Cas Ilgly Induces Apoptosis in Glioma C6 Cells In Vitro and In Vivo through Caspase-Dependent and Caspase-Independent Mechanisms

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

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Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

Current Proteomics ◽

10.2174/1570164615666180817100248 ◽

2019 ◽

Vol 16 (3) ◽

pp. 175-180

Author(s):

Fengjin Hao ◽

Yueqin Feng ◽

Yifu Guan

Keyword(s):

Biological Activity ◽

Botulinum Toxin ◽

Cell Membrane ◽

Western Blot ◽

Biological Function ◽

C6 Cells ◽

Green Fluorescence ◽

Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

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BCNU-loaded PEG–PLLA ultrafine fibers and their in vitro antitumor activity against Glioma C6 cells

Journal of Controlled Release ◽

10.1016/j.jconrel.2006.05.031 ◽

2006 ◽

Vol 114 (3) ◽

pp. 307-316 ◽

Cited By ~ 112

Author(s):

Xiuling Xu ◽

Xuesi Chen ◽

Xiaoyi Xu ◽

Tiancheng Lu ◽

Xin Wang ◽

...

Keyword(s):

Antitumor Activity ◽

C6 Cells ◽

Ultrafine Fibers ◽

Glioma C6

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Liposome-Mediated Gene Transfer into Established CNS Cell Lines, Primary Glial Cells, and in Vivo

Cell Transplantation ◽

10.1177/096368979800700212 ◽

1998 ◽

Vol 7 (2) ◽

pp. 175-185 ◽

Cited By ~ 9

Author(s):

Patrick Kofler ◽

Bettina Wiesenhofer ◽

Christine Rehrl ◽

Gottfried Baier ◽

Günter Stockhammer ◽

...

Keyword(s):

Gene Transfer ◽

Glial Cells ◽

Transfection Efficiency ◽

Primary Cultures ◽

C6 Glioma Cells ◽

C6 Cells ◽

Adult Rats ◽

Neuronal Precursor Cells

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a β-galactosidase (β-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 μg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A “boost-lipofection” markedly increased (twice) lipofection efficiency in C6 cells. Expression of β-GAL reached a maximum after 3-5 days. When the liposome–DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.

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Suicide Gene Therapy By Amphiphilic Copolymer Nanocarrier for Spinal Cord Tumor

Nanomaterials ◽

10.3390/nano9040573 ◽

2019 ◽

Vol 9 (4) ◽

pp. 573 ◽

Cited By ~ 3

Author(s):

So-Jung Gwak ◽

Jeoung Soo Lee

Keyword(s):

Spinal Cord ◽

Gene Therapy ◽

Spinal Cord Tumor ◽

Suicide Gene ◽

Amphiphilic Copolymer ◽

Suicide Gene Therapy ◽

Gene Carrier ◽

C6 Cells

Spinal cord tumors (SCT) are uncommon neoplasms characterized by irregular growth of tissue inside the spinal cord that can result in non-mechanical back pain. Current treatments for SCT include surgery, radiation therapy, and chemotherapy, but these conventional therapies have many limitations. Suicide gene therapy using plasmid encoding herpes simplex virus-thymidine kinase (pHSV-TK) and ganciclovir (GCV) has been an alternative approach to overcome the limitations of current therapies. However, there is a need to develop a carrier that can deliver both pHSV-TK and GCV for improving therapeutic efficacy. Our group developed a cationic, amphiphilic copolymer, poly (lactide-co-glycolide) -graft-polyethylenimine (PgP), and demonstrated its efficacy as a drug and gene carrier in both cell culture studies and animal models. In this study, we evaluated PgP as a gene carrier and demonstrate that PgP can efficiently deliver reporter genes, pGFP in rat glioma (C6) cells in vitro, and pβ-gal in a rat T5 SCT model in vivo. We also show that PgP/pHSV-TK with GCV treatment showed significantly higher anticancer activity in C6 cells compared to PgP/pHSV-TK without GCV treatment. Finally, we demonstrate that PgP/pHSV-TK with GCV treatment increases the suicide effect and apoptosis of tumor cells and reduces tumor size in a rat T5 SCT model.

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Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of α-tubulin

Biochemical Journal ◽

10.1042/bj20030776 ◽

2003 ◽

Vol 375 (1) ◽

pp. 121-129 ◽

Cited By ~ 8

Author(s):

Silvia A. PURRO ◽

C. Gastón BISIG ◽

María A. CONTIN ◽

Héctor S. BARRA ◽

Carlos A. ARCE

Keyword(s):

Cultured Cells ◽

In Vivo Studies ◽

Brain Extract ◽

Similar Degree ◽

C6 Cells ◽

Post Translational Modification ◽

Α Subunit ◽

C Terminus

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the α-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of α-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group. Azatyrosine competitively excluded incorporation of [14C]tyrosine into tubulin of soluble brain extract. A newly developed rabbit antibody specific to C-terminal azatyrosine was used to study incorporation of azatyrosine in cultured cells. When added to the culture medium (Ham's F12K), azatyrosine was incorporated into tubulin of glioma-derived C6 cells. This incorporation was reversible, i.e. after withdrawal of azatyrosine, tubulin lost azatyrosine and reincorporated tyrosine. Azatyrosinated tubulin self-assembled into microtubules to a similar degree as total tubulin both in vitro and in vivo. Studies by other groups have shown that treatment of certain types of cultured cancer cells with azatyrosine leads to reversion of phenotype to normal, and that administration of azatyrosine into animals harbouring human proto-oncogenic c-Ha-ras prevents tumour formation. These interesting observations led us to study this phenomenon in relation to tubulin status. Under conditions in which tubulin was mostly azatyrosinated, C6 cells remained viable but did not proliferate. After 7–10 days under these conditions, morphology changed from a fused, elongated shape to a rounded soma with thin processes. Incorporation of azatyrosine into the C-terminus of α-tubulin is proposed as one possible cause of reversion of the malignant phenotype.

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Inhibitory effect of antisense epidermal growth factor receptor RNA on the proliferation of rat C6 glioma cells in vitro and in vivo

Journal of Neurosurgery ◽

10.3171/jns.2000.92.1.0132 ◽

2000 ◽

Vol 92 (1) ◽

pp. 132-139 ◽

Cited By ~ 30

Author(s):

Peiyu Pu ◽

Xuwen Liu ◽

Aixue Liu ◽

Jianling Cui ◽

Yunting Zhang

Keyword(s):

Survival Time ◽

Glioma Cells ◽

C6 Glioma ◽

C6 Glioma Cells ◽

C6 Cells ◽

Content Type ◽

Proliferation Activity ◽

Fine Print

Object. The goal of this study was to evaluate the effect of antisense epidermal growth factor receptor (EGFR) RNA on the growth of rat glioma cells in vitro and in vivo and to determine the feasibility of targeting the EGFR gene for gene therapy in gliomas.Methods. Antisense EGFR complementary (c)DNA was transfected into C6 glioma cells by using lipofectamine. In vitro studies, Southern and Northern blot analyses, in situ hybridization, and immunohistochemical staining were designed to examine the integration and expression of antisense EGFR constructs. The 3′(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay and the average number of argyrophilic nuclear organizer regions (Ag-NORs) were used to evaluate cell proliferation, whereas the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method and microscopy were used to observe cell apoptosis. As part of the in vivo studies, parental C6 cells and C6 cells transfected with EGFR antisense cDNA were implanted stereotactically into the right caudate nucleus of Wistar rats (C6-injected animals and transfected C6-injected animals). Rats with well-established cerebral C6 glioma foci were treated intratumorally with either antisense EGFR cDNA or empty-vector DNA by using lipofectamine (treated-C6 and control treated group). The general behavior and survival of the rats, findings on magnetic resonance images of their brains, histopathological changes, proliferation activity, and apoptosis of the cerebral gliomas in each group of rats were examined.Exogenous antisense EGFR cDNA was integrated into the genome of C6 cells and expressed. In clones with a high expression of the antisense construct, there was a dramatic decrease in endogenous EGFR messenger RNA and protein levels, reduced proliferation activity, and induction of apoptosis in vitro. The mean survival time of rats injected with C6 cells was 17.3 days. The mean survival time of rats injected with C6 cells followed by treatment with empty vector in lipofectamine was 15.4 days. Survival time was significantly prolonged in 100% of the rats injected with antisense-transfected C6 cells and in two thirds of the rats injected with C6 cells followed by antisense EGFR cDNA. Magnetic resonance imaging revealed distinct cerebral tumor foci in C6-injected rats and in control rats of the treated group, but none were found in the rats injected with transfected C6 cells. Furthermore, tumor foci disappeared completely in C6-injected rats treated with antisense EGFR cDNA. The cerebral gliomas of the rats treated by injection of antisense EGFR RNA were characterized by reduced proliferation activity and the induction of apoptosis.Conclusions. The results of this study indicate that EGFR plays an important role in the genesis of malignant gliomas. It may, therefore, be an effective target of antisense gene therapy in patients with gliomas.

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In Vitro Characteristics of Glioma Cells Targeting by OX26-modified Liposomal Cisplatin

Letters in Drug Design & Discovery ◽

10.2174/1570180817999200330165213 ◽

2020 ◽

Vol 17 (9) ◽

pp. 1126-1138

Author(s):

Maryam Sadat Ashrafzadeh ◽

Amir Heydarinasab ◽

Azim Akbarzadeh ◽

Mehdi Ardjmand

Keyword(s):

Brain Tumor ◽

Biological Activities ◽

Drug Loading ◽

Therapeutic Effects ◽

C6 Cells ◽

Pegylated Liposomes ◽

Rat Glioma ◽

The Brain

Background: Drug delivery to the brain tumor is limited due to the presence of the blood-brain barrier (BBB). Objective: This study aimed to evaluate the therapeutic effects of cisplatin-loaded PEGylated liposomes, targeted with the OX26 antibody (targeted liposomal cisplatin) against transferrin receptor expressing rat glioma C6 cells in vitro. Method: The liposomes were synthesized using reverse phase evaporation method and were conjugated to the OX26 monoclonal antibody. They were characterized in terms of size, drug encapsulation efficiency, morphology and drug release experiments using dynamic light scattering, atomic absorption spectrometry, scanning electron microscopy, and dialysis membrane methods. Then, their biological activities were evaluated on targeting the BBB. Results: The characterization results showed that spherical nanodrug with a size of 157 nm and drug loading efficiency of 24% was synthesized, which released 64% of the loaded cisplatin after 72 h in a controlled release manner. The nanoparticles caused an increase in the cisplatin cytotoxicity effects by 1.7-, 1.8- and 1.8-fold, compared to cisplatin-loaded PEGylated liposomes (liposomal cisplatin) after 24, 48 and 72h incubation, respectively against C6 cells. Moreover, targeted liposomal cisplatin showed promising results in the transport of cisplatin across the BBB, in which it caused an increase in the cisplatin cytotoxicity on C6 cells by 2.7- and 2.4-fold, compared to cisplatin and liposomal cisplatin, respectively. Conclusion: Regarding the properties of the targeted liposomal cisplatin, it suggests that the potency of the formulation, to be evaluated, for the transport of cisplatin across the BBB, delivers it to the brain tumor in vivo.

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Modulation in vitro and in vivo of ACNU resistance in a subline of C6 glioma with reserpine

Journal of Neurosurgery ◽

10.3171/jns.1987.66.2.0251 ◽

1987 ◽

Vol 66 (2) ◽

pp. 251-255 ◽

Cited By ~ 3

Author(s):

Tatsuo Yoshida ◽

Keiji Shimizu ◽

Yukitaka Ushio ◽

Heitaro Mogami ◽

Yukiya Sakamoto

Keyword(s):

Combined Therapy ◽

Intrathecal Injection ◽

C6 Glioma ◽

Intracellular Uptake ◽

C6 Cells ◽

Content Type ◽

Meningeal Gliomatosis ◽

Fine Print

✓ Reserpine enhanced in vitro the cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in both the C6 glioma and its ACNU-resistant subline, C6/ACNU. Reserpine also enhanced the chemotherapeutic effect of ACNU in C6/ACNU-bearing (C6/ACNU-meningeal gliomatosis) rats, in which ACNU resistance could be modulated by combined ACNU and reserpine therapy. When 10 μM reserpine was added to ACNU in culture, the concentration of drug required for 50% inhibition of cell growth (IC50) of ACNU for C6/ACNU cells decreased to the level of that for C6 cells. When 20 μM reserpine was added to the culture, intracellular uptake of ACNU in C6/ACNU cells increased further and the efflux of the drug from the cells decreased. In in vivo experiments in rats, combined chemotherapy with ACNU (1 mg/kg) and reserpine (250 μg/kg) by intrathecal injection significantly increased the life span of the rats as compared to results with ACNU chemotherapy alone. The enhanced cytotoxicity of ACNU in ACNU-resistant glioma cells in vitro and in vivo may be explained by the increase of intracellular concentration of ACNU resulting from the inhibition of ACNU efflux from the resistant cells by reserpine. It was concluded that ACNU resistance could be modulated in vitro and in vivo by combined therapy with ACNU and reserpine.

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