Antimigratory effect of berberine in T98G, U87MG and primary glioma cell culture.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15045-e15045
Author(s):  
Irina V. Mezhevova ◽  
Svetlana Yu. Filippova ◽  
Sofia V. Timofeeva ◽  
Anastasia O. Sitkovskaya ◽  
Tatiana V. Shamova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Cell Cultures ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Surgical Removal ◽  
Experimental Sample ◽  
Wound Area ◽  
Migratory Activity ◽  
Primary Glioma

e15045 Background: Berberine is an alkaloid compound with a structure that is highly similar to that of intercalating agents. It affects numerous cell signaling pathways and is widely studied as potential anticancer drug. It is known that berberine affects cancer cells migration through metalloproteinase-2 inhibition, but this effect was never studied on glioma cells. Anti-migratory drugs are of special interest in brain cancer therapy since glioma's highly invasive nature makes total surgical removal of tumor practically impossible. The aim of the study was to evaluate berberine anti-migratory activity on glioma cells. Methods: Cell migration capacity of T98G and U87MG cell lines, as well as primary glioma cell culture established in our laboratory, was assessed via standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Prior to assay setting up cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. Cells were seeded at 250 000 cells per well on 24-well plates and incubated overnight in order to attach to plate bottom. After that a vertical wound was made manually in each well, and berberine was added to experimental wells to final concentration 50 mg/L. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 24 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: In our study we berberine exhibited anti-migratory activity in all cell cultures under study. In rather fast growing primary cell culture wound area decrease was 99.23%±0.62% in control sample and 91.75%±0.28% in experimental sample. The difference was small but significant at p < 0.001 level (df = 30). Popular permanent glioma cell lines T98G and U87MG showed more prominent decrease in studied parameter with higher degree of variance at the same time. In T98G wound area decrease was 71.6%±12.3% in control and 48.8%± 7.6% in experimental samples after 24 hours of cultivation in presence of 50 mg/L berberine. While U87MG demonstrated 60.28%±5.13% and 37.5%± 8.34% wound area decrease accordingly. The obtained difference between control and experimental groups in permanent cell cultures was statistically significant at the 0.05 level (df = 30). Conclusions: Our preliminary research proved berberine to be potent anti-migratory agent in glioma treatment. Further investigations are needed to evaluate its ability to inhibit glioma cell expansion in vivo.

scholarly journals An E3 Ubiquitin Ligase RNF139 Serves as a Tumor-Suppressor in Glioma

2021 ◽  
Author(s):  
Xiaofeng Chen ◽  
Weiping Kuang ◽  
Yong Zhu ◽  
Bin Zhou ◽  
Xiaosong Li ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Ubiquitin Ligase ◽  
Glioma Cell ◽  
Protein Modification ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Tissue Samples

AbstractGlioma is highly lethal because of its high malignancy. Ubiquitination, a type of ubiquitin-dependent protein modification, has been reported to play an oncogenic or tumor-suppressive role in glioma development, depending on the targets. Ring finger protein 139 (RNF139) is a membrane-bound E3 ubiquitin ligase serving as a tumor suppressor by ubiquitylation-dependently suppressing cell growth. Herein, we firstly confirmed the abnormal downregulation of RNF139 in glioma tissues and cell lines. In glioma cells, ectopic RNF139 overexpression could inhibit, whereas RNF139 knockdown could aggravate the aggressive behaviors of glioma cells, including hyperproliferation, migration, and invasion. Moreover, in two glioma cell lines, RNF139 overexpression inhibited, whereas RNF139 knockdown enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI3K) and AKT serine/threonine kinase 1 (AKT). In a word, we demonstrate the aberration in RNF139 expression in glioma tissue samples and cell lines. RNF139 serves as a tumor-suppressor in glioma by inhibiting glioma cell proliferation, migration, and invasion and promoting glioma cell apoptosis through regulating PI3K/AKT signaling.

scholarly journals LncRNA-XIST interacts with miR-29c to modulate the chemoresistance of glioma cell to TMZ through DNA mismatch repair pathway

2017 ◽  
Vol 37 (5) ◽  
Author(s):  
Peng Du ◽  
Haiting Zhao ◽  
Renjun Peng ◽  
Qing Liu ◽  
Jian Yuan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mismatch Repair ◽  
Cell Lines ◽  
Glioma Cell ◽  
Dna Mismatch Repair ◽  
Tumor Biology ◽  
Glioma Cells ◽  
Dna Mismatch ◽  
Cell Proliferation Inhibition ◽  
Glioma Cell Lines

Temozolomide (TMZ) is the most commonly used alkylating agent in glioma chemotherapy. However, growing resistance to TMZ remains a major challenge for clinicians. Recent evidence emphasizes the key regulatory roles of non-coding RNAs (lncRNAs and miRNAs) in tumor biology, including the chemoresistance of cancers. However, little is known about the role and regulation mechanisms of lncRNA cancer X-inactive specific transcripts (XIST) in glioma tumorigenesis and chemotherapy resistance. In the present study, higher XIST expression was observed in glioma tissues and cell lines, which was related to poorer clinicopathologic features and shorter survival time. XIST knockdown alone was sufficient to inhibit glioma cell proliferation and to amplify TMZ-induced cell proliferation inhibition. Moreover, XIST knockdown can sensitize TMZ-resistant glioma cells to TMZ. XIST can inhibit miR-29c expression by directly targetting TMZ-resistant glioma cells. DNA repair protein O6-methylguanine-DNA methytransferase (MGMT) plays a key role in TMZ resistance; transcription factor specificity protein 1 (SP1), a regulator of DNA mismatch repair (MMR) key protein MSH6, has been reported to be up-regulated in TMZ-resistant glioma cell lines. In the present study, we show that XIST/miR-29c coregulates SP1 and MGMT expression in TMZ-resistant glioma cell lines. Our data suggest that XIST can amplify the chemoresistance of glioma cell lines to TMZ through directly targetting miR-29c via SP1 and MGMT. XIST/miR-29c may be a potential therapeutic target for glioma treatment.

Correlation of DNA ploidy and morphological features of human glioma cell cultures with the establishment of cell lines

1988 ◽  
Vol 76 (5) ◽  
pp. 433-440 ◽  
Author(s):  
K. Onda ◽  
R. Tanaka ◽  
K. Washiyama ◽  
N. Takeda ◽  
T. Kumanishi
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Glioma Cell ◽  
Dna Ploidy ◽  
Morphological Features ◽  
Human Glioma ◽  
Human Glioma Cell

scholarly journals Casein Kinase 2 Interacting Protein-1 Suppresses Glioma Cell Proliferation via Regulating the AKT/GSK3β/β-Catenin Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Yan-Guo Xi ◽  
Deng-Peng Ren ◽  
Jing-Yun Jin ◽  
Lei Zhu ◽  
Tai-Long Yi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Brdu Incorporation ◽  
Rank Test ◽  
Interacting Protein ◽  
Glioma Cell Proliferation

Objective. Casein kinase 2 interacting protein-1 (CKIP-1) has exhibited multiple functions in regulating cell proliferation, apoptosis, differentiation, and cytoskeleton. CKIP-1 also plays an important role as a critical regulator in tumorigenesis. The aim of this study is to further examine the function of CKIP-1 in glioma cells. Methods. The expression level of CKIP-1 protein was determined in gliomas tissues and cell lines by immunohistochemistry stain and western blotting while the association of CKIP-1 expression with prognosis was analyzed by Kaplan-Meier method and compared by log-rank test. CKIP-1 was overexpressed or silenced in gliomas cell lines. CCK-8, colony formation assay, and BrdU incorporation assay were used to determine cell proliferation and DNA synthesis. Cell cycle and apoptosis rate were determined with fluorescence-activated cell sorting (FACS) method. Then, expression of key members in AKT/GSK3β/β-catenin pathway was detected by western blot analysis. Results. In the present study, we reported new evidence that CKIP-1 was reversely associated with the proliferation of glioma cells and survival in glioma patients. Additionally, the overexpressed CKIP-1 significantly inhibited glioma cell proliferation. Further experiments revealed that CKIP-1 functioned through its antiproliferative and proapoptotic activity in glioma cells. Importantly, mechanistic investigations suggested that CKIP-1 sharply suppressed the activity of AKT by inhibiting the phosphorylation, markedly downregulated the phosphorylated GSK3β at Ser9, and promoted β-catenin degradation. Conclusions. Overall, our results provided new insights into the clinical significance and molecular mechanism of CKIP-1 in glioma, which indicated CKIP1 might function as a therapeutic target for clinical treatment of glioma.

scholarly journals 2036 Extracellular matrix as a novel approach to glioma therapy

2018 ◽  
Vol 2 (S1) ◽  
pp. 11-12
Author(s):  
Mark H. Murdock ◽  
Jordan T. Chang ◽  
George S. Hussey ◽  
Nduka M. Amankulor ◽  
Johnathan A. Engh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Urinary Bladder ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Time Lapse ◽  
Cell Types ◽  
Neoplastic Cells ◽  
Novel Approach ◽  
Primary Glioma

OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and common primary tumor type in the central nervous system across all age groups; affected adults have a life expectancy of just 14 months. As glioma cells invade the surrounding normal parenchyma they remodel the composition and ultrastructure of the surrounding extracellular matrix (ECM), suggesting that the native (i.e., “normal”) microenvironment is not ideal for their survival and proliferation. Recent reports describe suppressive and/or lethal effects of mammalian ECM hydrogels derived from normal (nonneoplastic) sources upon various cancer types. ECM-based bioscaffolds placed at sites of neoplastic tissue resection in humans have never been reported to facilitate cancer recurrence. The objective of the present research is to evaluate mammalian ECM as a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECM hydrogels from porcine dermis, small intestine, and urinary bladder were produced as described previously. Primary glioma cells were graciously supplied by Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered through ATCC. Cells were plated onto tissue culture plastic at ~60% confluence and allowed to attach for 24 hours before treatment. The saline-soluble fraction (SSF) of ECM was obtained by mixing lyophilized, comminuted ECM with 0.9% saline for 24 hours then filtering the resulting mixture through a 10 kDa molecular weight cutoff column. All assays and kits were followed according to the manufacturer’s instructions. Cell viability was measured via MTT assay (Vybrant® MTT Cell Proliferation Assay, Invitrogen) and by live/dead staining (LIVE/DEAD® Cell Imaging Kit, Invitrogen). Time lapse videos were created by taking images every 20 minutes for 18 hours (phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagent was ordered from Biotium. Temozolomide was ordered through Abmole. All in vivo work was conducted according to protocols approved by the University of Pittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS: ECM hydrogels derived from porcine dermis, small intestine, or urinary bladder all decreased the viability of primary glioma cells in vitro, with urinary bladder extracellular matrix (UBM) having the most dramatic effects. The SSF of UBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, was sufficient to recapitulate this detrimental effect upon neoplastic cells in vitro and was used for the remainder of the experiments described herein. In a cell viability assay normalized to the media treatment, non-neoplastic CHME5 and N1E-115 cells scored 103% and 114% after 48 hours when treated with UBM-SSF and 2 primary high-grade glioma cell types scored 17% and 30.5% with UBM-SSF (n=2). Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence of UBM-SSF for 18 hours while most primary glioma cells shriveled and died within this time. Darkfield time-lapse video of wells containing Nucview dye, fluorescent upon cleavage by active caspase-3, confirmed that within 12 hours most primary glioma cells underwent apoptosis while CHME5 and HFF did not. In culture with primary astrocytes, high grade primary glioma cells, and U-87 MG glioma cells for 24 hours, UBM-SSF was found to significantly increase the population of primary astrocytes compared with media (p<0.05) while decreasing the 2 glioma cell types to approximately one-third as many cells as the media control (p<0.0001). A dose-response of temozolomide from 0 to 10,000 μM showed that when treating 2 non-neoplastic cell types (CHME5 and HFF) and 2 types of primary glioma cell there was no difference in survivability at any concentration. Contrasted to this, a dose-response of UBM-SSF from 350 to 7000 μg/mL showed that the non-neoplastic cells survived significantly better than the glioma cells at concentrations of 875 μg/mL and upward (p<0.05). In preliminary animal experiments, large primary glioma tumors in the flanks of athymic nude mice were resected and replaced with either UBM SSF or Matrigel (an ECM product of neoplastic cell origin). After 7 days the resection sites with UBM-SSF had little tumor regrowth if any compared with the dramatic recurrence seen in the Matrigel injection sites (n=2). In a separate survival study comparing PBS to UBM-SSF injections in the flank-resection model, all animals given PBS had to be sacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSF were sacrificed at 15, 24, and 39 days (n=3), indicating a moderate increase in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OF IMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZ in 2005, the standard of care for patients with glioblastoma multiforme has not improved. These findings indicate that non-neoplastic ECM contains potent bioactive regulators capable of abrogating malignancy. Our in vitro data suggest these molecules appear to have no deleterious effect on non-neoplastic cells while specifically inducing apoptosis in glioma cells. Our in vivo data suggest that these molecules may be useful in delaying glioma recurrence, thus resulting in extended lifespan. Delivering soluble fractions of ECM to a tumor site may represent a novel approach to glioma therapy, sidestepping traditional cytotoxic therapies in favor of utilizing putative endogenous anti-tumor pathways.

Tumor necrosis factor–related apoptosis-inducing ligand–mediated apoptosis in established and primary glioma cell lines

2002 ◽  
Vol 13 (3) ◽  
pp. 1-11 ◽  
Author(s):  
Jay Jaganathan ◽  
Joshua H. Petit ◽  
Barbara E. Lazio ◽  
Satyendra K. Singh ◽  
Lawrence S. Chin
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Cell Lines ◽  
Glioma Cell ◽  
Apoptotic Cell ◽  
Glioma Cells ◽  
Trail Receptors ◽  
Glioma Cell Lines ◽  
Caspase 7 ◽  
U373 Cells

Object Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF cytokine family, which mediates programmed cell death (apoptosis) selectively in tumor cells. The selective tumoricidal activity of TRAIL is believed to be modulated by agonistic (DR4 and DR5) and antagonistic receptors (DcR1 and DcR2), which appear to compete for ligand binding. Because TRAIL is expressed in a wide range of tissues, including brain, kidney, and spleen, and seems consistently to induce cell death in tumor cells, the cytokine has been identified as a promising approach for selectively inducing tumor cell death. In this study, the authors examine the importance of TRAIL's receptors in both its selectivity for tumor cells and its ability to induce apoptosis. Methods The authors first examined sensitivity to TRAIL and expression of TRAIL receptors in four established and four primary cultured glioma cell lines by using viability and fluorescent apoptosis assays. They then evaluated DR5 expression and JNK, caspase 3, and caspase 7 activation by conducting immunoblot analyses. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to study expression of DR4, DR5, DcR1, and DcR2. The DR5 transcripts from one TRAIL-sensitive, one partially TRAIL-resistant, and one TRAIL-resistant cell line were subsequently sequenced. The expression of TRAIL receptors in normal and glial brain tumor pathological specimens were then compared using immunohistochemistry. Finally, to study the direct effects of DR5 on glioma cells, the authors conducted transient and stable transfections of the fulllength DR5 transcript into glioma cells with and without preestablished overexpression of the antiapoptotic gene bcl-2. The established glioma cell lines T98G and U87MG, and all primary cell lines, were apoptotic at greater than or equal to 100 ng/ml TRAIL. The A172 cells, by contrast, were susceptible only with cycloheximide, whereas U373MG cells were not susceptible to TRAIL. The JNK, caspase 3, and caspase 7 activity evaluated after treatment with TRAIL showed that TRAIL-sensitive cell lines exhibited downstream caspase activation, whereas TRAIL-resistant cells did not. The DR5 sequences in T98G, A172, and U373MG cell lines were identical to published sequences despite these differences in sensitivity to TRAIL. The RT-PCR performed on extracts from the eight glioma cell lines showed that all expressed DR5. Immunohistochemistry revealed ubiquitous expression of DR5 in glioma specimens, with an associated lack of decoy receptor expression. Normal brain specimens, by contrast, stained positive for both DR5 and DcR1. Overexpression of DR5 under both transfection conditions resulted in cell death in all three cell lines. The previously seen resistance of U373 cells to TRAIL was not observed. Apoptotic cell death was confirmed using DNA fragmentation in T98G cell lines and fluorescent miscroscopy in all cell lines. The T98G cells stably transfected with bcl-2 before DR5 overexpression were protected from cell death. Conclusions The authors conclude that DR5 represents a promising new approach to directly activating the intrinsic caspase pathway in glioma cells. The fact that TRAIL-resistant gliomas do not express decoy receptors suggests a mechanism of resistance unique from that proposed for normal tissues. The overexpression of DR5 induced apoptotic cell death in glioma cells without TRAIL and was able to overcome the resistance to TRAIL demonstrated in U373 cells. The Bcl-2 protects cells from DR5 by acting downstream of the receptor, most likely at the level of caspase activation.

Resistance to growth inhibition by transforming growth factor—β in malignant glioma cells with functional receptors

1998 ◽  
Vol 88 (3) ◽  
pp. 529-534 ◽  
Author(s):  
Shiro Isoe ◽  
Hirofumi Naganuma ◽  
Shin Nakano ◽  
Atsushi Sasaki ◽  
Eiji Satoh ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Malignant Glioma ◽  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Malignant Glioma Cells ◽  
Tgf Β1 ◽  
Fine Print

Object. The aim of this study was to investigate the mechanism by which malignant glioma cells escape from growth inhibition mediated by transforming growth factor-β (TGF-β), a ubiquitous cytokine that inhibits cell proliferation by causing growth arrest in the G1 phase of the cell cycle. Methods. The authors measured the response of eight malignant glioma cell lines to the growth-inhibiting activity of TGF-β in vitro and the expression of TGF-β Types I and II receptors in malignant glioma cells. The effect of TGF-β on the expression of a p27Kip1 cyclin-dependent kinase inhibitor was also investigated to assess the downstream signal transmission from TGF-β receptors. All malignant glioma cell lines were insensitive to growth inhibition by TGF-β1 and TGF-β2. Analyses of TGF-β receptors by means of affinity labeling in which 125I-TGF-β1 was used showed that six glioma lines had both TGF-β Types I and II receptors on their cell surfaces, whereas two lines had very small amounts of TGF-β Type I and/or Type II receptors. Northern blot analysis showed that all tumor lines expressed variable levels of messenger RNAs for both TGF-β Types I and II receptors. Flow cytometric analyses revealed that treatment of malignant glioma cells with TGF-β1 significantly downregulated the expression of p27Kip1 protein in all malignant glioma cell lines except one. Conclusions. The authors suggest that most malignant glioma cells express TGF-β Types I and II receptors, which can transmit some signals downstream and that the loss of response to TGF-β growth inhibition may not be caused by an abnormality of the TGF-β receptors.

Differential effects of vincristine and phenytoin on the proliferation, migration, and invasion of human glioma cell lines

1995 ◽  
Vol 82 (6) ◽  
pp. 1035-1043 ◽  
Author(s):  
Jörg-Christian Tonn ◽  
Hans Kristian Haugland ◽  
Jaakko Saraste ◽  
Klaus Roosen ◽  
Ole Didrik Laerum
Keyword(s):  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Dose Dependent ◽  
Fine Print

✓ The aim of this study was to investigate the antimigratory and antiinvasive potential of vincristine sulfate (VCR) on human glioma cells and to analyze whether phenytoin (5,5-diphenylhydantoin; DPH) might act synergistically with VCR. Vincristine affects the cytoplasmic microtubules; DPH has been reported to enhance VCR cytotoxicity in murine cells. In two human glioma cell lines, GaMG and D-37MG, we found VCR to reduce monolayer growth and colony formation in a dose-dependent fashion at concentrations of 10 ng/ml and above. Phenytoin increased the cytotoxic and cystostatic effects of VCR in monolayer cells but not in spheroids. Multicellular spheroids were used to investigate directional migration. A coculture system of GaMG and D-37MG spheroids with fetal rat brain aggregates was used to analyze and quantify tumor cell invasion. A dose-dependent inhibition of migration and invasion by VCR was observed in both cell lines without further enhancement by DPH. Immunofluorescence microscopy with antibodies against α-tubulin revealed dose-dependent morphological alterations in the microtubules when the cells were exposed to VCR but not after incubation with DPH. Based on the combination of standardized in vitro model systems currently in use and the present data, the authors strongly suggest that VCR inhibits migration and invasion of human glioma cells. This is not altered by DPH, which inhibits cell proliferation in combination with VCR.

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