U87MG Cells Research Articles

Enhanced Cytotoxic Effects of Cold Plasma Deposition of Topotecan: A Novel Approach for Local Cancer Drug Delivery to Glioblastoma Cells.

Despite the numerous advances in glioblastoma multiforme (GBM) treatment, GBM remains as the most malignant and aggressive form of brain cancer, characterized by a very poor outcome, highlighting the ongoing need for the development of new therapeutic strategies. A novel intervention using plasma-assisted local delivery of oncology drugs was developed to mediate the drug delivery, which might improve drug uptake and/or chemotherapeutic action. Topotecan (TPT), a water-soluble topoisomerase I inhibitor with major cytotoxic effects during the S-phase of the cell cycle, was selected as the candidate drug because despite its potent antitumor activity, the systemic administration to the brain is limited due to low crossing of the blood-brain barrier. For these reasons, TPT may be repurposed for local combined therapies. We aimed to explore options for the local treatment of GBM where systematic delivery is challenging, using a combination between plasma-based technologies and TPT on a human brain cancer cell line (U-251mg). The evaluation of direct TPT plasma deposition using a helium plasma jet (J-Plasma, Apyx Medical) with a nebulizer onto U-251mg cells grown in 2D or 3D culture showed a reduction in the metabolic activity and cell mass and decreased long-term survival, indicating synergistic effects between the drug and the plasma treatment. The plasma-assisted approach was confirmed using temozolomide (TMZ) as a standard drug for glioblastoma treatment, as well as with two skin cancer cell lines. These results revealed a pathway for new combinations and approaches to local drug application for a range of cancers.

Adiponectin facilitates the cell cycle, inhibits cell apoptosis and induces temozolomide resistance in glioblastoma via the Akt/mTOR pathway.

Adiponectin (ADN) regulates DNA synthesis, cell apoptosis and cell cycle to participate in the pathology and progression of glioblastoma. The present study aimed to further explore the effect of ADN on temozolomide (TMZ) resistance in glioblastoma and the underlying mechanism of action. Glioblastoma cell lines (U251 and U87-MG cells) were treated with ADN and TMZ at different concentrations; subsequently, 3.0 µg/ml ADN and 1.0 mM TMZ were selected as the optimal concentrations for the experimental conditions. LY294002 (a PI3K inhibitor) was added to ADN or ADN + TMZ-treated glioblastoma cell lines. Cell growth rate was determined using the Cell Counting Kit-8 assay, the apoptotic rate and cell cycle were evaluated using Annexin V/propidium iodide and cell cycle assays, and p-Akt (Thr308), p-Akt (Ser473), Akt, p-mTOR, c-caspase 3, caspase 3, Bax, cyclin B1 and cyclin D1 expression was determined by western blotting. Adiponectin receptor (ADIPOR) 1 and ADIPOR2 were expressed in glioblastoma cell lines. The glioblastoma cell line growth rate was increased by ADN in a concentration- and time-dependent manner. ADN inhibited glioblastoma cell line apoptosis and facilitated cell cycle. Of note, ADN activated the Akt/mTOR pathway and the addition of LY294002 reversed the effect of ADN, indicating that ADN activated the Akt/mTOR pathway to suppress apoptosis and promote cell cycle in glioblastoma cell lines. Notably, TMZ inhibited glioblastoma cell line growth, promoted apoptosis and increased G2 phase cell cycle arrest. However, the addition of ADN reversed the effect of TMZ in glioblastoma cell lines, disclosing that ADN induced TMZ resistance. Markedly, ADN-mediated TMZ resistance was further attenuated by LY294002, suggesting that ADN activated the Akt/mTOR pathway to induce TMZ resistance in glioblastoma cell lines. In conclusion, ADN activated the Akt/mTOR pathway to facilitate cell cycle, inhibit cell apoptosis and induce TMZ resistance in glioblastoma.

Comprehensive evaluation of EGFR and AKT targeting efficacy of resveratrol loaded PEGylated liposomes for the glioblastoma management: In silico, in vitro BBB permeation studies.

Red grapes contain resveratrol (Resv), a polyphenol with anti-inflammatory, anti-diabetic, and anticancer properties. In this study, in silico molecular docking was used to assess the binding affinity of Resv to target proteins. Resv was encapsulated in PEGylated liposomes (LNPs) using Phospholipon 90G, cholesterol, and DSPE-mPEG2000. The particle size, surface charge, and structural details of the Res-LNPs and the Blank LNPs were determined. The effects of Res-LNPs and pure Resv were examined in vitro in C6 (rat glioma) and U87 MG (human glioblastoma) cell lines to evaluate cell survival, uptake, wound healing, and apoptosis. BBB permeability of the Res-LNPs was assessed using an in vitro BBB model with hCMEC/D3 cells. EGFR and AKT 1 and 2 expression levels in Resv-treated U87 MG cells were analyzed by RT-qPCR. Res-LNPs had a particle size of 155.0±1.62nm and an encapsulation efficiency (% EE) of 76.62±3.43. FTIR, DSC, and XRD analyses confirmed the complete entrapment of Resv within the LNPs, displaying a unilamellar spherical morphology, as verified by SEM and TEM. In vitro studies on C6 and U87 MG cell lines showed that Res-LNPs significantly improved cell viability, uptake, migration, and apoptosis compared with Resv. An in vitro BBB model demonstrated that Res-LNPs efficiently crossed the BBB and accumulated in brain cancer cells. RT-qPCR results indicated that Resv treatment reduced EGFR and AKT 1 and 2 gene expression in U87 MG cells. These results suggest that Res-LNPs effectively crossed BBB and inhibited EGFR and its downstream pathways in glioma cell lines.

Effects of intentionally-treated water on cell migration of human glioblastoma cells.

This study investigated if human glioblastoma cancer cells (U87MG cell line) cultured in intentionally treated water could reduce cell migration, a prerequisite for metastasis, as compared to the same cells cultured in untreated (control) water. Three Buddhist monks entered a meditative state and directed their awareness to bottles of ultrapure water while holding the intention that the water would cause beneficial changes in U87MG. The study was conducted double-blind whereby all aspects of the study involving cell growth and migration measures, as well as all subsequent statistical evaluations, were performed without knowledge of the type of water being used. Cell cultures were incubated in growth mediums prepared with treated and untreated water, and a wound healing assay was employed to measure cell migration. U87MG cells incubated with treated water migrated less efficiently than the same cells in untreated water. A repeated measures ANOVA, spanning four time periods (0, 3, 6, and 9 h), determined that the time × water condition interaction was associated with p < 0.005. Intentioned awareness appeared to change as-yet unknown properties of ultrapure water, resulting in a reduction of U87MG cells migration activity. Further research is warranted to replicate these results and to investigate the underlying protein expression mechanisms in influencing cell migration.

Isobolographic interactions of cannabidiol and AM 1172 with cisplatin in human neuroblastoma and glioblastoma cell lines: An in vitro study.

Glioblastoma is the most aggressive brain cancer in humans with very poor prognosis and high mortality rate. Despite advances in treatment, glioblastoma almost always recurs and new therapeutic methods are urgently needed. This study aimed at assessing the cytotoxic and antiproliferative effects of AM 1172 and cannabidiol (two cannabinoid receptor ligands) in vitro, when used alone and in combination with cisplatin (a standard cytotoxic drug), in various human neuroblastoma (CHP-134, KELLY), human glioblastoma (U-87MG and T98G) and rat glioblastoma (C6) cell lines. Our experiments showed that AM 1172 and cannabidiol inhibited cell proliferation with IC50 values in the range of 2.29-17.21μM (for AM 1172) and 11.61-20.35μM (for cannabidiol), respectively. The selectivity index for AM 1172 ranged from 0.61 to 4.60 and that for cannabidiol ranged from 1.45 to 2.55 in the studied glioblastoma and neuroblastoma cell lines. With isobolographic analysis, it was found that AM 1172 combined with cisplatin exerted a synergistic interaction in the CHP-134cell line (p<0.01). In contrast, AM 1172 when combined with cisplatin produced an antagonistic interaction in the C6 cell line (p<0.01). The remaining combinations of AM 1172 with cisplatin in the U-87MG, KELLY and T98G cell lines were additive. In case of cannabidiol, its combination with cisplatin produced an antagonistic interaction in the T98G cell line (p<0.0001), whereas the combinations of cannabidiol with cisplatin in the CHP-134, U-87MG, KELLY, and C6 cell lines were additive in nature. The synergistic and additive interactions for the combination of AM 1172 and cannabidiol with cisplatin seem to be a promising direction in glioblastoma therapy. Unfortunately, the combinations producing antagonistic interactions (AM 1172+cisplatin in C6, and cannabidiol+cisplatin in T98G cell lines) should be avoided due to the antagonistic antiproliferative effect of two-drug mixtures.

Radiofrequency Induced Time-Dependent Alterations in Gene Expression and Apoptosis in Glioblastoma Cell Line.

The widespread use of wireless communication technologies has increased human exposure to radiofrequency electromagnetic fields (RF-EMFs). Considering the brain's close proximity to mobile phones and its entirely electrical transmission network, it emerges as the organ most profoundly impacted by the RF field. This study aims to investigate the potential effects of RF radiation on cell viability, apoptosis, and gene expressions in glioblastoma cells (U118-MG) at different exposure times (1, 24, and 48 h). To achieve this, we designed and implemented an in vitro RF exposure system operating at a frequency of 2.1 GHz, specifically for cell culture studies, with an average specific absorption rate (SAR) of 1.12 ± 0.18 W/kg determined through numerical dosimetry calculations. Results reveal a significant influence of a 48 h exposure to a 2.1 GHz RF field on U118-MG cell viability, gene expression, and the induction of caspase (CASP) dependent apoptosis. Notably, increased CASP3, CASP8, and CASP9 mRNA levels were observed after 24 and 48 h of RF treatment. However, only the 48 h RF exposure resulted in apoptotic cell death and a significant elevation in the BAX/BCL-2 ratio. This observed effect may be influenced by extended exposure durations surpassing the cell's doubling time. The increased BAX/BCL-2 ratio, which acts as a key switch for apoptosis, and the heterogeneous morphology of the astrocyte-derived U118-MG cell line may also play a role in this effect.

Arachidonoylethanolamide promotes cellular senescence in a human glioblastoma cell line.

Glioblastomas are the most common and deadly primary brain tumors, with high mortality rates despite aggressive therapies. Cellular senescence is important for cancer development, as it limits tumor progression; however, it may also stimulate inflammation at the tumor microenvironment, promoting tumor development. Hence, modulation of senescence is an important target for cancer therapy. Endocannabinoids modulate energy metabolism and the functions of the immune and nervous systems and have shown significant anti-tumor effects in experimental conditions, inhibiting cell growth and proliferation, while promoting apoptosis. Altered endocannabinoid concentrations are related to development of different types of cancer, and recent studies have shown that endocannabinoids and their synthetic analogs are capable of modulating senescence in multiple tissues, affecting cell proliferation and survival. Nonetheless, their effects on cellular senescence in cancer have not been defined. This study explored the effect of the endocannabinoid arachidonoylethanolamide on the induction of cellular senescence in human glioblastoma cell line U-87MG. Our results show that direct supplementation of AEA decreases cell cycle progression, while increasing beta-galactosidase activity and expression of p21, in U-87MG cells, in a dose- and time-dependent manner. Our data suggest that arachidonoylethanolamide may be useful for the modulation of glioblastoma senescence and should be explored further as an adjuvant for cancer therapy.

Bullatine A suppresses glioma cell growth by targeting SIRT6.

Gliomas are the most common primary tumors of the nervous system, which is generally treated using adjuvant chemotherapy following surgical resection. However, patient survival time is still short, and there is currently no successful treatment for highly malignant gliomas. Bullatine A (BLA) is a diterpenoid alkaloid of the genus Aconitum which antirheumatic and anti-inflammatory pharmacological properties. The effects of BLA on gliomas have not yet been elucidated. In this study, we investigated the effects of BLA on human brain malignant glioblastoma cells. Our results showed that BLA inhibited the proliferation of U87MG and U251cells in a dose-dependent manner and decreased their survival rate. BLA dose-dependently induced apoptosis in U87MG cells, upregulated the expression of cleaved caspase-9, cleaved caspase-3 pro-apoptotic protein, and Bax protein, and downregulated the expression of Bcl-2 anti-apoptotic protein. Moreover, BLA dose-dependently induced U87MG and U251cell cycle arrest in the G2/M phase, and downregulated the expression of p-ERK and Myc proteins. Further, BLA significantly inhibited the acetylation of histones H3K9 and H3K56, and upregulated the expression of the protein deacetylase SIRT6. Mechanistic studies revealed that the effect of BLA on inducing apoptosis and inhibiting the proliferation of glioma cells was blocked by SIRT6 knockout. In summary, our study indicated that BLA is a potential therapeutic agent for glioma that targets SIRT6 to inhibit glioma cell proliferation and induce apoptosis.

Sulforaphane Wrapped in Self-Assembled Nanomicelle Enhances the Effect of Sonodynamic Therapy on Glioma.

Background/Objectives: The two obstacles for treating glioma are the skull and the blood brain-barrier (BBB), the first of which forms a physical shield that increases the difficulties of traditional surgery or radiotherapy, while the latter prevents antitumor drugs reaching tumor sites. To conquer these issues, we take advantage of the high penetrating ability of sonodynamic therapy (SDT), combined with a novel nanocomplex that can easily pass the BBB. Methods: Through ultrasonic polymerization, the amphiphilic peptides (C18GR7RGDS) were self-assembled as a spherical shell encapsulating a sonosensitizer Rose Bengal (RB) and a plant-derived compound, sulforaphane (SFN), to form the nanocomplex SFN@RB@SPM. Results/Conclusions: SFN@RB@SPM can be internalized by the glioma cells through the tumor-targeting motif RGDS (abbreviated for the peptide sequence composed of arginine, glycine, aspartic acid, and serine), and further executes antitumor function during SDT. Also, SFN@RB@SPM could be easily taken up by U87-MG cells and cross the BBB in glioma-bearing mice during SDT. The mechanism investigation revealed that, compared with the SFN-free nanocomplex (RB@SPM), SFN@RB@SPM induced much more apoptosis of U87-MG cells in an ROS-dependent manner through the depletion of glutathione by SFN and the cavitation effect by SDT. In animal experiments, besides a significant reduction in tumor volume and a delay in losing body weight, H&E staining showed a massive infiltration of neutrophils adjacent to the tumor sites, indicating this novel nanocomplex SFN@RB@SPM can synergistically augment SDT efficacy, partially by enhancing the antitumor function of innate immunity.

Exploring the Therapeutic Potential of Cannabidiol in U87MG Cells: Effects on Autophagy and NRF2 Pathway.

Cannabinoids include both endogenous endocannabinoids and exogenous phytocannabinoids, such as cannabidiol (CBD), and have potential as therapeutic agents in cancer treatment due to their selective anticancer activities. CBD exhibits both antioxidant and pro-oxidant effects depending on its concentration and cell types. These properties allow CBD to influence oxidative stress responses and potentially enhance the efficacy of antitumor therapies. In this study, we treated U87MG glioma cells with low dose (1 μM) CBD and evaluated its molecular effects. Our findings indicate that CBD reduced cell viability by 20% (p < 0.05) through the alteration of mitochondrial membrane potential. The alteration of redox status by CBD caused an attempt to rescue mitochondrial functionality through nuclear localization of the GABP transcription factor involved in mitochondria biogenesis. Moreover, CBD treatment caused an increase in autophagic flux, as supported by the increase in Beclin-1 and the ratio of LC3-II/LC3-I. Due to mitochondria functionality alteration, pro-apoptotic proteins were induced without activating apoptotic effectors Caspase-3 or Caspase-7. The study of the transcription factor NRF2 and the ubiquitin-binding protein p62 expression revealed an increase in their levels in CBD-treated cells. In conclusion, low-dose CBD makes U87MG cells more vulnerable to cytotoxic effects, reducing cell viability and mitochondrial dynamics while increasing autophagic flux and redox systems. This explains the mechanisms by which glioma cells respond to CBD treatment. These findings highlight the therapeutic potential of CBD, suggesting that modulating NRF2 and autophagy pathways could represent a promising strategy for glioblastoma treatment.

PEGylated Nanoliposomal Doxorubicin Conjugated with Specific TREM2 Peptides for Glioma-Targeting Therapy.

PEGylated liposomes can deliver anti-cancer drugs to brain tumors, and achieve enhanced permeability and retention effects. Triggering receptor expressed on myeloid cells 2 (TREM2) is an excellent biomarker for precise therapyof glioma. The present study is aimed at designing PEGylatednanoliposomal doxorubicin (PLD) conjugated with peptides targeting TREM2 for glioma-targeting therapy. The specific peptides are designed with the Rosetta Peptiderive Protocol. Schrodinger's peptide-specific version of Glide is used for molecular docking.PLD modified with peptides (peptide-PLD) are engineered and prepared. Cell cycle, apoptosis, cell invasion and migration, cell viability, and colony-formation assays are performed to analyze glioma cell functions. The anti-tumor effects of peptide-PLD are validated in an intracranial U87-MG cells orthotopic glioma model. The targeting peptides HLRKLRKR and LRKLRLRL showed specific affinity for TREM2 and better cellular uptake in U87-MG cells. PLD with peptide modification demonstrated stable doxorubicin loading, small sizes (<60nm), and enrichment in the mouse brain. Peptide-PLD treatment inhibited the Akt/GSK3β/β-catenin pathway, thereby inhibiting cell invasion and migration, and colony-forming ability in U87-MG cells. The peptide modification of PLD achieved better suppression of glioma development than PLD.Overall, TREM2-targeting peptides are successfully designed, and peptide-PLD served as a potent drug delivery carrier for glioma-targeting therapy.

LAT4 drives temozolomide induced radiotherapy resistance in glioblastoma by enhancing mTOR pathway activation

BackgroundGlioblastoma multiforme (GBM) represents the most prevalent form of primary malignant tumor within the central nervous system. The emergence of resistance to radiotherapy and chemotherapy represents a significant impediment to advancements in glioma treatment.MethodsWe established temozolomide (TMZ)-resistant GBM cell lines by chronically exposing U87MG cell lines to TMZ, and dimethyl sulfoxide (DMSO) was used as placebo control. In vivo and in vitro experiments verified the resistance of resistant cells to chemotherapy and radiotherapy. LAT4 was identified by transcriptomics to be associated with GBM treatment resistance and relapse. The relationship between LAT4 and mTOR pathway activity was also analyzed. Finally, the effect of BCH (LAT inhibitor) combined with radiotherapy on GBM prognosis was verified in vivo.ResultsWe have first confirmed that TMZ not only induces resistance to chemotherapy in GBM cells but also enhances their resistance to radiotherapy, which is a significant finding in the process of building TMZ-resistant U87MG GBM cell lines. We then performed comprehensive transcriptomic analysis and identified amino acid metabolism as a potential key factor in radiotherapy resistance. Specifically, we confirmed that the upregulation of LAT4 following chemotherapy enhances leucine metabolism within tumors in vitro and in vivo, thereby modulating the mechanistic target of mTOR pathway and leading to radiotherapy resistance. Of note, the application of inhibitors targeting leucine metabolism was shown to restore the sensitivity of these cells to radiotherapy, highlighting a potential therapeutic strategy for overcoming resistance in GBM.ConclusionsOur study links tumor sensitivity to chemotherapy and radiotherapy and highlights the critical role of LAT4 in activating the mTOR pathway and GBM radiotherapy resistance. It suggests ways to improve radiotherapy sensitivity to GBM.

177Lu Radiolabeled Polydopamine Decorated with Fibroblast Activation Protein Inhibitor for Locoregional Treatment of Glioma.

Radionuclide therapy is expected to be a powerful tool for glioma treatment. Here, we introduce a novel nuclear nanomedicine based on polydopamine (PDA), incorporating fibroblast activation protein inhibitor (FAPI) and macrocyclic chelator (DOTA) for specific cancer targeting and 177Lu labeling. The synthesized nanoradiopharmaceutical, 177Lu-DOTA-PEG-PDA-FAPI, exhibits good stability in serum, saline and PBS over 5 days. 177Lu-DOTA-PEG-PDA-FAPI shows efficient specific uptake and internalization when incubated with U87MG cells. In vivo distribution visualized prominent accumulation and long retention ability of 177Lu-DOTA-PEG-PDA-FAPI at tumor sites after local administration. Moreover, 177Lu-DOTA-PEG-PDA-FAPI has satisfactory antitumor ability without apparent toxic and side effects observed from therapy assay and H&E staining. This study highlights the feasibility of using PDA as a nanocarrier for glioma endoradiotherapy by targeting fibroblast activation protein.

CXCL12 restricts tumor growth by suppressing the Ras, ERK1/2, c-Myc, and the immune checkpoint PD-L1 pathways

Cytokines constitute a family of proteins that modulate the immune system and are secreted by many cells. CXCL12, along with its receptor CXCR4, are essential players in numerous processes. Dysregulation of their function underlie the mechanism(s) of several pathologies, including malignancies. Here, we demonstrate an unexpected effect of the cytokine and its receptor: In both cells and animal models, CXCL12 restricts tumorigenicity of the human glioblastoma cells U87-MG and U-118, and of a cell line derived from PyMT mouse breast cancer. Overexpression of CXCL12 inhibits activation of the oncogene Ras which results in downregulation of its proliferative signals, such as reduced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2), inhibition of c-Myc expression, and subsequent inhibition of cell cycle. Furthermore, CXCL12 induces downregulation of the growth differentiation factor 15 (GDF15), insulin-like growth factor-binding protein 6 (IGFBP6), and matrix metalloproteinase-3 (MMP3), which are implicated in sending metastases. Indeed, monitoring cell migration in vitro and generation of metastases in mice demonstrate that CXCL12 slows the migration of U87-MG and PyMT cells. Remarkably, overexpression of CXCL12 also downregulates the cell surface immune checkpoint protein programmed cell death-ligand 1 (PD-L1), resulting in recruitment of cytotoxic CD8 T cells into xenografts accompanied by their shrinkage. Overall, CXCL12 inhibits tumor growth through several distinct mechanisms: inhibition of cell cycle and migration, as well as impairment of immune checkpoint, thereby stimulating a strong host's immune response. The mechanism(s) that renders CXCL12 a tumor-promoting factor in certain cells and a suppressor in others has remained elusive.

Pharmacognostic Evaluation of Parmelia sulcata Taylor and its Cytotoxic Effects on A Glioblastoma Cell Line

Objectives The current study aims to establish the pharmacognostic profile and evaluate the cytotoxic potential of Parmelia sulcata Talyor in the glioblastoma cell line. Materials and Methods In this experimental investigation, pharmacognostic evaluation has been conducted via standard procedures, and active fractions obtained through column chromatography, further identified by liquid chromatography–mass spectrometry (LC–MS). The total antioxidant capacity of the ethanolic extract of the lichen was obtained via the phosphomolybdate method, and a reducing power assay was conducted to determine its in vitro antioxidant potential. The glioblastoma U87-MG cell lines were cultivated in minimum essential medium supplemented with nonessential amino acids, and 10% fetal bovine serum was maintained under controlled conditions. 3-(4,5-Dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide assay was used to test the cytotoxicity of ethanolic extract of lichen and its isolated active fraction. Results The pharmacognostic profile of P. sulcata inhabiting in lithophytic life form has been established. Morphological parameters aided in studying the physical attributes of lichen, whereas microscopic parameters helped to determine the histological characteristics. LC–MS analysis of purified fractions confirms the presence of phenolics in the lichens responsible for anticancer activity. Pharmacological data on U87-MG cell lines revealed the possible usage of this lichen in the treatment of glioblastoma and its associated pathological conditions. Lichen extract and its purified fraction (L16–L21) significantly ( p &lt; 0.05) inhibited the proliferation of U87 glioblastoma cells in a dose-dependent manner. Conclusion This study reported that the shield lichen has promising cytotoxic activities in U87-MG cell lines, which was marked by a reduction in cancer cell proliferation. Further pharmacognostic and phytochemical data confirmed its identity and facilitated its inclusion in various Pharmacopoeias.

Beta-Caryophyllene Augments Radiotherapy Efficacy in GBM by Modulating Cell Apoptosis and DNA Damage Repair via PPARγ and NF-κB Pathways.

Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy with limited treatment options. Radiotherapy (RT) is often used for treating unresectable GBM; however, the outcomes are often limited due to the radioresistance of GBM. Therefore, the discovery of potential radiosensitizers to enhance GBM responses to RT is crucial. Beta-caryophyllene (BCP), a natural cannabinoid, promotes cancer apoptosis by upregulating the PPARγ signaling pathway and can cross the blood-brain barrier due to its lipophilic nature. This study aimed to evaluate the radiosensitizing potential of BCP in GBM cells. U87MG and GL261 cells and a GL261 tumor-bearing model were treated with RT, BCP, or both. Treatment efficacy was assessed using the MTT assay and tumor growth tracking, and the underlying mechanisms were investigated using western blotting, immunofluorescence staining, and other analyses. BCP synergistically enhanced the efficacy of RT in cell culture, as evidenced by the combination index determined through the MTT assay. This enhancement was mediated by the BCP-induced deceleration of DNA damage repair, as demonstrated by sustained γH2AX signal, upregulated PPARγ levels, and reduced expression of pAKT, pERK, and NF-κB, indicating apoptosis induction and inhibition of survival pathways. BCP significantly inhibited tumor growth in GL261 tumor-bearing mice with no discernible side effects. These findings indicate that BCP may serve as a potential radiosensitizer for improving RT outcomes in GBM by inhibiting DNA repair, inducing apoptosis, and suppressing anti-apoptotic and survival pathways.

Preparation and Optimization of Gemcitabine Loaded PLGA Nanoparticle Using Box-Behnken Design for Targeting to Brain: In Vitro Characterization, Cytotoxicity and Apoptosis Study

Background: Treatment of glioma with conventional approaches remains a far-reaching target to provide the desired outcome. This study aimed to develop and optimize Gemcitabine hydrochloride- loaded PLGA nanoparticles (GNPs) using the Box-Behnken design methodology. The independent variables chosen for this study included the quantity of Polymer (PLGA) (X1), Tween 80 (X2), and Sonication time (X3), whereas the dependent variables were Particle size (Y1) EE % (Y2) and PDI (Y3). The optimized biodegradable nanoparticles were investigated for their anticancer effectiveness in U87MG human glioblastoma cells in vitro. Method: The formulation process involved two steps. Initially, emulsification was carried out by combining the organic polymer solution with the aqueous surfactant solution. Subsequently, in the second step, the organic solvent was evaporated, resulting in the precipitation of the polymer and the formation of nanoparticles. The quantity of PLGA, Tween 80, and PVA (at a constant concentration) was adjusted based on the experimental trial approach. Subsequently, the PLGA-based nanoparticles underwent characterization, wherein their particle size, encapsulation efficiency, polydispersity index (PDI), and cumulative release were assessed. The optimal formulation composition was determined as 200 mg of PLGA, 4 ml of Tween 80, and 2 mg of PVA. Further, the optimized GNPs were evaluated for their anti-cancer effectiveness on U87 MG cells by MTT and apoptosis assay. Results: The results demonstrated that the optimized GNPs exhibited an encapsulation efficiency of 81.66 %, a particle size of 140.1 nm, and a PDI of 0.37. The morphology of the Opt-GNPs was observed to be spherical through transmission electron microscopy (TEM). Conclusion: The Apoptosis study further confirmed the observations of MTT assay as the Opt- GNPs significantly enhanced the apoptosis in U-87 MG cells than the Standard marketed formulation.

Extracellular adenosine oppositely regulates the purinome machinery in glioblastoma and mesenchymal stem cells.

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed-back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB-conditioned medium or co-cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell-based strategies using MSCs.

In Silico and In Vitro Study of mRNA Biomarkers for Glioblastoma Multiforme Resistance to Temozolomide (TMZ): The Association with Stemness.

Glioblastoma multiforme (GBM) is an aggressive brain tumor that primarily affects adults. The Stupp Protocol, which includes surgical resection, chemoradiation, and monotherapy with temozolomide (TMZ), is the standard treatment regimen for GBM. However, repeated use of TMZ leads to resistance in GBM cells, resulting in a poor prognosis for patients. This resistance is driven by several intrinsic factors. This study aims to identify potential biomarkers of resistance associated with stemness. We utilized datasets from GEO, performed Venn diagram intersection analysis, conducted GO enrichment analysis using DAVID and ENRICHR, carried out pathway enrichment analysis with KEGG and REACTOME, and executed survival analysis with GEPIA. Additionally, we compared mRNA expression using the Human Protein Atlas and validated our findings with qRT-PCR. We identified that PAQR6 and ITPKB mRNA expression was consistently higher in TMZ-resistant T98G cells, but TGFBI mRNA expression was found to be significantly higher in TMZ-resistant T98G cells compared to U87MG cells. In addition, a significantly higher CD133 mRNA expression as a stemness marker was found in T98G cells compared to U87MG cells. It is hoped that the acquired disease-related resistance biomarker candidates will be able to be used at the clinical level in terms of non-invasive early detection in GBM patients. However, additional research is required to validate the findings of this preliminary biomarker discovery study.

Investigation of the protective and therapeutic effects of Diphenhydramine in an in vitro Parkinson’s model

Objective: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons. Due to the rising prevalence of PD, the need for neuroprotective treatments is increasing, and these are being investigated as a means to slow the disease’s progression. Diphenhydramine (DPH), acting as a histamine 1 receptor antagonist, crosses the blood-brain barrier and exerts effects on the central nervous system. Our aim in this study is to evaluate the neuroprotective and therapeutic effects of DPH in an in vitro PD model induced by 6-hydroxydopamine (6-OHDA). Materials and Methods: An in vitro PD model was established in Glioblastoma (U-118 MG) cells using 6-OHDA. DPH was applied at three different concentrations before and after 6-OHDA application. The protective effect of DPH was evaluated by assessing cell viability using the XTT cell proliferation assay. The results were analyzed using statistical analysis methods. Results: Our study demonstrated that dose-controlled administration of DPH has both neuroprotective and therapeutic effects on an in vitro Parkinson’s model established with 6-OHDA in the U-118MG cell line. According to our findings, DPH at concentrations of 1, 10, and 100 µM significantly increased cell viability compared to the 6-OHDA control group. DPH at 1 and 10 µM concentrations showed important potential for therapeutic and neuroprotective use. Conclusions: The in vitro study indicates that DPH has neuroprotective and therapeutic effects on PD-modeled U-118MG neuronal cells by increasing cell viability. Nevertheless, in vivo studies are needed to evaluate the effects of DPH on animal models of PD.