Proliferation Of T98G Cells Research Articles

Chinese herb related molecules Catechins, Caudatin and Cucurbitacin-I inhibit the proliferation of glioblastoma by activating KDELR2-mediated endoplasmic reticulum stress

Brain gliomas are difficult in the field of tumor therapy because of their high recurrence rate, high mortality rate, and low selectivity of therapeutic agents. The efficacy of Traditional Chinese Medicine (TCM) in the treatment for tumours has been widely recognized. Here, three Chinese herb related molecules, namely Catechins, Caudatin and Cucurbitacin-I, were screened by bioinformatic means, and were found to inhibit the proliferation of glioblastoma T98G cells using Colony-forming and CCK-8 assays. Notably, the simultaneous use of all three molecules could more significantly inhibit the proliferation of glioma cells. Consistent with this, temozolomide, each in the combination with three molecules, could synergistically inhibit the proliferation of T98G cells. Results of qPCR assay was also showed that this inhibition was through the activation of the KDELR2-mediated endoplasmic reticulum stress (ER) pathway. Molecular docking experiments further revealed that Catechins, Caudatin and Cucurbitacin-I could activate ER stress might by targeting KDELR2. Taken together, these results suggest that these herbal molecules have the potential to inhibit the growth of glioma cells and could provide a reference for clinical therapeutic drug selection.

Theranostic Potential of Adaptive Cold Atmospheric Plasma with Temozolomide to Checkmate Glioblastoma: An In Vitro Study

Simple SummaryGlioblastoma (GBM) is an aggressive form of brain cancer. Here, we present a combination therapy of cold atmospheric plasma (CAP) and temozolomide (TMZ) to treat GBM in vitro. We analyze the effects of the co-treatment in two GBM (TMZ-resistant and -sensitive) cell lines. The aim of this study is mainly to sensitize these cells using CAP so that they respond well to TMZ. We further found that the removal of cell culture media after CAP treatment does not affect the sensitivity of CAP to cancer cells but enhances the effects of TMZ. However, it was observed in our study that keeping the CAP-treated media for a shorter time did not significantly inhibit T98G cells. Interestingly, keeping the same plasma-treated media for a longer duration resulted in a decrease in cell viability. On the contrary, TMZ-sensitive cell A172 responded well to the co-treatment. This could be a potential reason for the sensitization of the combination therapy.Cold atmospheric plasma (CAP) has been used for the treatment of various cancers. The anti-cancer properties of CAP are mainly due to the reactive species generated from it. Here, we analyze the efficacy of CAP in combination with temozolomide (TMZ) in two different human glioblastoma cell lines, T98G and A172, in vitro using various conditions. We also establish an optimized dose of the co-treatment to study potential sensitization in TMZ-resistant cells. The removal of cell culture media after CAP treatment did not affect the sensitivity of CAP to cancer cells. However, keeping the CAP-treated media for a shorter time helped in the slight proliferation of T98G cells, while keeping the same media for longer durations resulted in a decrease in its survivability. This could be a potential reason for the sensitization of the cells in combination treatment. Co-treatment effectively increased the lactate dehydrogenase (LDH) activity, indicating cytotoxicity. Furthermore, apoptosis and caspase-3 activity also significantly increased in both cell lines, implying the anticancer nature of the combination. The microscopic analysis of the cells post-treatment indicated nuclear fragmentation, and caspase activity demonstrated apoptosis. Therefore, a combination treatment of CAP and TMZ may be a potent therapeutic modality to treat glioblastoma. This could also indicate that a pre-treatment with CAP causes the cells to be more sensitive to chemotherapy treatment.

Brain endothelial cells-derived extracellular vesicles overexpressing ECRG4 inhibit glioma proliferation through suppressing inflammation and angiogenesis.

Esophageal cancer related gene-4 (ECRG4) has been shown to be a candidate tumor suppressor in many tumors, but its role in glioma remains poorly understood. This study aimed to explore whether extracellular vesicles (EVs) derived from brain endothelial cells which overexpressed ECRG4 have anti-tumor effect on gliomas in vivo and in vitro, as well as the possible mechanism. A constructed lentivirus expressing the ECRG4 gene was transfected into the hCMEC/D3 cell line. The EVs were isolated from the cells and characterized by Western blot with exosome markers of CD9, CD63, CD81, Alix. RT-PCR and Western blot were performed to verify ECRG4 expression. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clone formation assays were applied to detect the proliferation of glioma cells incubated with EVs expressing the ECRG4 (ECRG4-exo). The level of inflammatory cytokines and angiogenesis related factors, including nuclear factor kappa-B (NF-κB), interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) levels were detected by ELISA. The T98G cell xenograft mouse model was established and treated with ECRG4-EV. The tumor volume and weight were recorded. p38-MAPK, p-p38-MAPK proteins were determined by Western blot in tumor tissues. As a result, EVs can be internalized into U87MG and T98G cells. ECRG4-EV inhibited U87MG and T98G cell proliferation. ECRG4-EV also inhibited the expression of factors involved in inflammation and angiogenesis. In addition, ECRG4-EVs suppressed tumor growth and decreased the production of inflammatory cytokines through inactivation of p38-MAPK signal pathway. In conclusion, ECRG4-EVsuppresses glioma proliferation through modulating the inflammation and angiogenesis.

GOLPH3 Regulates EGFR in T98G Glioblastoma Cells by Modulating Its Glycosylation and Ubiquitylation.

Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.

Abstract 6128: Evaluation of therapeutic efficacy of combination of 4HPR and SAHA for inhibition of growth of human glioblastoma T98G xenografts in zebrafish

Abstract Glioblastoma is the most aggressive and deadly primary brain cancer in humans. Despite the current treatments using surgery, radiotherapy, and chemotherapy, glioblastoma reappears due to drug resistance. New approaches for treatment of glioblastoma suggest that combination of two drugs may overcome the drug resistance. In this study, we have evaluated the efficacy of the combination of N-(4-hydroxphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA) for inhibition of human glioblastoma T98G cells in a zebrafish xenograft model in our laboratory. Both 4HPR and SAHA have been approved by the United States Food and Drug Administration for treatment of cancers in the clinics. The synthetic retinoid 4HPR works via retinoic acid receptor signaling pathways to inhibit cancer cell proliferation and induce apoptosis, while SAHA is a histone deacetylase (HDAC) inhibitor that targets class I, II, and IV HDACs for controlling growth of cancer cells. Although rodent models have provided valuable insights into the biology and pathology of glioblastoma, these models are frankly unsuitable and increasingly unaffordable for high-throughput screening and profiling of novel therapeutic agents for rapid translation to the clinics. The optical transparency of zebrafish embryos and larvae provides the unique opportunity to track tumors in a way that is impossible in rodent models. Small size of zebrafish embryos allows for study design in small plates, making them a more useful laboratory system than other cancer models. Zebrafish lack a fully functioning adaptive immune response until 4 days post-fertilization, allowing rejection-free xenografts of human tumors during this period. Recently, zebrafish xenograft models have emerged as a new powerful tool for studying different types of human tumors including glioblastoma and for evaluating therapeutic efficacy of different drugs alone and in combination. Organ systems and cell types are common in human and zebrafish. Tumors that occur in the same organs in human and zebrafish look and behave alike, often share common genetic makeups, and employ basic mechanisms for tumor formation, expansion, and spread. Zebrafish xenograft model of human glioblastoma offers us a major opportunity for discovering key pathways of this tumor growth and for evaluating efficacy of a novel combination therapy. We loaded exponentially grown T98G cells labeled with CellTrace™ Red into the glass needle (0.75 mm internal diameter without filament, World Precision Instruments, FL) at a density of about 1 × 106 cells/200 µl. Using a micro-injector, each anesthetized zebrafish larva was microinjected with about 100 T98G cells into the duct of Cuvier at 1.5 - 3.0 psi. After injection, larvae were transferred to fresh egg water for recovery during 1 h at 28 ± 0.5°C and then incubated at 33°C for the rest of the experiment. After 2 days of tumor growth, we treated zebrafish larvae with 4 µM 4HPR and 8 µM SAHA alone and in combination and incubated for 3 days. Results showed that 4HPR and SAHA alone inhibited growth of T98G xenografts in zebrafish, but the combination of the two drugs most significantly reduced T98G cell proliferation inside of the zebrafish. Our work suggested that the zebrafish xenograft model could be useful to identify the therapeutic efficacy of combination of drugs in the treatment of glioblastoma. Citation Format: Firas Khathayer, Katie L. Kathrein, Mitzi Nagarkatti, Swapan K. Ray. Evaluation of therapeutic efficacy of combination of 4HPR and SAHA for inhibition of growth of human glioblastoma T98G xenografts in zebrafish [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6128.

The expression and clinical significance of forkhead box protein K1 in glioma tissues

Objective Explore of the expression and significance of forkhead box protein K1 (FOXK1) in glioma. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting analysis of mRNA and protein expression levels of FOXK1 in glioma tissues; Kaplan-Meier analysis of the relationship between FOXK1 and prognosis parameters; RNA interference on the expression of FOXK1 in glioblastoma cell. Transwell migration assay for the migration of LN18 cells. Cell counting kit-8 (CCK-8) counting for T98G cell proliferation. Results RT-qPCR and Western blotting showed that FOXK1 mRNA and protein is highly expressed in glioma tissues. The expression of FOXK1 was not significantly correlated with gender (P>0.05) and age of glioma patients (P>0.05), while FOXK1 expression was significantly with WHO classification (P<0.01). Kaplan-Meier analysis found that patients with high expression of FOXK1 had a poor prognosis (P<0.05). Transwell migration assay results indicate that FOXO1 promotes cancer cell metastasis. The CCK-8 cell count results indicate that FOXK1 promotes glioma cell proliferation. Conclusion FOXK1 is highly expressed in glioma patients and is associated with glioma development, progression, and metastasis. Key words: Forkhead box protein K1; Glioblastoma; Metastasis

Novel small molecule protein kinase CK2 inhibitors exert potent antitumor effects on T98G and SEGA cells in vitro.

Tumours of astroglial origin, both malignant glioblastoma (GBM) and benign subependymal giant cell astrocytoma (SEGA), pose a serious medical problem. Casein kinase 2 (CK2), a member of the serine/threonine kinase family, has antiapoptotic properties and plays a vital role in glial tumour cell survival. It contributes to invasive cell growth and is often upregulated in malignant neoplastic cells; however, its role in benign tumours of astrocytic origin is less understood. In the present study we investigated the effects of small molecule CK2 inhibitors on proliferation and viability of glioma cells in vitro. The experiments were conducted on commercial T98G malignant glioma cell line and the SEGA cell line, derived from a paediatric case of tuberous sclerosis complex (TSC). Cell cultures were incubated with selected CK2 inhibitors: 4,5,6,7-tetrabromo-1H-benzimidazole (TBI), 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) at 0.1, 1, 10, 25, 50, 75 and 100 µM concentrations for 24 and 48 hours. Cell proliferation was assessed using a cell counter and cell viability was evaluated by MTT assay. TBB at 75 µM and 100 µM, and TBI starting from 25 µM, both reduced T98G cell proliferation after 24 hours, while DMAT was ineffective. All tested small molecule CK2 inhibitors appear to reduce T98G cell growth and viability after 48 hours, although TBI appeared to be the most effective and reduced cell growth in the 50-100 µM dose range. TBI also showed potential efficacy in reducing the number and viability of SEGA cells after 48 hours. Proliferation and viability of SEGA cells have proven resistant to TBB treatment. DMAT only reduced the viability of SEGA cells at 24 (at 100 µM) and 48 hours (10-100 µM). Importantly, normal human astrocyte cells were found to be moderately resistant to TBB, while their viability was mildly reduced at higher doses of DMAT and TBI. In conclusion, CK2 appears to play a role not only in malignant glioma cells but it can also sustain the viability and proliferation of benign astrocytoma cells. The obtained antitumor effects of CK2 inhibitors significantly exceeded their mild or no effect on normal astrocytes in control, which supports the therapeutic potential of these compounds against gliomas.

Adaptogens in chemobrain (Part II): Effect of plant extracts on chemotherapy-induced cytotoxicity in neuroglia cells

BackgroundCancer chemotherapy-induced cognitive impairments are apparently associated with harmful effects on physiological functions of brain cells. Adaptogens, are known to exhibit neuroprotective effects and to increase cognitive functions in clinical studies. In our previous study (Seo et al., 2018), we demonstrated that selected adaptogenic extracts significantly attenuate cytostatic-induced regulation of more than 100 genes involved in the activation of neuronal death and inhibiting neurogenesis. Neuroprotective and cytoprotective activities of adaptogens rise the question about their possible impact on cytostatic effects of a chemotherapeutic combination of 5-fluorouracil, epirubicin and cyclophosphamide (FEC). AimThe aim of this study was to assess the effects of selected adaptogenic herbal extracts, namely of andrographolide (AND), Herba Andrographidis (AP), Radix Eleutherococci (ES) genuine extracts, their fixed combination (AE), and the combination of three adaptogenic herbs, Rhodiola Radix, Shisandra Fructus and Eleutherococci Radix (RSE) on the cytotoxicity of a fixed combination of 5-fluorouracil, epirubicin and cyclophosphamide (FEC) on neuroglia cells. MethodsCytotoxicity of FEC, adaptogenic extracts and their combination with FEC was tested on isolated T98G cells in a wide range of concentrations of all tested compounds. ResultsFEC reproducibly inhibited the proliferation of T98G cells by 50% at concentrations of 5 × 10−1 µg/ml epirubicin, 500 × 10−1 µg/ml 5-fluorouracil and 20 × 10−1 µg/ml 4-hydroperoxycyclophosphamide after 24 h incubation of cells. These concentrations were subsequently used for experiments with adaptogenic extracts. The cytotoxic activity of FEC was not significantly changed in the presence of AND, ES and AE. Furthermore, it was potentiated by AP extract and RSE in concentrations of 0.06–6 µg/ml and 17.6–26.4 µg/ml. ConclusionThe neuroprotective effect of adaptogens did not attenuate the cytotoxic activity of FEC. Application of cytostatic drugs in combination with adaptogenic plant extracts likely have no impact in cytotoxic effect of FEC. Furthermore, AP and RSE potentiated the cytotoxic effects of FEC.

In vitro Influence of Different Fatty Acids on the Pharmacological Effect of Temozolomide

Introduction: Glioblastoma multiforme is the most malignant cancer in the human body with limited treatment options, in which Temozolomide (TMZ) is the conventional chemotherapeutic agent (CA) often prescribed for this condition. Fatty acids (FAs) per se, including polyunsaturated fatty acids (PUFAs), have been prescribed as adjuvants molecules for general cancer treatment. However, little is known about potential interactions between these lipids and the CAs. TMZ was combined with different FAs to access the nature of the interactions between these compounds. Methods: Two in vitro barrier models of the gut-brain axis (GBA) and the blood-brain barrier (BBB) were used. In each of them, 2-hydroxyoleic acid (2-OHOA); gamma-linolenic acid (GLA); and fish oil (FO) which contains both docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were used as treatments. Results: In both GBA and BBB models, the combination of TMZ with each FA elicited antagonistic effects, interfering with the transepithelial electrical resistance (TEER) and producing less or no response in the inhibition of T98G cell proliferation. This was associated with alterations in cell morphology, β-catenin and E-cadherin protein expression, and induction of lipid droplets. Conclusion: Using our in vitro barrier models, we concluded that the FA might negatively interfere with TMZ`s cytotoxic effect. On the other hand, TMZ also may play a negative role in the antitumor action of specific FA like GLA or 2-OHOA, as observed in the GBA model. These results call into question the clinical utilization of these FA as nutritional adjuvants for patients under the use of TMZ.

FOXO1 is crucial in glioblastoma cell tumorigenesis and regulates the expression of SIRT1 to suppress senescence in the brain.

In the present study, the role of Forkhead Box O1 (FOXO1) in glioblastoma (GBM) cell tumorigenesis was examined and the underlying mechanisms were investigated. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to analyze the expression of FOXO1 in GBM cell lines (LN18 and T98G) and tissues. Compared with the control groups, FOXO1 was significantly downregulated in the GBM tissues and GBM cell lines (P<0.05). The effects of the expression of FOXO1 on GBM cell proliferation and cell cycle were examined using flow cytometry. The overexpression of FOXO1 markedly inhibited LN18 and T98G cell proliferation and arrested cell cycle at the G0/G1 phase. In addition, FOXO1 facilitated cell senescence through regulation of the expression of sirtuin1. Epithelial‑mesenchymal transition (EMT) is a complex process, which affects cell growth, invasion and metastasis. The results of the present study revealed that FOXO1 inhibited EMT and metastasis in GBM. These finding revealed a novel mechanism of FOXO1 in the suppression of tumorigenesis and metastasis of GBM cells and suggested that FOXO1 may be a potential therapeutic target for treating GBM.

P08.58 Impact of selected pentabromobenzylisothiourea (ZKK-3) combined with hyperbaric oxygen therapy on therapeutic response and oxygenation status of T98G glioblastoma cell line

Abstract Introduction: Glioblastoma (GBM), the primary brain tumour derived from astroglial cells, is the most frequently diagnosed intracranial malignancy in adults. Poor prognosis for GBM patients results from high invasiveness of neoplastic cells related with faulty angiogenesis and persistent hypoxia states. In such conditions glioma cells develop radio- and chemotherapy-resistant phenotype. It is postulated that application of hyperbaric oxygen (HBO) may improve therapeutic response via refinement of tumour tissue oxygenation. The more effective therapeutic strategies are also required. One of the novel compounds that exhibit anti-tumour properties are pentabromobenzylisothioureas. The aim of this study was to examine whether HBO may enhance anti-tumour efficacy of selected pentabromobenzylisothiourea when both agents were administered as combined ZKK-3/HBO therapy. Materials and Methods: Human glioblastoma T98G cell line was cultured in medium supplemented with novel isothiourea derivative - ZKK-3. The proliferation and viability of glioma cells cultured in hypoxia or hyperbaric oxygen conditions were examined. Cell proliferation was tested 24 hours after ZKK-3 addition using Nikon Inverted Microscope and Multisizer 3 Beckman Coulter. The viability assay was taken 24 and 48 hours post ZKK-3 supplementation by CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega). Hypoxia state of T98G cells was determined via the evaluation of HIF-1α protein expression using HIF-1A ELISA Kit (Thermo Scientific). Tests were performed on cells treated with ZKK-3 and cultured in various oxygen conditions: 1/ normoxia (24 hours), 2/ hypoxia (24 hours), 3/ HBO (2 ATA, 1 hour followed by 23 hours of normoxia), 4/ double hypoxia, 5/ hypoxia/HBO. Results: Proliferation of T98G cells treated with ZKK-3 was significantly decreased after hyperbaric oxygenation in comparison to hypoxia conditions. Administration of HBO resulted also in statistically significant diminution of glioma cells’ viability. Expression of HIF-1α in T98G cells strongly depended on oxygen conditions: in hypoxia and double hypoxia the level of protein was significantly elevated compared with normoxia, while after hyperbaric oxygenation no changes or even decrease of HIF-1α level was observed. Comparison of double hypoxia and hypoxia/HBO groups showed that HBO addition after ZKK-3 supplementation caused significant reduction of HIF-1α expression. Conclusions: Treatment of malignant glioma cells with selected isothiourea derivative is significantly more efficient when combined with hyperbaric oxygen administration. Beneficial effect of HBO on decreasing HIF-1α expression allows to consider hyperbaric oxygenation as the way to overcome tumour hypoxia.ACKNOWLEDGEMENT: The research was supported by the KNOW-MMRC project and Foundation for the Development of Diagnostic and Therapy.

PT93, a novel caffeic acid amide derivative, suppresses glioblastoma cells migration, proliferation and MMP-2/-9 expression.

Glioblastoma multiforme (GBM) is the most malignant type of primary brain tumor in adults and can diffusely infiltrate adjacent normal tissue. GBM is therefore rarely cured by surgery or radiation therapy. Matrix metalloproteinases (MMPs) are involved in tissue remodeling and numerous other physiological progresses. The MMPs MMP-2 and MMP-9 are associated with the invasion ability of GBM. PT93 is a novel caffeic acid amide derivative that was first synthesized in 2013. In the present study, the human GBM T98G, U87 and U251 cell lines and the normal mouse neuron HT22 cell line were used to investigate the anticancer and cytotoxic effects of PT93 in vitro. The cytotoxicity of PT93 was measured using MTT and lactate dehydrogenase assays. The anti-proliferation effect was tested using a cell colony formation assay. Gelatin zymography analysis and a scratch test were used to investigate the anti-migration mechanism of PT93. Western blot analysis was used to measure the expression of MMP-2/-9. The experimental results showed that PT93 suppressed the proliferation of T98G cells, and showed cytotoxicity effects at high concentration in T98G, U87, U251 and HT22 cell lines. Furthermore, PT93 limited the migration ability of the cells and inhibited the extracellular MMP-2 and MMP-9 activity of T98G and U251 cells. Finally, the present study confirmed that PT93 affects the level of MMP-2/-9 expression in T98G cells in a concentration-dependent manner. The present study indicates that PT93, as a novel caffeic acid amide derivative, may be used in the treatment of GBM.

Derivatives of the pyrazolo[1,5-a]pyrimidine acetamide DPA-713 as translocator protein (TSPO) ligands and pro-apoptotic agents in human glioblastoma

The 18kDa translocator protein (TSPO) is a target for novel glioblastoma therapies due to its upregulation in this cancer and relatively low levels of expression in the healthy cortex. The pyrazolo[1,5-a]pyrimidine acetamides, exemplified by DPA-713 and DPA-714, are a class of high affinity TSPO ligands with selectivity over the central benzodiazepine receptor. In this study we have explored the potential anti-glioblastoma activity of a library of DPA-713 and DPA-714 analogues, and investigated the effect of amending the alkyl ether chain on TSPO affinity and functional potential. All ligands demonstrated nanomolar affinity for TSPO, but showed diverse functional activity, for example DPA-713 and DPA-714 did not affect the proliferation or viability of human T98G glioblastoma cells, while the hexyl ether and benzyl ether derivatives decreased proliferation of T98G cells without affecting proliferation in human fetal glial SVGp12 cells. These ligands also induced apoptosis and dissipated T98G mitochondrial membrane potential. This suggests that the nature of the alkyl ether chain of pyrazolo[1,5-a]pyrimidine acetamides has little influence on TSPO affinity but is important for functional activity of this class of TSPO ligands.

ATPS-15CANNABIDIOL REDUCES GLIOMA CELLS PROLIFERATION AND VIABILITY

Phytocannabinoids exert antineoplastic activities in several types of cancers, including gliomas. However, psychotropic effects, such as dizziness and physical dependence produced by the major active ingredient of cannabis Δ9-tetrahydrocannabinol, limit their clinical application. Emerging evidence suggests that cannabidiol (CBD), a non-psychoactive component of plant cannabis, also reduces tumor growth in several glioma models; however, the molecular and cellular mechanisms by which CBD produces antitumor activities are yet to be further explored. To better understand how CBD produces antitumor actions, we studied its effects on the viability and proliferation of human GBM cell line T98G cells using cell proliferation reagents WST-1 and [3H]-thymidine incorporation assay, respectively. Multiple doses and time points were used to assess the dose response and time course of the antitumor actions produced by CBD and the DNA alkylating agent carmustine (BCNU) as comparator. Both CBD and BCNU produced a dose-dependent suppression on the viability of T98G cells. Specifically, the half maximal inhibitory concentration of CBD (IC50 = 1.55 µM) is approximately 30 times lower than BCNU (IC50 = 47.17 µM) at 72hr time point, indicating that CBD is more potent than BCNU in reducing cell viability. Both CBD and BCNU at their maximal effective dose suppressed proliferation of T98G cells in a time-dependent manner. Specifically, the inhibitory effects of CBD (1 µM) on cell viability and proliferation started at 24hr, whereas the inhibitory effects of BCNU (1mM) on cell viability and proliferation started at 4hr. The differences between the two compounds on viability and proliferation are consistent with the notion that they act through distinct mechanism of action. The strong sensitivity of T98G cells to both CBD and BCNU suggest that it is a suitable model system for the study of the molecular and cellular mechanisms of action and potential interaction of these compounds. Supported by {type:entrez-nucleotide,attrs:{text:DA026430,term_id:78765130,term_text:DA026430}}DA026430 (NS).

Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.

Cannabidiol, a Non-Psychoactive Cannabinoid Compound, Inhibits Proliferation and Invasion in U87-MG and T98G Glioma Cells through a Multitarget Effect

In the present study, we found that CBD inhibited U87-MG and T98G cell proliferation and invasiveness in vitro and caused a decrease in the expression of a set of proteins specifically involved in growth, invasion and angiogenesis. In addition, CBD treatment caused a dose-related down-regulation of ERK and Akt prosurvival signaling pathways in U87-MG and T98G cells and decreased hypoxia inducible factor HIF-1α expression in U87-MG cells. Taken together, these results provide new insights into the antitumor action of CBD, showing that this cannabinoid affects multiple tumoral features and molecular pathways. As CBD is a non-psychoactive phytocannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anti-cancer drug in the management of gliomas.

Diazepam Inhibits Proliferation of Human Glioblastoma Cells Through Triggering a G0/G1 Cell Cycle Arrest

Glioblastoma (GBM), the most common primary brain tumor, is the most aggressive malignancy in humans. Its rapid proliferation is a major obstacle to successful treatment. Patients with GBM often suffer from psychological disturbances associated with poor prognosis and physical discomfort. Diazepam is one of the most frequently used benzodiazepines (BZs) in cancer patients for its desirable psychotropic effects. The central effects of BZs are mediated by the activation of central BZ receptors. This study investigates whether diazepam has inhibitory effect on proliferation of GBM cell line T98G and explores its possible mechanism. Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell cycle distribution was examined by flow cytometry. Western blot with specific protein antibodies was used to detect regulatory proteins involved in cell cycle regulation. Diazepam significantly decreased the proliferation of T98G cells in a dose-dependent and time-dependent manner. This effect was not reversed by the central BZ receptor antagonist flumazenil or the peripheral BZ receptor antagonist PK11195, indicating that it was not mediated by BZ receptors. Flow cytometry indicated that diazepam caused a cell accumulation in G0/G1 phase, thereby contributing to cell proliferation inhibition. Furthermore, our findings showed that lessened phosphorylation of Rb accounted for diazepam-induced G0/G1 phase arrest. Diazepam inhibits the proliferation of human GBM T98G cells by inducing G0/G1 phase arrest. Diazepam has potential to be a lead for new drugs in GBM therapy because of its antitumor activity.

MicroRNA-214-mediated UBC9 expression in glioma

It has been reported that ubiquitin-conjugating enzyme 9 (Ubc9), the unique enzyme2 in the sumoylation pathway, is up-regulated in many cancers. However, the expression and regulation of UBC9 in glioma remains unknown. In this study, we found that Ubc9 was up-regulated in glioma tissues and cell lines compared to a normal control. UBC9 knockdown by small interfering RNA (siRNA) affected cell proliferation and apoptosis in T98G cells. Further experiments revealed that microRNA (miR)-214 directly targeted the 3' untranslated region (UTR) of UBC9 and that there was an inverse relationship between the expression levels of miR-214 and UBC9 protein in glioma tissues and cells. miR-214 overexpression suppressed the endogenous UBC9 protein and affected T98G cell proliferation. These findings suggest that miR-214 reduction facilitates UBC9 expression and is involved in the regulation of glioma cell proliferation. [BMB Reports 2012; 45(11): 641-646]

Microtubule-Severing ATPase Spastin in Glioblastoma: Increased Expression in Human Glioblastoma Cell Lines and Inverse Roles in Cell Motility and Proliferation

We studied the expression and distribution of the microtubule-severing enzyme spastin in 3 human glioblastoma cell lines (U87MG, U138MG, and T98G) and in clinical tissue samples representative of all grades of diffuse astrocytic gliomas (n = 45). In adult human brains, spastin was distributed predominantly in neuronsand neuropil puncta and, to a lesser extent, in glia. Compared with normal mature brain tissues, spastin expression and cellular distribution were increased in neoplastic glial phenotypes, especiallyin glioblastoma (p < 0.05 vs low-grade diffuse astrocytomas). Overlapping punctate and diffuse patterns of localization wereidentified in tumor cells in tissues and in interphase and mitotic cells ofglioblastoma cell lines. There was enrichment of spastin in the leading edges of cells in T98G glioblastoma cell cultures and in neoplastic cell populations in tumor specimens. Real-time polymerase chain reaction and immunoblotting experiments revealed greater levels of spastin messenger RNA and protein expression in theglioblastoma cell lines versus normal human astrocytes. Functional experiments indicated that spastin depletion resulted in reduced cell motility and higher cell proliferation of T98G cells. Toour knowledge, this is the first report of spastin involvement incellmotility. Collectively, our results indicate that spastinexpression in glioblastomas might be linked to tumor cell motility, migration, and invasion.

A comparative study of morphine stimulation and biphalin inhibition of human glioblastoma T98G cell proliferation in vitro

Biphalin is a new type of opioid peptide analog with high analgesic potency that is over 1000-fold greater than morphine. Because of its less addictive nature, biphalin has been suggested as a prospective new analgesic drug. Its high analgesic activity may be related to synergic interaction with all three types of opioid receptors (μ, δ, and κ). Earlier data implicating involvement of opioid receptors, particularly MOR (μ opioid receptor) and KOR (κ opioid receptor), in cell cycle regulation prompted us to investigate the effect of biphalin and morphine on human glioma T98G cell proliferation in vitro. We have documented an inhibitory effect of biphalin on tumor cell growth related to a decreased proliferation rate, decline of cell ability to form colonies, and modulation of the Ki-67 proliferation index. Morphine displayed the opposite effect and triggered stimulation of T98G cell proliferation. Our experiments have shown that biphalin might constitute an alternative solution for morphine application in anti-pain and anti-cancer therapy.