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. 2013 Aug;114(1):33-42.
doi: 10.1007/s11060-013-1158-7. Epub 2013 May 22.

Increased glutamate uptake in astrocytes via propentofylline results in increased tumor cell apoptosis using the CNS-1 glioma model

Valerie L Jacobs  1 , Joyce A De Leo
Affiliations

Increased glutamate uptake in astrocytes via propentofylline results in increased tumor cell apoptosis using the CNS-1 glioma model

Valerie L Jacobs et al. J Neurooncol. 2013 Aug.

Abstract

Glioblastoma multiform is one of the most common and aggressive primary brain tumors in adults. High glutamate levels are thought to contribute to glioma growth. While research has focused on understanding glutamate signaling in glioma cells, little is known about the role of glutamate between glioma and astrocyte interactions. To study the relationship between astrocytes and tumor cells, the CNS-1 rodent glioma cell line was used. We hypothesized increased glutamate uptake by astrocytes would negatively affect CNS-1 cell growth. Primary rodent astrocytes and CNS-1 cells were co-cultured for 7 days in a Boyden chamber in the presence of 5 mM glutamate. Cells were treated with propentofylline, an atypical synthetic methylxanthine known to increase glutamate transporter expression in astrocytes. Our results indicate astrocytes can increase glutamate uptake through the GLT-1 transporter, leading to less glutamate available for CNS-1 cells, ultimately resulting in increased CNS-1 cell apoptosis. These data suggest that astrocytes in the tumor microenvironment can be _targeted by the drug, propentofylline, affecting tumor cell growth.

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Conflict of interest statement

Disclosure: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CNS-1 cells proliferate and preferentially grow in glutamate containing media. (a) Graphical representation of the percent of live CNS-1 cells following 3 days of growth in varying glutamate concentrations determined by trypan blue staining. Increasing glutamate resulted in a significant increase in CNS-1 cell number (* = p < 0.05). (b) CNS-1 cells were CFSE labeled on day 0 and analyzed by FACS analysis following 3 days of growth in varying glutamate concentrations. (c) Graphical representation of the percent of live CNS-1 cells following 7 days of growth in 5 mM glutamate, replaced with varying glutamate concentrations overnight and analyzed by trypan blue staining on day 8. Removal of glutamate resulted in a significant decrease in CNS-1 viability (* = p < 0.05).
Figure 2
Figure 2
Propentofylline increases Annexin V expression on CNS-1 cells when co-cultured with astrocytes. (a) Graphical representation of percent Annexin V+ CNS-1 cells co-cultured with astrocytes for 7 days and treated with PPF. (* = p < 0.05) (b) Representative FACS plots (media and 10 μM PPF) of Annexin V and Propidium Iodide staining in CNS-1 cells. (c) Graphical representation of percent Annexin V+ CNS-1 cells cultured alone for 7 days and treated with PPF. (d) Representative FACS plots (media and 10 μM PPF) of Annexin V and Propidium Iodide staining in CNS-1 cells. (e) Graphical representation of percent of Annexin V+ primary astrocytes cultured alone for 7 days and treated with PPF. (f) Representative FACS plots (media and 10 μM PPF) of Annexin V and Propidium Iodide staining in primary astrocytes. Data are representative of 3 replicates/experimental group; experiment was repeated three times.
Figure 3
Figure 3
Propentofylline decreases residual glutamate in media. (a) Graphical representation of percent glutamate remaining in media compared to untreated (media) group in astrocyte and CNS-1 co-culture treated with varying PPF dosages (* = p < 0.05). (b) Graphical representation of percent glutamate remaining in media compared to untreated (media) group in astrocyte cultures treated with varying PPF doses (* = p < 0.05). (c) Graphical representation of percent glutamate remaining in media compared to untreated (media) group in CNS-1 culture treated with varying PPF dosages. Data are representative of 3 replicates/experimental group; experiment was repeated twice. (d) Propentofylline’s effects on apoptosis are inhibited with GLT-1 blockade in astrocytes. Astrocytes were treated with GLT-1 or GLAST siRNA while co-cultured with CNS-1 cells for 7 days in 5 mM glutamate and treated with PPF (10 μM). Apoptosis of CNS-1 cells was significantly reduced with GLT-1 blockade in astrocytes (* = p < 0.05). Data are representative of 3 replicates/experimental group; experiment was repeated twice.
Figure 4
Figure 4
Propentofylline increases Annexin V expression on U251 cells when co-cultured with human astrocytes. (a) Graphical representation of percent Annexin V+ U-251 cells co-cultured with human astrocytes for 7 days and treated with PPF. (* = p < 0.05) (b) Representative FACS plots (media and 10 μM PPF) of Annexin V and Propidium Iodide staining in U-251 cells and human astrocytes. (c) Graphical representation of percent Annexin V+ U-251 cells cultured alone for 7 days and treated with PPF. (d) Representative FACS plots (media and 10 μM PPF) of Annexin V and Propidium Iodide staining in U-251 cells. (e) Graphical representation of percent Annexin V+ primary human astrocytes cultured alone for 7 days and treated with PPF. (f) Representative FACS plots (media and 10 μM PPF) of Annexin V and Propidium Iodide staining in primary human astrocytes. Data are representative of 3 replicates/experimental group; experiment was repeated twice.
Figure 5
Figure 5
Propentofylline decreases residual glutamate in the media of human astrocytes. (a) Graphical representation of percent glutamate remaining in media compared to untreated (media) group in astrocyte and U251 co-culture treated with varying PPF dosages (* = p < 0.05). (b) Graphical representation of percent glutamate remaining in media compared to untreated (media) group in astrocyte culture treated with varying PPF dosages (* = p < 0.05). (c) Graphical representation of percent glutamate remaining in media compared to untreated (media) group in U251 culture treated with varying PPF dosages. Data are representative of 3 replicates/experimental group; experiment was repeated twice.
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