Human Glioblastoma U87MG Cells Research Articles

Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

Hortensins, Type 1 Ribosome-Inactivating Proteins from Seeds of Red Mountain Spinach: Isolation, Characterization, and Their Effect on Glioblastoma Cells.

Ribosome inactivating proteins (RIPs) are specific N-β-glycosylases that are well-characterized in plants. Their enzymatic action is to damage ribosomes, thereby blocking protein translation. Recently, several research groups have been working on the screening for these toxins in edible plants to facilitate the use of RIPs as biotechnological tools and biopesticides and to overcome public prejudice. Here, four novel monomeric (type 1) RIPs have been isolated from the seeds of Atriplex hortensis L. var. rubra, which is commonly known as edible red mountain spinach. These enzymes, named hortensins 1, 2, 4, and 5, are able to release the β-fragment and, like many other RIPs, adenines from salmon sperm DNA, thus, acting as polynucleotide:adenosine glycosidases. Structurally, hortensins have a different molecular weight and are purified with different yields (hortensin 1, ~29.5 kDa, 0.28 mg per 100 g; hortensin 2, ~29 kDa, 0.29 mg per 100 g; hortensin 4, ~28.5 kDa, 0.71 mg per 100 g; and hortensin 5, ~30 kDa, 0.65 mg per 100 g); only hortensins 2 and 4 are glycosylated. Furthermore, the major isoforms (hortensins 4 and 5) are cytotoxic toward human continuous glioblastoma U87MG cell line. In addition, the morphological change in U87MG cells in the presence of these toxins is indicative of cell death triggered by the apoptotic pathway, as revealed by nuclear DNA fragmentation (TUNEL assay).

Implantable SDF-1α-loaded silk fibroin hyaluronic acid aerogel sponges as an instructive component of the glioblastoma ecosystem: Between chemoattraction and tumor shaping into resection cavities

In view of inevitable recurrences despite resection, glioblastoma (GB) is still an unmet clinical need. Dealing with the stromal-cell derived factor 1-alpha (SDF-1α)/CXCR4 axis as a hallmark of infiltrative GB tumors and with the resection cavity situation, the present study described the effects and relevance of a new engineered micro-nanostructured SF-HA-Hep aerogel sponges, made of silk fibroin (SF), hyaluronic acid (HA) and heparin (Hep) and loaded with SDF-1α, to interfere with the GB ecosystem and residual GB cells, attracting and confining them in a controlled area before elimination. 70 µm-pore sponges were designed as an implantable scaffold to trap GB cells. They presented shape memory and fit brain cavities. Histological results after implantation in brain immunocompetent Fischer rats revealed that SF-HA-Hep sponges are well tolerated for more than 3 months while moderately and reversibly colonized by immuno-inflammatory cells. The use of human U87MG GB cells overexpressing the CXCR4 receptor (U87MG-CXCR4+) and responding to SDF-1α allowed demonstrating directional GB cell attraction and colonization of the device in vitro and in vivo in orthotopic resection cavities in Nude rats. Not modifying global survival, aerogel sponge implantation strongly shaped U87MG-CXCR4+ tumors in cavities in contrast to random infiltrative growth in controls. Overall, those results support the interest of SF-HA-Hep sponges as modifiers of the GB ecosystem dynamics acting as “cell meeting rooms” and biocompatible niches whose properties deserve to be considered toward the development of new clinical procedures. Statement of SignificanceBrain tumor glioblastoma (GB) is one of the worst unmet clinical needs. To prevent the relapse in the resection cavity situation, new implantable biopolymer aerogel sponges loaded with a chemoattractant molecule were designed and preclinically tested as a prototype targeting the interaction between the initial tumor location and its attraction by the peritumoral environment. While not modifying global survival, biocompatible SDF1-loaded hyaluronic acid and silk fibroin sponges induce directional GB cell attraction and colonization in vitro and in rats in vivo. Interestingly, they strongly shaped GB tumors in contrast to random infiltrative growth in controls. These results provide original findings on application of exogenous engineered niches that shape tumors and serve as cell meeting rooms for further clinical developments.

Comparative analysis of α-pinene alone and combined with temozolomide in human glioblastoma cells

α-Pinene (PEN) is a phyto compound present in terpene plants. In traditional medicine, PEN has been used for its anti-inflammatory, pain-relieving, and bronchodilator properties. The effect of PEN in combination with temozolomide (TMZ) in glioblastoma multiforme (GBM) cells has been evaluated. The action of the PEN + TMZ combination on cell migration, soft-agar, and cell death was determined in LN229 and U87MG human glioblastoma cells. In combination, PEN with TMZ showed a synergistic inhibitory effect in the GBM cells. The PEN + TMZ treatment showed a higher fluorescent intensity and reduced the percentage of wound area closure compared to the compound alone. The compounds in combination also resulted in a reduction in single-cell colony formation. To conclude, the study showed that plant-derived PEN enhanced the effectiveness of standard chemotherapeutic, TMZ, in LN229 and U87MG cells.

Preparation and Characterization of Hydrogel Films and Nanoparticles Based on Low-Esterified Pectin for Anticancer Applications.

Prospective adjuvant anticancer therapy development includes the establishing of drug delivery systems based on biocompatible and biodegradable carriers. We have designed films and nanoparticles (NPs) based on low-esterified pectin hydrogel using the ionic gelation method. We investigated morphology, nanomechanical properties, biocompatibility and anticancer activity. Hydrogel films are characterized by tunable viscoelastic properties and surface nanoarchitectonics through pectin concentration and esterification degree (DE), expressed in variable pore frequency and diameter. An in vitro study showed a significant reduction in metabolic activity and the proliferation of the U87MG human glioblastoma cell line, probably affected via the adhesion mechanism. Glioma cells formed neurosphere-like conglomerates with a small number of neurites when cultured on fully de-esterified pectin films and they did not produce neurites on the films prepared on 50% esterified pectin. Pectin NPs were examined in terms of size distribution and nanomechanical properties. The NPs' shapes were proved spherical with a mean diameter varying in the range of 90-115 nm, and a negative zeta potential from -8.30 to -7.86 mV, which indicated their stability. The NPs did not demonstrate toxic effect on cells or metabolism inhibition, indicating good biocompatibility. Nanostructured biomaterials prepared on low-esterified pectins could be of interest for biomedical applications in adjuvant anticancer therapy and for designing drug delivery systems.

Anti-cancer effect of Cissus quadrangularis on human glioblastoma cells

Abstract Objectives Glioblastoma multiforme (GBM) is a common and fatal brain tumour in the central nervous system with a poor survival rate and a median survival time of 15 months only. The standard treatment is aggressive surgical resection followed by radiotherapy and chemotherapy. However, effective drugs available in chemotherapy are limited. This study was designed to evaluate, for the first time, the potential therapeutic effect of Cissus quadrangularis (CQ) in human glioblastoma cells and to investigate its possible mechanisms of action. Methods In this study, we examined the anticancer activity of CQ in human glioblastoma U87 MG cells by cell viability assay, cell migration assay, immunofluorescence staining and Western blot. Results Our results demonstrated that CQ treatment induced U87 cytotoxicity, cell cycle arrest and cell death. The cytotoxicity of CQ mediates ER stress, autophagy and mitochondrial apoptosis by suppressing pro-survival signalling pathways (extracellular signal-regulated kinase and signal transducer and activator of transcription 3 pathways). Conclusions The findings of this study imply that CQ is a promising anti-cancer candidate for the treatment of GBM.

The Combination of Baicalin with Knockdown of mir148a Gene Suppresses Cell Viability and Proliferation and Induces the Apoptosis and Autophagy of Human Glioblastoma Multiforme T98G and U87MG Cells.

Glioblastoma multiforme (GBM) is a heterogeneous and highly vascularized brain tumor that avoids apoptosis due to P-glycoprotein (P-gp) mediated multidrug resistance. Therefore, the development of new therapeutic strategies that induce apoptosis and inhibit proliferation is urgently warranted. We examined the efficacy of the combination of baicalin (BAI) and knockdown of miR-148a gene in human glioblastoma T98G and U87MG cell lines. T98G and U87MG cells were transfected with miR148a siRNA. The influence of miR- 148a siRNA in combination with BAI on T98G and U87MG cell viability, proliferation, apoptosis, and autophagy was evaluated as well. Alterations in the mRNA expression of autophagy-related genes were analyzed using RT-qPCR. The transfection of T98G and U87MG cells with miR148a specific siRNA and exposition on baicalin led to a significant reduction in cell viability and proliferation, the accumulation of sub G1-phase cells and a reduced population of cells in the S and G2/M phases (only in U87MG cell line), increased population of cells in the S phase in T98G cell line and apoptosis or necrosis induction and induction of autophagy for both cell lines. The siRNA-induced miR-148a mRNA knockdown in combination with baicalin may offer a novel therapeutic strategy to more effectively control the growth of human GBM cells. Thus, knockdown of this gene in combination with baicalin inhibits proliferation (cell cycle arrest in the S phase in T98G but not in U87MG cells), induces apoptosis, and regulates autophagy in T98G and U87MG cells. However, further studies are urgently needed to confirm a positive phenomenon for the treatment of GBM.

Chemo-radiation therapy of U87-MG glioblastoma cells using SPIO@AuNP-Cisplatin-Alginate nanocomplex

Megavoltage radiotherapy and cisplatin-based chemotherapy are the primary glioblastoma treatments. Novel nanoparticles have been designed to reduce adverse effects and boost therapeutic effectiveness. In the present study, we synthesized the SPIO@AuNP-Cisplatin-Alginate (SACA) nanocomplex, composed of a SPIO core, a gold shell, and an alginate coating. SACA was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). U87-MG human glioblastoma cells and the HGF cell line (a healthy primary gingival fibroblast) were treated in multiple groups by a combination of SACA, cisplatin, and 6 MV X-ray. The MTT assay was used to assess the cytotoxicity of cisplatin and SACA (at various concentrations and for 4 h). Following the treatments, apoptosis and cell viability were evaluated in each treatment group using flow cytometry and the MTT assay, respectively. The findings demonstrated that the combination of SACA and 6 MV X-rays (at the doses of 2 and 4 Gy) drastically decreased the viability of U87MG cells, whereas the viability of HGF cells remained unchanged. Moreover, U87MG cells treated with SACA in combination with radiation exhibited a significant increase in apoptosis, demonstrating that this nanocomplex effectively boosted the radiosensitivity of cancer cells. Even though additional in vivo studies are needed, these findings suggest that SACA might be used as a radiosensitizer nanoparticle in the therapy of brain tumors.

Resveratrol inhibits glioblastoma cells and chemoresistance progression through blockade P-glycoprotein and targeting AKT/PTEN signaling pathway

Malignant gliomas have been categorized as a debilitating class of brain tumors that are resistant to radiation and chemotherapeutic drugs, and have a poor prognosis. Hyper-activation of PI3K/AKT pathway and overexpression of p-glycoprotein transporter contributes to enhanced glioblastoma survival and chemoresistance. Resveratrol which possibly inhibits PI3K pathway, has been thus investigated for a potential therapeutic role in glioma. In the present study, the effect of resveratrol on human U87MG and doxorubicin-resistant glioblastoma cells (U87MG/DOX) survival evaluated by MTT. The ability of resveratrol to overcome doxorubicin resistance in glioblastoma cells was also explored with Rhodamines 123 uptake and ELISA assays. Resveratrol reduced cell survival in a PTEN and P53-dependent manner which was an effect associated with the inhibition of PI3K signaling pathway and via the activation of P-glycoprotein. Our finding showed that resveratrol, as a glioblastoma cell growth inhibitor and chemosensitizer, could be promising if used in the treatment of brain cancer. Resveratrol inhibits the progression of glioblastoma cells and reverses chemoresistance by upregulating PTEN, and suppressing AKT and P-glycoprotein. Targeting PTEN with resveratrol may offer a novel therapeutic approach for the chemo-sensitization of glioblastoma cells.

Nanoarchitectured conjugates targeting angiogenesis: investigating heparin-taurocholate acid conjugates (LHT7) as an advanced anti-angiogenic therapy for brain tumor treatment

BackgroundGlioblastoma is a highly malignant brain tumor associated with poor prognosis. Conventional therapeutic approaches have limitations due to their toxic effects on normal tissue and the development of tumor cell resistance. This study aimed to explore alternative mechanisms for glioblastoma treatment by targeting angiogenesis.MethodsThe study investigated the anti-angiogenic properties of heparin in glioblastoma treatment. To overcome the limitations of heparin, a heparin-taurocholate conjugate (LHT7) was synthesized by conjugating heparin to taurocholic acid. The study utilized the U87MG human glioblastoma cell line and human umbilical vein endothelial cells (HUVEC) as experimental models. Cell viability assays and sprouting assays were performed to assess the effects of LHT7. Additionally, phosphorylation of angiogenesis-related proteins, such as phospho-ERK and phospho-VEGFR2, was measured. The anti-angiogenic effects of LHT7 were further evaluated using a glioblastoma orthotopic mouse model.ResultsTreatment with LHT7 resulted in a dose-dependent reduction in cell viability in U87MG human glioblastoma cells. The sprouting of HUVEC cells was significantly decreased upon LHT7 treatment. Furthermore, LHT7 treatment led to a decrease in the phosphorylation of angiogenesis-related proteins, including phospho-ERK and phospho-VEGFR2. In the glioblastoma orthotopic mouse model, LHT7 exhibited anti-angiogenic effects, supporting its potential as a therapeutic agent.ConclusionsThe conjugation of heparin and taurocholic acid to create LHT7 offers several advantages over conventional therapeutic approaches for glioblastoma. LHT7 demonstrated anti-angiogenic properties, as evidenced by the reduction in cell viability and inhibition of endothelial cell sprouting. Moreover, LHT7 modulated the phosphorylation of angiogenesis-related proteins. These findings suggest that LHT7 holds promise as a medication for glioblastoma treatment, offering potential implications for improving patient outcomes.Graphical

Added Value of Scintillating Element in Cerenkov-Induced Photodynamic Therapy.

Cerenkov-induced photodynamic therapy (CR-PDT) with the use of Gallium-68 (68Ga) as an unsealed radioactive source has been proposed as an alternative strategy to X-ray-induced photodynamic therapy (X-PDT). This new strategy still aims to produce a photodynamic effect with the use of nanoparticles, namely, AGuIX. Recently, we replaced Gd from the AGuIX@ platform with Terbium (Tb) as a nanoscintillator and added 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenylporphyrin (P1) as a photosensitizer (referred to as AGuIX@Tb-P1). Although Cerenkov luminescence from 68Ga positrons is involved in nanoscintillator and photosensitizer activation, the cytotoxic effect obtained by PDT remains controversial. Herein, we tested whether free 68Ga could substitute X-rays of X-PDT to obtain a cytotoxic phototherapeutic effect. Results were compared with those obtained with AGuIX@Gd-P1 nanoparticles. We showed, by Monte Carlo simulations, the contribution of Tb scintillation in P1 activation by an energy transfer between Tb and P1 after Cerenkov radiation, compared to the Gd-based nanoparticles. We confirmed the involvement of the type II PDT reaction during 68Ga-mediated Cerenkov luminescence, id est, the transfer of photon to AGuIX@Tb-P1 which, in turn, generated P1-mediated singlet oxygen. The effect of 68Ga on cell survival was studied by clonogenic assays using human glioblastoma U-251 MG cells. Exposure of pre-treated cells with AGuIX@Tb-P1 to 68Ga resulted in the decrease in cell clone formation, unlike AGuIX@Gd-P1. We conclude that CR-PDT could be an alternative of X-PDT.

Metabolic Adjustments following Glutaminase Inhibition by CB-839 in Glioblastoma Cell Lines.

Most tumor cells can use glutamine (Gln) for energy generation and biosynthetic purposes. Glutaminases (GAs) convert Gln into glutamate and ammonium. In humans, GAs are encoded by two genes: GLS and GLS2. In glioblastoma, GLS is commonly overexpressed and considered pro-oncogenic. We studied the metabolic effects of inhibiting GLS activity in T98G, LN229, and U87MG human glioblastoma cell lines by using the inhibitor CB-839. We performed metabolomics and isotope tracing experiments using U-13C-labeled Gln, as well as 15N-labeled Gln in the amide group, to determine the metabolic fates of Gln carbon and nitrogen atoms. In the presence of the inhibitor, the results showed an accumulation of Gln and lower levels of tricarboxylic acid cycle intermediates, and aspartate, along with a decreased oxidative labeling and diminished reductive carboxylation-related labeling of these metabolites. Additionally, CB-839 treatment caused decreased levels of metabolites from pyrimidine biosynthesis and an accumulation of intermediate metabolites in the de novo purine nucleotide biosynthesis pathway. The levels of some acetylated and methylated metabolites were significantly increased, including acetyl-carnitine, trimethyl-lysine, and 5-methylcytosine. In conclusion, we analyzed the metabolic landscape caused by the GLS inhibition of CB-839 in human glioma cells, which might lead to the future development of new combination therapies with CB-839.

CBMS-3 SEARCH FOR COMPOUNDS WITH ANTI-TUMOUR EFFECTS AGAINST GLIOBLASTOMA

Abstract Purpose Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and the resistance to temozolomide, the standard treatment for GBM. Therefore, the development of new therapeutic agents for GBM is desired. We searched our compound library for compounds with anti-tumor activity against GBM and identified Curcumin (Cur) derivatives, Compound A and B. The purpose of this study was to investigate the antitumor activity of Compound A and B against GBM. Methods and Results To evaluate the antitumor activity of Compound A and B against GBM, we performed the MTT assay. Human glioblastoma cell lines, U87-MG and U251 cells, were treated with Compound A and B. After 96 hours, cell viability was evaluated using CCK-8, and the IC50 values of Compound A and B were calculated. In U87-MG, the IC50 of Cur was 9.78 μM, whereas the that of Compound A was 2.42 μM and that of Compound B was 1.28 μM. In U251 cells, the IC50 of Cur was 9.50 μM, whereas that of Compound A was 2.27 μM and that of Compound B was 0.64 μM. Next, to examine the effects of Compound A and B on normal cells, we performed the same MTT assay using primary cultured rat astrocytes. At the concentrations at which antitumor effects were observed (Compound A; 3 μM, B; 1.5 μM), there was no reduction in cell viability in primary rat cultured astrocytes. Discussion The present study shows that Compound A and B exhibit antitumor activity against human glioblastoma cells at lower concentrations than Cur without affecting normal cells. We will examine the effects of Compound A and B in a mouse model of brain tumor transplanted with U87-RFP cells in the future, which may lead to the development of noble brain tumor therapeutics.

ANGI-04. DEPLETION OF CHITINASE-3-LIKE PROTEIN 1 (CHI3L1) IN HUMAN U87-MG GLIOMA CELLS AFFECTS TUMOUR GROWTH AND NEOVASCULATURE IN INTRACRANIAL MOUSE MODEL

Abstract Chitinase-3-like protein 1 (CHI3L1) plays multiple roles in inflammation, tissue remodelling, and wound healing. It is a new, potentially druggable target for the treatment of cancer due to its association with both extracellular matrix (ECM) modifications and potential involvement in neoangiogenesis. Expression of CHI3L1 is upregulated in glioblastoma (GBM), the most common and aggressive primary brain tumour. The role of CHI3L1 in GBM progression is not fully understood and calls for further research. We generated a knockout (KO) of CHI3L1 in human glioblastoma U87-MG cells. To evaluate the effects of CHI3L1 KO on tumour growth, wild-type (WT) or CHI3L1 KO cells were intracranially implanted into athymic mice and tumour volume was assessed with magnetic resonance imaging. We have measured the serum level of CHI3L1 in both groups and correlated it with the tumour burden. Immunofluorescent staining against endothelium marker – von Willebrand factor (vWF), and perivascular water channel – aquaporin 4 (AQP4) were employed to evaluate the tumour vasculature in both groups. We found that CHI3L1 KO in glioblastoma cells significantly reduced volume of established tumors. Blood serum level of CHI3L1 positively correlated with tumour burden of experimental animals, which corroborates its role as a biomarker. Immunostaining for vWF revealed that in CHI3L1 KO gliomas there were more non-capillary blood vessels than in controls. We observed a higher level of AQP4 in CHI3L1 KO tumours, which suggests a difference in the structure of perivascular space, and thus vasculature functionality, in CHI3L1 depleted gliomas. In conclusion, we demonstrated the involvement of glioma-derived CHI3L1 in the growth and neovasculature of experimental human gliomas. Our findings suggest that the depletion of CHI3L1 in glioma cells results in a higher number and possibly different structure of non-capillary blood vessels in the tumour. Studies supported by the grant N 2020/39/B/NZ4/02683 from the National Science Center.

Carborane-Containing Folic Acid bis-Amides: Synthesis and In Vitro Evaluation of Novel Promising Agents for Boron Delivery to Tumour Cells.

The design of highly selective low-toxic, low-molecular weight agents for boron delivery to tumour cells is of decisive importance for the development of boron neutron capture therapy (BNCT), a modern efficient combined method for cancer treatment. In this work, we developed a simple method for the preparation of new closo- and nido-carborane-containing folic acid bis-amides containing 18-20 boron atoms per molecule. Folic acid derivatives containing nido-carborane residues were characterised by high water solubility, low cytotoxicity, and demonstrated a good ability to deliver boron to tumour cells in in vitro experiments (up to 7.0 µg B/106 cells in the case of U87 MG human glioblastoma cells). The results obtained demonstrate the high potential of folic acid-nido-carborane conjugates as boron delivery agents to tumour cells for application in BNCT.

Voxtalisib and low intensity pulsed ultrasound combinatorial effect on glioblastoma multiforme cancer stem cells via PI3K/AKT/mTOR.

Glioblastoma (GBM) is the most aggressive brain tumor contributed to tumor growth by cancer stem cells (CSCs). Targeting CSCs is vital to preventing differentiation into cancer cells, their proliferation, and treatment resistance. According to research, PI3K/AKT/mTOR signaling is active in GBM and GBMCSCs. Anticancer medications combined with ultrasound application have been proposed as a strategy to increase the drug intake of cancer cells. This study aims to investigate the effects of inhibition of PI3K/ AKT/ mTOR pathway with dual inhibitor Voxtalisib (Vox; also known as XL765) and low intensity pulsed ultrasound (LIPUS) combinations in GBM and GBMCSCs in the point of cell survival. F-actin was also used to evaluate cell motility. GBMCSCs were isolated from the human glioblastoma U87 MG cell line using the fluorescence-activated cell sorting (FACS) method. Cells were exposed to various concentrations of Vox, LIPUS, and their combinations. Cell count and viability assay was used to determine drug delivery doses. F-actin and mTOR immunofluorescence staining were used to identify cytoskeletal alterations and PI3K/AKT/mTOR signal pathway suppression, respectively. Additionally, the migration capacity of cells was shown with standard wound-healing experiments. High doses of Vox+LIPUS inhibited mTOR and decreased the viability in both cell groups. Inhibiting mTOR activated autophagy, and LIPUS increased autophagy in GBM cells. However, GBMCSCs were resistant to autophagy even at high drug dosages. Both in GBM and GBMCSCs, combinations of Vox and LIPUS were observed to decrease F-actin density and cell motility. The combination of Vox+LIPUS has increased drug effectiveness in targeted GBM and GBMCSCs. Combinatory treatment with PI3K/AKT/mTOR signaling pathway and LIPUS has been thought to help develop more effective therapeutic approaches for GBM.

Spironolactone Induces Apoptotic Cell Death in Human Glioblastoma U87-MG Cancer Cells

Background: Spironolactone is a conventional drug widely in use for the treatment of heart failure and hypertension patients. On the other side recent studies have reported spironolactone can prevent growth and drug resistance in cancer stem cells (CSCs), by inhibiting DNA double-strand break (DSB) repair; suggesting its potential application in cancer therapy. Objectives: Our study aimed at assessing the potential cytotoxicity of spironolactone in human U87-MG glioblastoma cells. Methods: Different concentrations of spironolactone (0 - 50 μM) for 48 and 72 h were used for treatment. Cell viability assay was carried out by the 4, 5-dimethylthiazole-2-yl, 2, 5-diphenyl tetrazolium (MTT) method. Apoptosis was evaluated using annexin V/PI staining and flow cytometry and colorimetric measurement of caspase 8 and 9 activity. Results: Our findings showed a significant dose-dependent cytotoxic effect of spironolactone with maximum effect in 30 μM (P-value < 0.05). Spironolactone can induce approximately 20% apoptotic cell death in U87-MG cancer cells which were mainly related to early apoptotic cells. Indeed, the activity of caspase 8 and 9 was significantly elevated in spironolactone-treated cells compared to mock control. Conclusions: Findings showed the cytotoxic effect of spironolactone in U87-MG glioblastoma cancer cells in a mechanism dependent on apoptosis cell death induction. Our findings suggest the potential application of spironolactone in the treatment of glioblastoma in vitro.

Synthesis, X-ray diffraction and anti-proliferative biological activity of hispolon derivatives and their (η6-p-cymene)(Hispolonato)Ruthenium[II] chloride complexes

Hispolon is a natural product extracted from Phellinus igniarius and Phellinus linteus fungi that has previously shown antitumor activity. In this study we present the synthesis, chemical characterization and in vitro anti-proliferative activity of three [(η6-p-cymene)Ru(L)Cl] neutral complexes, where L = hispolon derivatives. The single crystal X-ray structures of the three Ru complexes all have the expected piano stool geometry with the p-cymene ligand at the apex of the piano stool and occupying three of the sites in a distorted-octahedral arrangement. Completing the octahedral coordination sphere are two β-diketone oxygen atoms of the deprotonated hispolon derivatives which are coordinated in a bidentate fashionand a chlorine atom. The cytotoxicity of the three hispolon derivatives along with their corresponding complexes was studied for A549 lung, HCT116 colon and U87 glioblastoma cell lines. In the glioblastoma cell line, increase of biological activity (lower IC50) is seen for all three hispolons after arene-ruthenium complexation: 5.1 times higher for 2,3,4-trimethoxy-hispolon (Hisp8), and 1.5 times for both 3-methoxy,4-hydroxy-hispolon (Hisp1) and 3,4-dimethoxy-hispolon (Hisp4). This effect was less pronounced in the colon HCT116 cell line with complexation having 1.2–1.4 times higher biological activity. For the lung cell line A549, there was no such effect and complex formation slightly decreased the biological activity. Regardless, in the glioblastoma cell line, the presence of Hisp8, the most liposoluble agent, either as the pure compound or as ligand in the η6-p-cymene-Ru complex, proved effective as an antitumor agent. More specifically, the [(η6-p-cymene)Ru(Hisp8)Cl] complex showed a decrease in IC50 value for all 3 cell lines, when compared to Hisp8. We also performed molecular mechanics, DFT and docking calculations to determine the inhibitory ability of hispolon analogs towards aldehyde dehydrogenase, which targets glioblastoma stem cells. All three [(η6-p-cymene)Ru(Hisp)Cl] coordination complexes of hispolon derivatives are more effective than the related neutral hispolons in the U87 MG human glioblastoma cell line. Our computational studies clearly indicate an important role for aldehyde dehydrogenase Arg139 amino acid. In addition, computational results indicate hispolonato anions are much better inhibitors of aldehyde dehydrogenase and improve the anti-glioblastoma biological activity, consistent with IC50 data.

Correlation of biomechanics and cancer cell phenotype by combined Brillouin and Raman spectroscopy of U87-MG glioblastoma cells.

The elucidation of biomechanics furthers our understanding of brain tumour biology. Brillouin spectroscopy is a new optical method that addresses viscoelastic properties down to subcellular resolution in a contact-free manner. Moreover, it can be combined with Raman spectroscopy to obtain co-localized biochemical information. Here, we applied co-registered Brillouin and Raman spectroscopy to U87-MG human glioblastoma cells in vitro. Using two-dimensional and three-dimensional cultures, we related biomechanical properties to local biochemical composition at the subcellular level, as well as the cell phenotype. Brillouin and Raman mapping of adherent cells showed that the nucleus and nucleoli are stiffer than the perinuclear region and the cytoplasm. The biomechanics of the cell cytoplasm is affected by culturing conditions, i.e. cells grown as spheroids are stiffer than adherent cells. Inside the spheroids, the presence of lipid droplets as assessed by Raman spectroscopy revealed higher Brillouin shifts that are not related to a local increase in stiffness, but are due to a higher refractive index combined with a lower mass density. This highlights the importance of locally defined biochemical reference data for a correct interpretation of the Brillouin shift of cells and tissues in future studies investigating the biomechanics of brain tumour models by Brillouin spectroscopy.

Abstract 3916: Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome

Abstract Coibamide A is a cyclopeptide natural product with potent antiproliferative activity against cultured human cancer cells and subcutaneous U87-MG glioblastoma tumors in mice. We have recently determined that coibamide A binds to the Sec61 translocon channel at a site that is close, but not identical, to the binding site of the peptide/polyketide natural product apratoxin A. As the majority of secreted proteins in mammalian cells are trafficked through the conventional secretory pathway via the Sec61 translocon, this raised the possibility that coibamide A and apratoxin A block import and secretion of different Sec61 client proteins. In the present study, we analyzed and compared the secretome of human U87-MG glioblastoma cells in the presence and absence of both marine natural products. U87-MG glioblastoma cells were treated with coibamide A (5 nM), apratoxin A (5 nM), or vehicle for 24 h. The growth media was then replaced with serum-free media for a further 24 h. Tryptic proteomic samples were generated from cell-free conditioned media and analyzed on a LC-coupled Orbitrap Fusion mass spectrometer. The high-resolution Orbitrap mass analyzer was used to both identify precursor ions for tandem mass (MS2) analysis in the ion trap mass analyzer and to quantitate those ions (MS1). Secreted proteins were identified from peptide–spectrum matches to the human protein database using a decoy database to assign an FDR and quantified from their MS1 ion intensities. Comparison of mass spectrometric data against the human secretome database revealed 175 proteins in the U87-MG cell secretome that were identified with high confidence. Differences between coibamide A and apratoxin A treatment were not significant, however both Sec61 inhibitors induced significant suppression of the U87-MG secretome relative to vehicle-treated cells. Treatment with coibamide A or apratoxin A resulted in the reduction of 75% of quantifiable secretome (131 proteins). Of the remaining 44 proteins, 34 are directed to intracellular organelles or contain a motif associated with retention in the endoplasmic reticulum and 10 were not suppressed with treatment but were hypothesized to be Sec61 clients. Malignant glioblastoma cells secrete proteins into the extracellular space and use complex intercellular signaling mechanisms for a survival advantage. Our results indicate that broad-acting inhibitors of Sec61-dependent co-translational translocation block the progression of a significant percentage of the U87-MG glioblastoma cell secretome including several proteins that have previously been detected as biomarkers in the cerebrospinal fluid, blood, or urine of brain tumor patients. Taken together, our work illustrates the feasibility of inducing a reversible suppression of numerous (undrugged) extracellular signals by targeting Sec61-dependent protein biogenesis in the secretory pathway with drug-like molecules. Citation Format: Daphne R. Mattos, Jeffrey D. Serrill, Philip R. Gafken, Kerry L. McPhail, Walter K. Vogel, Jane E. Ishmael. Cyanobacterial natural product Sec61α inhibitors induce broad suppression of the glioma cell secretome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3916.