Induction of apoptosis in rat C6 glioma cells by panaxydol

Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. Previous studies have shown that panaxydol inhibited the growth of various kinds of malignant cell lines. To date, there has been no report concerning the effect of panaxydol on cell growth inhibition in glioma cells. In this paper, we examined panaxydol's antiproliferation and proapoptotic effects on rat C6 glioma cells and investigated its mechanism. Cell growth inhibition of panaxydol was determined by MTT reduction assay. Apoptosis of cells was measured by both Hoechst 33258 staining and Annexin V analysis. It was found that panaxydol markedly inhibited proliferation of C6 cells in a dose-dependent manner with ID(50) of 40 microM. The cell apoptosis was observed at 48 h in the presence of panaxydol. In concert with these findings, Western blot analysis showed a decreased expression of bcl-2 and increased levels of Bax and caspase-3 in C6 cells treated by panaxydol. In conclusion, panaxydol has profound effects on growth and apoptosis of C6 cells, suggesting that panaxydol may be a potential candidate for the treatment of malignant gliomas.     

Induction of apoptosis by penta-acetyl geniposide in rat C6 glioma cells

Penta-acetyl geniposide, (Ac)(5)-GP, was produced by acetylation of a glycoside, isolated from an extract of Gardenia fructus. Previously, we have reported that C6 glioma cells could be inhibited in culturing as well as in bearing rats by treating with (Ac)(5)-GP. In this study, the effect and action of (Ac)(5)-GP on inducing cell death was examined in rat C6 glioma cells. Treatment of C6 glioma cells with (Ac)(5)-GP caused cell death, chromatin condensation, and internucleosomal DNA ladder. Also, cell cycle arrest at G(0)/G(1) phase revealed that (Ac)(5)-GP-induced cell death appears to be mediated by apoptosis. In addition, the results also showed that p53 and c-Myc increased due to treatment of (Ac)(5)-GP in a dose-response and time-dependent manner. Concomitant with the expression of p53 and c-Myc, decreased level of Bcl-2 and increased level of Bax protein were observed. These results suggest that cell death caused by (Ac)(5)-GP through apoptosis and cell cycle arrest at G(0)/G(1) may be associated with the induction of p53, c-Myc and may be mediated with apoptosis-related Bcl-2 family proteins.     

Effects of the Ca ionophore A23187 on zinc-induced apoptosis in C6 glioma cells

Zinc ions are essential, but at elevated concentrations, they also have toxic effects on mammalian cells. Zinc plays a crucial role in cell proliferation and differentiation and it even protects cells against apoptosis caused by various reagents. On the other hand, zinc at high concentrations causes cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, and breakdown of the mitochondrial membrane potential. In the present work, a clone of rat C6 glioma cells that was resistant to toxic effects of ZnCl₂ up to 250 μM was employed to study the effect of the ionophore A23187 on zinc-induced apoptosis. Neither 150 μM Zn²⁺ nor 100 nM A23187 alone caused apoptosis as measured by internucleosomal DNA fragmentation. However, combined exposure of C6 cells to 100 nM A23187 and 150 μM Zn²⁺ for 48 h was effective in inducing apoptosis. Because the so-called calcium ionophore A23187 is not specific for Ca²⁺ ions but also transports Zn²⁺ with high selectivity over Ca²⁺, we investigated whether this substance promoted the uptake of Zn²⁺ ions into C6 cells. Employing the zinc-specific fluorescence probe Zinquin, we observed that the very low concentration of 1.9 nM A23187 significantly and rapidly raised the intracellular mobile Zn²⁺ content. Analysis by atomic absorption spectroscopy revealed that incubation with 1.9 nM A23187 caused a doubling of the total intracellular zinc level within 60 min. We conclude that the apoptosis evoked by the combined action of Zn²⁺ and A23187 was the result of enhanced Zn²⁺ influx evoked by the ionophore, resulting in higher intracellular zinc levels.     

Ultrastructure of nuclei of cisplatin-treated C6 glioma cells undergoing apoptosis

C6 glioma cells, treated with a cytostatic dose of cisplatin (1.66 x 10(-5) M) ceased dividing by 24 h and, most of them had undergone apoptosis by 72-96 h. The reactive cells were classified into 5 types (T-I to V), according to the ultrastructure of nuclei. At 4 h, 20.4% of cells (T-I) showed minute condensation and margination of chromatin. The nuclear envelope (NE) formed slim and deep invaginations consisting of the inner or both membranes. The later kind of NE invaginations often extended to the enlarged nucleoli and contained nucleolus-like material at its cytoplasmic side. Some nuclear pores were covered with a dome-shaped "cap" formed by fine filamentous material. The number of T-I cells increased to 53.3% by 72 h. In T-II cells, which appeared at 24 h, the chromatin was condensed into dense irregular masses separated from the NE by a lucent space with filamentous structures preventing complete margination of chromatin. Nucleoli of T-II cells were small and showed partial segregation of their components. The "capped" pores were absent in these apparently more damaged cells. From 24 h, cells with large and lobulated nuclei (T-III) started to increase in number and peaked at 72 h (6.6%). Except for some small lobules, the chromatin of T-III cells was moderately aggregated and the NE was well preserved. Typical apoptotic cells with highly condensed and marginated chromatin (T-IV) peaked at 48-72 h (2.4-4.8%). They appeared in 2 varieties, including cells with wrinkled nuclei with less condensed and incompletely marginated chromatin or more lobulated forms with highly condensed marginated chromatin suggesting their origin from T-II or T-III cells. T-IV cells, as well as their fragments, underwent phagocytosis and secondary necrosis (T-V cells, 48.6% at 96 h). Two alternative routes of nuclear changes leading to cisplatin-triggered apoptosis, as represented by the sequence T-I --> T-III --> T-IV/V or T-I --> T-II --> T-IV/V, may explain the initially less or more damaged cells. These alternatives, together with progressive recruitment of reactive cells, suggest intrapopulation differences in the sensitivity of cells or in the cell cycle perturbations induced by cisplatin. Except for the T-IV and T-V cells, observed alterations of cytoplasmic organelles, including mitochondria, were fewer than reported in previous studies on cisplatin.     

Cadmium-induced apoptosis in C6 glioma cells: Mediation by caspase 9-activation

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC₅₀-value of 0.7 M, while human A549 adenocarcinoma cells were relatively resistant with an IC₅₀-value of 164 M CdCl₂. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl₂ and was concentration-dependent between 1–100 M CdCl₂, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 M CdCl₂. Furthermore, cadmium (1 M, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd²⁺, other toxic heavy metal ions (Hg²⁺, Pb²⁺, Ni²⁺, Fe²⁺, CrO₄ ^2–, Cu²⁺ or Co²⁺) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn²⁺ which caused apotosis at high concentrations (>150 M) whereas it protected against cadmium-induced apoptosis at low concentrations (10–50 M).     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Dexamethasone inhibits apoptosis in C6 glioma cells through increased expression of Bcl-XL

The glucocorticoid dexamethasone (Dex) has been reported to modulate a number of signaling pathways and physiological processes, including apoptosis. This study was carried out to investigate the cytoprotective mechanism of Dex in C6 glioma cells. Pre-treatment of cells with Dex inhibited apoptosis induced by staurosporine, etoposide and thapsigargin. Apoptosis inhibition correlated with blockade of mitochondrial cytochrome c release, abolition of caspase-3 activity along with inhibition of caspase-9 and PARP cleavage. Dex-mediated cytoprotection coincided with the induction of the anti-apoptotic protein, Bcl-X_L. The specific glucocorticoid receptor antagonist, RU486, reversed the anti-apoptotic effect of Dex and prevented Bcl-X_L induction. Here, we show for the first time that knockdown of Bcl-X_L expression with siRNA reversed the protective effects of the glucocorticoid in glioma cells. We conclude that Dex-mediated inhibition of apoptosis in C6 glioma cells is through induction of Bcl-X_L.     

Glucocorticoids enhance serum deprivation- but not calcium-induced cytotoxicity in rat C6 glioma cells

While glucocorticoids have been shown to exacerbate calcium-induced neuronal damage, little is known about the effects of these hormones on calcium-induced damage to glial cells. Here we examine the effect of synthetic glucocorticoid dexamethasone on calcium ionophore A23187 and serum deprivation-induced damage to rat C6 glioma cells. Treatment of the glioma cells with A23187 reduced cell viability, similar in extent to that observed with serum deprivation. Both A23187 and serum deprivation caused cell damage without degradation of the genomic DNA into nucleosomic fragments. In addition, the reduction in cell viability caused by A23187 was not significantly altered by DEX at concentrations enhancing serum deprivation-induced cell death. These results suggest that the cytotoxic effect of A23187 on glial cells may be mediated through a mechanism different from that underlying serum deprivation-induced cell death, and that, in contrast to calcium-induced neuronal damage, calcium-induced damage to glial cells is likely to be insensitive to glucocorticoids.     

Adrenocorticoid receptors in C6 glioma cells: Effects on cell growth

Rat C6 glioma cells contain two receptors for adrenocorticoids—the predominant glucocorticoid receptor and low densities of the Type I corticosteroid (mineralocorticoid) receptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosteceptor. Nanomolar concentrations of deoxycorticosterone, corticosterone and aldosterone, which fully occupy Type I receptors, produced a slight stimulatory effect on C6 cell growth in serum-free media. However, spironolactone, a Type I receptor antagonist, and pregnenolone, which does not bind to Type I receptors, had similar effects. Therefore, the slight growth stimulation produced by low steroid concentrations is not mediated by Type I or glucocorticoid receptors, but may be due to an effect on cell membrane properties or other receptor-independent action. Occupation of glucocorticoid receptors by higher concentrations of corticosteroids inhibited C6 cell growth.     

Heat shock induction of a 65 kDa ATP—binding proteinase in rat C6 glioma cells

The 45,55,65 and 100kDa ATP-binding proteinases(ATP-BPases) of the heat-shocked (44℃ for 30 min,recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography.Their molecular masses,isoelectric points (pI),pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9amino acid sequence was determined by Edman degradation,but no homologies to other proteins in the protein data bases were found.30 and 31kDa proteinases can be cleaved from the 45,55 and 65 kDa proteinases to which they are linked.A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.     

Growth inhibition and differentiation of C6 glioma cells on treatment with hmba.

HMBA, a differentiation inducer belonging to the class of hybrid polar compounds, is known to induce terminal differentiation of a number of leukemic and solid tumour cell lines. In this report we have shown that HMBA markedly inhibits growth of C6 glioma cells at non-cytotoxic concentrations ranging from 2.5 m m to 10 m m in a dose-dependent manner. The growth inhibitory effect can be detected as early as 18--24 h. By the sixth day the growth inhibition decreases at all the concentrations tested. Treatment with HMBA results in an accumulation of C6 cells in G0/G1 phase along with a decrease in the number of cells in S phase. HMBA induces morphological differentiation of C6 cells and increases expression of glial fibriliary acidic protein (GFAP), a marker for mature astrocytes. HMBA induces c-fos and represses cycloheximide-induced c-jun and fra-1 expression. HMBA-induced growth inhibition of C6 cells is accompanied by a decrease in Cdk4 protein levels. However, HMBA fails to sustain low Cdk4 levels, which may be responsible for HMBA's failure to sustain the growth inhibitory effect.     

Zinc induces apoptosis that can be suppressed by lanthanum in C6 rat glioma cells

Zinc ions have both essential and toxic effects on mammalian cells. Here we report the ability of zinc to act as an inducer of apoptosis in C6 rat glioma cells. Incubation with 150 to 300 microM ZnCl2 caused cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, nuclear fragmentation and breakdown of the mitochondrial membrane potential. On the other hand, zinc deprivation by the membrane permeable chelator TPEN [N,N,N',N',-tetrakis (2-pyridyl-methyl)-ethylenediamine] also induced programmed death in this cell line, indicating the existence of intracellular zinc levels below and above which apoptosis is induced. Zinc-induced apoptosis in C6 cells was independent of major signaling pathways (protein kinase C, mitogen activated protein kinase and guanylate cyclase) and protein synthesis, but was increased by facilitating zinc uptake with the ionophore pyrithione. Lanthanum(III)chloride was also able to increase the net zinc uptake, but nevertheless apoptotic features and zinc toxicity were reduced. Remarkably, lanthanum suppressed the zinc-induced breakdown of the mitochondrial membrane potential. We conclude that in C6 cells lanthanum acts in two different ways, as a promoter of net zinc uptake and as a suppressor of zinc-induced apoptosis.     

Volatile anesthetics affect the morphology of rat glioma C6 cells via RhoA, ERK, and Akt activation

Treatment of rat glioma C6 cells with the beta-receptor agonist isoproterenol induces a massive increase in cAMP. Concomitantly the cells change their morphology from a fibroblast-type to an astrocyte-like (stellated) cell shape. The stellated morphology can be completely reverted by thrombin and sphingosine-1-phosphate (S-1-P) but also to a certain extent by clinical concentrations of volatile anesthetics. The anesthetic-induced reversion of the stellated cell shape seems to be mediated by a number of cellular alterations. Central to the effect is most likely a RhoA/Rho-kinase activation, but also the MAPKK/MEK and the Akt/protein kinase B pathway are activated by the anesthetics. With the use of specific inhibitors we were able to show that activation of the MAPKK/MEK pathway inhibits, whereas activation of the Akt/protein kinase B pathway stimulates the reversal of the stellated cell shape by the anesthetics. In summary, volatile anesthetics affect the morphology of rat glioma C6 cells by activation of the RhoA/Rho kinase, the MAPKK/MEK, and the Akt/protein kinase B signaling pathways.     

Resveratrol increases glutamate uptake and glutamine synthetase activity in C6 glioma cells

Resveratrol, a phytoalexin found mainly in grapes, is a promising natural product with anti-cancer and cardio-protective activities. Here, we investigated, in C6 glioma cells, the effect of resveratrol on some specific parameters of astrocyte activity (glutamate uptake, glutamine synthetase and secretion of S100B, a neurotrophic cytokine) commonly associated with the protective role of these cells. Cell proliferation was significantly decreased by 8% and 26%, following 24h of treatment with 100 and 250 microM resveratrol. Extracellular S100B increased after 48 h of resveratrol exposure. Short-term resveratrol exposure (from 1 to 100 microM) induced a linear increase in glutamate uptake (up to 50% at 100 microM resveratrol) and in glutamine synthetase activity. Changes in these glial activities can contribute to the protective role of astrocytes in brain injury conditions, reinforcing the putative use of this compound in the therapeutic arsenal against neurodegenerative diseases and ischemic disorders.     

Ivermectin inhibits the growth of glioma cells by inducing cell cycle arrest and apoptosis in vitro and in vivo

Glioma, the most predominant primary malignant brain tumor, remains uncured due to the absence of effective treatments. Hence, it is imperative to develop successful therapeutic agents. This study aimed to explore the antitumor effects and mechanisms of ivermectin (IVM) in glioma cells in vitro and in vivo. The effects of IVM on cell viability, cell cycle arrest, apoptosis rate, and morphological characteristics were determined respectively by MTT assay/colony formation assay, flow cytometry, and transmission electron microscope. In addition, the expression levels of cycle-related and apoptosis-associated proteins were individually examined by Western blot analysis. Moreover, cell proliferation and apoptosis analyses were carried out by TUNEL, Ki-67, cleaved caspase-3, and cleaved caspase-9 immunostaining assay. Our results demonstrated that IVM has a potential dosage-dependent inhibition effect on the apoptosis rate of glioma cells. Meanwhile, the results also revealed that IVM induced apoptosis by increasing caspase-3 and caspase-9 activity, upregulating the expressions of p53 and Bax, downregulating Bcl-2, activating cleaved caspase-3 and cleaved caspase-9, and blocking cell cycle in G0/G1 phase by downregulating levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E. These findings suggest that IVM has an inhibition effect on the proliferation of glioma cells by triggering cell cycle arrest and inducing cell apoptosis in vitro and in vivo, and probably represents promising agent for treating glioma.     

2‐Hexadecenal inhibits growth of C6 glioma cells

2‐Hexadecenal (2HD) formation in the organism occurs via irreversible enzymatic degradation of sphingosine‐1‐phosphate or nonenzymatic γ‐, UV‐, or HOCl‐induced destruction of a number of sphingolipids including S1P. The current research focuses on the study of 2HD effects on C6 glioma cells growth. The results obtained show that 2HD causes a dose‐dependent decrease in proliferative and mitotic indices. The change in the mitotic index is due to the redistribution of cells in the different phases of mitosis. These processes are accompanied by cytoskeleton rearrangement and changes in cell morphology, which are expressed in F‐actin redistribution, change in the number and type of filopodia and fibrils, leading to cell shape changes, decrease in intercellular contacts and monolayer rarefaction. Cells treatment with 2HD leads to apoptosis induction and signalling pathways modification, including activation of JNK, p38, and ERK1/2 MAPK but not PI3K. The effects observed are not related to the cytotoxicity of 2HD. Significance of the study: 2HD—an unsaturated aldehyde, which level can rise under conditions of oxidative stress as a result of nonenzymatic sphingolipids' destruction. The mechanisms of 2HD action on various cell types have not been sufficiently studied. Therefore, the study on functional role of this aldehyde in different cell types that may be its target is relevant. This study demonstrated that 2HD inhibits growth of C6 glioma cells due to modification of intracellular processes of signal transduction, cytoskeleton rearrangement, change in the mitotic regimen and apoptosis induction.     

Intracellular zinc distribution and transport in C6 rat glioma cells

In mammalian cells, the intracellular availability of zinc influences numerous crucial processes. Its distribution has previously been visualized with several fluorescent probes, but it was unclear how these probes are compartmentalized within the cell. Here, we show that in C6 cells the zinc-specific probe Zinquin is evenly distributed. Thus, the significantly lower level of fluorescence in the nucleus and a punctuate vesicular staining are real differences in the concentrations of zinc. Chemical perturbation of the steady state by releasing intracellular protein-bound zinc with the sulfhydryl-reactive N-ethylmaleimide (NEM) resulted in a vanadate sensitive transport of zinc out of the nucleus and into zincosomes. If the zinc-release was performed with the histidine-reactive diethylpyrocarbonate, sequestration was reduced compared to treatment with NEM, indicating the importance of histidine within membrane zinc transporters. Another major factor regulating the zinc homeostasis is ion export. As determined by atomic absorption spectroscopy, up to 50% of the cellular zinc was exported by a mechanism sensitive to lanthanum ions. We conclude that different concentrations of labile zinc exist in different cellular compartments, which are maintained by export and intracellular transport of zinc.