Human Glioblastoma Cell Line A172 Research Articles

Comparative Study of the Anticancer Effects of Selenium Nanoparticles and Selenium Nanorods: Regulation of Ca2+ Signaling, ER Stress and Apoptosis

The anti-cancer effects of selenium sources are well known. Among other things, selenium has been shown to have a pleiotropic effect, causing cancer cell death without affecting the healthy cell’s viability, or, in the case of brain cells, has a cytoprotective effect. This feature of selenium determined its use in medicine and its use as part of dietary supplements. In recent years, selenium in the form of nanoparticles has received increased attention. Selenium nanoparticles also have anti-cancer effects, and their use appears to be more effective at significantly lower doses compared to other sources of selenium. The shape and size of nanoparticles largely affect the efficiency of nanoselenium application. We obtained two different types of selenium nanoparticles via the laser ablation technique—spherical selenium nanoparticles (SeNPs) about 100 nm in diameter and grown selenium nanorods (SeNrs) about 1 μm long and about 100 nm thick. We compared the anti-cancer efficacy of these two types of selenium nanoparticles using inhibitory analysis, PCR analysis and fluorescence microscopy. It turned out that both types of nanoparticles with high efficiency dose-dependently activate apoptosis in the human glioblastoma cell line A-172, as the most aggressive type of brain tumor. Apoptosis induction was determined not only by the concentration of nanoparticles, but also by the time. It was shown that SeNrs induce the process of apoptosis in glioblastoma cells more efficiently during 24 h of exposure and their effect is enhanced after 48 h without activation of necrosis, whereas the use of spherical SeNPs after 48 h of exposure can cause necrosis in some glioblastoma cells. It has been shown that Ca2+ signals of glioblastoma cells are significantly different for SeNPs and SeNrs. SeNPs cause a dose-dependent transient increase in the number of Ca2+ ions in the cell cytosol ([Ca2+]i), while SeNrs cause a slow rise in [Ca2+]i reaching a new stationary level, which may determine the cytotoxic effects of nanoparticles. It turned out that SeNPs and SeNrs cause depletion of the Ca2+ depot of the endoplasmic reticulum and ER-stress, which correlates with increased expression of genes encoding proapoptotic proteins. In our study, it was found that SeNPs do not activate the Ca2+ signaling system of healthy L-929 mouse fibroblast cells, while SeNrs activate a moderate slow growth in [Ca2+]i. That fact could indicate a lower selectivity of the SeNrs action.

Investigation of the Role of Induced Overexpression of the Isolated Secreted Klotho on the A-172 Human Glioblastoma Cells.

Klotho gene, identified in 1997 as an anti-aging gene, can manufacture two protein products: transmembrane and secreted forms. The later research revealed the involvement of klotho in carcinogenesis. However, little is known about the action of different Klotho forms on antitumor effects is still. The purpose of this article is to evaluate the effect of isolated secreted Klotho overexpression on the growth features of human glioblastoma cell line A-172.A-172 was transfected by a plasmid vector incorporating secreted Klotho sequence by the liposomal method. Overexpression assay was carried out quantitatively on both mRNA and protein using RT-qPCR and ELISA, correspondingly. It was shown that the relative expression of secreted Klotho in the experimental group was significantly higher than in the untransfected group by both methods (p < 0.001). At the same time, the growth curves and MTT proliferation assay demonstrated significantly decreased values under induced overexpression (p < 0.01). The increased amount of cells with activated caspases and annexin V (p < 0.001) corresponded with the expression of secreted Klotho. This mechanism, as suggested, maybe causative of the observed effects.

AMPK increases expression of ATM through transcriptional factor Sp1 and induces radioresistance under severe hypoxia in glioblastoma cell lines

We have previously reported that severe hypoxia increases expression and activity of the DNA damage sensor ATM by activation of the key energy sensor AMPK. Here, to elucidate molecular mechanisms underlying increased expression and activity of ATM by AMPK under severe hypoxia, we investigated roles of transcriptional factors Sp1 and FoxO3a using human glioblastoma cell lines T98G and A172. Severe hypoxia increased expression of ATM, AMPKα and Sp1 but not that of FoxO3a. Knockdown of AMPKα suppressed expression of ATM and Sp1 and suppressed cellular radioresistance under severe hypoxia without affecting cell cycle distribution. Knockdown of Sp1 suppressed expression of ATM. These results suggest that increased expression and activity of AMPK under severe hypoxia induce cellular radioresistance through AMPK/Sp1/ATM pathway.

Bis-Bibenzyls from the Liverwort Pellia endiviifolia and Their Biological Activity.

Based on previous investigations where bis-bibenzyls isolated from liverworts showed various biological activities (cytotoxic, antimicrobial, and antiviral), we investigated their cytotoxic activity in several human cancer cell lines. From the methylene-chloride/methanol extract of the liverwort Pellia endiviifolia, three bis-bibenzyls of the perrottetin type were isolated, namely perrottetin E, 10′-hydroxyperrottetin E, and 10,10′-dihydroxyperrottetin E. The last two were found for the first time in this species. Their structures were resolved using 1D and 2D NMR, as well as by comparison with data in the literature. Cytotoxic activity of the isolated compounds was tested on three human leukemia cell lines, HL-60 (acute promyelocytic leukemia cells), U-937 (acute monocytic leukemia cells), and K-562 (human chronic myelogenous leukemia cells), as well as on human embryonal teratocarcinoma cell line (NT2/D1) and human glioblastoma cell lines A-172 and U-251, and compared to the previously isolated bis-bibenzyls (perrottetins) of similar structure. The isolated compounds exhibited modest activity against leukemia cells and significant activity against NT2/D1 and A-172. Overall, the most active cytotoxic compounds in this investigation were perrottetin E (1), isolated in this work from Pellia endiviifolia, and perrottetin F phenanthrene derivative (7), previously isolated from Lunularia cruciata and added for a comparison of their cytotoxic activity.

Involvement of CD73 and A2B Receptor in Radiation-Induced DNA Damage Response and Cell Migration in Human Glioblastoma A172 Cells.

Glioblastoma is the most common malignant tumor of the central nervous system and is treated with a combination of surgery, radiation and chemotherapy. However, the tumor often acquires radiation resistance, which is characterized by an increased DNA damage response (DDR). Here, we show that CD73, which generates extracellular adenosine from ATP, and A2B receptor, which is activated by adenosine, are involved in the γ-radiation-induced DDR and the enhanced migration ability of human glioblastoma cell line A172. To investigate DDR, we evaluated ataxia telangiectasia mutated (ATM) activation and focus formation of histone H2A isoform γ (γH2AX) and p53-binding protein 1 (53BP1) in the nucleus of A172 cells after γ-irradiation. Antagonists of A2B receptor and CD73, or knockdown with small interfering RNA (siRNA), suppressed γ-radiation-induced DDR and promoted γ-radiation-induced cell death, as well as suppressing γ-radiation-induced cell migration and actin remodeling. These results suggest that activation of A2B receptor by extracellular adenosine generated via CD73 promotes γ-radiation-induced DDR, leading to recovery from DNA damage, and also enhances cell migration and actin remodeling. The CD73-A2B receptor pathway may be a promising target for overcoming radiation resistance and the acquisition of malignant phenotypes during radiotherapy of glioblastoma.

β-Elemene Inhibits the Proliferation and Migration of Human Glioblastoma Cell Lines via Suppressing Ring Finger Protein 135.

β-Elemene is commonly used as an anti-cancer agent in different types of cancers and its effects on glioblastoma have been studied through different pathways. However, its effect through ring finger protein 135 (RNF135, OMIM 611358) (RNF135), which is upregulated in glioblastomas, has not yet been explored. The current study is focused on the effects of β-elemene on human glioblastoma cell lines U251, U118, A172 and U87 through RNF13 5. A cell counting kit-8 assay and wound healing assay have been utilized to test the proliferation and migration of the cells. Western blot and quantitative real-time-polymerase chain reaction (qRT-PCR) were used to evaluate the level of expression of RNF135. A model of nude mice was used to explore progression of the tumor in vivo. It was observed that increasing treatment time or dose of β-elemene remarkably decreased viability of the cells. The cells that were treated with β-elemene had a much lower speed of moving toward the gap in comparison to untreated cell lines. β-Elemene-treated cells showed a much lower level of expression of RNF135 mRNA than control groups (p <0.05) and the levels of RNF135 protein were lower in the cells treated with β-elemene than in control groups (p <0.05). Moreover, tumor progression in subcutaneous xenograft nude mice was delayed with the injection of β-elemene. Altogether, our findings suggest that β-elemene inhibits proliferation, migration and tumorigenicity of human glioblastoma cells through suppressing RNF135.

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.

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.

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.

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.

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.

The relationship of the expression level of glycosphingolipids in human glioblastoma cell line A172, U251 and U251/VM-26 and drug resistance

Objective To study the relationship of the drug resistance and glycosphingolipids in glioma.Methods The sensitivity of the teniposide in A172 and U251 cells were detected by MTT,and their glycosphingolipids were detected by ESI-LIT-MS.The drug resistance cell line U251/VM-26 was established by culture with gradual increased dose of VM-26.Then the sensitivity of the teniposide in U251 and U251/VM-26 were checked by MTT,the drug resistance associated gene MDR1 and MRP1 were detected by real-time PCR,and their glycosphingolipids were detected by ESI-LIT-MS.Results The drug resistant of A172 were higher than U251 cells.Meanwhile,C18-LacCer,C18-Gb3,C18-Gb4,are highly accumulated in A172 cells than in U251 cells.The drug resistant of U251/VM-26 cells was 100-folds higher than U251 cells after co-cuturing with VM-26,then the levels of C18-Gb4 are significantly increased in U251/VM-26 cells,C18-LacCer and C18-Gb3 are also increased.Real-time PCR data show that MRP1 in A172 cells are higher than U251 cells,the level of MDR1 and MRP1 gene are also highly accumulated in U251/VM-26 cells.Conclusions Glioma-associated glycosphingolipids,C18-LacCer、C18-Gb3 and C18-Gb4,are correlated with drug resistance of VM-26,and MDR1 and MRP1 gene are involved in the drug resistant of those glioma ceils. Key words: Glioma; Teniposide; Drug resistance; Glycosphingolipids; Mass spectrum

MiR-708 acts as a tumor suppressor in human glioblastoma cells

Glioblastoma (GBM) is one of the most lethal forms of human cancer, and new clinical biomarkers and therapeutic targets are urgently required. microRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the post-transcriptional and/or translational level by binding the 3' untranslated regions (3' UTRs) of target mRNAs. The dysregulated expression of several miRNAs has been reported to modulate glioma progression. In the present study, we defined the expression and function of miR-708, which, based on real-time PCR analysis, were downregulated in GBM cells. The overexpression of miR-708 inhibited cell proliferation and invasion and induced apoptosis in the human GBM cell lines A172 and T98G. Furthermore, the overexpression of miR-708 reduced the expression of Akt1, CCND1, MMP2, EZH2, Parp-1 and Bcl2 in A172 and T98G cells. Taken together, our study suggests that miR-708 affects GBM cell proliferation and invasion, and induces apoptosis. It is suggested that miR-708 may play an important role as a tumor suppressor in GBM and it may be an attractive target for therapeutic intervention in GBM.

A transfection method for short interfering RNA with the lipid-like self-assembling nanotube, A6K

The aim of the present study was to develop a novel transfection method for short interfering RNA (siRNA). A nanotube with surfactant activity, A6K, consisting of six alanine residues and a hydrophilic head, lysine, was compared to the conventional cationic transfectant reagents siFECTOR and Lipofectamine 2000. Cytotoxicity for the human glioblastoma cell lines U87MG, A172, and T98G was examined with the MTS assay. Transfection efficiency was analyzed with FITC-labeled siRNA targeting matrix metalloproteinase (MMP)-2 mRNA by fluorescent activity on microscopy. The ultrastructure of A6K was evaluated by electron microscopy. The level of cytotoxicity associated with A6K in the U87MG cells was significantly lower than with siFECTOR and Lipofectamine 2000. Transfection efficiency for siRNA was increased in a dose- and time-dependent fashion. The relative expression of MMP-2 mRNA to β-actin was reduced in a dose-dependent manner by real-time RT-PCR analysis. The ultrastructure of the A6K was transformed to micelle formation when mixed with the siRNA. The lipid-like self-assembling peptide, A6K, has genes in the micelle associated with the hydrophilic tail. This transfection method is a novel and stable technique with lower cytotoxicity than the current standard methods.

Protease-activated receptor-2 (PAR2) mediates VEGF production through the ERK1/2 pathway in human glioblastoma cell lines

Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.

PO-79 Protease-activated receptor-2 (PAR2) mediates VEGF production through the ERK1/2 pathway in human glioblastoma cell lines

Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.

Effect of Rosehip (&amp;lt;i&amp;gt;Rosa Canina&amp;lt;/i&amp;gt;) Extracts on Human Brain Tumor Cell Proliferation and Apoptosis

Rosehips are blossoms from the wild rose (Rosa canina) and are commonly used as an herbal remedy. Previous reports have shown that extracts made from rosehip plants are able to reduce cell proliferation of cancer cells. In this study, we investigated the efficacy of rosehip extracts in preventing cell proliferation of three human glioblastoma cell lines A-172, U-251 MG and U-1242 MG cell lines. Each of the glioblastoma cell lines treated with rosehip extracts (1 mg/mL-25 ng/mL) demonstrated a significant decrease in cell proliferation. The rosehip extract-mediated decrease in cell proliferation was equal to or better than the decrease of cell proliferation observed when inhibitors of the MAPK (U0126, 10 μM) or AKT (LY294002, 20 μM) signaling pathways were utilized. Additionally, pretreatment of the these cell lines with Rosehip extracts (1 mg/mL-25 ng/mL) selectively decreased AKT, MAPK, and p70S6K phosphorylation suggesting these extracts prevent glioblastoma multiforme cell proliferation by blocking both the MAPK and AKT signaling mechanisms. Results from colorimetric cell death assays, cell cycle analysis by flow cytometry, as well as western blot studies demonstrate that rosehip extracts inhibit cell proliferation but do not promote apoptosis. Moreover, rosehip extracts were able to increase the efficacy of Temozolomide, a chemotherapeutic agent used to treat patients with glioblastomas. Surprisingly, rosehip extracts demonstrated a greater inhibition of cell proliferation than in combination with Temozolomide (100 μM) or Temozolomide as a single agent. Taken together these data suggest that rosehip extracts are capable of decreasing glioblastoma cell proliferation without promoting apoptosis and demonstrate a greater cell proliferation inhibitory effect than Temozolomide. More importantly, rosehip extracts may serve as an alternative or compliment to current chemotherapeutic regimens for glioblastomas.

Influence of miR -451 on biological characters of glioma cell line A172

Objective To investigate the influence of miR -451 on biological characters of gliomas cells A172. Method The oligonucleotide of miR -451 mimics was transfected into human glioblastoma cell line A172. The expression of miRNA -451 was identified by real - time PCR after transfection, and targeting protein expression was evaluated by Western blot. The cell proliferation was determined by flow cytometry and MTT assay. Moreover,cell cycle molecules were detected by Western blot analysis. Results The miR -451was markedly up -regulated after transfection of miRNA -451 mimics group. As compared with control and scramble group, the cell cycle was significantly arrested over 17.4% in G0/G1 phase by flow cytometry assay,meanwhile the protein expression of C - myc and regulatory molecules for cell cycle progression including CDK2, CDK4, Cyclin D1 and Cyclin E were all down -regulated. The proliferation was significantly decreased by the MTT assay at the same time. Conclusions MiR - 451 could inhibit the proliferation activity of A172 glioma cell. It suggests that miR -451 is a tumor suppressor gene in glioma. Key words: MicroRNA -451; Glioma; Cell proliferation; Cell cycle

Characterization of inhibitors of phosphodiesterase 1C on a human cellular system

Different inhibitors of the Ca(2+)/calmodulin-stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured K(m) values (cAMP, 1.7 microm; cGMP, 1.3 microm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca(2+) ionophore ionomycin increases intracellular Ca(2+) in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca(2+) concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca(2+)-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration-dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 microm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodiesterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin-induced decline of forskolin-induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1-mediated functions.