Citrus flavonoid 3,5,6,7,8,3′,4′-heptamethoxyflavone induces BDNF via cAMP/ERK/CREB signaling and reduces phosphodiesterase activity in C6 cells

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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Faculty Opinions recommendation of Increased serum phosphodiesterase activity in women with pre-eclampsia.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1032788.374883 ◽

2006 ◽

Author(s):

Euan Wallace

Keyword(s):

Phosphodiesterase Activity

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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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TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis

Journal of Dental Research ◽

10.1177/0022034519871890 ◽

2019 ◽

Vol 98 (12) ◽

pp. 1386-1396 ◽

Cited By ~ 3

Author(s):

X. Hong ◽

S.N. Min ◽

Y.Y. Zhang ◽

Y.T. Lin ◽

F. Wang ◽

...

Keyword(s):

Acinar Cell ◽

Cell Injury ◽

Ex Vivo ◽

Secretory Granules ◽

Acinar Cells ◽

Autophagic Flux ◽

C6 Cells ◽

Tnf Α ◽

Α Level

IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor–α (TNF-α) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-α level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-α level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients’ SMGs. TNF-α treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-α treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-α–treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-α also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-α treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-α increased the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-α–induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-α is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.

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Disruption of the Complex between GAPDH and Hsp70 Sensitizes C6 Glioblastoma Cells to Hypoxic Stress

International Journal of Molecular Sciences ◽

10.3390/ijms22041520 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1520

Author(s):

Marina A. Mikeladze ◽

Elizaveta A. Dutysheva ◽

Victor G. Kartsev ◽

Boris A. Margulis ◽

Irina V. Guzhova ◽

...

Keyword(s):

Cell Death ◽

Hypoxic Stress ◽

C6 Cells ◽

Tumor Resistance ◽

Glioblastoma Cells ◽

Enzyme Expression ◽

Hsp70 Chaperone ◽

Therapeutic Tools ◽

Enzyme Molecules

Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools.

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α-Conotoxins and α-Cobratoxin Promote, while Lipoxygenase and Cyclooxygenase Inhibitors Suppress the Proliferation of Glioma C6 Cells

Marine Drugs ◽

10.3390/md19020118 ◽

2021 ◽

Vol 19 (2) ◽

pp. 118

Author(s):

Tatiana I. Terpinskaya ◽

Alexey V. Osipov ◽

Elena V. Kryukova ◽

Denis S. Kudryavtsev ◽

Nina V. Kopylova ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Glioma Cells ◽

Nordihydroguaiaretic Acid ◽

C6 Glioma ◽

Cyclooxygenase Inhibitors ◽

C6 Glioma Cells ◽

C6 Cells ◽

Lipoxygenase Inhibitor ◽

Glioma C6

Among the brain tumors, glioma is the most common. In general, different biochemical mechanisms, involving nicotinic acetylcholine receptors (nAChRs) and the arachidonic acid cascade are involved in oncogenesis. Although the engagement of the latter in survival and proliferation of rat C6 glioma has been shown, there are practically no data about the presence and the role of nAChRs in C6 cells. In this work we studied the effects of nAChR antagonists, marine snail α-conotoxins and snake α-cobratoxin, on the survival and proliferation of C6 glioma cells. The effects of the lipoxygenase and cyclooxygenase inhibitors either alone or together with α-conotoxins and α-cobratoxin were studied in parallel. It was found that α-conotoxins and α-cobratoxin promoted the proliferation of C6 glioma cells, while nicotine had practically no effect at concentrations below 1 µL/mL. Nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor, and baicalein, a 12-lipoxygenase inhibitor, exerted antiproliferative and cytotoxic effects on C6 cells. nAChR inhibitors weaken this effect after 24 h cultivation but produced no effects at longer times. Quantitative real-time polymerase chain reaction showed that mRNA for α4, α7, β2 and β4 subunits of nAChR were expressed in C6 glioma cells. This is the first indication for involvement of nAChRs in mechanisms of glioma cell proliferation.

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