T98G Glioblastoma Cells Research Articles

Diosgenin as a Novel Alternative Therapy for Inhibition of Growth, Invasion, and Angiogenesis Abilities of Different Glioblastoma Cell Lines.

Fenugreek (Trigonella foenum-graecum) seeds and roots of wild yam (Dioscorea villosa) possess nutritional and medicinal properties and have been used for centuries in traditional medicine to treat different diseases and inflammatory responses. Diosgenin is a natural steroidal sapogenin extracted from fenugreek and wild yam and it is one of the major bioactive compounds used in the treatment of diabetes, hypercholesterolemia, and inflammation. Recent studies have shown a promising effect of diosgenin as ananti-tumor agent for inhibition of cell proliferation and induction of apoptosis in many cancers such as colon cancer, leukemia, breast cancer, and liver cancer. We examined the effects of different concentrations (5, 10, 15, 20, and 25µM) of diosgenin on proliferation of rat C6 and human T98G glioblastoma cell lines. We noticed that diosgenin had a high inhibitory effect on the growth of both C6 and T98G cell lines. Diosgenin induced the differentiation of glioblastoma cells, as determined by the increasein the expression of the differentiation marker glial fibrillary acidic protein (GFAP); and decreased the dedifferentiation of the cells, as shown by the decreasein the abundance of the dedifferentiation marker proteins Id2, N-Myc, telomerase reverse transcriptase (TERT), and Notch-1. It also induced apoptosis in C6 and T98G cell lines and themolecular mechanisms involved in the induction of apoptosis included increase in pro-apoptotic Bax protein and decrease in anti-apoptotic Bcl-2 protein. Further, the diosgenin-induced suppression of cell migration was correlated with the decrease in expressionof matrix metalloproteinase 2 (MMP2) and MMP9; and the inhibition of angiogenesis, as determined by the tube formation assay, was correlated with a decrease in the protein levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). In conclusion, diosgenin showed anti-tumoreffects in glioblastoma cells by induction of differentiation and apoptosis and inhibition of migration,invasion, and angiogenesis.

Cytotoxic effect of unclassified group K rotaviruses on T98G and U87MG glioblastoma cells in vitro.

e15597 Background: Oncolytic virotherapy is developing intensively in modern oncology. Viruses demonstrate the ability to the direct oncolysis and to the stimulation of antitumor immune activity; this experiment was aimed at solving the question of the prevalence of one of them. Glial tumors are the most common brain tumors; oncolytic viruses show certain prospects in their treatment due to the ability to penetrate the blood-brain barrier. The aim of the study was to determine the possible oncolytic effect of new unclassified group K rotaviruses (RVK) on T98G and U87MG glioblastoma cells in vitro. Methods: T98G and U87MG cell cultures were received from Russian banks of cell lines of human and animal tissues. Standard culturing was performed with attenuated apatogenic RVK strains No. 100 and No. 228 at a concentration of 108, 107, 106 and 105 particles/mL. The cytotoxic effect was determined with MTT and Annexin V assays, cell morphology was evaluated by the light-optical method. Results: Both RVK strains demonstrated a dose-dependent cytotoxic activity; the maximal effect was observed in strain No.100 at a dose of 108 particles/mL on U87MG cells (predominantly apoptosis). Studies of cell morphology showed a pronounced effect of RVK on the cell culture: significant degenerative changes in cells, a tendency to a decrease in cluster size, a change in their shape and granularity. Cluster formation in culturing in the serum-free medium is considered in the literature as a property of cancer stem cells responsible in vivo for tumor recurrence and its chemo- and radio-resistance. T98G cells demonstrated morphological changes: nuclear segmentation, diffused cytoplasm, indistinct cell borders with signs of syncytium formation. Conclusions: The established oncolytic effect of RVK strain No. 100 in vitro on glioma cells, presumably with tumor stem cells, indicates a significant potential for the use of these rotaviruses in treatment of glial tumors.

Epidermal Growth Factor (EGF) Augments the Invasive Potential of Human Glioblastoma Multiforme Cells via the Activation of Collaborative EGFR/ROS-Dependent Signaling.

Abnormal secretion of epidermal growth factor (EGF) by non-neuronal cells (e.g., glioma-associated microglia) establishes a feedback loop between glioblastoma multiforme (GBM) invasion and a functional disruption of brain tissue. Considering the postulated significance of this vicious circle for GBM progression, we scrutinized mechanisms of EGF-dependent pro-invasive signaling in terms of its interrelations with energy metabolism and reactive oxygen species (ROS) production. The effects of EGF on the invasiveness of human glioblastoma T98G cells were estimated using time-lapse video microscopy, immunocytochemistry, cell cycle assay, immunoblot analyses, and Transwell® assay. These techniques were followed by quantification of the effect of EGFR (Epidermal Growth Factor Receptor) and ROS inhibitors on the EGF-induced T98G invasiveness and intracellular ROS, ATP, and lactate levels and mitochondrial metabolism. The EGF remarkably augmented the proliferation and motility of the T98G cells. Responses of these cells were accompanied by cellular rear–front polarization, translocation of vinculin to the leading lamellae, and increased promptness of penetration of micropore barriers. Erlotinib (the EGFR inhibitor) significantly attenuated the EGF-induced T98G invasiveness and metabolic reprogramming of the T98G cells, otherwise illustrated by the increased mitochondrial activity, glycolysis, and ROS production in the EGF-treated cells. In turn, ROS inhibition by N-acetyl-L-cysteine (NAC) had no effect on T98G morphology, but considerably attenuated EGF-induced cell motility. Our data confirmed the EGFR/ROS-dependent pro-neoplastic and pro-invasive activity of EGF in human GBM. These EGF effects may depend on metabolic reprogramming of GBM cells and are executed by alternative ROS-dependent/-independent pathways. The EGF may thus preserve bioenergetic homeostasis of GBM cells in hypoxic regions of brain tissue.

Pt(O,O'-acac)(γ-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca2+]i, in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca2+ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca2+ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca2+]i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca2+]i. These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

Lavender Volatile Oil: A New Solvent for Propolis Extraction, Chemical Composition, Antioxidant Activity and Cytotoxicity on T98G Glioblastoma Cell Line

Glioblastoma is the most aggressive and common brain tumor. The survival of the patient suffering from glioblastoma is not so long. Recently, natural products including propolis have been reported as chemo/radiosensitizers in glioblastoma treatment. Propolis is a resinous complex collected from different parts of medicinal plants. This complex structured bee product can be used after extraction of its biologically active compounds and ethanol is reported to be the best solvent. There is an urgent need to find out new, healthier, and compatible administration tools of propolis intake, especially for cancer patients. The main aim of this study is to find out the solubility of propolis in lavender volatile oil. Total phenolic content and antioxidant activity of new propolis extract was determined. Cytotoxic activity of the obtained extract was also tested on T98G glioblastoma cell lines in vitro. Results showed that the total phenolic content of new propolis extract was determined as 10.16 mg GAE/mL. DPPH radical scavenging activity (SC50) was found as 0.34 ± 0.012 mg/mL. The cytotoxic effects appeared with concentrations of 100 μg/ml and more (p<0.01). It could be concluded that lavender essential oil is as efficient solvent as ethanol and might be a new administration tool of propolis, especially for cancer patients.

Deglucohellebrin: A Potent Agent for Glioblastoma Treatment.

Glioblastoma is the most common primary brain tumor in adults with a dismal prognosis. To date, several anticancer agents have been isolated from plants. Helleborus odorus subsp. Cyclophyllus is an endemic plant of the Balcan flora. Herewith, we investigated for the first time, the anti-glioma effect of deglucohellebrin (DGH) extracted from the roots of Helleborus. We investigated the effect of DGH in U251MG, T98G and U87G glioblastoma cell lines. We selected the T98G cells because of their inherent temozolomide resistance. The IC50 value of reduced viability for DGH was 7x10-5M in U251MG cells, 5x10-5M for the T98G cells and 4x10-5M in U87G cells during 72h treatment. DGH induced G2/M cell cycle arrest, caspace-8 activation and significant mitochondrial membrane depolarization, suggesting the activation of the intrinsic, mitochondrial- dependent apoptotic pathway. DGH and temozolomide induced changes in CDs' expression in U251MG and T98G cells. In zebrafish, DGH did not induce toxicity or behavioral alterations. The present study is the first to determine the anti-glioma activity of DGH. DGH may be a potent agent for glioblastoma treatment and further studies are needed.

Adipose mesenchymal stromal/stem cells expanded by a GMP compatible protocol displayed improved adhesion on cancer cells in flow conditions

BackgroundAdipose tissue derived mesenchymal stromal/stem cells (ASC) can be expanded using supernatant rich in growth factors (SRGF) as Good Manufacturing Practice compatible additive, instead of fetal bovine serum (FBS). After transendothelial migration, ASC can migrate to cancer masses where they can release active substances. Due to their homing and secretion properties ASC can be used as targeted drug delivery vehicles. Nevertheless, the fraction of ASC actually reaching the tumor target is limited. The impact of culture conditions on ASC homing potential on cancer cells is unknown.MethodsIn dynamic in vitro conditions, we perfused FBS or SRGF ASC in flow chambers coated with collagen type I and fibronectin or seeded with endothelial cells or with HT1080, T98G and Huh7 cancer cells. Expression of selected adhesion molecules was evaluated by standard cytofluorimetry. Dynamic intracellular calcium concentration changes were evaluated in microfluidic and static conditions.ResultsWhen compared to FBS ASC, not specific adhesion of SRGF ASC on collagen type I and fibronectin was lower (−33.9%±12.2% and −45.3%±16.9%), while on-target binding on HT1080 and T98G was enhanced (+147%±8% and 120.5%±5.2%). Adhesion of both FBS and SRGF ASC on Huh7 cells was negligible. As confirmed by citofluorimetry and by function-blocking antibody, SRGF mediated decrease of CD49a expression accounted for lower SRGF-ASC avidity for matrix proteins. Upon stimulation with calcium ionophore in static conditions, mobilization of intracellular calcium in SRGF ASC was greater than in FBS ASC. In dynamic conditions, upon adhesion on matrix proteins and HT1080 cells, SRGF ASC showed marked oscillatory calcium concentration changes.ConclusionsSRGF can enhance specific ASC binding capacity on selected cancer cells as HT1080 (fibrosarcoma) and T98G (glioblastoma) cells. Upon cell-cell adhesion, SRGF ASC activate intracellular responses potentially improving cell secretion functions. SRGF ASC could be considered as suitable drug delivery vehicle for cancer therapy.

Cerebrospinal fluid of chronic osteoarthritic patients induced interleukin-6 release in human glial cell-line T98G

BackgroundChronic osteoarthritic pain is not well understood in terms of its pathophysiological mechanism. Activated glial cells are thought to play a role in the maintenance of chronic pain. T98G glioblastoma cell line was previously observed to release higher amounts of interleukin-6 (IL-6) when treated with cerebrospinal fluid (CSF) from patients with another chronic pain condition, post-herpetic neuralgia. In this study, we investigated the ability of CSF from patients diagnosed with knee osteoarthritis suffering from chronic pain, to trigger the release of pro-inflammatory cytokines, IL-6, IL-1beta and tumour necrosis factor alpha (TNF-α) from T98G. Characterization of upstream signalling was also explored.MethodsFifteen osteoarthritis patients undergoing total knee replacement due to chronic knee pain and 15 patients without pain undergoing other surgeries with spinal anaesthesia were prospectively recruited. CSF was collected during anaesthesia. CSF were added to cultured T98G cells in the presence of lipopolysaccharide. IL-6, IL-1β and TNF-α release from T98G cells were measured using enzyme immunoassay. Antibody array and western blotting were performed using CSF-triggered T98G cell lysates to identify possible signalling targets. Age, gender and pain scores were recorded. Mann-Whitney U test was used to compare IL-6 release and protein expression between groups. Association between IL-6 and pain score was analysed using linear regression.ResultsSignificant higher levels of IL-6 were released by T98G cells when induced by osteoarthritis patients’ CSF in the presence of LPS. The IL-6 levels showed positive association with pain score (adjusted B estimate = 10.1 (95% Confidence Interval 4.3–15.9); p = 0.001). Antibody array conducted with 6 pooled T98G cell lysate induced with osteoarthritis pain patient CSF identified greater than 2-fold proteins including STE20-related kinase adaptor protein and spleen tyrosine kinase. Further validation done using western blotting of individual CSF-triggered T98G cell lysate showed non-significant increase.ConclusionHigher IL-6 release from T98G when triggered by OA-CSF, in the presence of LPS, suggest the presence of “unknown molecule” in CSF that may be crucial in the maintenance phase of chronic pain in our osteoarthritis population. Further studies on the signalling pathways involved in pain and relevance of IL-6 release from T98G cells in other pain models are needed.

Hypoxia-induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase-1.

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia-induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl2 ). Both the hypoxic gas mixture (1% O2 ) and chemical hypoxia-induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia-mediated activation of caspase-1. Active caspase-1 induced the classical caspase-dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase-3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase-1/classical caspase-dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase-3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.

Synergism of 4HPR and SAHA increases anti-tumor actions in glioblastoma cells.

Glioblastoma is the most malignant and prevalent brain tumor in adults. It can grow and spread quickly causing harm to the brain health. One of the major challenges in treatment of glioblastoma is drug resistance. Use of synergistic combination of two drugs with different anti-tumor effects is nowadays highly considered in the development of effective therapeutic strategies for many malignancies. In the present study, we showed synergistic therapeutic efficacies of two chemical compounds, N-(4-hydroxyphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA), for significant reduction in cell viability of rat C6 and human T98G glioblastoma cells. These compounds (4HPR and SAHA)were used alone or in synergistic combination for evaluating their various anti-tumor effects. The results showed that combination of 4HPR and SAHA significantly induced morphological and molecular features of astrocytic differentiation in C6 and T98G glioblastoma cells. Combination of 4HPR and SAHA proved to be an important therapeutic strategy for inhibiting cell growth and inducing differentiation in glioblastoma cells. Furthermore, combination of the two drugs showed more efficacies than either dug alone in reducing in vitro cell invasion (transwell assay), cell migration (wound healing assay), and angiogenesis (tube formation assay) due to down regulation of the molecules involved in these processes. The ultimate of goal of using this combination of drugs was induction of apoptosis. The results showed that these drugs in synergistic combination contributed highly to increases in morphological and molecular features of apoptotic death in the tumor cells. The results from molecular studies indicated that cell death occurred via activation of the extrinsic and intrinsic pathways of apoptosis in both C6 and T98G cells. The drugs in combination also contributed to dramatic inhibition of histone deacetylase 1, an important epigenetic player in promoting growth in glioblastoma cells. This novel combination of drugs should also be considered as a promising therapeutic strategy for the treatment of glioblastoma in vivo.

EFFECT OF PULSED MAGNETIC FIELDS ON THE EXPRESSION LEVELS OF TUMOR SUPPRESSOR GENES IN HUMAN T98G GLYOBLASTOMA CELL LINE

Aim: to study the effect of a pulsed magnetic field (PMF) on the expression of key tumor suppressor genes, such as aPc, MLH, and MGMt in human t98G glioblastoma cell line. material and methods . the PMF with the intensity of 15 and 300 mt was used alone and in combination with ionizing radiation at a single dose of 10 Gy. to perform ionizing radiation, theratron Equinox 60 co unit Best theratronics Ltd., Ottawa, canada) was used. the source of the pulsed magnetic field was Neuro-Ms / D therapeutic advanced device of the Neurosoft company. Live and dead cells were determined in NanoEntekJuliFl cell counter (Korea) using a 0.4 % trypan blue solution to stain dead cells. total RNa was extracted according to the protocol of the manufacturer trizol with changes: the aqueous phase was separated with trizol reagent twice. the quantitative measurement of the isolated RNa was carried out on a Qubit 2.0 fluorimeter using a kit of reagents with the Quant-it RNa assayKit RNa intercalating dye (Life technologies, usa). the expression of MLH, aPc, and MGMt genes was evaluated by Rt-PcR using a cFx96 amplifier (BioRad, usa). Data were analyzed using the cycle threshold (ct) method with normalization for tBP gene expression in each sample. Relative expression of the genetic locus (Exp) was calculated by the 2-Δct method. statistical analysis of the results was carried out using the statictica v10 software package. Results . One day after exposure to PMF, significant differences in the MGMt expression level compared to the control were found (p<0.05). a significant decrease in the transcriptional activity of the MGMt gene in glioblastoma cells was observed with PMF intensity of 15 mt, and correlated with the cell mortality rate. No changes in the mortality rate were observed after radiation exposure combined with 15 mt PMF. However, the mortality rate decreased from 18.7 % to 15 % after radiation exposure combined with 300 mt PMF. Conclusion . the effect of reduction in the transcriptional activity of MGMt in t98G glioblastoma cells and the effect of PMF as a monofactor on their viability characterize the magnetic susceptibility of tumor cell mechanisms. Given the multidirectional nature of the combined interaction of ionizing radiation and PMF, it is necessary to emphasize the importance of choosing and justifying the role of biotropic parameters of PMF in order to exclude a negative effect on the treatment.

3-Bromofascaplysin is a prospective chemical compound for developing new chemotherapy agents in glioblastoma treatment.

The study used rat glioma C6 cell line (ATCC®; cat no CCL-107), U-87MG cell line (ATCC; cat no. HTB-14™) and human glioblastoma T98-G cells (ATCC® CRL-1690™). Cell culture method, experimental pharmacological trials and γ-radiation in vitro, as well as flow cytofluorometry were used in the study. Cytotoxic effect of the tested compounds is stronger than the effect of unsubstituted fascaplysin, and appears to be dose-dependent and time-dependent. 3-bromofascaplysin is more efficient for cancer cells elimination, and by the end of the experiment the amount of living cancer cells in G0 phase remained at its lowest. Cytotoxic effect of 3-bromofascaplysin on glioblastoma T98-G cells is inferior to that of TMZ, and in case of preliminary radiation treatment of cancer cells with 48Gy the effect of the compound matches the TMZ treatment results. 3-Bromofascaplysin is a prospective chemical compound for development of new anti-cancer chemotherapeutic agents.

Targeting Cancer Cell Metabolism with Metformin, Dichloroacetate and Memantine in Glioblastoma (GBM).

To investigate the effects of metformin, dichloroacetate (DCA), and memantine on T98G and U87-MG human glioblastoma (GBM) cells to target tumor cell metabolism in a multi-directional manner. IC50 levels for metformin, DCA, metformin+DCA and memantine were determined by MTT assay in T98G and U87-MG cells in vitro. Casp3, Bcl-2, Bax, c-Myc and GSK-3B protein expressions were investigated post treatments. Fifteen GBM+ tumor tissues were assessed for Casp-3, Bcl-2, Bad, Bax for apoptotic protein expression patterns. Cancer cell metabolism targeting drugs metformin, DCA, metformin+DCA and memantine induced cytotoxicity in a dose-dependent manner in T98G and U87-MG cells. IC50 for memantine is found as 0.5 mM (p < 0.01) which is nearly 10 times lower concentration than that of metformin. Fifteen GBM+ tumor tissues had differential apoptotic protein expressions. Memantine exerted anti-cancer mechanism of action in T98G and U87-MG cells, however, such a mechanism requires deeper investigation for GBM treatment.

In vitro Comparison of Anticancer and Immunomodulatory Activities of Resveratrol and its Oligomers

Stilbenoids are a group of naturally occurring phenolic compounds produced de novo in many plant species to protect against fungal infection and toxins. Resveratrol (3,4’,5-trihydroxystilbene), a stilbenoid isolated from grapes and many species of plants, is widely known for its extensive bioactivities, such as antioxidation, anti-inflammatory and anticancer effects. Recently, many novel resveratrol oligomers have been isolated from various plants. While their structures are diverse, these oligomers are characterized by the polymerization of two or more resveratrol units. Little is known regarding the bioactivities of these novel resveratrol oligomers. We hypothesized that these oligomers might have similar or superior bioactivities to resveratrol. In this study, we designed in vitro models to compare resveratrol side by side with natural dimers (ε-viniferin and gnetin H) and trimers (suffruticosol B and C) from plants for their bioactivities. Our data showed that these oligomers, similar to resveratrol, had many activities such as downregulation of cancer cell proliferation, upregulation of normal keratinocyte growth, and the modulation of the expression of pro- and anti-inflammatory cytokines in immune cells. Furthermore, these oligomers appeared to have higher cytotoxicity than resveratrol in normal human blood cells; the order was the trimers (suffruticosol B and C) &amp;gt; dimers (ε-viniferin and gnetin H) &amp;gt; monomer (resveratrol). However, only suffruticosol C, gnetin H and resveratrol showed anticancer activity against human T98G glioblastoma cells at lower concentrations. No correlation between anticancer or immunomodulating effects and the degree of polymerization was observed. Our results provide some preliminary evidences that resveratrol oligomers could be potential preventive or therapeutic agents for cancers and other immune-related diseases. As far as any superiority of efficacy of these stilbenoids is concerned, more extensive studies are definitely needed.

Modulation of glioma-inflammation crosstalk profiles in human glioblastoma cells by indirubin-3’-(2,3 dihydroxypropyl)-oximether (E804) and 7-bromoindirubin-3′-oxime (7BIO)

Indirubins E804 (indirubin-3’-(2,3 dihydroxypropyl)-oximether) and 7BIO (7-Bromoindirubin-3′-oxime) are synthetic derivatives of natural indirubin, the active compound in Danggui Longhui Wan, a traditional Chinese remedy for cancer and inflammation. Herein, we explore E804 and 7BIO for their potential to modulate key pro-inflammatory genes and cytokines in LN-18 and T98G glioblastoma cells. High grade gliomas typically secrete large amounts of inflammatory cytokines and growth factors that promote tumor growth in an autocrine fashion. Inflammation is emerging as a key concern in the success of new treatment modalities for glioblastomas. Studies indicate that select indirubin derivatives bind and activate signaling of the AHR pathway, as well as inhibit cyclin-dependent kinases and STAT3 signaling. AHR signaling is involved in hematopoiesis, immune function, cell cycling, and inflammation, and thus may be a possible target for glioma treatment. To determine the significance of the AHR pathway in LN-18 and T98G glioma inflammatory profiles, and on the effects of E804 and 7BIO on these profiles, we used 6,2′,4′-trimethoxyflavone (TMF), a putative selective AHR antagonist. It was confirmed that E804 and 7BIO activates the AHR leading to cyp1b1 expression, and that TMF antagonizes expression. We then employed a commercial cancer inflammation and immunity crosstalk qRT-PCR array to screen for anti-inflammatory related properties. TMF alone inhibited expression of ifng, ptsg2, il12b, tnfa, il10, il13, the balance between pd1 and pdl1, and even expression of mhc1a/b. E804 was very potent in suppressing many pro-inflammatory genes, including il1a, il1b, il12a, ptgs2, tlr4, and others. E804 also affected expression of il6, vegfa, and stat3. Conversely, 7BIO induced cox2, but suppressed a different selection of pro-inflammatory genes including nos2, tnfa, and igf1. Secretion of IL-6 protein, an iconic inflammatory cytokine, was decreased by E804. VEGF (vascular endothelial growth factor) protein secretion was upregulated by 7BIO, yet downregulated by E804 and E804 plus TMF. Thus, E804 is both an AHR ligand and regulator of important pro-inflammatory cytokines such as IL-6 and oncogene STAT3, among others. Our results point to the use of E804 and TMF in combination as a promising new treatment for glioblastoma.

P11.14 The addition of hyperbaric oxygen to the treatment of T98G glioblastoma cell cultures with selected CK2 inhibitor TDB enhances its anti-tumour effect

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive glial neoplasm characterized by infiltrative cell growth in adults. The current standard of care for high-grade glioma patients includes surgery, radiotherapy and chemotherapy using temozolomide and other alkylating agents. Despite this, median survival of the patients with GBM is less than two years. One of the main factors related to the progression of neoplastic cells is hyperactivity of protein kinases, including CK2 kinase and a high level of tumor hypoxia, both of which also contribute to tumour resistance to treatment. Our previous study indicated that some CK2 inhibitors exhibit anti-proliferative effect on glioma cells. The aim of this study was to examine whether HBO may enhance the anti-tumour efficacy of 1-(β-D-2’-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) - CK2 inhibitor, under combination HBO/TDB therapy. MATERIAL AND METHODS Studies were performed on human glioblastoma T98G cell line (ATCC). Cells were cultured in MEM medium supplemented with 1-(β-D-2’-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole in concentrations of 0.1 μM; 1 μM, 25 μM, 50 μM, 75μM, 100 μM. Cultures were sustained in various oxygen conditions: normoxia for 24 and 48 hours or1 hour of hyperbaric oxygen (HBO, 97,5% O2/2,5% CO2, pressure of 2 ATA) followed by 23 hours of normoxia (21% O2/5% CO2/78% N2). Control groups included the cultures sustained in normoxia conditions. The viability of glioma cell lines was established at 24 and 48 hours post TDB treatment by Cell Titer 96®A Queous One Solution Cell Proliferation Assay. RESULTS The study evidenced that TDB inhibited the viability of neoplastic cells at a concentration of 75 μM after 24 and 48 hours. Administration of hyperbaric oxygen alone also caused a reduction in viability of glioblastoma cells. The cytotoxic effect of the tested CK2 inhibitor was significantly enhanced when tested compound was combined with HBO. What’s more, under the influence of hyperbaric oxygen, TDB significantly reduced the viability of tumor cells already at a concentration of 25 μM after 24 hours. CONCLUSIONS Hyperbaric oxygen administration significantly reduces the viability of T98G line cells and increases their sensitivity to the tested CK2 kinase inhibitor. The beneficial cytotoxic effect of TDB/HBO can be obtained at lower concentrations of the tested compound and after shorter exposure time as compared to its administration without HBO. Acknowledgement: The research was supported by the Foundation for the Development of Diagnostic and Therapy, Warsaw

Temozolomide has anti-tumor effects through the phosphorylation of cPLA2 on glioblastoma cells

Temozolomide is an alkylating agent used as the first line of treatment for glioblastoma. However, chemoresistance to temozolomide is common in glioma patients. In addition, there are likely many unknown mechanisms for the anti-tumor effects of temozolomide. It is known that an alkylating agent, sulfur mustard, activates cytosolic phospholipase A2 (cPLA₂) releasing arachidonic acid to suppress tumors. The present study was performed to elucidate the involvement of cPLA2 in the anti-tumor mechanisms of temozolomide.In three glioblastoma cell lines (GL261, U251MG and T98G), we performed several evaluations including cell viability, cell migration and apoptosis, to study temozolomide-induced anti-tumor effects. Further, we evaluated tumor size in the murine orthotropic glioblastoma model after oral administration of temozolomide. Finally, we investigated the phosphorylation of cPLA2 in GL261 cells treated with temozolomide, and clarified whether phosphorylation of cPLA2 affects cell growth.Temozolomide suppressed cell growth and cell migration in glioblastoma cells in vitro and showed anti-tumor effect in the murine orthotopic glioblastoma model in vivo. Furthermore, temozolomide increased phosphorylation of cPLA2, which was associated with suppression of cell growth. However, in MGMT high-expressing glioblastoma T98G cells, temozolomide could not suppress cell growth or cause phosphorylation of cPLA2.These findings indicate that temozolomide suppressed cell growth partly by phosphorylation of cPLA2 in glioblastoma cells. In addition, because temozolomide did not cause phosphorylation of cPLA2 in MGMT high-expressing glioblastoma T98G cells, phosphorylation of cPLA2 may be caused by DNA alkylation of temozolomide.

Exosomal L1CAM Stimulates Glioblastoma Cell Motility, Proliferation, and Invasiveness.

Immunoglobulin superfamily protein L1CAM (L1, CD171) normally facilitates neuronal migration, differentiation, and axon guidance during development. Many types of cancers, including glioblastoma (GBM), also abnormally express L1, and this has been associated with poor prognosis due to increased cell proliferation, invasiveness, or metastasis. We showed previously that the soluble L1 ectodomain, which is proteolyzed from the transmembrane form, can stimulate proliferation and motility of GBM cells in vitro by acting through integrins and fibroblast growth factor receptors (FGFRs). Minute L1-decorated exosomal vesicles also are released by GBM cells and potentially could stimulate cell motility, proliferation, and invasiveness, but this needed to be demonstrated. In the present study, we aimed to determine if minute L1-decorated extracellular vesicles (exosomes) were capable of stimulating GBM cell motility, proliferation, and invasiveness. L1-decorated exosomes were isolated from the conditioned media of the human T98G GBM cell line and were evaluated for their effects on the behavior of glioma cell lines and primary tumor cells. L1-decorated exosomes significantly increased cell velocity in the three human glioma cells tested (T98G/shL1, U-118 MG, and primary GBM cells) in a highly quantitative SuperScratch assay compared to L1-reduced exosomes from L1-attenuated T98G/shL1 cells. They also caused a marked increase in cell proliferation as determined by DNA cell cycle analysis and cell counting. In addition, L1-decorated exosomes facilitated initial GBM cell invasion when mixed with non-invasive T98G/shL1 cells in our chick embryo brain tumor model, whereas mixing with L1-reduced exosomes did not. Chemical inhibitors against focal adhesion kinase (FAK) and fibroblast growth factor receptor (FGFR) decreased L1-mediated motility and proliferation to varying degrees. These novel data show that L1-decoratred exosomes stimulate motility, proliferation and invasion to influence GBM cell behavior, which adds to the complexity of how L1 stimulates cancer cells through not only soluble ectodomain but also through exosomes.

TNFR, TRAF2, NF-κB mRNA Levels of Glioblastoma Multiforme Cells Treated by Conditioned Medium of Umbilical Cord-derived Mesenchymal Stem Cells

BACKGROUND: Glioblastoma multiforme (GBM) is a human malignant brain tumor which is arise from glial cells. Our previous study proved that GBM cells proliferation increased after treating by conditioned medium of umbilical cord-derived mesenchymal stem cells (CM-UCSCs). Cells proliferation is probably mediated by tumor necrosis factor (TNF)-α which could bind to membrane receptor and induce signaling pathway. Therefore, this research was intended to analyze the mRNA expression of TNF-α signaling pathway molecules on CM-treated GBM cells by measuring TNF receptor 1 and 2 (TNFR1 and TNFR2), TNFR associated factor 2 (TRAF2), nuclear factor kappa B (NF-κB) mRNA level, and TNFR2 protein level.METHODS: UCSCs and human glioblastoma T98G cells were cultured and harvested after 80% confluence. CM was prepared by growing UCSCs in serum alpha Minimum Essential Media (α-MEM) for 24 hours. Fifty percent concentration of CM-UCSCs was used to treat T98G cells for 24 hours. TNF-α level in CM-UCSC was detected using enzyme linked-immunosorbent assay (ELISA), while the expression of TNFR1, TNFR2, TRAF2 and NF-κB were detected using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR), and TNFR2 protein level was detected using sandwich ELISA.RESULTS: TNF-α level was detected in CM-UCSCs 4.4 pg/mL. Moreover, the expression of TNFR1, TNFR2, TRAF2 and NF-κB were significantly 1.4-fold, 4.9-fold, 5.6-fold, 1.8-fold respectively higher in T98G treated cells than control. TNFR2 protein level in T98G treated cells was 11.57 pg/mg protein higher than control.CONCLUSION: The expression of molecules involved in TNF-α signaling pathway were up regulated in T98G cells treated by CM-UCSCs.KEYWORDS: CM-UCSCs, TNFR1, TNFR2, TRAF2, NF-κB, T98G cells

Human bone marrow-derived mesenchymal stem cell-secreted exosomes overexpressing microRNA-34a ameliorate glioblastoma development via down-regulating MYCN.

Exosomes play important roles in intercellular communication through signaling pathways affecting tumor microenvironment modulation and tumor proliferation, including those in glioblastoma (GBM). As yet, however, limited studies have been conducted on the inhibitory effect of human bone marrow-derived mesenchymal stem cell (hBMSC)-derived exosomes on GBM development. Therefore, we set out to assess the role of hBMSC secreted exosomes, in particular those carrying microRNA-34a (miR-34a), in the development of GBM. Microarray-based expression analysis was employed to identify differentially expressed genes and to predict miRNAs regulating MYCN expression. Next, hBMSCs were transfected with a miR-34a mimic or inhibitor after which exosomes were isolated. Proliferation, apoptosis, migration, invasion and temozolomide (TMZ) chemosensitivity of exosome-exposed GBM cells (T-98G, LN229 and A-172) were measured in vitro. The mechanism underlying MYCN regulation was investigated using lentiviral transfections. The in vivo inhibitory effect of exosomal miR-34a was measured in nude mice xenografted with GBM cells through subcutaneous injection of hBMSCs with an upregulated miR34a content. We found that poorly-expressed miR-34a specifically targeted and negatively regulated the expression of MYCN in GBM cells. In addition we found that miR-34a was delivered to T-98G, LN229 and A-172 GBM cells via hBMSC-derived exosomes. Exogenous overexpression of miR-34a in hBMSC-derived exosomes resulted in inhibition of GBM cell proliferation, invasion, migration and tumorigenesis in vitro and in vivo, while promoting the chemosensitivity of GBM cells to TMZ by silencing MYCN. From our data we conclude that hBMSC-derived exosomes overexpressing miR-34a may be instrumental for the therapeutic targeting and clinical management of GBM.