Human T98G Glioma Cells Research Articles

The effect of resveratrol, its naturally occurring derivatives and tannic acid on the induction of cell cycle arrest and apoptosis in rat C6 and human T98G glioma cell lines

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a potent chemopreventive and potentially cancer therapeutic agent. Since rapid metabolism limits resveratrol bioavailability, derivatives less prone to metabolic transformation are being sought and tested.We evaluated the effect of resveratrol, and its analogs (pterostilbene and 3,5,4′-trimethoxystilbene) along with tannic acid, on cell cycle and apoptosis in rat C6 and human T98G glioma cells. At concentration ranges both lower and higher than IC50 calculated based on MTT assay, all these polyphenols affected the cell cycle distribution. However, resveratrol and pterostilbene increased the percentage of the cells in S phase, while trimethoxystilbene (TMS) caused a massive accumulation of cells at the G2/M phase of the cell cycle. Tannic acid had no effect on cell cycle distribution in C6 cells, but increased the number of dead cells in both glioma cell lines. The ability to induce apoptosis by tannic acid and stilbenes was confirmed by phosphatidylserine externalization, the loss of mitochondrial membrane potential and the level of cleaved caspase-3. The apoptosis rate was most significantly increased by TMS and this was related to p53 induction.These results indicate that methoxylated stilbenes are efficient inhibitors of glioma cell proliferation and apoptosis inducers and might be considered adjuvants in glioma therapy.

Beta-adrenoceptor-mediated cyclic AMP signal in different types of cultured nerve cells in normoxic and hypoxic conditions

β-adrenergic neurotransmission is an important factor regulating brain activity such as neuronal and glial survival, plasticity, membrane transport or cellular metabolism. Intracellular β-adrenergic signaling, via a stimulatory G protein (Gs), activates two major down-stream effectors, i.e., adenylyl cyclase (AC) and cAMP-dependent protein kinase A (PKA). The aim of this work was to study the ability of endogenous (adrenaline and noradrenaline) and exogenous (isoprenaline) β-adrenergic receptor agonists to increase cAMP in different types of nerve cells. Moreover, we wanted to precisely identify the receptor isoform involved in the observed phenomenon using selective β1-, β2- β3-adrenoceptor blockers. In an additional study, the negative influence of hypoxia on the AC/cAMP intracellular signaling system was tested. The study was conducted in parallel on rat primary glial (astrocytes) cultures, primary neuronal cultures, C6 glioma cells and human T98G glioma cells. The formation of [^(3)H] cAMP by agonists and antagonists was measured in [^(3)H] adenine prelabeled cells under normoxic and hypoxic conditions. The obtained results revealed that adrenaline, noradrenaline and isoprenaline strongly stimulated cAMP production in all tested cell types (with highest potency in C6 glioma cells). In glial and neuronal cells the adrenaline-evoked cAMP effect was mediated mainly by the β1-adrenoceptor, whereas in tumor cells the effect was probably mediated by all three β-subtype specific drugs. The AC/cAMP intracellular signaling system is affected by hypoxic conditions. Considering both physiological and therapeutic importance of β-family receptors the present work characterized the β-adrenoceptor-mediated cAMP signal transduction pathway in different nerve cells in normoxic and hypoxic conditions. The proposed in vitro model of hypoxic conditions may serve as a good model system to study the biological effects of endogenous catecholamines as well as potential therapeutics targeting adrenergic receptors, which are impaired during ischemia in vivo.

Characterization of nuclear betaII-tubulin in tumor cells: a possible novel target for taxol.

As the subunits of microtubules, alpha- and beta-tubulins have been thought to only exist in the cytoplasm where they are incorporated into microtubules. However, the beta(II) isotype of tubulin has recently been observed in the nuclei of rat kidney mesangial cells [Walss et al., 1999: Cell Motil. Cytoskeleton 42:274-284]. In this study, we detected nuclear beta(II)-tubulin in rat C6 glioma cells, human T98G glioma cells, human MCF-7 breast carcinoma cells, human MDA-MB-435 breast carcinoma cells, and human Hela cervix carcinoma cells. In addition, nuclear beta(II)-tubulin in these cells was found to exist as alphabeta(II) dimers instead of assembled microtubules and appeared to be particularly concentrated in the nucleoli. Several anti-tubulin drugs were used to treat C6 cells to determine their influence on nuclear beta(II)-tubulin. Taxol, a tubulin drug with higher specificity for beta(II)-tubulin than for other beta-tubulin isotypes, irreversibly decreased nuclear beta(II) content in a concentration-dependent manner in C6 cells. Meanwhile, cells were found to be apoptotic as was suggested by the presence of multiple micronuclei and DNA fragmentation. On the other hand, no depletion of nuclear beta(II)-tubulin was observed when C6 cells were incubated with colchicine or nocodazole, two anti-tubulin drugs with higher specificity for the alphabeta(IV) isotype, supporting the hypothesis that drugs with higher specificity for beta(II)-tubulin deplete nuclear beta(II)-tubulin.

In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance

Glial cells release a variety of molecules that support neuronal function. Because heat shock proteins (Hsps) are important in the survival of neurons subjected to metabolic stress, the possibility that glia can release the inducible form of the 70 kDa Hsp (Hsp70) was examined. Additionally, the ability of neuronal cells to show increased stress tolerance by taking up a mixture of constitutive and inducible forms of Hsp70 (Hsc/Hsp70) added to the extracellular fluid was tested. Human T98G glioma cells and differentiated LA-N-5 neuroblastoma cells were used as model glia and neurons to investigate these points. Hsp70 was analyzed using affinity chromatography, Western blotting, and immunofluorescence microscopy. The glioma cells were shown to export Hsp70 into the culture medium whether under normal conditions or subjected to heat shock. The amount of glial Hsp70 released ranged from 5 to 15 pg per 106 cells per day, being greater following heat shock. Neuroblastoma cells took up biotinylated Hsc/Hsp70 within 1 h after it was added to the culture medium and it made them more resistant to heat shock (44°C) and to staurosporine-induced apoptosis. This increased stress tolerance was especially important in neuroblastoma cells induced to differentiate with phorbol ester because those ‘mature neurons’ showed a 10-fold decline in endogenous Hsp70, which was accompanied by increased susceptibility to heat shock and staurosporine-induced apoptosis. These results suggest that extracellular Hsp70 may provide a means by which glia can affect neuronal function, perhaps enhancing neuronal stress tolerance.

Enhancing effect of electric stimulation on cytotoxicity of anticancer agents against rat and human glioma cells

Electropermeabilization, or electroporation, has been used to deliver genes or drugs into the cytoplasm through micropores in the cell membrane caused by electric stimulation. The cytotoxic effect of a combination of anticancer agents with electric stimulation on rat C6 and human T98G glioma cells was examined in vitro. Electric pulses of 100 μsec square waves (eight cycles at 1 Hz) at various electric fields were delivered to C6 or T98G glioma cell suspensions in combination with several anticancer agents. Cell growth was evaluated 48–72 h after treatment. Measurement of cell lysis by electric stimulation was used to assess the optimum field strength for electroporation. Electric stimulation enhanced significantly the cytotoxicity of bleomycin to both C6 and T98G cells by more than 1000-fold using an electric field of 1750 V/cm for C6 cells and 1000 V/cm for T98G cells. The enhancement disappeared when bleomycin concentration was reduced to 100 pg/ml. The cytotoxicity of carboplatin was weakly but significantly enhanced by electric stimulation when a high dose of carboplatin was used. However, there was no enhancement of the cytotoxicity of nimustine hydrochloride (ACNU), etoposide, and vincristine. These results indicate that the combination of bleomycin and electroporation is the most potent candidate for electrochemotherapy in vivo.

Quantitative Analysis of Glioma Cell Invasion by Confocal Laser Scanning Microscopy in a Novel Brain Slice Model

To quantitatively analyze the spatial extent of glioma cell migration in an organotypic brain slice culture, we developed a new invasion model with the aid of confocal laser scanning microscopy (CLSM). CLSM allowed not only for three-dimensional visualization of the invasive pattern of human T98G glioma cells in the living brain slice but also for serial analysis of the invasive process over several weeks. Twenty-four hours after the T98G glioma spheroid was initiated to coculture with a brain slice, the glioma cells detached themselves from the spheroid and spontaneously continued to migrate on the surface of the brain slice, while they diffusely invaded into the slice by migrating to a deeper site. Immunohistochemical analysis revealed that these migrating glioma cells much more strongly immunostained for matrix metalloproteinase (MMP)-2 and -9 than the tumor spheroid which remained at the implanted site. Treatment of the T98G glioma spheroid with 1,10-phenanthroline, a specific inhibitor of MMPs, significantly inhibited not only the cell migration on the surface of the brain slice but also the invasion of the glioma cells into the slice. The present version of the glioma invasion model using CLSM makes it possible to spatially and serially analyze the extent of glioma cell invasion in the living brain slice for several weeks, making it a very useful tool for investigating the cellular and molecular mechanisms of glioma invasion under conditions most analogous to those of normal brains in vivo.

The antitumor effect of MX2, a new morpholino anthracycline, against malignant glioma cell lines and its subcellular distribution.

The chemotherapeutic effect of MX2 (3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarminomycin), a new lipophilic morpholino anthracycline, against rat C6 and human T98G glioma cells, was examined in vitro and in vivo. The subcellular distribution of MX2 was also studied. The drug concentrations of MX2 required for the 50% inhibition of cell growth (IC50) for C6 and T98G cells were 25.5 +/- 1.3 ng/ml and 70.6 +/- 6.8 ng/ml, respectively, which were much lower than the IC50 values for nimustine (ACNU). A C6 subline resistant to ACNU, C6/ACNU, was established by continuous exposure to graded concentrations of ACNU. The IC50 of MX2 for C6/ACNU was 28.3 +/- 2.2 ng/ml, indicating no cross-resistance to MX2. In the rats bearing the intracerebral C6 tumors, the life span was increased by about 40 to 100% after intravenous administration of MX2 at doses ranging from 1 to 3 mg/kg of body weight. Confocal laser scanning microscopy demonstrated visually the good accumulation of MX2 in the implanted intracerebral C6 tumors, as well as its predominant distribution in the cytoplasm over the nucleus in both cell lines in vitro. Ultrastructural studies also demonstrated the cytotoxic effects of MX2 against glioma cells. Our results suggest that MX2 may be a useful chemotherapeutic agent in the treatment of malignant gliomas and that confocal laser scanning microscopy is useful for the study of the cellular pharmacokinetics of anthracycline derivatives, such as MX2.

Subcellular localization and cellular pharmacokinetics of MX2, a new morpholino anthracycline in glioma cells using confocal laser scanning microscopy

The cellular uptake, subcellular distribution and retention of MX2, a new morpholino anthracycline, were compared with those of adriamycin (ADM) using confocal laser scanning microscopy (CLSM) in rat C6 and human T98G glioma cell lines. The tumor cells were exposed to 1-30 micrograms ml-1 of MX2 and ADM for 120 min and further incubated without drugs for 120 min after washing twice with medium. During incubation, real-time subcellular distribution of MX2 and ADM in living tumour cells were observed at various intervals using CLSM. For analysis of the in vivo uptake. Wistar rats bearing the C6 glioma were intravenously administered MX2 at a dose of 5 mg per kg body weight 60 min before sacrifice. The fluorescence of MX2 was predominantly seen in the cytoplasm in both C6 cells and T98G cells, although it was also present in the nucleus. In contrast, that of ADM was mainly confined to the nucleus in both cell lines. The fluorescent intensity of ADM in the nucleus after 120 min of exposure was approximately 1.5-fold higher than that of MX2 at the same dose exposure, probably indicating a greater amount of ADM accumulated in the nucleus than MX2. The influx and efflux of MX2 were much more rapid and greater than those of ADM in both cell lines. There was almost no difference in subcellular distribution among the doses tested in this study. The subcellular distribution of MX2 in vivo was almost similar to that of MX2 in vitro. These results suggest other mechanisms by which MX2 exerts its cytotoxic effects on tumour cells together with the inhibition of DNA topoisomerase II, which has been reported previously. It is considered that the CLSM technique is useful for the study of the cellular pharmacokinetics of antitumour agents such as anthracycline derivatives.