U87MG Cell Lines Research Articles

Cryptolepine-transferrin nanosystem preparation and the anticancer effects on human malignant glioblastoma U87MG cells.

In this study, the synthesis and characterization of the human transferrin (hTf)-cryptolepine complex nanostructure were performed. The interaction of hTf in the forms of native and nanostructures with cryptolepine was evaluated through various spectroscopic methods, molecular docking, and molecular dynamic (MD) simulation studies. Finally, the anticancer mechanisms of the prepared complex nanostructure on the Human malignant glioblastoma U87MG cell line and normal astrocytes were assessed using cell viability, RT-PCR, and ELISA assays. The data showed that hTf and hTf-cryptolepine complex nanostructures exhibited particle sizes of 149.37±28.98 and 161.85±33.44nm, and average zeta potential values of -11.85±1.98 and -23.87±2.202mV, respectively. It was also found that the drug release behavior from the hTf-cryptolepine complex nanostructures displayed a pH-sensitive pattern, demonstrating a strong correlation with the Higuchi model and a moderate correlation with the Korsmeyer-Peppas model. Spectroscopy and MD simulation studies revealed that the interaction of hTf and cryptolepine did not induce significant changes in the structure of the protein. Moreover, binding parameters were determined to be greater for hTf-cryptolepine complex nanostructures compared to the hTf-cryptolepine complex. Cellular assays showed that the calculated IC50 concentrations of cryptolepine, hTf-cryptolepine complex, and hTf-cryptolepine complex nanostructures in Human malignant glioblastoma U87MG cells were 16.1, 14.31, and 4.60μM, respectively. Furthermore, it was demonstrated that Human malignant glioblastoma U87MG cells treated with hTf-cryptolepine complex nanostructures had a more significant increase in caspase-3 expression and activity compared to free cryptolepine or the hTf-cryptolepine complex. In conclusion, this paper may hold great promise for the introduction of bionanotherapeutics, which require further evaluation in future studies.

Quercetin Suppresses Glioma Stem Cells via Activating p16-INK4 Gene Expression through Epigenetic Regulation.

Our study aimed to explore the effects of quercetin on glioma stem cells in patients with brain tumors. Human glioblastoma cell line, U373MG, or glioma stem cell lines, were treated with quercetin. Cell viability was determined by using the cell counting kit 8 assays. Cell apoptosis was determined by using the Annexin-V reagent. Western blotting and qPCR were used to detect the protein and mRNA levels of cyclindependent kinase inhibitor 2A (p16INK4a). Chromatin immunoprecipitation analysis was used to determine the enrichment of H3K27me3 on the p16-INK4 locus with or without quercetin. Treatment with quercetin inhibited cell viability and induced cell apoptosis in U373MG cells. Moreover, treatment with quercetin inhibited the cell viability of four glioma stem cell lines (G3, G10, G15, and G17) from brain tumor samples at high concentrations while having no obvious effects for the other two glioma stem cell lines (G9 and G21). Treatment with quercetin increased the mRNA and protein levels of p16- INK4 in glioma stem cell lines. The study of the underlying mechanism revealed that treatment with quercetin reduced H3K27me3 (an epigenetic modification to the DNA packaging protein histone H3) levels at the p16-INK4 locus. In conclusion, quercetin inhibits glioma cell growth by activating p16-INK4 gene expression through epigenetic regulation.

Combined Effect of Conventional Chemotherapy with Epigenetic Modulators on Glioblastoma

Background/Objectives: Glioblastoma is the most common malignant primary brain tumor, characterized by necrosis, uncontrolled proliferation, infiltration, angiogenesis, apoptosis resistance, and genomic instability. Epigenetic modifiers hold promise as adjuvant therapies for gliomas, with synergistic combinations being explored to enhance efficacy and reduce toxicity. This study aimed to evaluate the effects of single or combined treatments with various anticancer drugs (Carboplatin, Paclitaxel, Avastin), natural compounds (Quercetin), and epigenetic modulators (suberoylanilide hydroxamic acid and 5-Azacytidine) on the expression of some long noncoding RNAs and methylation drivers or some functional features in the U87-MG cell line. Methods: Treated and untreated U87-MG cells were used for the evaluation of drug-induced cytotoxicity, apoptotic events, and distribution in cell cycle phases, detection of cytokine release, and assessment of gene expression and global methylation. Results: Cytotoxicity assays led to the selection of drug concentrations to be used in further experiments. Expression analysis revealed distinct downregulation of nearly all investigated genes and long noncoding RNAs following treatments. All treatments resulted in a higher percentage of global methylation compared to untreated controls. All treatments effectively increased levels of apoptosis, while the epigenetic modulators exhibited a lower proliferation profile, with combined treatments showing elevated values of cell lysis. Conclusions: The results indicate a link between Carboplatin and Avastin treatments and DNA methylation mechanisms involving EZH2, DNMT3A, and DNMT3B, with Avastin’s direct impact on these enzymes warranting further study. This research underscores the promise of platinum-based therapies combined with epigenetic drugs to reactivate silenced tumor suppressor genes and optimize methylation profiles.

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.

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.

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.

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.

Synergistic effect of cold atmospheric plasma and methylene blue loaded nano micelles on treating human glioblastoma cells: An in vitro and molecular dynamics study.

One of the most recent cancer treatment methods is cold atmospheric plasma (CAP), which destroys cancer cells without affecting healthy cells. Also, the created photons in the CAP flame can be used to excite a proper photosensitizing agent (PS). Therefore, using nano micelle systems containing a proper photosensitizer may be beneficial in raising the treatment efficacy of CAP. In this study, we utilized molecular dynamics (MD) simulation to optimize a nano micellar system containing methylene blue to take advantage of the induced photodynamic effect of a CAP generator with helium gas on a glioblastoma cell line. Some micelle properties were first determined and optimized by MD with GROMACS software. Then, micelles containing methylene blue (Micelle-MB) and free methylene blue (MB) at various concentrations were prepared. Singlet oxygen dosimetry using 1,3-diphenylisobenzofuran (DPBF)was performed in the presence and absence of Micelle-MB and MB. Subsequently, the cytotoxicity of MB and Micelle-MB was evaluated on U87-MG cancer cells, and their half-maximal inhibitory concentrations (IC50) were determined. After 48 hr of treatment, the percentage of cell survival was determined using the MTT test. The experiments were repeated at least three times. The synergy index was selected to compare the results. Treatment with CAP and MB reduced the survival rate compared to the PS-free group with CAP. Results of singlet oxygen dosimetry showed that Micelle-MB might be more efficient in producing ROS. CAP treatment with Micelle-MB resulted in more cell death than free MB. In addition, cell viability decreased in Micelle-MB groups with increasing irradiation time in the three investigated irradiation times. Using Micelle-MB in the CAP treatment improves treatment efficiency in the U87-MG cell line.

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.

Antimicrobial and Antiproliferative Properties of 2-Phenyl-N-(Pyridin-2-yl)acetamides.

Infectious diseases, including bacterial, fungal, and viral, have once again gained urgency in the drug development pipeline after the recent COVID-19 pandemic. Tuberculosis (TB) is an old infectious disease for which eradication has not yet been successful. Novel agents are required to have potential activity against both drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB. In this study, we present a series of 2-phenyl-N-(pyridin-2-yl)acetamides in an attempt to investigate their possible antimycobacterial activity, cytotoxicity on the HepG2 liver cancer cell line, and-as complementary testing-their antibacterial and antifungal properties against a panel of clinically important pathogens. This screening resulted in one compound with promising antimycobacterial activity-compound 12, MICMtb H37Ra = 15.625 μg/mL (56.26 μM). Compounds 17, 24, and 26 were further screened for their antiproliferative activity against human epithelial kidney cancer cell line A498, human prostate cancer cell line PC-3, and human glioblastoma cell line U-87MG, where they were found to possess interesting activity worth further exploration in the future.

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.

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.

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.

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.

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.

Discovery of novel Macrocyclic small molecules Based on 2-Amino-4-thiazolylpyridineas selective EGFR inhibitors with high Blood-Brain barrier penetration for the treatment of glioblastoma

Because epidermal growth factor receptor (EGFR) is the most commonly mutated oncogene in glioblastoma (GBM), the development of EGFR inhibitors has become a promising direction for the treatment of GBM. However, due to factors such as limited blood-brain barrier (BBB) permeability and pathway compensation mechanisms, current EGFR inhibitors targeting GBM are not satisfactory. In the previous study, we obtained compound 10c with strong anti-cell proliferation activity. Since macrocyclization can effectively change the physical and chemical properties of molecules, and optimize their selectivity. Therefore, a series of 2-amino-4-thiazolyl pyridine scaffold macrocyclic derivatives were designed and synthesized using compound 10c as the lead compound in this study. Compound 3a, which inhibited the growth of glioblastoma cell lines U87MG and U87-EGFRVIII, had average IC50 values of 4.69µM and 4.98µM, respectively. Compound 3a was highly selective to 9 kinases in the ErbB family, including ErbB2 and ErbB4. In addition, compound 3a demonstrated good blood-brain barrier permeability in mice, the blood-brain concentration of the drug remained above 20% within 5-60min following intravenous administration in mice. In conclusion, compound 3a is a promising candidate for novel EGFR inhibitors targeting GBM.

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.

Effect of human reovirus strain R-92 on tumor cell lines

Background. Among all the new methods and approaches, virotherapy with oncolytic viruses, both in combination with immunotherapy and without it, shows high efficiency in various phases of clinical trials and good tolerance by patients.Aim. To study the sensitivity of some immortalized cancer cell lines to the R-92 strain of human reovirus with cell characteristics at the ultrastructural level.Materials and methods. The study was carried out on cell lines of HeLa, A549, U87MG. Cells were planted in an amount of 15 thousand per well of a 96-well plate and after adhesion, the virus was inoculated by adding a medium containing virus particles in 4 tenfold dilutions (approximately 10 9 –106 particles per ml). Next, the cells were cultured for 24 h, after which the number of living cells in the wells was determined indirectly using the methyl tetrazolium test, which was carried out according to standard methods. To study the ultrastructure of infected cells, cells were seeded into a T25 flask and inoculated with the virus at the maximum concentration. After 24 h of cultivation, the cells were fixed in a 2.5 % glutaraldehyde solution in phosphate buffer for 1 h, after which they were washed three times in phosphate buffer and samples were processed for TEM according to standard methods.Results. Diluting the virus 1000 times led to a decrease in the cytostatic effect in all three cultures to a level practically no different from the control. HeLa turned out to be the most sensitive culture to reovirus. In the experiment, the number of living cells decreased to 60.4 ± 10.2 % compared to the control during incubation with the maximum number of viral particles and to 63.7 ± 16.2 % with a tenfold dilution of the virus. This indicator was significantly lower than in the other two studied cultures under these cultivation conditions (p &lt;0.001).In addition, at the maximum virus concentration, the A549 culture was less sensitive than the U87MG culture (p &lt;0.01). At lower concentrations of viral particles, the average viability of the studied cell lines did not differ significantly from each other. Analysis of electron diffraction patterns showed that the virus successfully replicates in the cytoplasm of the studied cultures, but is not released from the cell, which is apparently due to the short incubation period. TEM also showed cell damage characteristic of apoptosis or necroptosis, uniformly expressed in all studied cultures.Conclusion. Cell lines A549, HeLa and U87MG, according to the results of the methyl tetrazolium test, demonstrate different sensitivity to the human reovirus strain P-92. The TEM picture of cells from infected cultures showed signs of the development of apoptosis or necroptosis.

Co-delivery of temozolomide and quercetin with folic acid-conjugated exosomes in glioblastoma treatment.

Aim: The study aims to improve glioblastoma multiforme (GBM) treatment by combining temozolomide (TMZ) and quercetin (Qct), using folic acid (FA)-conjugated exosomes to overcome TMZ resistance and enhance blood-brain barrier (BBB) penetration.Methods: Exosomes were isolated and after characterizing and modifying their surfaces with FA, drug loading of TMZ and Qct into exosomes was done. In vitro assays, including cell viability tests, RT-PCR, Western-blotting and flow-cytometry, were performed using U87MG and U251MG GBM cell lines. In vivo analysis included administering exosome-drug formulations to glioblastoma-bearing Wistar rats, monitored through optical imaging and PET scans, followed by post-mortem immunohistochemistry and histological examination.Results: The results showed successful exosome isolation and FA conjugation, with drug release studies indicating accelerated release of TMZ and Qct in acidic conditions, enhancing cytotoxicity. Immunofluorescence indicated greater exosome uptake in GBM cells due to FA conjugation. Cell viability assays demonstrated increased toxicity of the combination therapy, correlating with elevated apoptosis. In vivo studies revealed significant tumor size reduction, alongside increased apoptosis and reduced angiogenesis, particularly in the TMZ-Qct-Exo-FA group.Conclusion: FA-conjugated exosomes loaded with TMZ and Qct represent a promising strategy to enhance GBM treatment efficacy by improving drug delivery, apoptosis induction and inhibiting the PI3K/Akt/mTOR pathway.

MiR-155-5p regulates autophagy and apoptosis of glioma cells through RICTOR.

Glioma characterized by the high degree of drug resistance and the poor prognosis is the most common primary malignant tumors of the brain. And miRNA is involved in a variety of biological behaviors of tumors, enhancing or inhibiting the occurrence and development of tumors. Therefore, the present study aims to explore whether miR-155-5p can regulate autophagy and apoptosis of glioma through RICTOR. The significantly differential gene miR-155-5p was identified from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) databases GSE165937 and GSE138764 using bioinformatics analysis, and its expression was validated by quantitative real-time polymerase chain reaction (qRT-PCR). The putative target genes of miR-155-5p were predicted through interrogation of relevant databases, followed by identification of key target genes. Subsequently, core target genes were selected for functional enrichment analysis. The U87MG cell line was utilized as the experimental model and divided into Negative Control1 (NC1) group, Mimic group, Negative Control2 (NC2) group, Inhibitor group, and NC + 3-methyladenine (3-MA) group. The expression levels of miR-155-5p, RICTOR, P62, LC-3, Bax, Bcl-2, and Caspase-3 were assessed using qRT-PCR, cellular fluorescence imaging, and Western blotting; while apoptosis in the U87MG cell line was evaluated via flow cytometry. The results showed that miR-155-5P was highly expressed in glioma cells, which could inhibit the expression of Bax, Caspase-3, LCII/LCI and Beclin-1, and increase the expression of Bcl2 and P62. Flow cytometry and cell fluorescence were used to verify the above results. Moreover, when U87MG cells treated with miR-155-5p inhibitor were inhibited by 3-MA, the results showed that miR-155-5p enhanced the anti-apoptotic ability of U87MG cells by regulating autophagy. In addition, the bioinformatics results show that miR-155-5p survival prognosis in glioma into a strong negative correlation, while the survival prognosis of RICTOR in glioma showed a strong positive correlation. The core target genes Kyoto Encyclopedia of Genes and Genomes (KEGG) mainly occurred in PI3K-AKT signaling pathway; in addition, qRT-PCR and Western blot confirmed the regulatory effect of miR-155-5P on RICTOR. MiR-155-5p regulates autophagy and apoptosis-related proteins in glioma cells through RICTOR, affecting the occurrence and development of glioma.