Comparative Effects of Deferiprone and Salinomycin on Lead-Induced Disturbance in the Homeostasis of Intrarenal Essential Elements in Mice

Lead (Pb) exposure induces severe nephrotoxic effects in humans and animals. Herein, we compare the effects of two chelating agents, salinomycin and deferiprone, on Pb-induced renal alterations in mice and in the homeostasis of essential elements. Adult male mice (Institute of Cancer Research (ICR)) were randomized into four groups: control (Ctrl)—untreated mice administered distilled water for 28 days; Pb-exposed group (Pb)—mice administered orally an average daily dose of 80 mg/kg body weight (BW) lead (II) nitrate (Pb(NO₃)₂) during the first two weeks of the experimental protocol followed by the administration of distilled water for another two weeks; salinomycin-treated (Pb + Sal) group—Pb-exposed mice, administered an average daily dose of 16 mg/kg BW salinomycin for two weeks; deferiprone-treated (Pb + Def) group—Pb-exposed mice, administered an average daily dose of 20 mg/kg BW deferiprone for 14 days. The exposure of mice to Pb induced significant accumulation of the toxic metal in the kidneys and elicited inflammation with leukocyte infiltrations near the glomerulus. Biochemical analysis of the sera revealed that Pb significantly altered the renal function markers. Pb-induced renal toxicity was accompanied by a significant decrease in the endogenous renal concentrations of phosphorous (P), calcium (Ca), copper (Cu) and selenium (Se). In contrast to deferiprone, salinomycin significantly improved renal morphology in Pb-treated mice and decreased the Pb content by 13.62% compared to the Pb-exposed group. There was also a mild decrease in the renal endogenous concentration of magnesium (Mg) and elevation of the renal concentration of iron (Fe) in the salinomycin-treated group compared to controls. Overall, the results demonstrated that salinomycin is a more effective chelating agent for the treatment of Pb-induced alterations in renal morphology compared to deferiprone.     

Physical Forces in Glioblastoma Migration: A Systematic Review

The invasive capabilities of glioblastoma (GBM) define the cancer’s aggressiveness, treatment resistance, and overall mortality. The tumor microenvironment influences the molecular behavior of cells, both epigenetically and genetically. Current forces being studied include properties of the extracellular matrix (ECM), such as stiffness and “sensing” capabilities. There is currently limited data on the physical forces in GBM—both relating to how they influence their environment and how their environment influences them. This review outlines the advances that have been made in the field. It is our hope that further investigation of the physical forces involved in GBM will highlight new therapeutic options and increase patient survival. A search of the PubMed database was conducted through to 23 March 2022 with the following search terms: (glioblastoma) AND (physical forces OR pressure OR shear forces OR compression OR tension OR torsion) AND (migration OR invasion). Our review yielded 11 external/applied/mechanical forces and 2 tumor microenvironment (TME) forces that affect the ability of GBM to locally migrate and invade. Both external forces and forces within the tumor microenvironment have been implicated in GBM migration, invasion, and treatment resistance. We endorse further research in this area to target the physical forces affecting the migration and invasion of GBM.     

Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells

About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.     

TRPML2 Mucolipin Channels Drive the Response of Glioma Stem Cells to Temozolomide and Affect the Overall Survival in Glioblastoma Patients

The survival of patients with glioblastoma (GBM) is poor. The main cause is the presence of glioma stem cells (GSCs), exceptionally resistant to temozolomide (TMZ) treatment. This last may be related to the heterogeneous expression of ion channels, among them TRPML2. Its mRNA expression was evaluated in two different neural stem cell (NS/PC) lines and sixteen GBM stem-like cells by qRT-PCR. The response to TMZ was evaluated in undifferentiated or differentiated GSCs, and in TRPML2-induced or silenced GSCs. The relationship between TRPML2 expression and responsiveness to TMZ treatment was evaluated by MTT assay showing that increased TRPML2 mRNA levels are associated with resistance to TMZ. This research was deepened by qRT-PCR and western blot analysis. PI3K/AKT and JAK/STAT pathways as well as ABC and SLC drug transporters were involved. Finally, the relationship between TRPML2 expression and overall survival (OS) and progression-free survival (PFS) in patient-derived GSCs was evaluated by Kaplan–Meier analysis. The expression of TRPML2 mRNA correlates with worse OS and PFS in GBM patients. Thus, the expression of TRPML2 in GSCs influences the responsiveness to TMZ in vitro and affects OS and PFS in GBM patients.     

Demethoxycurcumin Suppresses Human Brain Glioblastoma Multiforme GBM 8401 Cell Xenograft Tumor in Nude Mice In Vivo

Demethoxycurcumin (DMC), a derivate of curcumin, has been shown to induce apoptotic cell death in human glioblastoma multiforme GBM 8401 cells via cell cycle arrest and induction of cell apoptosis. However, there is no report showing DMC suppresses glioblastoma multiforme cells in vivo. In the present study, we investigated the effects of DMC on GBM8401 cells in vivo. At first, we established a luciferase-expressing stable clone named GBM 8401/luc2. Second, mice were inoculated subcutaneously with GBM 8401/luc2 cells to generate a xenograft tumor mice model. After inoculation, tumor volume reached 100–120 mm³, and all mice were randomly divided into three groups: Group I was treated with 110 µL phosphate-buffered solution (PBS) containing 0.1% dimethyl sulfoxide, Group II with 30 mg/kg of DMC, and Group III with 60 mg/kg of DMC. Mice from each group were given the oral treatment of DMC by gavage for 21 days. The body weight and tumor volume were recorded every 3 days. DMC significantly decreased the tumor volumes, and 60 mg/kg treatment showed a higher decrease in tumor volumes than that of 30 mg/kg, However, DMC did not affect the body weights. The photons emitted from mice tumors were detected with Xenogen IVIS imaging system, DMC at both doses decreased the total photon flux and 60 mg/kg treatment of DMC has low total photon flux than that of 30 mg/kg. The tumor volumes and weights in 60 mg/kg treatment of DMC were lower than that of 30 mg/kg. Immunohistochemical analysis was used to measure protein expression of tumors and results showed that DMC treatment led to lightly staining with anti-Bcl-2 and -XIAP and 60 mg/kg treatment of DMC has lighter staining with anti-Bcl-2 and -XIAP than that of 30 mg/kg. The higher dose (60 mg/kg) of DMC has higher signals of cleaved-caspase-3 than that of the lower dose (30 mg/kg). Furthermore, the hematoxylin and eosin (H&E) staining of liver tissues showed no significant difference between DMC-treated and control-groups. Overall, these observations showed that DMC suppressed tumor properties in vivo and DMC may be used against human glioblastoma multiforme in the future.     

Bisdemethoxycurcumin Induces Cell Apoptosis and Inhibits Human Brain Glioblastoma GBM 8401/Luc2 Cell Xenograft Tumor in Subcutaneous Nude Mice In Vivo

Bisdemethoxycurcumin (BDMC) has biological activities, including anticancer effects in vitro; however, its anticancer effects in human glioblastoma (GBM) cells have not been examined yet. This study aimed to evaluate the tumor inhibitory effect and molecular mechanism of BDMC on human GBM 8401/luc2 cells in vitro and in vivo. In vitro studies have shown that BDMC significantly reduced cell viability and induced cell apoptosis in GBM 8401/luc2 cells. Furthermore, BDMC induced apoptosis via inhibited Bcl-2 (anti-apoptotic protein) and increased Bax (pro-apoptotic proteins) and cytochrome c release in GBM 8401/luc2 cells in vitro. Then, twelve BALB/c-nude mice were xenografted with human glioblastoma GBM 8401/luc2 cancer cells subcutaneously, and the xenograft nude mice were treated without and with BDMC (30 and 60 mg/kg of BDMC treatment) every 3 days. GBM 8401/luc2 cell xenografts experiment showed that the growth of the tumors was significantly suppressed by BDMC administration at both doses based on the reduction of tumor size and weights. BDMC did not change the body weight and the H&E histopathology analysis of liver samples, indicating that BDMC did not induce systemic toxicity. Meanwhile, treatment with BDMC up-regulated the expressions of BAX and cleaved caspase-3, while it down-regulated the protein expressions of Bcl-2 and XIAP in the tumor tissues compared with the control group. This study has demonstrated that BDMC presents potent anticancer activity on the human glioblastoma GBM 8401/luc2 cell xenograft model by inducing apoptosis and inhibiting tumor cell proliferation and shows the potential for further development to the anti-GBM cancer drug.     

NBM-BMX,an HDAC8 Inhibitor,Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the β-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of β-catenin was reversed by proteasome inhibitor via the β-catenin/ GSK3β signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the β-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.     

Myricanol Induces Apoptotic Cell Death and Anti-Tumor Activity in Non-Small Cell Lung Carcinoma in Vivo

This study explored the inhibiting effect and mechanism of myricanol on lung adenocarcinoma A549 xenografts in nude mice. Forty nude mice with subcutaneous A549 xenografts were randomly divided into five groups: high-dose myricanol (40 mg/kg body weight) group; middle-dose myricanol (20 mg/kg body weight) group; low-dose myricanol (10 mg/kg body weight) group; polyethylene glycol 400 vehicle group (1 mL/kg); and tumor model group. Nude mice were sacrificed after 14 days of treatment and the tumor inhibition rate (TIR, %) was then calculated. The relative mRNA expression levels of Bax, Bcl-2, VEGF, HIF-1α, and survivin in the tumor tissues were determined by real-time PCR. TUNEL assay was applied to determine cellular apoptosis, while IHC test was performed to detect the protein expression levels of Bax, Bcl-2, VEGF, HIF-1α, and survivin. The TIR of the three myricanol-treated groups ranged from 14.9% to 38.5%. The IHC results showed that the protein expression of Bcl-2, VEGF, HIF-1α, and survivin were consistently downregulated, whereas that of Bax was upregulated after myricanol treatment. Myricanol also significantly upregulated the mRNA expression of Bax and downregulated that of Bcl-2, VEGF, HIF-1α, and survivin in a dose-dependent manner (p < 0.05 to 0.001). These results are consistent with those of IHC. The TUNEL assay results indicated that apoptotic-positive cells significantly increased in the myricanol-treated tumor tissues compared with the cells of the vehicle control group (p < 0.01 to 0.001). These data suggest that myricanol could significantly decelerate tumor growth in vivo by inducing apoptosis.     

Glycine Nano-Selenium Enhances Immunoglobulin and Cytokine Production in Mice Immunized with H9N2 Avian Influenza Virus Vaccine

This study was performed to investigate the immune enhancement effect of glycine nano-selenium, a microelement on H9N2 avian influenza virus vaccine (H9N2 AIV vaccine) in mice. Fifty (50) Specific Pathogen Free Kunming mice aged 4–6 weeks (18–20 g Body weight) were randomly divided into five groups: control normal group, which received no immunization + 0.5 mL 0.9% normal saline, positive control group, which received H9N2 AIV vaccine + 0.5 mL 0.9% normal saline, 0.25 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 0.25 mg/kg selenium solution, 0.5 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 0.5 mg/kg selenium solution, and 1 mg/kg selenium group, which received H9N2 AIV vaccine + 0.5 mL 1 mg/kg selenium solution. Hematoxylin and eosin staining, enzyme linked immunosorbent assay (ELISA), and quantitative real time polymerase chain reaction (qRT-PCR) methods were used to investigate the pathological changes, immunoglobulin levels, and cytokine gene expressions in this study. The results showed that all tested doses (0.25 mg/kg, 0.5 mg/kg and 1.00 mg/kg) of glycine nano-selenium did not lead to poisoning in mice. In addition, when compared to the positive control group, glycine nano-selenium increased the immunoglobin indexes (IgA, IgG, IgM and AIV-H9 IgG in serum) as well as the mRNA levels of IL-1β, IL-6 and INF-γ in the liver, lungs, and spleen (p < 0.05). In summary, glycine nano-selenium could enhance the efficacy of avian influenza vaccine.     

Up-Regulation of Cyclooxygenase-2 (COX-2) Expression by Temozolomide (TMZ) in Human Glioblastoma (GBM) Cell Lines

TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte^® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.     

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.     

Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

Glioblastoma multiforme (GBM) is a malignant primary brain tumor. The 5-year relative survival rate of patients with GBM remains <30% on average despite aggressive treatments, and secondary therapy fails in 90% of patients. In chemotherapeutic failure, detoxification proteins are crucial to the activity of chemotherapy drugs. Usually, glutathione S-transferase (GST) superfamily members act as detoxification enzymes by activating xenobiotic metabolites through conjugation with glutathione in healthy cells. However, some overexpressed GSTs not only increase GST activity but also trigger chemotherapy resistance and tumorigenesis-related signaling transductions. Whether GSTM3 is involved in GBM chemoresistance remains unclear. In the current study, we found that T98G, a GBM cell line with pre-existing temozolomide (TMZ) resistance, has high glycolysis and GSTM3 expression. GSTM3 knockdown in T98G decreased glycolysis ability through lactate dehydrogenase A activity reduction. Moreover, it increased TMZ toxicity and decreased invasion ability. Furthermore, we provide next-generation sequencing–based identification of significantly changed messenger RNAs of T98G cells with GSTM3 knockdown for further research. GSTM3 was downregulated in intrinsic TMZ-resistant T98G with a change in the expression levels of some essential glycolysis-related genes. Thus, GSTM3 was associated with glycolysis in chemotherapeutic resistance in T98G cells. Our findings provide new insight into the GSTM3 mechanism in recurring GBM.     

O6-Methylguanine-Methyltransferase (MGMT) Promoter Methylation Status in Glioma Stem-Like Cells is Correlated to Temozolomide Sensitivity Under Differentiation-Promoting Conditions

Glioblastoma (GBM) is the most malignant type of primary brain tumor with a very poor prognosis. The actual standard protocol of treatment for GBM patients consists of radiotherapy and concomitant temozolomide (TMZ). However, the therapeutic efficacy of this treatment is limited due to tumor recurrence and TMZ resistance. Recently isolated, glioma stem-like cells (GSCs) are thought to represent the population of tumorigenic cells responsible for GBM resistance and recurrence following surgery and chemotherapy. In addition, MGMT (O6-methylguanine-methyltransferase) methylation is considered as one of the principal mechanisms contributing to TMZ sensitivity of GBM. In this study we have isolated GSCs from 10 adult GBM patients and investigated the relationship between MGMT methylation status and Temozolomide (TMZ) sensitivity of these lines grown either in stem-like or differentiation promoting conditions. Sensitivity to TMZ was significantly associated with MGMT methylation status in cells committed to differentiation but not in stem-like cells. In addition, patients harboring highly methylated MGMT promoters had a longer overall survival. These results reveal the importance of the differentiation process when considering the predictive value of MGMT status in GSCs for clinical response to TMZ.     

Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner

Glioblastoma multiforme (GBM) recurrences after temozolomide (TMZ) treatment result from the expansion of drug-resistant and potentially invasive GBM cells. This process is facilitated by O6-Methylguanine-DNA Methyltransferase (MGMT), which counteracts alkylating TMZ activity. We traced the expansion of invasive cell lineages under persistent chemotherapeutic stress in MGMT^low (U87) and MGMT^high (T98G) GBM populations to look into the mechanisms of TMZ-induced microevolution of GBM invasiveness. TMZ treatment induced short-term, pro-invasive phenotypic shifts of U87 cells, in the absence of Snail-1 activation. They were illustrated by a transient induction of their motility and followed by the hypertrophy and the signs of senescence in scarce U87 sub-populations that survived long-term TMZ stress. In turn, MGMT^high T98G cells reacted to the long-term TMZ treatment with the permanent induction of invasiveness. Ectopic Snail-1 down-regulation attenuated this effect, whereas its up-regulation augmented T98G invasiveness. MGMT^low and MGMT^high cells both reacted to the long-term TMZ stress with the induction of Cx43 expression. However, only in MGMT^high T98G populations, Cx43 was directly involved in the induction of invasiveness, as manifested by the induction of T98G invasiveness after ectopic Cx43 up-regulation and by the opposite effect after Cx43 down-regulation. Collectively, Snail-1/Cx43-dependent signaling participates in the long-term TMZ-induced microevolution of the invasive GBM front. High MGMT activity remains a prerequisite for this process, even though MGMT-related GBM chemoresistance is not necessary for its initiation.     

Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy

Tumor recurrence is the main challenge in glioblastoma (GBM) treatment. Gold standard therapy temozolomide (TMZ) is known to induce upregulation of IL8/CXCL2/CXCR2 signaling that promotes tumor progression and angiogenesis. Our aim was to verify the alterations on this signaling pathway in human GBM recurrence and to investigate the impact of TMZ in particular. Furthermore, a combi-therapy of TMZ and CXCR2 antagonization was established to assess the efficacy and tolerability. First, we analyzed 76 matched primary and recurrent GBM samples with regard to various histological aspects with a focus on the role of TMZ treatment and the assessment of predictors of overall survival (OS). Second, the combi-therapy with TMZ and CXCR2-antagonization was evaluated in a syngeneic mouse tumor model with in-depth immunohistological investigations and subsequent gene expression analyses. We observed a significantly decreased infiltration of tumor-associated microglia/macrophages (TAM) in recurrent tumors, while a high TAM infiltration in primary tumors was associated with a reduced OS. Additionally, more patients expressed IL8 in recurrent tumors and TMZ therapy maintained CXCL2 expression. In mice, enhanced anti-tumoral effects were observed after combi-therapy. In conclusion, high TAM infiltration predicts a survival disadvantage, supporting findings of the tumor-promoting phenotype of TAMs. Furthermore, the combination therapy seemed to be promising to overcome CXCR2-mediated resistance.     

Polyunsaturated Fatty Acid-Enriched Lipid Fingerprint of Glioblastoma Proliferative Regions Is Differentially Regulated According to Glioblastoma Molecular Subtype

Glioblastoma (GBM) represents one of the deadliest tumors owing to a lack of effective treatments. The adverse outcomes are worsened by high rates of treatment discontinuation, caused by the severe side effects of temozolomide (TMZ), the reference treatment. Therefore, understanding TMZ’s effects on GBM and healthy brain tissue could reveal new approaches to address chemotherapy side effects. In this context, we have previously demonstrated the membrane lipidome is highly cell type-specific and very sensitive to pathophysiological states. However, little remains known as to how membrane lipids participate in GBM onset and progression. Hence, we employed an ex vivo model to assess the impact of TMZ treatment on healthy and GBM lipidome, which was established through imaging mass spectrometry techniques. This approach revealed that bioactive lipid metabolic hubs (phosphatidylinositol and phosphatidylethanolamine plasmalogen species) were altered in healthy brain tissue treated with TMZ. To better understand these changes, we interrogated RNA expression and DNA methylation datasets of the Cancer Genome Atlas database. The results enabled GBM subtypes and patient survival to be linked with the expression of enzymes accounting for the observed lipidome, thus proving that exploring the lipid changes could reveal promising therapeutic approaches for GBM, and ways to ameliorate TMZ side effects.     

Analysis of miR-9-5p,miR-124-3p,miR-21-5p,miR-138-5p,and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM’s diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.     

MicroRNA 3928 Suppresses Glioblastoma through Downregulation of Several Oncogenes and Upregulation of p53

Glioblastoma (GBM) is the most frequent and lethal primary malignant brain tumor. Despite decades of research, therapeutic advances that significantly prolong life are non-existent. In recent years, microRNAs (miRNAs) have been a focus of study in the pathobiology of cancer because of their ability to simultaneously regulate multiple genes. The aim of this study was to determine the functional and mechanistic effects of miR-3928 in GBM both in vitro and in vivo. To the best of our knowledge, this is the first article investigating the role of miR-3928 in GBM. We measured endogenous miR-3928 expression levels in a panel of patient-derived GBM tissue samples and cell lines. We found that GBM tissue samples and cell lines express lower levels of miR-3928 than normal brain cortex and astrocytes, respectively. Therefore, we hypothesized that miR-3928 is a tumor suppressive microRNA. We verified this hypothesis by showing that exogenous expression of miR-3928 has a strong inhibitory effect on both cell growth and invasiveness of GBM cells. Stable ex vivo overexpression of miR-3928 in GBM cells led to a reduction in tumor size in nude mice xenografts. We identified many targets (MDM2, CD44, DDX3X, HMGA2, CCND1, BRAF, ATOH8, and BMI1) of miR-3928. Interestingly, inhibition of the oncogene MDM2 also led to an upregulation of wild-type p53 expression and phosphorylation. In conclusion, we find that miR-3928, through the downregulation of several oncogenes and upregulation and activation of wild-type p53, is a strong tumor suppressor in GBM. Furthermore, the fact that miR-3928 can target many important dysregulated proteins in GBM suggests it might be a “master” regulatory microRNA that could be therapeutically exploited.     

The E3 Ubiquitin Ligase NEDD4-1 Mediates Temozolomide-Resistant Glioblastoma through PTEN Attenuation and Redox Imbalance in Nrf2–HO-1 Axis

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. It is highly resistant to chemotherapy, and tumor recurrence is common. Neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ligase that controls embryonic development and animal growth. NEDD4-1 regulates the tumor suppressor phosphatase and tensin homolog (PTEN), one of the major regulators of the PI3K/AKT/mTOR signaling axis, as well as the response to oxidative stress. Methods: The expression levels of NEDD4-1 in GBM tissues and different cell lines were determined by quantitative real-time polymerase chain reaction and immunohistochemistry. In vitro and in vivo assays were performed to explore the biological effects of NEDD4-1 on GBM cells. Temozolomide (TMZ)-resistant U87MG and U251 cell lines were specifically established to determine NEDD4-1 upregulation and its effects on the tumorigenicity of GBM cells. Subsequently, miRNA expression in TMZ-resistant cell lines was investigated to determine the dysregulated miRNA underlying the overexpression of NEDD4-1. Indole-3-carbinol (I3C) was used to inhibit NEDD4-1 activity, and its effect on chemoresistance to TMZ was verified. Results: NEDD4-1 was significantly overexpressed in the GBM and TMZ-resistant cells and clinical samples. NEDD4-1 was demonstrated to be a key oncoprotein associated with TMZ resistance, inducing oncogenicity and tumorigenesis of TMZ-resistant GBM cells compared with TMZ-responsive cells. Mechanistically, TMZ-resistant cells exhibited dysregulated expression of miR-3129-5p and miR-199b-3p, resulting in the induced NEDD4-1 mRNA-expression level. The upregulation of NEDD4-1 attenuated PTEN expression and promoted the AKT/NRF2/HO-1 oxidative stress signaling axis, which in turn conferred amplified defense against reactive oxygen species (ROS) and eventually higher resistance against TMZ treatment. The combination treatment of I3C, a known inhibitor of NEDD4-1, with TMZ resulted in a synergistic effect and re-sensitized TMZ-resistant tumor cells both in vitro and in vivo. Conclusions: These findings demonstrate the critical role of NEDD4-1 in regulating the redox imbalance in TMZ-resistant GBM cells via the degradation of PTEN and the upregulation of the AKT/NRF2/HO-1 signaling pathway. Targeting this regulatory axis may help eliminate TMZ-resistant glioblastoma.     

Extracellular Sphingosine-1-Phosphate Downstream of EGFR Increases Human Glioblastoma Cell Survival

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.