U87 Glioblastoma Cells Research Articles

A Comprehensive Method to Analyze Single Cell Vibrations.

All living cells vibrate depending on metabolism. It has been hypothesized that vibrations are unique for a given phenotype and thereby suitable to diagnose cancer type, stage, and pre-assess the effectiveness of pharmaceutical treatments in real time. However, cells exhibit highly variable vibrational signals, can be subject to environmental noise, and may be challenging to differentiate, having so far limited the phenomenon's applicability. Here, we combined the sensitive method of force-spectroscopy using optical tweezers (OT) with comprehensive statistical analysis. After data acquisition, the signal was decomposed into its spectral components via Fast Fourier Transform (FFT). Peaks were parameterized and subjected to Principal Component Analysis (PCA), to perform an unbiased multivariate statistical evaluation. This method, which we term Cell Vibrational Profiling (CVP), systematically assesses cellular vibrations. To validate CVP technique, we conducted experiments on five U251 glioblastoma (GBM) cells, using 8-10 μm polystyrene beads as a control for comparison. We collected raw data using OT, segmenting into 150+ five-second intervals. Each segment was converted into power spectra (PS) representing a frequency resolution of 10,000 Hz for both cells and controls. U251 GBM cells exhibited significant vibrations at 402.6, 1254.6, 1909.0, 2169.4, and 3462.8 Hz (p<0.0001). This method was further verified with PCA modelling, which revealed that in cell-cell comparisons using the selected frequencies, overlap frequently occurred, and clustering was difficult to discern. In contrast, comparison between cell-bead models showed that clustering was easily distinguishable. Our paper establishes CVP as an unbiased, comprehensive technique to analyze cell vibrations. This technique effectively differentiates between cell types and evaluates cellular responses to therapeutic interventions. Notably, CVP is a versatile, cell-agnostic technique requiring minimal sample preparation and no labelling or external interference. By enabling definitive phenotypic assessments, CVP holds promise as a diagnostic tool and could significantly enhance the evaluation of pharmaceutical treatments.

Napabucasin Inhibits Proliferation and Migration of Glioblastoma Cells (U87) by Regulating JAK2/STAT3 Signaling Pathway.

Background and Objectives: Napabucasin (NP) was discovered as a natural compound that suppresses cancer stemness by inhibiting the signal transducer and activator of the transcription 3 (STAT3) signaling pathway. In this study, the anti-proliferative and apoptotic effects of NP and the chemotherapy agent doxorubicin (DX), a natural compound, on glioblastoma cells (U87) were investigated. Materials and Methods: In this study, the effects of NP and DX on cell viability on the glioblastoma U87 cell line were determined by MTT test. Expressions of Jak2/Stat3 genes were examined by qRT-PCR. Apoptosis was evaluated by Hoescht 33258 staining. Moreover, NP, its antagonistic-synergistic effects and IC50 doses of the combined treatment of DX were determined. Results: Napabucacin and doxorubicin were found to inhibit glioblastoma U87 cell proliferation. It was determined that NP applied in the range of 0.3-1 µM and its combination with DX killed almost all of the glioblastoma cells in 48 h of application. Additionally, it was observed that Jak2/Stat3 expressions downregulated. Conclusions: These results show that NP suppresses the proliferation of glioblastoma cells. It was shown that the combination of NP and DX can prevent invasion of the U87 cell line due to its Jak2/Stat3 inhibitory effect. Since it can suppress Jak2/Stat3, an important cancer cell proliferation pathway in glioblastoma, the combination of NP and DX can be used as an alternative treatment agent. But no synergistic effect of NP and DX on the U87 cells of the glioblastoma cell line was observed.

Antiproliferative Effects of Naja anchietae and Naja senegalensis Venom Peptides on Glioblastoma Cell Lines.

This study explores the potential of natural bioactive peptides from animal venoms as targeted anti-cancer agents with reduced toxicity. Initially, we screened a broad collection of animal venoms for their antiproliferative activity against cancer cell lines. From this collection, we selected venoms from Naja anchietae and Naja senegalensis due to their promising activity. Utilizing reverse- phase high-performance liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF MS and MALDI-TOF TOF MSMS), and Edman degradation sequencing, we isolated and characterized three peptides named CTNanc1, CTNanc2, and CTNanc3 from Naja anchietae, and three others named CTNsen1, CTNsen2, and CTNsen3 from Naja senegalensis, each with a molecular weight of around 7 kDa. These purified peptides demonstrated inhibition of U87 glioblastoma cell proliferation, but not of U251 and T98G cells, in cell viability assays. To assess the impact of these treatments on cell viability, apoptosis, and necrosis, flow cytometry assays were conducted on U87 cells at 72 h. The results showed a decrease in cell viability and an increase in dead cells, suggesting that the treatments not only promote apoptosis, but may also lead to increased necrosis or late-stage apoptosis as the exposure time increases. These findings suggest that these peptides could be developed as leads for cancer therapy.

High loading and sustained-release system of doxorubicin-carbon dots as nanocarriers for cancer therapeutics

Nanocarriers for drugs have been investigated for decades, yet it is still challenging to achieve sustained release from nanomaterials due to drug loading inefficiency and burst release. In this study, we developed novel functional carbon dots (CDs) and investigated the therapeutic efficacy by studying the loading efficiency and release behavior of the anticancer drug doxorubicin (DOX). CDs were successfully synthesized using a one-step pyrolysis method with varying concentrations of citric acid (CA) and thiourea (TU). Functional groups, morphology, particle size, and zeta potential of synthesized CT-CDs and DOX loaded CT-CDs were investigated by UV-visible, Fluorescence, dynamic light scattering, Zeta Potential measurements, FTIR, and transmission electron microscopy. The zeta potential data revealed DOX loading onto CT-CDs by charge difference, i.e. -24.6 ± 0.44 mV (CT-CDs) and 20.57 ± 0.55 mV (DOX-CT-CDs). DOX was loaded on CDs with a loading efficiency of 88.67 ± 0.36%.In vitrodrug release studies confirmed pH-dependent biphasic drug release, with an initial burst effect and sustained release of DOX was found to be 21.42 ± 0.28% (pH 5), 13.30 ± 0.03% (pH 7.4), and 13.95 ± 0.18% (pH 9) even after 144 h at 37 °C. The CT-CDs were non-toxic and biocompatible with L929 Fibroblasts cells. The cytotoxic effect of DOX-CT-CDs showed a concentration-dependent effect after 48 h with Glioblastoma U251 cells. Flow cytometry was used to examine the cellular uptake of CT-CDs and DOX-CT-CDs in L929 and U251 cells. It was observed that the maximum CT-CDs uptake was around 75% at the end of 24 h. This study showed that the synthesized fluorescent CT-CDs demonstrated a high drug loading capacity, pH-dependent sustained release of DOX, and high cellular uptake by mammalian cells. We believe this work provides practical and biocompatible CDs for chemotherapeutic drug delivery that can be applied to other drugs for certain therapeutic aims.

Long-term exposure of human U87 glioblastoma cells to polyethylene microplastics: Investigating the potential cancer progression

Precancerous cells are present in all human bodies. Various environmental triggers can promote the development of cancer. Microplastics, an emerging concern, may potentially act as one such trigger, contributing to cancer initiation or progression. Studies have confirmed the presence of microplastics within the human body. This raises concerns about their potential toxicity and health risks. In the present study, we aimed to investigate the impact of polyethylene microplastics (PE-MPs) within the size range of 37–75 microns on glioblastoma cancer cells. Initially, we assessed the short-term effects of six different concentrations of PE-MPs (20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, and 0.62 mg/mL) on the U87 glioblastoma cell line. The results demonstrated that PE-MPs exposure led to an increase in cell proliferation compared to the untreated control group. Based on these findings, we decided to further explore the long-term effects of PE-MPs on U87 cancer cells. To evaluate the long-term effects, U87 glioblastoma cells were continuously exposed to 0.005 g of PE-MPs over an extended period of 26 days. Chronic exposure to PE-MPs significantly increased the proliferative and migratory capacities of U87 cells compared to the unexposed control group. Furthermore, continuous PE-MPs exposure altered the behavior and morphological characteristics of U87 cells. These cells exhibited a propensity to aggregate and form colonies within the culture flask. The formation of spheroid structures was also observed in the PE-MPs-exposed cell population. The results of this research indicate that polyethylene microplastics can promote the progression of glioblastoma cancer.

Cancer Chemotherapeutic Effect of Vernonia Amygdalina Delile on Glioblastoma Brain Cancer Cell.

This study is targeted at assessing the chemotherapy factor of the ethanol extract of Vernonia amygdalina Delile (VAD). U87 glioblastoma cells were treated with extract and a fraction of Vernonia amygdalina Delile (VAD) was harvested from the herbarium. Cytotoxicity was evaluated to determine the IC50 through microscopic observation followed by an MTT assay. Subsequently, flow cytometry with a FACS type was employed to conduct cell cycle and apoptosis analyses. Annexin V/PI and PI markers were used to assess apoptosis and cell cycle progression. The ethanol extract and ethyl acetate fraction of VAD showed promising effects as cancer chemotherapy in glioblastoma cells. The IC50 values for the extract and fraction were notably low, at 37.65 µg/ml and 10.12 µg/ml, respectively, for U87 cells. Analysis of apoptosis using FACS revealed a more pronounced apoptotic effect of the 15 µg/ml fraction of VAD on both early and late apoptosis compared to the 75 µg/ml extract of VAD. Although some differences in cell cycle properties were observed, there were no significant differences in cell cycle analysis between the extract and fraction. These findings underscore the efficacy of VAD's ethanol extract and ethyl acetate fraction as chemotherapeutic agents against U87 cancer cells. The low IC50 values and significant induction of early apoptosis highlight the cytotoxic effects of these treatments on U87 cells.

Relaxometric properties and biocompatibility of a novel nanostructured fluorinated gadolinium metal-organic framework.

A novel Gd-MOF based on tetrafluoro-terephthalic acid has been synthesized and its structure has been solved using X-ray single crystal diffraction data. The compound, with the formula [Gd2(F4BDC)3·H2O]·DMF, is isostructural with other Ln-MOFs based on the same ligand and has been recently reported. Its crystals were also reduced to nanometer size by employing acetic acid or cetyltrimethylammonium bromide (CTAB) as a modulator. The relaxometric properties of the nanoparticles were evaluated in solution by measuring 1H T1 and T2 as a function of the applied magnetic field and temperature. The biocompatibility of Gd-MOFs was evaluated on murine microglial BV-2 and human glioblastoma U251 cell lines. In both cell lines, Gd-MOFs do not modify the cell cycle profile or the activation levels of ERK1/2 and Akt, which are protein-serine/threonine kinases that participate in many signal transduction pathways. These pathways are fundamental in the regulation of a large variety of processes such as cell migration, cell cycle progression, differentiation, cell survival, metabolism, transcription, tumour progression and others. These data indicate that Gd-MOF nanoparticles exhibit high biocompatibility, making them potentially valuable for diagnostic and biomedical applications.

Magnetic lipid-poly(lactic-co-glycolic acid) nanoparticles conjugated with epidermal growth factor receptor antibody for dual-targeted delivery of CPT-11

To entrap sparingly water-soluble drugs like CPT-11 (irinotecan), the poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) is highly favored due to its low cytotoxicity and approval for clinical use. On the other hand, entrapping hydrophobic oleic acid-coated iron oxide magnetic nanoparticles (OMNP) in PLGA NP can provide a nanovehicle for magnetically targeted drug delivery. Our goal in this study is to develop a new dual-targeted magnetic lipid-polymer NP for the delivery of CPT-11. We first co-entrap OMNP and CPT-11 in self-assembled lipid-PLGA NP to prepare OLNP@CPT-11. The OLNP@CPT-11 surface was modified with an epidermal growth factor receptor (EGFR) antibody Cetuximab (CET), which can actively target the overexpressed EGFR on the U87 glioblastoma cell surface. The OLNP-CET@CPT-11 enables dual targeting through both external magnetic guidance and CET-mediated active targeting. The NP was characterized for physicochemical properties using various analytical techniques. In vitro study confirms ligand-receptor interaction results in enhanced endocytosis of OLNP-CET@CPT-11 by U87 cells, which offers increased cytotoxicity and elevated cell apoptosis rates. Furthermore, magnetic guidance of OLNP-CET@CPT-11 to U87 cells can induce cell death exclusively in the magnetically targeted zone. The dual-targeted strategy also provides the best therapeutic efficacy against subcutaneously implanted U87 tumors in nude mice with intravenously delivered OLNP-CET@CPT-11.

Comparison of the effects of photodynamic exposure with the use of chlorine E6 on glioblastoma cells of the U251 line and human embryonic kidney cells of the HEK293 line in vitro

Malignant gliomas of the brain are a global medical and social problem with a trend toward a steady increase in morbidity and mortality rates. A method that enables the visual identification of tumor tissue and simultaneously selectively destroys it is photodynamic therapy, which involves the introduction of a photosensitizer (PS) followed by its activation at a certain wavelength of light. The selectivity of the accumulation of PS in the tumor tissue of the malignant gliomas is one of the key issues in the problem of increasing the effectiveness of photodynamic therapy. Objective: to compare the effects of photodynamic exposure using PS chlorin E6 on human glioblastoma (GB) cells of the U251 line and non-malignant human embryonic kidney cells of the HEK293 line. Material and methods. Groups of cell cultures were formed depending on the conditions of cultivation and exogenous influence: 1) control - cultivated in a standard nutrient medium (Modified Eagle's Medium (MEM)) with L-glutamine, 1 mmol of sodium pyruvate, 10% fetal bovine serum) and experimental: 2) cultivated under the conditions of adding chlorin E6 (concentrations 1.0 and 2.0 μg/ml); 3) cultivated on a nutrient medium without the addition of PS and exposed to laser irradiation (LI) (λ=660 nm, power in the range 0.4-0.6 W, dose in the range 10-75 J/cm2, continuous or pulse mode); 4) cultured under conditions of chlorin E6 addition and subsequent exposure to LI (power in the range 0.4-0.6 W, dose in the range 10-75 J/cm2, continuous or pulse mode). After exposure to the specified experimental factors, dynamic observation with microphotographic registration was performed for 24 h, followed by microscopic and micrometric studies (number of viable cells, total number of cells, mitotic index (MI,%)). Results. PS chlorin E6 is incorporated into the cytoplasm of cells of U251 and HEK293 cell lines, the intensity of fluorescence is comparable. Upon exposure to chlorin E6 (1.0 and 2.0 μg/ml), cytodestructive and antimitotic effects are increased in a dose-dependent manner in the culture of human GB cells of the U251 line. The cytodestructive effect of chlorin E6 on cell cultures of the HEK293 line is less pronounced, but the antimitotic effect is comparable in both types of cell cultures. Under the influence of LI, cytodestructive and antimitotic effects increase in a dose-dependent manner in the culture of human GB cells of the U251 line. The level of cytodestructive and antimitotic effects is significantly lower in the cultures of non-neoplastic HEK293 cells. The most significant drop in the mitotic activity of GB U251 cells (~100%) was recorded at the lowest LI dose of 25 J/cm2, power of 0.6 W in pulse mode. For HEK293 cells, the most significant decrease in mitotic activity (~80%) was recorded at LI with a power of 0.6 W and dose of 75 J/cm2 in continuous mode. Under the combined effect of chlorin E6 (1 and 2 μg/ml, pre-incubation of 4 h) and LI in different modes, the viability of tumor cells in U251 culture decreases in a dose-dependent manner; the smallest dose of LI to achieve the maximum cytotoxic effect is 25 J/cm2, with a power of 0.6 W in pulse mode when using chlorin E6 at a concentration of 2 μg/ml. The specified characteristics of photodynamic exposure do not cause irreversible effects in HEK293 cultures (reference cells). Conclusions. An effective mode of photodynamic exposure to achieve a cytodestructive and antimitotic effect in the culture of human GB cells of the U251 line, which is relatively safe for non-malignant cells, has been established: the combined application of a laser irradiation dose of 25 J/cm2, with a power of 0.6 W in pulse mode during the preliminary incubation of the cell culture with chlorin E6 at a concentration of 2 μg/ml for 4 h.

Mechanisms of Glioblastoma Replication: Ca2+ Flares and Cl- Currents.

Glioblastoma (GBM) is amongst the deadliest types of cancers, with no resolutive cure currently available. GBM cell proliferation in the patient's brain is a complex phenomenon controlled by multiple mechanisms. The aim of this study was to determine whether the ionic fluxes controlling cell duplication could represent a target for GBM therapy. In this work, we combined multi-channel Ca2+ and Cl- imaging, optical tweezers, electrophysiology, and immunohistochemistry to describe the role of ion fluxes in mediating the cell volume changes that accompany mitosis of U87 GBM cells. We identified three main steps: (i) in round GBM cells undergoing mitosis, during the transition from anaphase to telophase and cytokinesis, large Ca2+ flares occur, reaching values of 0.5 to 1 μmol/L; (ii) these Ca2+ flares activate Ca2+-dependent Cl- channels, allowing the entry of Cl- ions; and (iii) to maintain osmotic balance, GBM cells swell to complete mitosis. This sequence of steps was validated by electrophysiological experiments showing that Cl- channels are activated either directly or indirectly by Ca2+, and by additional live-cell imaging experiments. Cl- channel blockers with different molecular structures, such as niflumic acid and carbenoxolone, blocked GBM replication by arresting GBM cells in a round configuration. These results describe the central role of Ca2+ flares and Cl- fluxes during mitosis and show that inhibition of Ca2+-activated Cl- channels blocks GBM replication, opening the way to new approaches for the clinical treatment of GBM. Implications: Our work identifies ionic fluxes occurring during cell division as targets for devising novel therapies for glioblastoma treatment.

ZnO-Salen NPs Employed as Chemosensor for Detection of Al3+ and K+ in Aqueous Medium, Developing Human Cell Images.

ZnO nanoparticles (NPs) were prepared and characterized by different analytical methods and then they were used to decorate with N, N´-bis(salicylidene)ethylenediamine (salen) in order to perform as receptor for the metal ions in an aqueous medium. The results show that ZnO-salen selectively detects Al3+ ions in aqueous medium since the intensity of fluorescence has been enhanced significantly. However, the presence of K+ in the medium further intensified the fluorescence emission for the [ZnO-salen-Al3+] system. The above system has been applied to recognize Al3+ and K+ in cells by developing the cell images, for which, the fluorescence image is brightened if a human glioblastoma U251 cell contains [ZnO-salen-Al3+] + K+ ions, consisting of the fluorescence titration. The binding global constant for Al3+ and the subsequent recognition of K+ by ZnO-salen resulted in β2(Al3+) = 6.61 × 103 and β2(K+) = 3.71 × 103 with a detection limit of 36.51 µM for Al3+ and 17.39 µM for K+. In the cell toxicity analysis, the cell viability was over 85% for the ZnO-salen even in the concentration as high as 100 mM.

The Fluorinated NAD Precursors Enhance FK866 Cytotoxicity by Activating SARM1 in Glioblastoma Cells.

Glioblastoma, a formidable brain tumor characterized by dysregulated NAD metabolism, poses a significant therapeutic challenge. The NAMPT inhibitor FK866, which induces NAD depletion, has shown promise in controlling tumor proliferation and modifying the tumor microenvironment. However, the clinical efficacy of FK866 as a single drug therapy for glioma is limited. In this study, we aim to disrupt NAD metabolism using fluorinated NAD precursors and explore their synergistic effect with FK866 in inducing cytotoxicity in glioblastoma cells. The synthesized analogue of nicotinamide riboside (NR), ara-F nicotinamide riboside (F-NR), inhibits nicotinamide ribose kinase (NRK) activity in vitro, reduces cellular NAD levels, and enhances FK866's cytotoxicity in U251 glioblastoma cells, indicating a collaborative impact on cell death. Metabolic analyses reveal that F-NR undergoes conversion to fluorinated nicotinamide mononucleotide (F-NMN) and other metabolites, highlighting the intact NAD metabolic pathway in glioma cells. The activation of SARM1 by F-NMN, a potent NAD-consuming enzyme, is supported by the synergistic effect of CZ-48, a cell-permeable SARM1 activator. Temporal analysis underscores the sequential nature of events, establishing NAD depletion as a precursor to ATP depletion and eventual massive cell death. This study not only elucidates the molecular intricacies of glioblastoma cell death but also proposes a promising strategy to enhance FK866 efficacy through fluorinated NAD precursors, offering potential avenues for innovative therapeutic interventions in the challenging landscape of glioblastoma treatment.

Inhibition of GPR68 kills glioblastoma in zebrafish xenograft models

ObjectiveInhibition and knockdown of GPR68 negatively affects glioblastoma cell survival in vitro by inducing ferroptosis. Herein, we aimed to demonstrate that inhibition of GPR68 reduces the survival of glioblastoma cells in vivo using two orthotopic larval xenograft models in Danio rerio, using GBM cell lines U87-MG and U138-MG. In vivo survival of the cancer cells was assessed in the setting of GPR68 inhibition or knockdown.ResultsIn vitro, shRNA-mediated knockdown of GPR68 inhibition demonstrated potent cytotoxic effects against U87 and U138 glioblastoma cell lines. This effect was associated with increased intracellular lipid peroxidation, suggesting ferroptosis as the underlying mechanism of cell death. Translating these findings in vivo, we established a novel xenograft model in zebrafish by successfully grafting fluorescently labeled human glioblastoma cells, which were previously shown to overexpress GPR68. shRNA knockdown of GPR68 significantly reduced the viability of grafted GBM cells within this model. Additionally, treatment with ogremorphin (OGM), a highly specific small molecule inhibitor of GPR68, also reduced the viability of grafted GBM cells with limited toxicity to the developing zebrafish embryos. This study suggests that therapeutic targeting of GPR68 with small molecules like OGM represents a promising approach for the treatment of GBM.

Effect of Apis mellifera syriaca Bee Venom on Glioblastoma Cancer: In Vitro and In Vivo Studies.

Glioblastoma multiforme (GBM) is a highly aggressive and fatal primary brain tumor. The resistance of GBM to conventional treatments is attributed to factors such as the blood-brain barrier, tumor heterogeneity, and treatment-resistant stem cells. Current therapeutic efforts show limited survival benefits, emphasizing the urgent need for novel treatments. In this context, natural anti-cancer extracts and especially animal venoms have garnered attention for their potential therapeutic benefits. Bee venom in general and that of the Middle Eastern bee, Apis mellifera syriaca in particular, has been shown to have cytotoxic effects on various cancer cell types, but not glioblastoma. Therefore, this study aimed to explore the potential of A. mellifera syriaca venom as a selective anti-cancer agent for glioblastoma through in vitro and in vivo studies. Our results revealed a strong cytotoxic effect of A. mellifera syriaca venom on U87 glioblastoma cells, with an IC50 of 14.32 µg/mL using the MTT test and an IC50 of 7.49 µg/mL using the LDH test. Cells treated with the bee venom became permeable to propidium iodide without showing any signs of early apoptosis, suggesting compromised membrane integrity but not early apoptosis. In these cells, poly (ADP-ribose) polymerase (PARP) underwent proteolytic cleavage similar to that seen in necrosis. Subsequent in vivo investigations demonstrated a significant reduction in the number of U87 cells in mice following bee venom injection, accompanied by a significant increase in cells expressing caspase-3, suggesting the occurrence of cellular apoptosis. These findings highlight the potential of A. mellifera syriaca venom as a therapeutically useful tool in the search for new drug candidates against glioblastoma and give insights into the molecular mechanism through which the venom acts on cancer cells.

The Functional Interaction of KATP and BK Channels with Aquaporin-4 in the U87 Glioblastoma Cell.

K+ channels do play a role in cell shape changes observed during cell proliferation and apoptosis. Research suggested that the dynamics of the aggregation of Aquaporin-4 (AQP4) into AQP4-OAP isoforms can trigger cell shape changes in malignant glioma cells. Here, we investigated the relationship between AQP4 and some K+ channels in the malignant glioma U87 line. The U87 cells transfected with the human M1-AQP4 and M23-AQP4 isoforms were investigated for morphology, the gene expression of KCNJ8, KCNJ11, ABCC8, ABCC9, KCNMA1, and Cyclin genes by RT-PCR, recording the whole-cell K+ ion currents by patch-clamp experiments. AQP4 aggregation into OAPs increases the plasma membrane functional expression of the Kir6.2 and SUR2 subunits of the KATP channels and of the KCNMA1 of the BK channels in U87 cells leading to a large increase in inward and outward K+ ion currents. These changes were associated with changes in morphology, with a decrease in cell volume in the U87 cells and an increase in the ER density. These U87 cells accumulate in the mitotic and G2 cell cycle. The KATP channel blocker zoledronic acid reduced cell proliferation in both M23 AQP4-OAP and M1 AQP4-tetramer-transfected cells, leading to early and late apoptosis, respectively. The BK channel sustains the efflux of K+ ions associated with the M23 AQP4-OAP expression in the U87 cells, but it is downregulated in the M1 AQP4-tetramer cells. The KATP channels are effective in the M1 AQP4-tetramer and M23 AQP4-OAP cells. Zoledronic acid can be effective in targeting pathogenic M1 AQP4-tetramer cell phenotypes inhibiting KATP channels and inducing early apoptosis.

Pyrimidine compounds BY4003 and BY4008 inhibit glioblastoma cells growth via modulating JAK3/STAT3 signaling pathway

Glioblastoma (GBM) is a brain tumor characterized by its aggressive and invasive properties. It is found that STAT3 is abnormally activated in GBM, and inhibiting STAT3 signaling can effectively suppress tumor progression. In this study, novel pyrimidine compounds, BY4003 and BY4008, were synthesized to target the JAK3/STAT3 signaling pathway, and their therapeutic efficacy and mechanisms of action were evaluated and compared with Tofacitinib in U251, A172, LN428 and patient-derived glioblastoma cells. The ADP-Glo™ kinase assay was utilized to assessed the inhibitory effects of BY4003 and BY4008 on JAK3, a crucial member of the JAK family. The results showed that both compounds significantly inhibited JAK3 enzyme activity, with IC50 values in the nanomolar range. The antiproliferative effects of BY4003, BY4008, and Tofacitinib on GBM and patient-derived glioblastoma cells were evaluated by MTT and H&E assays. The impact of BY4003 and BY4008 on GBM cell migration and apoptosis induction was assessed through wound healing, transwell, and TUNEL assays. STAT3-regulated protein expression and relative mRNA levels were analyzed by western blotting, immunocytochemistry, immunofluorescence, and qRT-PCR. It was found that BY4003, BY4008 and Tofacitinib could inhibit U251, A172, LN428 and patient-derived glioblastoma cells growth and proliferation. Results showed decreased expression of STAT3-associated proteins, including p-STAT3, CyclinD1, and Bcl-2, and increased expression of Bax, a pro-apoptotic protein, as well as significant down-regulation of STAT3 and STAT3-related genes. These findings suggested that BY4003 and BY4008 could inhibit GBM growth by suppressing the JAK3/STAT3 signaling pathway, providing valuable insights into the therapeutic development of GBM.

Potential of GSPT1 as a novel target for glioblastoma therapy.

Glioblastoma is the most common malignant brain tumor in adults, the survival rate of which has not significantly improved over the past three decades. Therefore, there is an urgent need to develop novel treatment modalities. We previously reported that G1 to S phase transition 1 (GSPT1) depletion induces delayed cell cycle in primary astrocytes. Herein, we examined the potential of GSPT1 as a novel target for glioblastoma therapy. CC-885, a cereblon modulator that degrades GSPT1 by bridging GSPT1 to the CRL4 E3 ubiquitin ligase complex, was administered to nude mice with transplanted brain tumors of U87 glioblastoma cells. The survival period was significantly longer in CC-885 treated mice than in control mice. Furthermore, we generated GSPT1-knockout (KO) U87 cells and GSPT1-KO U87 cells with stable overexpression of FLAG-tagged GSPT1 (Rescued GSPT1-KO). Mice with transplanted GSPT1-KO U87 cells and Rescued GSPT1-KO U87 cells showed significantly longer and similar survival periods, respectively, as those with wild-type (WT) U87 cells. GSPT1-KO U87 cells showed enhanced apoptosis, detected by cleaved PARP1, compared to WT U87 cells. Brain tumors with transplantation of GSPT1-KO U87 cells also showed enhanced apoptosis compared to those with transplantation of WT and Rescued GSPT1-KO U87 cells. GSPT1 expression was confirmed in patients with glioblastoma. However, the clinical study using 87 glioblastoma samples showed that GSPT1 mRNA levels were not associated with overall survival. Taken together, we propose that GSPT1 is an essential protein for glioblastoma growth, but not its malignant characteristics, and that GSPT1 is a potential target for developing glioblastoma therapeutics.

Matrix Protein of Vesicular Stomatitis Virus Targets the Mitochondria, Reprograms Glucose Metabolism, and Sensitizes to 2-Deoxyglucose in Glioblastoma.

A potential therapeutic approach for cancer treatment is target oxidative phosphorylation and glycolysis simultaneously. The matrix protein of vesicular stomatitis virus (VSV MP) can target the surface of mitochondria, causing morphological changes that may be associated with mitochondrial dysfunction and oxidative phosphorylation inhibition. Previous research has shown that mitochondrial abnormalities can direct glucose metabolism toward glycolysis. Thus, after treatment with VSV MP, glycolysis inhibition is necessary to completely block glucose metabolism and eradicate cancer. Here, to inhibit glycolysis, the 2-deoxy-D-glucose (2-DG), a synthetic glucose analog was used to combine with VSV MP to treat cancer. This study aims to determine how VSV MP affects the glucose bioenergetic metabolism of cancer cells and to evaluate the synergistic effect of 2-DG when combined with VSV. Our results indicated that in U87 and C6 glioblastoma cell lines, VSV MP caused mitochondrial membrane potential loss, cytochrome c release, and glucose bioenergetics metabolism reprogramming. When combined with 2-DG, VSV MP synergistically aggravated cell viability, apoptosis, and G2/M phase arrest. Meanwhile, the combination therapy exacerbated ATP depletion, activated AMPK, and inhibited mammalian target of rapamycin signaling pathways. In addition, 2-DG treatment alone induced autophagy in glioblastoma cells; however, VSV MP inhibited the autophagy induced by 2-DG in combined treatment and finally contributed to the enhanced cytotoxic effect of the combination strategy in U87 and C6 cancer cells. In the orthotopic U87 glioblastoma model and subcutaneous C6 glioblastoma model, the combined treatment led to significant tumor regression and prolonged survival. A potent therapeutic approach for treating glioblastoma may be found in the combination of VSV MP and glycolytic inhibitors.

Fluspirilene exerts an anti-glioblastoma effect through suppression of the FOXM1-KIF20A axis.

Given the infiltrative nature of human glioblastoma (GBM), cocktail drug therapy will remain a vital tool for the treatment of the disease. We investigated fluspirilene, perphenazine, and sulpiride, three classic anti-schizophrenic drugs, as possible anti-GBM agents. The CCK-8 assay demonstrated that fluspirilene possesses the most outstanding anti-GBM effect. We performed molecular mechanisms studies in vitro and an orthotopic xenograft model in mice. Fluspirilene inhibited proliferation and migration in vitro in U87MG and U251 GBM cell lines. Flow cytometry demonstrated that treatment increased apoptosis and cells accumulated in the G2/M phase. Our analysis of publicly available expression data for several cell lines treated with the drug led to the identification of several genes, including KIF20A, that are downregulated by fluspirilene and lead to growth inhibition/apoptosis. We also demonstrated that siRNA knockdown of KIF20A, a member of the kinesin family, attenuated cell proliferation in GBM cells and an orthotopic xenograft model in mice. A regulator of KIF20A, the oncogenic transcription factor FOXM1, was identified using the String database, which harbors protein interaction networks. In fluspirilene-treated cells, FOXM1 protein was decreased, indicating that KIF20A was downregulated in the presence of the drug due to decreased FOXM1 protein. These results demonstrate that fluspirilene is an effective anti-GBM agent that works by suppressing the FOXM1-KIF20A oncogenic axis.

New ALKBH2 and ALKBH5 inhibitors for treating glioblastoma

PurposeGlioblastoma is an aggressive brain tumor with high mortality and a median survival of about 15 months. Starting from our previous published compound, MV1035 that inhibited migration and invasiveness of glioblastoma U87 cells, and also exhibited a synergic effect in combination with the alkylating agent Temozolomide, we identified new active molecules, with the aim to understand the key motifs responsible for MV1035 activity against the DNA repair protein ALKBH2 and the RNA demethylase ALKBH5. Materials and methodsWe modified the original structure of MV1035, synthesizing new molecules that have been tested through MTT assay, cell-free ALKBH2 assay and cell-free ALKBH5 assay. ResultsWe found that compound 6 is able to reduce the activity of both ALKBH2 and ALKBH5. ConclusionWe obtained important information about the key motifs responsible for the activity of these inhibitors. However, considering the complexity, heterogeneity and plasticity of GBM and GSC cells, the new identified compound will need to be validated in vivo.