sincalide and Glioma

Short stature homeobox 2 (SHOX2) is significant gene in the development and progression of multiple types of tumors. Nonetheless, the biological role of SHOX2 within pan-cancer datasets has not been investigated. Thus, comprehensive bioinformatics analyses of pan-cancer datasets were conducted to explore how SHOX2 regulates tumorigenesis.. A variety of tumor datasets and online analytical tools, including SangerBox, TIMER2, LinkedOmic, GEPIA2 and cBioPortal, were applied to explore SHOX2 expression in various tumors. To ascertain the connections between SHOX2 expression and genetic alterations, SHOX2-related genes and tumor immunity, the pan-cancer datasets were examined. In vitro assays were applied to verify the biological functions of SHOX2 in glioma cells via CCK-8, wound healing, Transwell and colony formation assays.. Analyses found that SHOX2 was overexpressed in multiple cancer types. SHOX2 expression level was significantly correlated with isocitrate dehydrogenase (IDH), 1p/19q, O. SHOX2 was overexpressed in multiple cancer types in TCGA cohort. SHOX2 knockdown inhibited glioma cell proliferation, migration and colony formation ability. Our study showed that SHOX2 may be an immunotherapeutic and promising prognostic biomarker in certain types of tumors.

Topics: Biomarkers; Cell Division; Glioma; Homeodomain Proteins; Humans; Prognosis; Sincalide

The prognosis for the WHO grade 4 IDH-mutant astrocytoma is better than IDH-wildtype glioblastoma (GBM) patients. The purpose of this study is to explore the potential mechanism of how IDH1 mutation can increase the efficacy of radiotherapy and to establish a risk-score model to predict the efficacy of radiotherapy in WHO grade 4 gliomas. First, we conducted experimental study on the effect of IDH1

Topics: Brain Neoplasms; Calmodulin-Binding Proteins; Glioblastoma; Glioma; Humans; Isocitrate Dehydrogenase; Mutation; Prognosis; Sincalide; World Health Organization

Previous studies have suggested an important role for N6-methyladenosine (m6A) modification in the proliferation of glioma cells. N6, 2'-O-dimethyladenosine (m6Am) is another methylated form affecting the fate and function of most RNA. PCIF1 has recently been identified as the sole m6Am methyltransferase in mammalian mRNA. However, it remains unknown about the role of PCIF1 in the growth and survival of glioma cells.. We constructed glioma cell lines that stably downregulated/upregulated PCIF1, established intracranial xenograft models using these cell lines, and employed the following methods for investigations: CCK-8, EdU, colony formation, flow cytometry, qRT-PCR, Western blot, and immunohistochemistry.. Downregulating PCIF1 promoted glioma cell proliferation, while overexpressing PCIF1 showed the opposite effects. Overexpression of PCIF1 blocked cell cycle progression and induced apoptosis in glioma cells, which was further confirmed by alterations in the expression of cell checkpoint proteins and apoptotic markers. Interestingly, disruption of PCIF1 methyltransferase activity slightly reversed the effect of PCIF1 overexpression on cell proliferation, but had no significant reversal effects on cell cycle progression or apoptosis. Knockdown of PCIF1 promoted the growth of gliomas, while overexpressing PCIF1 inhibited tumor growth and prolonged the survival time of tumor-bearing mice. In addition, the mRNA and protein levels of PCIF1 were gradually decreased with the increase of WHO grade in glioma tissues, but there was no significant correlation with patient survival.. These results indicated that PCIF1 played a suppressing role in glioma growth and survival, which may not entirely depend on its methyltransferase activity.

Topics: Adaptor Proteins, Signal Transducing; Animals; Brain Neoplasms; Cell Proliferation; Glioma; Humans; Mammals; Methyltransferases; Mice; Nuclear Proteins; RNA, Messenger; Sincalide

3D collagen scaffold culture is a good tool to study glioma metastasis and recurrence in vitro.. The effect of 3D collagen culture on the colony formation, the sphere formation, and drug sensitivity of glioma cells was observed by soft-agar colony formation assays, sphere formation assays, and CCK-8 assays, respectively. 3D-glioma-drug genes were identified by previous results and online databases. Gene enrichment and PPI analyses were performed by R software and Metacsape. Hub 3D-glioma-drug genes were screened by STRING and Cytoscape. TCGA and CGGA databases and R software were used to analyze the distribution of hub genes in glioma and their effects on the prognosis. Western Blot was used to verify the effect of 3D collagen culture on the expression of hub genes. miRNAs targeting hub genes were predicted by ENCORI.. 3D collagen scaffold culture promoted colony formation, sphere formation, and drug resistance of glioma cells. There were 77 3D-glioma-drug genes screened, and the pathways enriched in the protein interaction network mainly included responses to stressors, DNA damage and repair, and drug metabolism. Hub 3D-glioma-drug genes were AKT1, ATM, CASP3, CCND1, EGFR, PARP1, and TP53. These genes and predicted miRNAs were expressed differentially in glioma samples and partially affected the prognosis of patients with glioma. These findings suggested these hub genes and miRNAs may play a key role in the effects generated by the 3D culture model and become new markers for glioma diagnosis and treatment.

Topics: Agar; Caspase 3; Collagen; Drug Resistance; ErbB Receptors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; Sincalide

Echinacoside (ECH) is a phenylethanoid extracted from the stems of Cistanches salsa, an herb used in Chinese medicine formulations, and is effective against glioblastoma multiforme (GBM). Epithelial-mesenchymal transition (EMT) is the cornerstone of tumorigenesis and metastasis, and increases the malignant behavior of GBM cells. The S phase kinase-related protein 2 (skp2), an oncoprotein associated with EMT, is highly expressed in GBM and significantly associated with drug resistance, tumor grade and dismal prognosis. The aim of this study was to explore the inhibitory effects of ECH against GBM development and skp2-induced EMT.. CCK-8, EdU incorporation, transwell, colony formation and sphere formation assays were used to determine the effects of ECH on GBM cell viability, proliferation, migration and invasion in vitro. The in vivo anti-glioma effects of ECH were examined using a U87 xenograft model. The expression levels of skp2 protein, EMT-associated markers (vimentin and snail) and stemness markers (Nestin and sox2) were analyzed by immunofluorescence staining and western blotting experiments.. ECH suppressed the proliferation, invasiveness and migration of GBM cells in vitro, as well as the growth of U87 xenograft in vivo. In addition, ECH downregulated the skp2 protein, EMT-related markers (vimentin and snail) and stemness markers (sox2 and Nestin). The inhibitory effects of ECH were augmented in the skp2-knockdown GBM cells, and reversed in cells with ectopic expression of skp2.. ECH inhibits glioma development by suppressing skp2-induced EMT of GBM cells.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioblastoma; Glioma; Glycosides; Humans; Nestin; S-Phase Kinase-Associated Proteins; Sincalide; Vimentin

Numerous studies indicated that long non-coding RNAs (lncRNAs) play critical roles in glioma initiation and progression. SNHG25 is a newly identified lncRNA. And the functional role and molecular mechanism of SNHG25 in glioma cells have not been investigated. In this study, we found that SNHG25 was upregulated in glioma cells and tissues. CCK-8, EDU, and colony formation assays demonstrated that SNHG25 knockdown markedly inhibited glioma cell proliferation. In vivo studies showed that SNHG25 knockdown significantly inhibited tumor growth. Further studies indicated that SNHG25 positively regulated MAP2K2 through sponging miR-579-5p. High expression of SNHG25 activated MAPK signaling through MAP2K2. These data suggest that SNHG25 is a potential target and biomarker for glioma.

Topics: Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioma; Humans; MicroRNAs; RNA, Long Noncoding; Sincalide

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Calcium-Binding Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Glioma; Humans; Mitochondria; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Saponins; Signal Transduction; Sincalide; Starfish

MicroRNAs (miRNAs) are a class of small noncoding RNAs that play important roles in tumor development and progression. miR-20a acts as an oncogene in many cancers; however, the underlying role of miR-20a in human glioma remains unknown.. Glioma tissue samples were obtained from 32 patients with primary glioma who had undergone surgery at the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Twenty-two normal brain tissue samples used as controls were obtained by internal decompression in patients who had undergone surgery for cerebral injury and cerebral hemorrhage at the same hospital.. Quantitative reverse transcription polymerase chain reaction showed upregulation of miR-20a in glioma tissues and cell lines compared with normal brain tissue and normal human astrocytes. Functional assays showed that miR-20a promotes proliferation and invasion and inhibits apoptosis in glioma cells. The bioinformatic analysis showed that CELF2 (CUGBP Elav-like family member 2) is a direct target gene of miR-20a, which was confirmed using a luciferase reporter assay. Downregulation of CELF2 reversed the effects of inhibiting miR-20a expression.. Collectively, these results suggest a critical role for miR-20a in glioma cell apoptosis, proliferation, and invasion via the direct targeting of CELF2 and indicate its potential application in cancer therapy.

Topics: Apoptosis; Brain Neoplasms; CELF Proteins; Cell Line, Tumor; Cell Proliferation; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioma; Humans; In Situ Nick-End Labeling; Male; MicroRNAs; Neoplasm Invasiveness; Nerve Tissue Proteins; RNA, Messenger; RNA, Small Interfering; Sincalide; Transfection

Glioma is a highly aggressive and lethal brain tumor. Signal transducers and activators of transcription (STAT) pathway are widely implicated in glioma carcinogenesis. Our previous study found that the Fynrelated kinase (FRK) gene, plays as a tumor suppressor in the development and progression of glioma. This study aimed to investigate the role of FRK in the activation pathway of STATs and its effect on the growth of glioma.. The U251 and U87 cells with stable FRK overexpression were generated by lentivirus technique. The effects of FRK on the related proteins of STAT signaling pathway were detected by western blotting. Coimmunoprecipitation was used to detect the association of FRK and STAT1. The effects of STAT1 on the proliferation of glioma cells were detected by CCK8 or Edu cell proliferation assays. The expressions and correlation of FRK and p-STAT1 in glioma tissues were detectd by western blotting or immunohistochemistry. The effect of FRK on the growth of glioma was investigated in vivo mouse model.. The level of p-JAK2 and p-STAT1 increased after FRK overexpression, while they decreased after FRK downregulation both in U251 and U87 cells. However, FRK had no effect on STAT3 phosphorylation. FRK-induced STAT1 activation was not dependent on JAK2. FRK associated with STAT1, induced STAT1 nuclear translocation and regulated the expressions of STAT1-related target genes. STAT1 overexpression suppressed the proliferation of glioma cells. In contrast, STAT1 knockdown by siRNA promoted glioma cell growth. Importantly, down-regulation of STAT1 partially attenuated FRK-induced growth suppression. The clinical sample-based study indicated that the expression of FRK was significantly correlated with the expression of p-STAT1. FRK significantly inhibited glioma tumor growth in vivo.. Our findings highlighted a critical role of FRK in tumor suppression ability through promoting STAT1 activation, and provided a potential therapeutic target for glioma.

Topics: Active Transport, Cell Nucleus; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Disease Progression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioma; HEK293 Cells; Humans; Janus Kinase 2; Male; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Phosphorylation; Protein-Tyrosine Kinases; Signal Transduction; Sincalide; STAT1 Transcription Factor; Tumor Burden

Gliomas are highly malignant tumors, the most common of which are astrocytomas. A growing number of studies suggest that dysregulation of miRNAs is a frequent event contributing to the pathogenesis of gliomas. In this study, we found that over-expression of miR-132 inhibited cell proliferation and migration and triggered apoptosis, while knockdown of miR-132 showed opposite effects. PEA-15 was identified as a direct target of miR-132. Reintroduction of PEA-15 without 3'UTR region reversed the inhibitory effects of miR-132 on cell proliferation, migration, and apoptosis. MiR-132 was inversely correlated with the PEA-15 expression. CREB (cAMP response element binding protein) and KLF (Krüppel-like factor 8) were conformed as transcription factors of miR-132, which bidirectionally regulate the expression of miR-132. Our study suggests that miR-132 is an important tumor suppressor of astrocytoma progression by targeting PEA-15, while CREB and KLF can modulate the expression of miR-132, thus providing new insight into the molecular mechanisms underlying astrocytoma progression in vitro.

Topics: Analysis of Variance; Apoptosis; Apoptosis Regulatory Proteins; Astrocytoma; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; CREB-Binding Protein; Disease Progression; Gene Expression Regulation, Neoplastic; Glioma; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Kruppel-Like Transcription Factors; MicroRNAs; Phosphoproteins; Repressor Proteins; RNA, Messenger; Sincalide; Time Factors; Transfection

Extensive oxidative stress has been considered a primary pathological factor for many neurodegenerative disorders (NDDs). We speculated that the oxidative damage to brain cells can be managed by promoting the endogenous cellular antioxidants through the RNA interference (RNAi) against Keap1 (kelch-like ECH-associated protein). Keap1 acts as a negative regulator of Nrf2 (NF-E2-related factor 2) that represses the activation of the antioxidant responsive element (ARE). Here, we investigated whether Keap1 knockdown enhances the cellular antioxidant capacity and provides the neuroprotection against oxidative stress from hydrogen peroxide and beta-amyloid (Aβ) peptide in U87mg cells. We found that the Keap1 siRNA pre-treated group displayed higher expression of diverse antioxidant genes and an increased antioxidant capacity compared to the control group. Moreover, the Keap1 RNAi exerted a cytoprotective effect against H2O2 treatment. In Aβ peptide treatment experiments, the Keap1 siRNA pre-treated groups maintained acceptable cell viability, relatively intact cellular morphology, and controlled oxidative damage levels while the control groups suffered from Aβ peptide-mediated neurotoxicity. Keap1 RNAi also attenuated the oxidative stress-mediated autophagy as well. These findings suggest that Keap1 RNAi can serve as a therapeutic strategy for relieving oxidative stress-associated symptoms in many NDDs.

Topics: Amyloid beta-Peptides; Analysis of Variance; Autophagy; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioma; Humans; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Kelch-Like ECH-Associated Protein 1; Malondialdehyde; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidants; Oxidative Stress; Peptide Fragments; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sincalide; Superoxide Dismutase; Superoxide Dismutase-1

RBQ3, also known as RBBP5 (RB-binding protein 5), was an RB-binding protein. Besides, it was one of core components of MLL1 (mixed lineage leukemia 1), which were required for H3K4 methyltransferase activity. MLL1 dysfunction was found to be associated with the progression of some cancers such as acute leukemias. However, the precise role of RBQ3 in tumor progression remains obscure. In this study, we explored the expression and clinical role of RBQ3 in gliomas. Our results showed that RBQ3 was significantly upregulated in clinical glioma specimens by Western blot and immunohistochemistry. Moreover, its level was significantly associated with the pathology grades. High RBQ3 expression was suggested to be an independent prognostic factor for glioma patients' survival by univariate and multivariate analyses. Serum starvation and refeeding assay indicated that the expression of RBQ3 increased 8h after serum-stimulation, together with percentage of cells at S phase. In addition, knockdown of RBQ inhibited U87-MG cell proliferation with CCK8 kit, flow cytometry assays and colony formation analyses; while the depletion of RBQ3 induced the apoptosis of U87-MG cells. All the findings suggested that RBQ3 might play an important role in glioma, and RBQ3 inhibitors might be novel anti-tumor agents.

Topics: Adult; Apoptosis; Brain; Brain Neoplasms; Cell Line, Tumor; Chi-Square Distribution; Colony-Forming Units Assay; Culture Media, Serum-Free; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Ki-67 Antigen; Male; Middle Aged; Nuclear Proteins; RNA, Small Interfering; Sincalide; Transfection

Studies have shown that several miRNAs play important roles in regulating a variety of cellular processes in gliomas. In these reports, upregulation of miR-193b has been found to be associated with a poor prognosis for glioma, but its functional mechanism in glioma remains unclear. This study investigates the roles of miR-193b in glioma tumor growth. We first showed that the expression of miR-193b was elevated in both glioma samples and glioma cells. Furthermore, downregulation of miR-193b by inhibitors was statistically correlated with a decrease in cell growth and a restored G1 accumulation. Luciferase assay and Western blot analysis revealed that Smad3 is a direct target of miR-193b. To prove that miR-193b regulated cell growth through the transforming growth factor-β (TGF-β) pathway in glioma cells by regulating Smad3, we tested endogenous targets of the TGF-β pathway by measuring the accumulation of p21 mRNAs after downregulation of miR-193b. The results confirmed that induction of p21 was promoted by miR-193b inhibitors in glioma cells, although this induction disappeared when Smad3 was knocked down with siRNA. Moreover, downregulation of Smad3 mitigates the miR-193b suppression of glioma proliferation. In conclusion, these results suggest that miR-193b regulated cell growth in glioma through the TGF-β pathway by regulating Smad3. Thus, our study indicates that miR-193b promotes cell proliferation by targeting Smad3 in human glioma, which may serve as a potentially useful target for development of miRNA-based therapies in the future.

Topics: Adult; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Glioma; HEK293 Cells; Humans; Male; MicroRNAs; Oligonucleotides; RNA, Small Interfering; Sincalide; Smad3 Protein; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Young Adult

The purposes of our study were to elucidate the role of BRG1 in the development of human glioma and to determine the effect of BRG1 on glioma cell growth, migration and invasion.. Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 190 glioma tissues, 8 normal brain tissues and 8 tumor adjacent normal brain tissues. We studied glioma cell proliferative ability with reduced BRG1 expression by siRNA using CCK-8 cell proliferation assay and cell cycle analysis. We studied the role of BRG1 in glioma cell migration and invasion by cell migration assay and matrigel invasion assay. We performed western blot to detect cyclin D1, cyclin B1 and MMP-2 protein expression. We also detected MMP-2 enzyme activity by gelatin zymography.. Our results showed that BRG1 expression was increased in benign tumor and malignant tumor compared with tumor adjacent normal brain tissue (P < 0.01 for both). We did not find any correlation between BRG1 expression and clinicopathological parameters. In addition, we found that knockdown of BRG1 in glioma cell lines inhibits cell growth due to the G1 phase arrest by downregulating cyclin D1. We further demonstrated that silencing of BRG1 in glioma cells inhibited the cell migration and invasion abilities, and downregulation of MMP-2 expression greatly contributed to the reduced cell invasion and migration abilities.. Our data indicated that BRG1 expression is significantly increased in human glioma and it may be involved in the process of glioma cell proliferation, migration and invasion.

Topics: Brain Neoplasms; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Cyclin B1; Cyclin D1; DNA Helicases; Female; G1 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Gene Silencing; Glioma; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Invasiveness; Nuclear Proteins; RNA, Small Interfering; Sincalide; Transcription Factors

Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma C6 cells with CCK-8S resulted in activation of protein kinase C isozymes betaI, betaII, gamma and zeta. The participation of protein kinase C in the CCK-8S-induced effect on C6 cell growth was demonstrated by measurement of [3H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C6 cells by a protein kinase C-dependent mechanism.

Topics: Animals; Blotting, Western; Carcinogens; Enzyme Activation; Glioma; Isoenzymes; Protein Kinase C; Protein Kinase C beta; Rats; Signal Transduction; Sincalide; Tetradecanoylphorbol Acetate; Thymidine; Tritium; Tumor Cells, Cultured

Cholecystokinin (CCK) is known to stimulate cell proliferation but involvement of CCKB type receptors has not been exactly demonstrated so far. We examined the effect of CCK-8S and two receptor agonists on rat glioma C6 cells when using different CCKB receptor agonists and antagonists. Both CCK-8S and CCKB receptor agonists BC 264 and Suc-Trp-N(Me)Nle-Asp-Phe-NH2 stimulate [3H]thymidine incorporation. These effects were inhibited by CCKB receptor antagonist L-365,260 over 100-fold more effectively than it was seen by using CCKA receptor antagonist L-364,718. The data indicate that CCKB receptor agonists are potent stimulants of rat glioma C6 cell DNA synthesis suggesting that CCKB receptor activation is involved in cell proliferation within the central nervous system.

Topics: Amino Acid Sequence; Animals; Benzodiazepinones; Cell Division; Cholecystokinin; Devazepide; DNA; Glioma; Hormone Antagonists; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Phenylurea Compounds; Rats; Receptors, Cholecystokinin; Sincalide; Thymidine; Tritium; Tumor Cells, Cultured

We investigated the effect of sulphated cholecystokinin octapeptide (CCK-8S) on free intracellular calcium concentration, phosphatidylinositol metabolism, and protein phosphorylation in C6 cells, a rat glioma cell line which was shown to express CCKB type receptors. Increase in [Ca2+]i by both influx across the cell membrane and release from internal stores was demonstrated by utilizing a laser confocal imaging system. Because CCK-8S produced a transient elevation of inositol triphosphate level participation of InsP3 in calcium signaling in C6 cells is very likely. Protein kinase C seems to be involved in CCK-8S induced signaling in rat glioma C6 cells as demonstrated by using in vivo phosphorylation experiments.

Topics: Animals; Calcium; Electrophoresis, Polyacrylamide Gel; Glioma; Inositol Phosphates; Phosphatidylinositols; Phosphoproteins; Phosphorylation; Protein Kinase C; Rats; Signal Transduction; Sincalide; Tumor Cells, Cultured

The present study was undertaken to compare binding characteristics of CCKB-type receptors in guinea-pig cortex, Jurkat T-cells, GH3 cells and C6 cells. The rank order of potency of a variety of CCK agonists and antagonists in inhibiting specific [3H]CCK-8S binding was highly correlated for the 4 CCKB receptor models as demonstrated by a computer-assisted statistical analysis. Taking the ligand binding profiles as the criterion it is concluded that CCKB receptors in guinea-pig cortex, Jurkat T-cells, pituitary GH3 cells and rat glioma C6 cells share identical pharmacological properties.

Topics: Animals; Binding, Competitive; Cell Line; Cerebral Cortex; Cholecystokinin; Glioma; Guinea Pigs; Kinetics; Pituitary Gland, Anterior; Rats; Receptors, Cholecystokinin; Sincalide; Structure-Activity Relationship; Tritium; Tumor Cells, Cultured

To our knowledge, no brain derived cell line has been shown as yet to bear cholecystokinin(CCK)B receptors. In this paper, CCK binding sites were identified on rat C6 glioma cells. Pharmacological characterization demonstrated a single class of high affinity binding sites (Kd = 1.7 +/- 0.3 x 10(-10) M) and a binding capacity of 6.1 +/- 1.8 fmol/mg protein. These CCK binding sites displayed a typical CCKB pharmacological profile as shown in competition studies by using several CCK-related compounds and nonpeptide CCK antagonists discriminating between CCKA and CCKB sites. In order to demonstrate that CCK binding sites constitute a functional receptor CCK-8S induced mobilization of free intracellular calcium was investigated in single C6 cells by using a laser scanning confocal imaging system. Since rise in [Ca2+]i noted by stimulation of C6 cells with CCK-8S could be blocked by the CCKB receptor antagonist L-365,260 (100 nM) but not by the CCKA receptor antagonist L-364,718 (100 nM), CCK induced calcium signal is triggered by activation of CCKB receptors in C6 cells. The rat C6 glioma cell line may serve as a useful model for studying CCKB receptor in brain.

Topics: Animals; Binding Sites; Biological Transport; Calcium; Glioma; Indicators and Reagents; Intracellular Membranes; Rats; Receptors, Cholecystokinin; Sincalide; Succinimides; Tumor Cells, Cultured