Glioblastoma Cell Line A172 Research Articles

Suppressing Dazl modulates tumorigenicity and stemness in human glioblastoma cells

BackgroundGlioblastoma is devastating cancer with a high frequency of occurrence and poor survival rate and it is urgent to discover novel glioblastoma-specific antigens for the therapy. Cancer-germline genes are known to be related to the formation and progression of several cancer types by promoting tumor transformation. Dazl is one such germline gene and is up-regulated in a few germ cell cancers. In this study, we analyzed the expression of Dazl in human glioblastoma tissues and cells, and investigated its significance in proliferation, migration, invasion and chemoresistance of the glioblastoma cell lines.MethodsWe evaluated the expression of Dazl in different pathologic grades of glioblastoma tissues by immunohistochemistry. We assessed the expression of Dazl in glioblastoma cells and normal human astrocytes (NHA) cells by western blotting and RT-qPCR. Then we generated Dazl knockout glioblastoma cell lines using the CRISPR/Cas9 gene-editing technology to explore the cellular function of Dazl. We detected the proliferation and germline traits via CCK-8 assays and alkaline phosphatase staining, respectively. Boyden chamber assays were performed to measure glioblastoma cell migration and invasion. Crystal violet staining was used to determine the number of viable cells after the treatment of Doxorubicin and Temozolomide. Finally, we used subcutaneous xenograft studies to measure the growth of tumors in vivo.ResultsWe found that Dazl was upregulated in glioblastoma tissues and glioblastoma cell lines. Dazl knockdown glioblastoma cells showed decreased cellular proliferation, migration, invasion, and resistance in vitro, and inhibited the initiation of glioblastoma in vivo. The glioblastoma cell lines A172, U251, and LN229 were found to express stem cell markers CD133, Oct4, Nanog, and Sox2. The expression of these markers was downregulated in Dazl-deficient cells.ConclusionsOur results indicated that Dazl contributes to the tumorigenicity of glioblastoma via reducing cell stemness. Therefore, cancer-germline genes might represent a new paradigm of glioblastoma-initiating cells in the treatment of malignant tumors.

WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line.

Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1—baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line.

P1.14-20 Tarloxotinib as a Novel Therapeutic Strategy for Oncogenic Alterations Across the ErbB Family of Receptors

The ErbB family of receptors tyrosine kinases (EGFR, HER2, HER3, and HER4) have been implicated in multiple different tumor types. The implementation of comprehensive next generation sequencing has allowed the identification of diverse gene alterations that function as oncogene drivers in these receptors. Some of the non-common gene alterations identified are resistant to marketed EGFR/HER2 inhibitors. Tarloxotinib is a prodrug that generates a potent and irreversible pan-HER inhibitor (tarloxotinib-E) under hypoxic conditions associated with tumors. In this study we evaluated the effect of tarloxotinib on several types of oncogenic mutations and fusions that involve the ErbB family of receptors. cDNAs encoding EGFR kinase domain duplications (EGFR-EGFR), EGFR-RAD51 gene fusion, EGFR-ERBB4, ERBB2-GRB7 and EZR-ERBB4 gene fusion were expressed in Ba/F3 cells. Using spheroid assays we evaluated the proliferation of A172 glioblastoma cell line treated with tarloxotinib, tarloxotinib-E or 1st, 2ndor 3rdgeneration EGFR/HER2 TKIs. We analyze the on target and signaling effects elicited by tarloxotinib-E via immunoblots. Using a nude mice xenograft model of the human derived cell line CUTO17 wi the EGFR exon 20 insertion p.N771_H773dupNPH, we evaluated tumor, tissue and blood drug levels by mass spectrometry and the effect of tarloxotinib on tumor growth. Our results demonstrate that tarloxotinib-E inhibits phosphorylation of EGFR with a kinase duplication and inhibits proliferation in a spheroid invasion assay in A172 cells. In the CUTO17 EGFR exon 20 model, treatment with tarloxotinib inhibited tumor growth. Intratumor levels of tarloxotinib-E were ∼20 times higher than skin and ∼50 times higher than plasma demonstrating selective tumor conversion of tarloxotinib. Cell growth inhibition (EC50) of novel HER family fusions (EGFR-EGFR, EGFR-RAD51, EGFR-ERBB4, ERRB2-GRB7 and EZR-ERBB4) will be presented. Tarloxotinib is a potent irreversible inhibitor in vitro for cells that harbor oncogenic alterations across the ERBB gene family, including EGFR kinase domain duplications, ErbB fusions and exon-20 insertions. Tarloxotinib is selectively activated in hypoxic tumor regions demonstrating a novel mechanism to generate a therapeutic window and avoid on-target EGFR-related toxicities.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

Investigating the Selectivity of Novel Chalcones for Human Cancer Cells Compared to Non‐Cancerous Human Cell Lines

We previously presented data on the anticancer activity of a small library of synthetic flavonoid derivatives against glioblastoma and breast cancer lines in addition to several other human cancer cell lines. This data suggested that the chalcone structural backbone afforded more potent anticancer activity compared to its flavanone counterpart. Using this data, we selected three lead compounds: RK6, RK7, and RK15, to screen for activity against non‐cancerous cells to test for selectivity. Here we present updated IC50 values of these three analogs against MCF7 breast adenocarcinoma cells in addition to the previously reported data on the A‐172 glioblastoma cell line. To determine the selectivity of these lead compounds, RK6, RK7, and RK15 were screened against two non‐cancerous human cell lines: normal human astrocytes (NHA) and normal human mammary epithelial cells (NHME).The activity of our lead compounds against A‐172 and MCF7 cells were assessed using the XTT cell viability assay to determine respective IC50 values. As reported previously, the IC50 values of our lead compounds in the A‐172 cell line were 20 μM, 22 μM, and 3 μM for RK6, RK7, and RK15 respectively. Updated IC50 values of the compounds in the MCF7 cell line were 19 μM, 99 μM, and 12 μM for RK6, RK7, and RK15.In order to evaluate the selectivity of the lead compounds for cancerous vs. non‐cancerous cells, we utilized the Vialight cell viability assay to screen the chalcones in NHA and NHME cells. In the NHA cells, the determined IC50 values of RK6, RK7, and RK15 were 47 μM, 52 μM, and >200 μM respectively. IC50 values were 60 μM, >200 μM, and >200 μM for the NHME cells treated with RK6, RK7, and RK15 respectively. This data was used to calculate selectivity indices for our lead compounds against A‐172 cells compared to NHA cells and MCF7 cells compared to NHME cells. For RK6, there was a 2.3 fold selectivity for A‐172 cells and a 3.1 fold selectivity for MCF7 cells compared to their non‐cancerous comparator cells. The selectivity index for RK7 showed a 2.3 fold selectivity for A‐172 compared to the NHA. In these instances, RK6 and RK7 appear to be somewhat selective for cancerous vs. non‐cancerous cell lines. Given the IC50 values >200 μM for RK7 in NHA cells and RK15 in NHA and NHME cells, exact selectivity indices could not be determined. However, in these occurrences, it would appear that the compounds demonstrated a high degree of selectivity for cancerous vs. non‐cancerous cells.Collectively, these results suggest that our lead chalcone compounds are at least somewhat selective for cancerous vs. non‐cancerous cells. Interestingly, the most potent compound in the A‐172 and MCF7 cells, RK15, demonstrated the highest degree of selectivity. This is intriguing given that recent trends in anticancer drug discovery favor compounds that are selective for cancer cells. These findings may provide some insights into the yet to be elucidated mechanisms of cell death by these novel chalcone derivatives, and will form the basis of future studies.Support or Funding InformationThis work was supported in part by an AFPE Gateway Scholarship for the first author.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CHD7 promotes glioblastoma cell motility and invasiveness through transcriptional modulation of an invasion signature

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.

Long noncoding RNA-RNCR3 overexpression deleteriously affects the growth of glioblastoma cells through miR-185-5p/Krüppel-like factor 16 axis.

Glioblastoma (GBM) is a devastating and highly aggressive tumor, which is apoptosis resistant and difficult to cure. Recently, long noncoding RNAs have been shown to play a pivotal role in GBM progression. Evidence has suggested that retinal noncoding RNA3 (RNCR3) is a GBM-associated noncoding RNA and is under-expressed in GBM. However, the function and mechanism of RNCR3 on GBM cell growth and apoptosis are still uncertain. In the current study, we found that the level of RNCR3 is decreased in U87, U251, U373, and A172 GBM cell lines when compared with the normal human astrocytes. Elevating long noncoding RNA RNCR3 expression markedly inhibits U87 and U251 cell survival and proliferation. Further studies indicated that RNCR3 overexpression promotes U87 and U251 cell apoptosis and activity caspase-3/7. Moreover, we found that RNCR3 overexpression promotes Krüppel-like factor 16 (KLF16) expression through inhibiting the level of miR-185-5p. We demonstrated that KLF16 is a direct target of miR-185-5p. An increased miR-185-5p level by a miR-185-5p mimic or decreased KLF16 by KLF16 small interfering RNA both reversed the function of RNCR3 overexpression on GBM cell growth and apoptosis. In summary, this study focuses oninvestigating the key molecular mechanisms of RNCR3 involved in GBM cell growth and apoptosis. Our data indicated that RNCR3 overexpression inhibits cell growth and induces its apoptosis through the miR-185-5p/KLF16 axis.

Paraptosis in human glioblastoma cell line induced by curcumin

Curcumin is a polyphenol compound extracted from Curcuma longa plant, is a molecule with pleiotropic effects that suppresses transformation, proliferation and metastasis of malignant tumors. Curcumin can cause different kinds of cell death depending of its concentration on the exposed cell type. Here we show that exposure of the glioblastoma cell line A172 to curcumin at 50 μM, the IC50, causes morphological change characteristic of paraptosis cell-death. Vesicles derived from the endoplasmic reticulum (ER) and low membrane potential of the mitochondria were constantly found in the exposed cells. Furthermore, changes in expression of the ER Stress Response (ERSR) genes IRE1 and ATF6, and the microRNAs (miRNAs) miR-27a, miR-222, miR-449 was observed after exposure to curcumin. AKT-Insulin and p53-BCL2 networks were predicted being modulated by the affected miRNAs. Furthermore, AKT protein levels reduction was confirmed. Our data, strongly suggest that curcumin exerts its cell-death properties by affecting the integrity of the reticulum, leading to paraptosis in the glioblastoma cells. These data unveils the versatility of curcumin to control cancer progression.

Interactive effect of pressure and temperature on the preservation of rat primary-cultured astrocytes and human glioblastoma cell line A172

ABSTRACTEnvironmental factors such as temperature and pressure are important determinants of cell survival. Although the effect of temperature on cell preservation has been previously reported, the effects of pressure, an equally important thermodynamic parameter, have not been sufficiently investigated. In this study, we investigated the effect of temperature and pressure on cellular viability, morphology, adhesiveness, cell death, cell cycle and glucose metabolism in rat primary-cultured astrocytes and A172 human glioblastoma cell line subjected to 4-day preservation. It was revealed that under favorable preservation conditions (temperature: 15°C–20°C, pressure: 0.1–30 MPa) (1) cell morphology and adhesiveness of preserved cells were maintained similar to freshly isolated cells; (2) cell cycle was arrested; (3) glucose uptake and intra/extra-cellular pH decrease were suppressed. These results suggest that lowering temperature to 15°C–20°C or increasing pressure up to 30 MPa at temperatures of 20°C–25°C can reduce cellular metabolism and maintain cell-membrane fluidity, thus resulting in higher viability.

The Effect of Fotemustine on Human Glioblastoma Cell Lines

Glioblastomas are a heterogeneous group of tumors of neuroectodermal origin. Tumor cell cultures obtained from patients with glioblastomas differ in morphology and phenotype, as well as genetic characteristics. In the current work we used glioblastoma cell lines A172 and T98G (known since the 1970s) and the new glioblastoma lines R1 and T2 (obtained in 2010 from surgical material of patients). The purpose of this study was to evaluate the effect of the alkylating antitumor drug fotemustine (FM) after single and repeated exposure on the survival and proliferation of these glioblastoma cells, as well as the effect of FM on gene expression of growth factors, extracellular matrix proteins, mesenchymal cell markers, and transcription factor Zeb1. The effect of FM on A172 and R1 glioblastoma cells differs from its effect on T98G and T2 cells. LD50 was in an amount of 60 μg/mL for A172 cells and 250 μg/mL for T98G cells. Single exposure of A172 and R1 cell lines to 100 μg/mL of FM resulted in the death of all cells in culture. A172 and R1 cell exposure to sublethal doses of FM caused decreased activity of all studied genes. After incubation of T98G and T2 glioblastoma cells with FM in sublethal doses we identified two cell populations: proliferating cells and nonproliferating growth-arrested cells. Cell exposure to FM in doses over 300 μg/mL led to the predominance of growth-arrested cells in populations. These cells remained viable for 1.5 months. The effect of FM on T98G and T2 cell lines was accompanied by increased activity of all the studied genes. In conclusion, a population of growth-arrested, drug-resistant cells has been revealed in T98G and T2 cell cultures after exposure to FM. These cells exhibit high expression of genes encoding factors associated with the epithelial–mesenchymal transition. We discuss the possible impact of nonproliferating cells actively producing growth factors on the tumor microenvironment.

Targeting Glioblastoma Cells Expressing CD44 with Liposomes Encapsulating Doxorubicin and Displaying Chlorotoxin-IgG Fc Fusion Protein.

We recently have established a successful xenograft model of human glioblastoma cells by enriching hyaluronic acid-dependent spheroid-forming populations termed U251MG-P1 cells from U251MG cells. Since U251MG-P1 cells have been confirmed to express CD44 along with principal stemness marker genes, OCT3/4, SOX2, KLF4 and Nanog, this CD44 expressing population appeared to majorly consist of undifferentiated cells. Evaluating the sensitivity to anti-cancer agents, we found U251MG-P1 cells were sensitive to doxorubicin with IC50 at 200 nM. Although doxorubicin has serious side-effects, establishment of an efficient therapy targeting undifferentiated glioblastoma cell population is necessary. We previously designed a chlorotoxin peptide fused to human IgG Fc region without hinge sequence (M-CTX-Fc), which exhibited a stronger growth inhibitory effect on the glioblastoma cell line A172 than an original chlorotoxin peptide. Combining these results together, we designed M-CTX-Fc conjugated liposomes encapsulating doxorubicin and used U251MG-P1 cells as the target model in this study. The liposome modified with M-CTX-Fc was designed with a diameter of approximately 100–150 nm and showed high encapsulation efficiency, adequate loading capacity of anticancer drug, enhanced antitumor effects demonstrating increasing uptake into the cells in vitro; M-CTX-Fc-L-Dox shows great promise in its ability to suppress tumor growth in vivo and it could serve as a template for targeted delivery of other therapeutics.

Molecular mechanisms underlying the α-tomatine-directed apoptosis in human malignant glioblastoma cell lines A172 and U-118 MG.

In the present study, the molecular mechanisms involved in the α-tomatine-induced apoptosis in human glioblastoma cell lines A172 and U-118 MG were investigated. Wright staining and ApopTag assays were conducted to confirm the apoptosis induced by α-tomatine treatment. Fura-2 assay determined an enhancement in free Ca2+ intracellularly, indicating the occurrence of Ca2+-dependent apoptosis induction. Western blot experiments were also performed to predict the apoptosis by measuring the changes in the Bax:Bcl-2 ratio. Increase of calpain activity triggered caspase-12 expression, which in turn further activated caspase-9. In addition, an increase in the ratio of Bax:Bcl-2 accounted for the mitochondrial release of cytochrome c into the cytosol for caspase-3 and caspase-9 activation. Elevated activity of calpain and caspase-3 yielded spectrin breakdown products with 145 and 120 kDa, respectively. Caspase-3 activation further cleaved the inhibitor of caspase activated DNase, while the apoptosis-inducing factor detected in the cytosol suggested that apoptosis was independent of caspase. The apoptosis induction was further supported by decreased expression levels of nuclear factor-κB and increased expression of the inhibitor of nuclear factor, IκBα. In conclusion, the presented experimental results revealed the stimulation of different molecular mechanisms for α-tomatine-mediated apoptosis in A172 and U-118 MG human glioblastoma cell lines.

Abstract B17: Targeting MK2 to improve temozolomide efficacy in glioblastoma

Abstract Glioblastoma is the most prevalent and aggressive form of brain cancer. The heterogeneous nature and survival mechanisms that glioblastoma tumors develop impede chemotherapy efficacy of temozolomide (TMZ) and result in tumor relapse. The p38 MAPK and its downstream kinase MK2 have been well established in regulating signaling pathways influencing inflammation, cell division and differentiation, as well as cell motility in response to a wide range of extracellular stimuli. Recent studies have identified p38 MAPK-MK2 pathway as an effector kinase complex that is activated following DNA damage and results in cell cycle arrest, so that cells have the capacity to repair their DNA and continue to proliferate. Importantly, cells with p53 mutation/deficiency were shown to rely on MK2 pathway for G2/M arrest and survival after exposure to chemotherapy and thus highlighting MK2 as a potential therapeutic target. We sought to determine if inhibition of MK2 in glioblastoma cells would result in increased efficacy of TMZ and to delineate the underlying mechanisms. Using genetic knockdown and pharmacological inhibitors we examined whether MK2 inhibition improves temzolomide efficacy in glioblastoma cell lines and patient-derived tumor cells of different subtypes (classical, mesenchymal, proneural and neural). Viability and clonogenic assays were used to determine survival of tumor cells to combination treatment. Apoptosis was determined using Annexin-V staining, cell cycle analysis using flow cytometry, mitotic entry by p-HH3 staining, extent of DNA damage using γH2AX staining and senescence using β-galactosidase staining. Western blotting analysis was performed to study changes in protein expression. We show that MK2 inhibition significantly improved TMZ anti-proliferative efficacy in A172 glioblastoma cell line and primary cells of classical subtype. Furthermore, MK2 inhibition reduced ability of TMZ treated cells to form colonies in clonogenic assay. Mechanistic studies revealed that MK2 knockdown reduced G2/M arrest induced by TMZ and promoted G0/G1 arrest. These changes were associated with increase in TMZ-induced apoptosis and senescence. We show that MK2 knockdown does not promote premature mitosis and mitotic catastrophe due to accumulation of DNA damage, as evidenced by lack of p-HH3 and γH2AX staining. In summary, our results demonstrate that MK2 inhibition is a novel strategy to sensitize glioblastoma cells to chemotherapy. We propose that in glioblastoma cells, MK2 signaling does not contribute to G2/M arrest induced by TMZ and instead MK2 plays a crucial role in regulating G0/G1 arrest in response to DNA damaging agents. Citation Format: Fadi Gurgis, Bryan Day, Brett Stringer, Terrance G. Johns, Lenka Munoz. Targeting MK2 to improve temozolomide efficacy in glioblastoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B17.

MiR-204 suppresses the development and progression of human glioblastoma by targeting ATF2.

In human cancers, miRNAs are important regulators of multiple cellular processes, and aberrant miRNA expression has been observed, and their alterations contribute to multiple cancer development and progression. Till now, little has been known about the role of miR-204 in human glioblastoma (GBM). In the present study, we used in-vitro assays to investigate the mechanisms of miR-204 in GBM cell lines and 60 cases of GBM tissues. Here, we found that miR-204 expression is downregulated in both GBM cell lines A172, U87 and U251 cells and GBM tissues as compared with NHA cells and normal tissues (all p<0.001). In addition, the ectopic expression of miR-204 suppressed A172 and U87 cell proliferation, migration and invasion. Meanwhile, miR-204 over-expression extremely inhibited the protein expression of ATF2. Notably, the enforced expression of ATF2 in A172 and U87 cells with the over-expression of miR-204 attenuated the inhibitory effects of miR-204 on proliferation, migration and invasion. In conclusion, our findings suggest that miR-204 suppressed cell proliferation, migration and invasion through inhibition of ATF2, thus, miR-204 may function as a useful drug target in the treatment and diagnosis of GBM.

Extract of the sea cucumber, Holothuria scabra, induces apoptosis in human glioblastoma cell lines

Background: Glioblastoma is the most aggressive primary brain tumor resistant to conventional treatment, and has a frequent recurrence. Holothuria scabra is the most widely consumed sea cucumber in Thailand and Asian countries. Sea cucumber extracts have valuable bioactive ingredients that are utilized in Chinese traditional medicine for several therapeutic measures including anti-cancer. In this study, we aimed to test the anticancer activity of H. scabra body wall extract in ethyl acetate fraction against two human glioblastoma cell lines A172 and U87MG.Material and methods: The anticancer effect of the ethyl acetate fraction of body wall extracts from H. scabra (HSBWEA) was tested against two human glioblastoma cell lines A172 and U87MG to determine cell viability, apoptotic cell analysis, mitochondrial membrane potentials, nuclear condensation and expression of apoptotic molecules. The bioactive compound in the extract was characterized by LC-MS/MS.Results: The extract exhibited strong cytotoxic effect to A172 and U87MG cells with IC50 4.23 and 4.46 mg/ml, respectively. The extract also induced both early and late stages of apoptosis, loss of mitochondrial membrane potentials and nuclear condensation and fragmentation. After treatment with the extract for 24 h, the cells expressed increasing amounts of a pro-apoptotic Bax and caspase-3 while an anti-apoptotic Bcl-2 was decreased in a concentration-dependent manner. The major bioactive components in this extract are triterpene glycosides similar to those described previously in H. scabra as well as in other related sea cucumber species.Conclusions: The crude extract from the body wall of H. scabra exhibited anticancer activity against human glioblastoma cells through the mitochondrial-mediated apoptotic pathway, and the compounds in the extract might be a novel candidate for anti-glioblastoma.

Bee Venom Induces Unfolded Protein Response in A172 Glioblastoma Cell Line

Background: Glioblastoma is a type of brain tumor with poor response to available therapies, and shows high rate of mortality. Despite remarkable advancements in our knowledge about cytogenetic and pathophysiologic features of glioblastoma, current treatment strategies are mainly based on cytotoxic drugs; however, these therapeutic approaches are facing progressive failure because of the resistant nature of glioblastomas. In the recent years, however, promising results have emerged owing to targeted therapies toward molecular pathways within cancerous cells. Unfolded Protein Response (UPR) is a remarkable signaling pathway that triggers both apoptosis and survival pathways within cells, and therefore induces UPR-related apoptotic pathways in cancer cells by ER stress inducers. Objectives: Recently, the role of Bee venom (Bv), which contains powerful bioactive peptides, in inducing UPR-related apoptosis was revealed in cancer cell lines. Nevertheless, currently there are no reports of Bv potential ability in induction of UPR apoptotic routes in glioblastoma. The aim of current study was to evaluate possible role of Bee venome in inducing of UPR pathway within A172 glioblastoma cell line. Materials and Methods: We treated the A172 glioblastoma cell line with different Bv doses, and assessed UPR-related genes expression by real-time Polymerase Chain Reaction (PCR). Results: The IC50 of Bv for the studied cell line was 28 μg/mL. Furthermore, we observed that Bv can induce UPR target genes (Grp94 and Gadd153) over-expression through a dose-dependent mechanism. Conclusions: Our results suggest the potential role of Bv as a therapeutic agent for glioblastomas. Keywords: Glioblastoma; A172 Cell Line; Unfolded Protein Response; Bee Venom

Olanzapine inhibits proliferation, migration and anchorage-independent growth in human glioblastoma cell lines and enhances temozolomide's antiproliferative effect.

The poor prognosis of patients with glioblastoma fuels the search for more effective therapeutic compounds. We previously hypothesised that the neuroleptic olanzapine may enhance antineoplastic effects of temozolomide the standard chemotherapeutic agent used in this disease. This study tested this hypothesis. The anti-proliferative effect of olanzapine was examined by MTT assays and cell count analysis. Soft-agar assays were performed to examine colony-forming ability. In addition, the inhibitory effect of olanzapine on the migratory capacity of U87MG and A172 cells was analyzed by Transwell(®) assays. Moreover, staining for annexin V/propidium iodide or carboxyfluorescein succinimidyl ester was performed prior to flow cytometric analysis in order to better understand the subjacent cellular mechanism. Our initial hypothesis that olanzapine may enhance temozolomide's anti-tumor activity could be confirmed in U87MG and A172 glioblastoma cell lines. Moreover, treatment with olanzapine alone resulted in a marked anti-proliferative effect on U87MG, A172 and two glioma stem-like cells with IC50 values ranging from 25 to 79.9 µM. In U87MG cells, anchorage-independent growth was dose-dependently inhibited. In A172 cells, migration was also shown to be inhibited in a dose-dependent manner. In addition, olanzapine was shown to exert a cell line-dependent pleomorphism with respect to the induction of apoptosis, necrosis and/or cytostasis. Our data show that the neuroleptic olanzapine enhances the anti-tumor activity of temozolomide against glioblastoma cell lines. Moreover, this is the first study to show that olanzapine provides on its own anti-cancer activity in glioblastoma and thus may have potential for repurposing.

A targeting peptide improves adenovirus-mediated transduction of a glioblastoma cell line

The progress of the application of adenovirus in cancer gene therapy is hindered by the lack of expression of native adenovirus receptor on a variety of cancer types. Hence, strategies are needed to retarget the adenoviral vector to non-native cellular surface receptors. In the present study, a new peptide SWDIAWPPLKVP, capable of selectively targeting a human glioblastoma cell line A172, was identified by direct biopanning of phage-display peptide libraries. The binding activity of the phage displaying SWDIAWPPLKVP peptide in A172 was more than 10-fold higher than that of the control phage. We then inserted the selected peptide SWDIAWPPLKVP into adenoviral hexon protein, and observed that the modified Ad5 had increased infectivity in A172 cells, compared with that in control cell lines. These findings demonstrated that a peptide acquired through phage display can mediate cell-specific Ad retargeting when inserted into Ad hexon, suggesting an approach for targeting adenoviral infection to specific cancer cells.

Functional analysis of [methyl-3H]choline uptake in glioblastoma cells: Influence of anti-cancer and central nervous system drugs

Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with 11C- or 18F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-3H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-3H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na+-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-3H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-3H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-3H]choline uptake that relies on a directed H+ gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-3H]choline uptake, central nervous system drugs did not affect [methyl-3H]choline uptake.

Artesunate Enhances the Antiproliferative Effect of Temozolomide on U87MG and A172 Glioblastoma Cell Lines

As chemotherapy with temozolomide is far from providing satisfactory clinical outcomes for patients with glioblastoma, more efficient drugs and drug combinations are urgently needed. The anti-malarial artesunate was previously shown to exert a profound cytotoxic effect on various tumor cell lines including those derived from glioblastoma. In the current study, we sought to examine the antiproliferative effect of a combination of temozolomide and artesunate on two different established human glioblastoma cell lines. The IC50 and IC25 were determined for temozolomide and artesunate in U87MG and A172 glioblastoma cell lines after 144 h of continuous drug exposure. The antiproliferative effect of combining both agents at IC50/IC50 and IC25/IC25 was determined by a cell viability assay. Moreover, necrosis and apoptosis were analyzed by annexin V/PI staining and flow cytometric analysis. In addition, cytostatic effects were examined by carboxyfluorescein diacetate succinimidyl ester staining and subsequent flow cytometry. In both glioblastoma cell lines, artesunate was found to enhance the antiproliferative effect exerted by temozolomide. Moreover, artesunate acted in concert with temozolomide in terms of cytostatic and necrotizing effects. These observations suggest that a combination of artesunate and temozolomide might result in increased cytotoxicity in glioblastoma.