aimed to unravel the synergistic anti-tumor potential of a biguanide drug, Metformin (MET) and a polyphenol, Epigallocatechin gallate (EGCG) to enhance the anti-GBM efficacy of the standard drug. Methods: The anti-proliferative effect of TMZ, MET, and EGCG, individually and in combination was elucidated in U87MG (human) and C6 (rat) glioma cells using MTT assay, and combination index was used to determine synergism. Cytotoxicity of the drugs was carried out in HEK293T noncancerous cells. Apoptotic morphological changes in the cells were observed by AO/EtBr staining. Furthermore, the effects of drugs on antioxidant and apoptotic genes (SOD, CAT, Nrf-2, Caspase- 9, and Bcl-2) were evaluated using qRT-PCR, and the protein levels of Nrf-2 and Caspase-9 were evaluated using ELISA. Results: The triple-drug combination (TMZ+ MET+ EGCG) synergistically inhibited the proliferation of U87MG and C6 glioma cells in a dose-dependent manner and promoted the apoptosis of glioma cells. The triple-drug combination significantly up-regulated the expression of antioxidant and apoptotic genes and induced oxidative stress, suggesting a shift in equilibrium towards apoptosis. Conclusion: MET and EGCG in combination with TMZ synergistically promoted oxidative stressinduced apoptosis in glioma cells. Hence, the combination of TMZ, MET, and EGCG may be therapeutically exploited for improving the clinical outcomes of patients with GBM.

Streptonaphthyridine A, a new naphthyridine analogue with antiproliferative activity against human glioma cells from mariana trench-associated actinomycete Streptomyces sp. SY2111

A new naphthyridine analogue, named streptonaphthyridine A (1), together with eight previously reported compounds (2–9), were isolated from a Mariana Trench sediment-associated actinomycete Streptomyces sp. SY2111. Planar structure of streptonaphthyridine A was established by analyses of its HRESIMS data and extensive NMR spectra and its absolute configuration was determined by a combination of single crystal X-ray diffraction analysis and optical rotation calculations. Streptonaphthyridine A (1) had antiproliferative activity against human glioma U87MG and U251 cells with IC50 values of 7.9 ± 1.3 and 13.4 ± 2.7 µM, respectively, and the known compound monomethylsulochrin (7) showed more potent activity with IC50 values of 0.6 ± 0.1 µM for U87MG cells and 0.1 ± 0.0 µM for U251 cells.

Advanced quantification for single-cell adhesion by variable-angle TIRF nanoscopy

Over the last decades, several techniques have been developed to study cell adhesion; however, they present significant shortcomings. Such techniques mostly focus on strong adhesion related to specific protein-protein associations, such as ligand-receptor binding in focal adhesions. Therefore, weak adhesion, related to less specific or nonspecific cell-substrate interactions, are rarely addressed. Hence, we propose in this work a complete investigation of cell adhesion, from highly specific to nonspecific adhesiveness, using variable-angle total internal reflection fluorescence (vaTIRF) nanoscopy. This technique allows us to map in real time cell topography with a nanometric axial resolution, along with cell cortex refractive index. These two key parameters allow us to distinguish high and low adhesive cell-substrate contacts. Furthermore, vaTIRF provides cell-substrate binding energy, thus revealing a correlation between cell contractility and cell-substrate binding energy. Here, we highlight the quantitative measurements achieved by vaTIRF on U87MG glioma cells expressing different amounts of α5 integrins and distinct motility on fibronectin. Regarding integrin expression level, data extracted from vaTIRF measurements, such as the number and size of high adhesive contacts per cell, corroborate the adhesiveness of U87MG cells as intended. Interestingly enough, we found that cells overexpressing α5 integrins present a higher contractility and lower adhesion energy.

The effect of temozolomide on Hsp60 and Hsp70 expression in extracellular vesicles derived from U87MG glioma cells

Abstract Objectives Temozolomide (TMZ) is an effective drug for glioblastoma multiforme (GBM), but the mechanism underlying TMZ resistance is poorly understood. New evidence has revealed that the release of heat shock proteins (Hsps) derived from extracellular vesicles (EVs) play an important role in cancer progression by modulating tumor microenvironment and cellular cross-talk. This study aims to evaluate the effects of TMZ on the expression of EV-derived and cellular Hsps and cell motility in U87MG human glioblastoma cell line. Methods Glial-EVs were isolated from the culture medium and characterized by SEM and immunoblotting. The effect of TMZ treatments (25, 200 and 750 µM) on cell proliferation (MTT assay), migration (scratch assay), and Hsp60 and Hsp70 levels (immunoblotting) were evaluated. Results TMZ treatments led to an increase in intracellular Hsp70 while decreasing EV-derived Hsp70. Cellular Hsp60 level was elevated at the low dose of TMZ, but it reduced at higher TMZ concentrations. Hsp60 was also decreased in EVs secreted from TMZ-treated cells. Besides, TMZ treatment reduced the proliferation and migration of glioma cells in a dose-dependent manner. Conclusions Our results suggest that TMZ has the potential to target both EV-derived and cellular Hsps for GBM treatment, thus it may reduce cell motility.

Investigating the Effect of Yttrium Oxide Nanoparticle in U87MG Glioma and PC3 Prostate Cancer: Molecular Approaches

Yttrium oxide (Y2O3) nanoparticles have very wide application areas such as biological imaging, photodynamic therapy, the material sciences, in the chemical synthesis of inorganic compounds, additives in plastic, paint, steel, optics, and iron. Potential risks to human health and the environment should be evaluated in a multi-dimensional perspective when developing nanoparticles for those applications. Therefore, in this research, we aimed to investigate changes in gene expression profiles (genes involved in different biological pathways) influenced by commonly Yttrium oxide (Y2O3) nanoparticle in human U87MG glioma and PC3 prostate cancer cell lines in vitro. The study was planned to be carried out in two stages. In the first stage, cell viability and cytotoxicity parameters were studied using 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays, respectively, with human U87MG glioma and human PC3 prostate cancer cell cultures. In the second stage, to obtain a clear insight into the molecular events after exposing, we examined the effects of selected Y2O3 nanoparticle on the expression of genes in U87MG and PC3 cell cultures using RT2 Profiler PCR Arrays. Y2O3 nanoparticles have IC20 of 0,18 mg/L and 2,903 mg/L in PC3 and U87MG cell lines, respectively. Y2O3 nanoparticle induced up-regulation of 24 and down-regulation of 22 genes in PC3 cells and up-regulation of 53 and down-regulation of 27 genes in U87MG cells. This study of gene expression profiles affected by nanotoxicity provides critical information for the clinical and environmental applications of Y2O3 nanoparticles.

Evaluation of Histone Deacetylase Inhibitors as Radiosensitizers for Proton and Light Ion Radiotherapy.

Significant opportunities remain for pharmacologically enhancing the clinical effectiveness of proton and carbon ion-based radiotherapies to achieve both tumor cell radiosensitization and normal tissue radioprotection. We investigated whether pretreatment with the hydroxamate-based histone deacetylase inhibitors (HDACi) SAHA (vorinostat), M344, and PTACH impacts radiation-induced DNA double-strand break (DSB) induction and repair, cell killing, and transformation (acquisition of anchorage-independent growth in soft agar) in human normal and tumor cell lines following gamma ray and light ion irradiation. Treatment of normal NFF28 primary fibroblasts and U2OS osteosarcoma, A549 lung carcinoma, and U87MG glioma cells with 5–10 µM HDACi concentrations 18 h prior to cesium-137 gamma irradiation resulted in radiosensitization measured by clonogenic survival assays and increased levels of colocalized gamma-H2AX/53BP1 foci induction. We similarly tested these HDACi following irradiation with 200 MeV protons, 290 MeV/n carbon ions, and 350 MeV/n oxygen ions delivered in the Bragg plateau region. Unlike uniform gamma ray radiosensitization, effects of HDACi pretreatment were unexpectedly cell type and ion species-dependent with C-12 and O-16 ion irradiations showing enhanced G0/G1-phase fibroblast survival (radioprotection) and in some cases reduced or absent tumor cell radiosensitization. DSB-associated foci levels were similar for proton-irradiated DMSO control and SAHA-treated fibroblast cultures, while lower levels of induced foci were observed in SAHA-pretreated C-12 ion-irradiated fibroblasts. Fibroblast transformation frequencies measured for all radiation types were generally LET-dependent and lowest following proton irradiation; however, both gamma and proton exposures showed hyperlinear transformation induction at low doses (≤25 cGy). HDACi pretreatments led to overall lower transformation frequencies at low doses for all radiation types except O-16 ions but generally led to higher transformation frequencies at higher doses (>50 cGy). The results of these in vitro studies cast doubt on the clinical efficacy of using HDACi as radiosensitizers for light ion-based hadron radiotherapy given the mixed results on their radiosensitization effectiveness and related possibility of increased second cancer induction.

New polyhydroxanthones from the marine-associated fungus Penicillium sp. ZZ1750

Five polyhydroxanthones, including new natural product ergochrome C (1) and new compounds ergochromes D–G (2–5), together with 11 known compounds (6–16), were isolated from the marine-associated fungus Penicillium sp. ZZ1750. Structures of the polyhydroxanthones were determined based on their HRESIMS data, extensive NMR spectroscopic analyses, the results from single crystal X-ray diffraction, 13C NMR and ECD calculations. All isolated compounds were assayed for their antiproliferative activity against human glioma U87MG and U251 cells and antimicrobial activities in inhibiting the growth of methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Candida albicans. The five polyhydroxanthones of ergochromes C–G (1–5) had moderate antimicrobial activities against MRSA and C. albicans with MIC values of 16–36 μg/mL. Only known compound calyxanthone (9) showed weak antiproliferative activity (IC50: 46.6 μM) against glioma U87MG cells.

A Novel Oral Arginase 1/2 Inhibitor Enhances the Antitumor Effect of PD-1 Inhibition in Murine Experimental Gliomas by Altering the Immunosuppressive Environment.

Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.

Stromal and Tumor Glioma-Derived Cells with Similar Characteristics have Differences in α-Smooth Muscle Actin Expression and Localization

Gliomas are solid brain tumors composed of tumor cells and recruited heterogenic stromal component. The study of the interactions between the perivascular niche and its surrounding cells is of great value in unraveling mechanisms of drug resistance in malignant gliomas. In this study, we isolated the stromal diploid cell population from oligodendroglioma and a mixed population of tumor aneuploid and stromal diploid cells from astrocytoma specimens. The stromal cells expressed neural stem/progenitor and mesenchymal markers showing the same discordant phenotype that is typical for glioma cells. Moreover, some of the stromal cells expressed CD133. For the first time, we demonstrated that this type of stromal cells had the typical myofibroblastic phenotype as the α-SMA+ cells formed α-SMA fibers and exhibited the specific function to deposit extracellular matrix (ECM) proteins at least in vitro. Immunofluorescent analysis showed diffuse or focal α-SMA staining in the cytoplasm of the astrocytoma-derived, A172, T98G, and U251MG glioma cells. We could suggest that α-SMA may be one of the main molecules, bearing protective functions. Possible mechanisms and consequences of α-SMA disruptions in gliomas are discussed.

LOXL3 Silencing Affected Cell Adhesion and Invasion in U87MG Glioma Cells.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.