U87MG Cells Research Articles

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.

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.

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.

Higher isoform of hnRNPA1 confer Temozolomide resistance in U87MG & LN229 glioma cells.

Gliblastoma is a malignant brain tumor; despite available treatment modalities, the tumor reoccurrence rate persist in the currently prescribed Temozolomide chemotherapy. Study aimed to study the inquisitive role of RNA binding splice factor protein hnRNPA1 in promoting glioma resistance against Temozolomide drug and therapeutic insights. In this study two non-expressing O6-methylguanine-DNA methyltransferase (MGMT) glioma cell lines U87MG & LN229. U87MG cells were grown in Temozolomide from 50μM upto 400μM & LN229 cells grown upto 200μM, till then both these cells acquired Temozolomide resistance. Both of these cells were grown & maintained continously in its highest dose of Temozolomide (TMZ). Splice factor protein SF2/ASF1 was functionally correlated with abundance of hnRNPA1 protein in Temozolomide (TMZ) resistant cells using its specific siRNA transfection approach, in detrmining SF2/ASF1 mediated hnRNPA1 splicing and Temozolomide resistant reversal. U87MG TMZ resistance, results an increase in the expression of pre mRNA-splicing factor SF2/ASF1, Heterogeneous Ribonucleoprotein A1 (hnRNPA1) and O6-methylguanine-DNA methyltransferase (MGMT) protein. MGMT expression was not observed in LN229 TMZ resistant cells. Further, mRNA sequencing of hnRNPA1 confirmed the exclusive abundance of its higher isoform in TMZ- resistant cells along with increase in SF2/ASF1 expression. Knocking down of SF2/ASF1 using its specific siRNA reverted the higher isoform of hnRNPA1 isoform Var2 to its lower isoform hnRNPA1 Var1 in U87 TMZ resistant cells, reveals hnRNPA1 alternative higher isoform abundance is SF2/ASF1 splice factor dependent. Additionally, selective knock down of hnRNPA1 higher isoform Var2 in TMZ resistant U87MG & LN229 promotes apoptosis, was further specfically enhanced on Wortmannin (PI3Kinase inhibitor) treatment. Targeting higher isoform Var2 of hnRNPA1 specifically induces chemosensitization in MGMT expressed Temozolomide resistant U87MG as well as in MGMT non-expressed LN229 TMZ resistant cells.

Mesenchymal Properties of Glioma Cell Lines.

Screening of cell surface markers of three glioma cell lines (astrocytoma 1321N1, glioblastoma T98g, and glioblastoma astrocytoma U373 MG) was performed. Glioma cells expressed common mesenchymal cell markers, although the expression levels varied between the cell lines. The expression of proneural markers and glioma cancer stem cell markers was very low and also varied. Induction of differentiation towards the mesodermal cell lineages showed effective adipogenic and osteogenic differentiation for only the U373 MG cell line, while the 1321N1 and T98g lines demonstrated weak adipogenic potential and failed to undergo osteogenic differentiation. The obtained results point to the intratumor phenotypical heterogeneity of cells in gliomas and to the differences between the three studied types of gliomas with regard to the content of cells with mesenchymal phenotype.

EXTH-01. EFFICACY OF PERAMPANEL AGAINST GLIOBLASTOMA CELLS

Abstract We investigated the antitumor effect of perampanel, which is the AMPA-type glutamate receptor antagonist widely used as an antiepileptic drug in Japan, on glioblastoma cells both in vitro and vivo. Commercially available glioblastoma cell lines (A-172, AM-38, T98G, U-138MG, U-251MG and YH-13) and newly established glioblastoma cell line 0125-DGC were used in the experiments. First, the antitumor effect of perampanel was evaluated by cell counting assay. The number of surviving cells after administrating drugs (perampanel, carbamazepine, sodium valproate, levetiracetam, and temozolomide) was counted. The cell proliferation inhibitory effect was observed in all cell lines after administration of perampanel and temozolomide. The antitumor effect was greater when perampanel and temozolomide were used together than when they were used alone. Next, we investigated the mechanism of the antitumor effect of perampanel on glioblastoma cells using FACS, western blotting, RT-PCR, and transwell assay. The expression levels of apoptosis-related proteins and mRNAs on glioblastoma cells were increased after administration of perampanel. Furthermore, perampanel demonstrated the inhibitory effect on the migration of glioblastoma cells. Finally, we evaluated the antitumor effect of perampanel in vivo. U-87MG cells were transplanted into the brains of immunodeficient mice, and these mice were treated by perampanel and/or temozolomide. The transplanted mice were divided into an untreated control group, a perampanel treatment group, a temozolomide treatment group, and a combination treatment group. As a result, survival times were significantly prolonged in all treatment groups compared to the untreated control group. Notably, survival times in the combination therapy group was significantly longer than in the monotherapy group. These results revealed that perampanel exhibits antitumor effects on glioblastoma cells by inducing apoptosis and suppressing cell migration ability. Furthermore, perampanel may enhance the antitumor effect of temozolomide for glioblastoma when it used as combination therapy.

DDEL-02. PRECLINICAL EFFICACY OF IL13RΑ2-TARGETING POLYPEPTIDE-DRUG CONJUGATE (SELF-DEPOTTM ONCOPDC) IN GLIOBLASTOMA AND DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) are currently incurable cancers. Systemic chemotherapy shows limited efficacy because of the intact blood-brain barrier. Convection-enhanced delivery (CED) offers promise by concentrating high doses of chemotherapeutics directly within these tumors. However, drugs administered via CED often face challenges in confirming precise target delivery and are quickly cleared by increased interstitial fluid flow and drug efflux transporters. This highlights the urgent need for innovative drugs that demonstrate target specificity and prolonged retention capabilities. Intrinsically disordered polypeptides (IDPs), derived from tropoelastin, self-assemble into water-insoluble structures with extended retention properties triggered by body temperature. IL13Rα2, differentially amplified in U87MG GBM and SF8628 DIPG cells, serves as a target for therapeutic intervention. An IL13Rα2-targeting IDP (XM161) was developed by incorporating IL13Rα2 binding sequences into the IDP domains. XM161 demonstrated high selectivity in binding to IL13Rα2, with Kd values of 1,786 nM for IL13Rα1 and 12.8 nM for IL13Rα2. Conjugation of XM161 with SN38, a potent topoisomerase I inhibitor, resulted in XM161-SN38, which remained in a dissolved state up to 50°C but formed insoluble aggregates above 27°C in the presence of cerebrospinal fluid. XM161-SN38 exhibited strong cytotoxicity against U87MG and SF8628 cells, with IC50 values of 14.2 nM and 0.48 nM, respectively. Importantly, XM161-SN38 demonstrated efficacy in overcoming Temozolomide resistance in T98G GBM cells, with IC50 values of 3.35 nM compared to 10.87 nM for unconjugated SN38. In orthotopic xenograft models established in BALB/cSlc-nu/nu male mice using U87MG-Luc2 cells, three doses (3 x 0.95 μg SN38 equivalents) of XM161-SN38 delivered via CED were well tolerated and significantly extended median survival to over 65 days, providing a notable survival benefit compared to untreated controls (42 days) and Topotecan-treated group (42.5 days). These findings validate the feasibility and therapeutic potential of XM161-SN38 for GBM treatment. Ongoing studies are underway to investigate the safety and pharmacokinetics of XM161-SN38 in tumor-naive mouse brains.

Asymmetric Synthesis and Biological Evaluation of Both Enantiomers of 5- and 6-Boronotryptophan as Potential Boron Delivery Agents for Boron Neutron Capture Therapy.

This research investigates boronated tryptophans as potential boron delivery agents for boron neutron capture therapy (BNCT) of cancer. We synthesized both enantiomers of 5- and 6-boronotryptophans (1a and 1b) using simple and inexpensive methods. Their uptake was assessed in two human cancer cell lines, CAL27 (head and neck cancer) and U87-MG (brain cancer), and compared to l-p-boronophenylalanine (l-BPA) as a reference. To determine whether these tryptophan derivatives are substrates for large amino acid transporter 1, we performed molecular dynamics simulations to explore their transport mechanism. Our findings reveal differences in boron compound accumulation between the cancer cell lines, indicating that tryptophan derivatives could serve as effective boron carriers when the clinically used boron carrier, BPA, is ineffective.

STEM-03. TARGETING F-ACTIN DYNAMICS OF GLIOBLASTOMAS TO OVERCOME THERAPY RESISTANCE: IMPACTS ON DNA REPAIR AND GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is a highly aggressive brain tumor with high recurrence and resistance to therapies. The F-actin cytoskeleton in GBM is related to cell invasion and the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Our research identifies a novel role for the F-actin pathway in DNA repair, impacting genomic stability and contributing to therapy resistance. Understanding F-actin’s role is crucial for developing targeted strategies to improve GBM treatment outcomes. This study examines the effects of pharmacological F-actin depolymerization on reversing resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM spheroids. We established resistant sublines of GBM U87-MG cells through IR and TMZ cycles, observing significant resistance increases via MTT assays. Also, resistant cells had enhanced spheroid formation capacity, with higher size and proliferation. Alterations in DNA repair and F-actin pathway were observed, with intensified and disorganized F-actin polymerization and efficient repair in resistant cells. Elevated expression of stemness markers (Nestin, CD133, and CD44) indicated a GSC-like phenotype in resistant cells, corroborated by increased tumorigenicity and de novo spheroid formation. These markers were further enhanced by DNA damage. Treating cells with actin polymerization inhibitors reduced resistance to TMZ and IR, partly due to impaired DNA repair capacity. Additionally, F-actin disassembly affected stemness marker expression, reinforcing the link between cytoskeletal dynamics and the GSC phenotype. Our findings suggest that F-actin dynamic is crucial for resistance in GBM. Targeting F-actin could resensitize recurrent GBM tumours by diminishing DNA repair capabilities and affecting the GSC-like phenotype, offering new therapeutic avenues.

Paclitaxel loaded Capmul MCM and tristearin based nanostructured lipid carriers (NLCs) for glioblastoma treatment: screening of formulation components by quality by design (QbD) approach.

Paclitaxel (PTX), a naturally occurring diterpenoid isolated from Taxus brevifolia, is a first-line drug for the treatment of glioblastoma; however, it suffers from the disadvantages of poor water solubility and nonspecific biodistribution, which cause serious side effects in the human body. The marketed formulation suffers from serious side effects, such as allergic reactions, neutropenia, and neuropathy, which require safe and effective formulations of PTX. In the present study, PTX was entrapped in a solid-liquid lipid mixture with the aid of a surfactant using a modified solvent evaporation technique. Higher entrapment of the impressive stability of the formulation was achieved by employing quality design-based strategies. Optimized levels by employing a numerical optimization technique for each factor, that is, surfactant concentration (X1), lipid concentration (X2), and amount of organic solvent (X3) were 0.3%, 0.76% & 8.3 ml respectively. The resultant formulation exhibited a particle size of 121.44 nm, entrapment efficiency of 94.27%, and zeta potential of -20.21 mV with unimodal size distribution. A reduction in the % crystalline index from 48 to 3.4% ensured the amorphous form of the entrapped drug inside the formulation, which precludes the fear of leakage and instability of the formulation. Cell line studies conducted on U87MG Cell lines also suggested that the NLC of paclitaxel are more effective than those of pure PTX. In summary, PTXNLC seem to be a superior alternative carrier system for the formulation industry to obtain higher entrapment with excellent stability.

Novel 1,8-Naphthalimide Derivatives Inhibit Growth and Induce Apoptosis in Human Glioblastoma.

Given the rapid advancement of functional 1,8-Naphthalimide derivatives in anticancer research, we synthesized these two novel naphthalimide derivatives with diverse substituents and investigated the effect on glioblastoma multiforme (GBM) cells. Cytotoxicity, apoptosis, cell cycle, topoisomerase II and Western blotting assays were evaluated for these compounds against GBM in vitro. A human GBM xenograft mouse model established by subcutaneously injecting U87-MG cells and the treatment responses were assessed. Both compounds 3 and 4 exhibited significant antiproliferative activities, inducing apoptosis and cell death. Only compound 3 notably induced G2/M phase cell cycle arrest in the U87-MG GBM cells. Both compounds inhibited DNA topoisomerase II activity, resulting in DNA damage. The in vivo antiproliferative potential of compound 3 was further validated in a U87-MG GBM xenograft mouse model, without any discernible loss of body weight or kidney toxicity noted. This study presents novel findings demonstrating that 1,8-Naphthalimide derivatives exhibited significant GBM cell suppression in vitro and in vivo without causing adverse effects on body weight or kidney function. Further experiments, including investigations into mechanisms and pathways, as well as preclinical studies on the pharmacokinetics and pharmacodynamics, may be instrumental to the development of a new anti-GBM compound.

A pH-Sensitive cRGD-PEG-siRNA Conjugated Compound Targeting Glioblastoma.

Glioblastoma ranks among the most prevalent primary intracranial tumors, characterized by high mortality and poor prognosis. Chemotherapy remains a key treatment strategy for gliomas, though most current drugs suffer from limited efficacy and significant toxicity. This study focuses on a cRGD-siEGFR coupling compound synthesized in a previous stage. Prior research indicated that cRGD-siEGFR molecules exhibited certain targeting and antitumor properties but faced issues of inadequate targeting, low efficacy, and high renal toxicity. To enhance antitumor efficacy and mitigate side effects, a pH-responsive, long-circulating, and highly targeted siRNA delivery system, the cRGD-PEG-siEGFR conjugate, was developed. The targeting, antitumor effects, and biological distribution of cRGD-PEG-siEGFR were examined. The results demonstrated that cRGD-PEG-siEGFR was effectively taken up by αvβ3-positive U87MG cells, specifically silenced EGFR gene expression, and exhibited antitumor effects. In normal physiological conditions, it avoided uptake by normal cells, thereby reducing side effects. Furthermore, in vivo biodistribution experiments revealed that cRGD-PEG-siEGFR, compared to cRGD-siEGFR, significantly decreased renal accumulation and exhibited prolonged circulation. Consequently, cRGD-PEG-siRNA emerges as a promising drug candidate with attributes of long circulation, high targeting, pH responsiveness, and substantial antitumor efficacy.

Histone deacetylase inhibition disrupts the molecular signature of the glioblastoma secretome related to extracellular vesicle-associated proteins and targets RAB7a and RAB14 in vitro

Glioblastoma (GBM) is the most aggressive brain tumor with a poor prognosis. While Histone Deacetylase inhibitors have shown promising results in inhibiting cancer cell invasion and promoting apoptosis, their effects on GBM secretion, specifically focusing on extracellular vesicles (EVs) secretion, remain largely unexplored. Using label-free NANOLC-MS/MS methodology, we identified significant changes in the abundance of membrane traffic regulatory proteins in the secretome of U87MG cells after the treatment with the HDAC inhibitor Trichostatin A (TSA). In silico analysis showed that TSA treatment disrupted the secretion pattern of EVs-associated proteins and cellular signaling pathways, both qualitatively and quantitatively. Notably, RAB14/RAB7a interaction was only observed in the secretome of cells treated with TSA. In vitro assays revealed that TSA treatment of glioma cells increased EVs secretion and intracellular protein levels of RAB7a and RAB14 without affecting gene expression, suggesting a role of these two EVs-associated proteins in grade IV glioma cells. Additionally, an integrative approach using clinical data highlighted a correlation between DNA mutations affecting vesicle traffic coding-genes and clinical and phenotypic outcomes in glioma patients. These findings provide insights into the interplay between epigenetics and GBM intracellular trafficking, potentially leading to improved strategies for targeting and modifying the complex signaling network established between GBM cells and the tumor cell microenvironment.

Dual-targeted TfRA4-DNA1-Ag@AuNPs: An innovative radiosensitizer for enhancing radiotherapy in glioblastoma multiforme

Radiation therapy (RT) is one of the most effective and widely used treatment methods for glioblastoma multiforme (GBM). However, its efficacy is often compromised by the inherent radioresistance of tumor cells, while the restrictive nature of the blood-brain barrier (BBB) specifically impedes the delivery of radiosensitizer. Thus, we constructed and characterized polyethylene glycol (PEG)-functionalized silver-gold core-shell nanoparticles (PSGNPs) targeting both BBB (TfRA4) and GBM (DNA1) (TDSGNPs). Afterwards, studies conducted both in vitro and in vivo were employed to assess the BBB penetration capabilities, abilities of GBM targeting and radiosensitization effect. Transmission electron microscope images of PSGNPs showed a core-shell structure, and the results of ultraviolet-visible absorption spectroscopy and dynamic light scattering displayed that TDSGNPs were successfully constructed with excellent dispersion properties. TDSGNPs could be specifically taken up by U87MG cells and the uptake peaked at 24 h. TDSGNPs combined with RT obviously increased the apoptosis proportion of the cells. It was shown by the in vitro and in vivo investigations that TDSGNPs could target U87MG cells after crossing the BBB, and further study revealed that TDSGNPs showed an uptake peak in the tumor sites after 3 h intravenous injection. The radiosensitization of TDSGNPs was better than that of the nanoparticles modified with single aptamers and the median survival of tumor-bearing mice was greatly extended. This study demonstrated that TDSGNPs could penetrate BBB to target GBM, functioning as a promising radiosensitizer for the targeted therapy of GBM.

Combined machine learning models, docking analysis, ADMET studies and molecular dynamics simulations for the design of novel FAK inhibitors against glioblastoma

Gliomas, particularly glioblastoma (GBM), are highly aggressive brain tumors with poor prognosis and high recurrence rates. This underscores the urgent need for novel therapeutic approaches. One promising target is Focal adhesion kinase (FAK), a key regulator of tumor progression currently in clinical trials for glioma treatment. Drug development, however, is both challenging and costly, necessitating efficient strategies. Computer-Aided Drug Design (CADD), especially when combined with machine learning (ML), streamlines the processes of virtual screening and optimization, significantly enhancing the efficiency and accuracy of drug discovery. Our study integrates ML, docking analysis, ADMET (absorption, distribution, metabolism, elimination, and toxicity) studies to identify novel FAK inhibitors specific to GBM. Predictive models showed strong performance, with an R2 of 0.892, MAE of 0.331, and RMSE of 0.467 using protein-level IC50 data in combined CDK, CDK extended fingerprints, and substructure fingerprint counts derived from 1280 FAK inhibitors. Another model, based on IC50 data from 2608 compounds tested on U87-MG cells, achieved an R2 of 0.789, MAE of 0.395, and RMSE of 0.536. Using these models, we efficiently identified 275 potentially active compounds out of 5107 candidates. Subsequent ADMET analysis narrowed this down to 16 potential FAK inhibitors that meet the established drug-likeness criteria. Moreover, molecular dynamics (MD) simulations validated the stable binding interactions between the selected compounds and the FAK protein. This study highlights the effectiveness of combining ML, docking analysis, and ADMET studies to rapidly identify potential FAK inhibitors from large databases, providing valuable insights for the systematic design of FAK inhibitors.

Anticancer effect of the oncolytic Newcastle disease virus harboring the PTEN gene on glioblastoma.

Glioblastoma (GBM) is one of the most lethal types of human brain cancer and is characterized by rapid growth, an aggressive nature and a poor prognosis. GBM is highly heterogeneous, and often involves several genetic mutations and abnormalities. Genetic disorders or low expression of phosphatase and tensin homolog (PTEN) are associated with GBM occurrence, progression and poor prognosis of patients with GBM. However, effective delivery of PTEN for expression in GBM cells within the brain remains challenging. The aim of the present study was to develop a therapeutic strategy to restore PTEN expression in GBM cells by utilizing a recombinant Newcastle disease virus (rNDV) vector expressing the human PTEN gene (rNDV-PTEN). Methods included infection of U87-MG cells with rNDV-PTEN, followed by assessments of PTEN expression, and cell proliferation, migration and apoptosis. Additionally, an orthotopic GBM mouse model was used to evaluate the in vivo efficacy of rNDV-PTEN. Infection with recombinant rNDV-PTEN treatment increased PTEN protein expression in the cytoplasm of the U87-MG cells, reduced cell proliferation and migration, and induced apoptosis by inhibiting the AKT/mTOR signaling pathway. In the orthotopic GBM mouse model, rNDV-PTEN significantly reduced tumor size and improved survival rates. Magnetic resonance imaging and in vivo imaging analyses confirmed the targeted delivery and efficacy of rNDV-PTEN. These findings highlight the usefulness of rNDV-PTEN as a promising therapeutic agent for GBM, representing a potential advancement in treatment, especially for patients with PTEN deficiency.

STUDIES ON GLYCOGEN PHOSPHORYLASE AS A POTENTIAL MOLECULAR TARGET IN GLIOBLASTOMA

Abstract AIMS Glioblastoma (GB) is one of the most aggressive malignancies of the brain and spinal cord. Current standard of care has remained surgical resection of the tumour, followed by radiotherapy and/or chemotherapy. Crucial challenges in the effective treatment of GB include resistance to the main chemotherapeutic, temozolomide. Prognosis is poor with median survival remaining at 14.6 months. A now established hallmark of cancer is the ability of malignant cells to “modify, or reprogram, cellular metabolism”, such as glycolysis upregulation. This hallmark could suggest the potential of targeting a specific metabolic component within glycolysis, thus reduce energy production, resulting in possible apoptosis/suppressed proliferation. Glycogen phosphorylase (GP), an enzyme in glyconeolysis, could be inhibited for a therapeutic effect in GB. In this project, GP levels across the three different isoforms (PYGB, PYGL, and PYGM) and the effect of the GP inhibitor CP-91149 was investigated across three different GB cell lines. METHOD Cell viability of T98G, U251-MG, and U87-MG glioblastoma cell lines and SVGp19 human fetal glial cell line was measured after administration of CP-91149 for 24, 48, and 72 hours. Migration of T98G and U251 cells following treatment with CP-91149 was measured in a wound healing assay. Visualisation of the three GP isoforms was achieved through western blot and confirmed by immunocytochemistry. Flow cytometry was utilized to expand analysis on GP inhibition through assessing cell cycle before and after treatments. RESULTS Results showed CP-91149 had a significant dose-dependent effect in vitro on all cell lines. Inhibition of GP resulted in reduced cell migration, also in a dose-dependent manner. Western blot and immunocytochemistry analysis showed PYGL is the most predominant isoform of GP. CONCLUSION Future work in this project will aim to silence expression of GP in GB cell lines in order to compare the results with that yielded from cells treated with the GP inhibitor CP-91149.

Combination of heme oxygenase-1 inhibitors and temozolomide to improve the anticancer effect in glioblastoma

Heme oxygenase-1 (HO-1) is involved in the oncogenesis of glioblastoma (GBM). In this work, we investigated for the first time how the co-administration (combo) of the HO-1 inhibitors SI1/09 or LS6/42 with temozolomide (TMZ) or temozolomide acid (TMZ Ac) may influence the proliferation of U87MG GBM cell lines. Moreover, two novel TMZ/HO-1 inhibitors codrugs LS8/21 and LS8/24 were synthesised, characterised and tested. Results indicate that the combos TMZ or TMZ Ac with LS6/42, as well as the corresponding LS8/24, are more efficacious in reducing U87MG cell proliferation with respect to reference drugs, allowing a possible reduction of the TMZ dosage and related side effects in clinical practice. The chemical and enzymatic stability of the most potent codrug LS8/24 was evaluated. The observed high potency performed by both combos and LS8/24 in cells suggests that HO-1 inhibition may give additional contribution to the antiproliferative effect of TMZ.

Synthesis, characterization, and in-vitro bioimaging applications of green emissive AgInS2-based core multi and alloyed shell (AgInS2/CdS/ZnS and AgInS2/ZnS/CdS) nanocrystals

This study explores the synthesis, characterization, and preliminary bioimaging applications of green-emissive AgInS2-based nanocrystals (NCs) featuring core-multi and alloyed shell structures, specifically AgInS2/CdS/ZnS and AgInS2/ZnS/CdS. The nanocrystals were synthesized using a controlled hot-injection method, which ensured the formation of uniform core–shell structures. Detailed characterization through X-ray diffraction (XRD), transmission electron microscopy (TEM), and photoluminescence spectroscopy confirmed the successful development of these core-multi and alloyed shell architectures. The photoluminescence quantum yields (QY) were determined to be approximately 25 % for AgInS2/CdS/ZnS and 30 % for AgInS2/ZnS/CdS, highlighting their efficient luminescent properties. In preliminary bioimaging studies, these nanocrystals exhibited effective cellular imaging capabilities. They showed pronounced fluorescence with minimal background interference in A549 and U87MG cells, suggesting their potential for precise and targeted imaging applications. Despite these promising results, further investigation is needed to fully understand the specificity of these nanocrystals and their interaction mechanisms with various cell types. The observed quantum yields and targeted imaging capabilities indicate that AgInS2-based nanocrystals are promising candidates for advanced bioimaging technologies.