Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas

Abstract

L-Glutamate (Glu) plays a crucial role in the growth of malignant gliomas. We have established the feasibility of accelerating a naturally occurring brain to-blood Glu efflux by decreasing blood Glu levels with intravenous oxaloacetate, the respective Glu co-substrate of the blood resident enzyme humane glutamate–oxaloacetate transaminase(hGOT). We wished to demonstrate that blood Glu scavenging provides neuroprotection in the case of glioma.We now describe the neuroprotective effects of blood Glu scavenging in a fatal condition such as brain-implanted C6 glioma in rats and brain-implanted human U87 MG glioma in nude mice. Rat (C-6) or human (U87) glioma cells were grafted stereotactically in the brain of rats or mice. After development of tumors, the animals were drinking oxaloacetate with or without injections of hGOT. In addition, mice were treated with combination treatment, which included drinking oxaloacetate with intracutaneous injections of hGOT and intraperitoneal injection of Temozolomide. Animals drinking oxaloacetate with or without injections of hGOT displayed a smaller tumor volume, reduced invasiveness and prolonged survival than control animals drinking saline. These effects were significantly enhanced by Temozolomide in mice, which increased survival by 237%. This is the first demonstration of blood Glu scavenging in brain cancer, and because of its safety, is likely to be of clinical significance for the future treatment of human gliomas. As we demonstrated, the blood glutamate scavenging treatment in combination with TMZ could be a good candidate or as an alternative treatment to the patients that do not respond to TMZ.

Figures

Fig. 1

a. A representative example of MRI analysis of C6 glioma growth in vivo. At 7 days post-implantation and before the treatment, an MRI imaging was performed (left column, 1–2 pictures T2 imaging, 3–4 pictures T1 imaging). The rats were randomized and either treated with 0.2 M OxAc or 0.3 M NaCl for additional 14 days. The MRI imaging was performed again at the end of the treatment (21 days post glioma implantation, right column, 1–4 pictures T2 imaging). b. Tumor volume of the rats before and after the treatment was calculated in n = 14 for control group and in n = 15 for the treated group using MRIcro software. OxAc-treated rats show significantly reduced tumor growth compared to the control group. *p < 0.01 (repeated measures ANOVA test)

Fig. 2

Tumor growth over 21 days in CD1 nude mice. U87 GM bearing CD1 nude mice were treated on day 7 with OxAc+hGOT, TMZ, OxAc+ hGOT +TMZ combined treatment, and control. *p < 0.05; ***p < 0.001 (one-way ANOVA with Newman-Keuls post test, n = 4) versus control group treated with 0.3 M NaCl

Fig. 3

Morphology of tumors derived from U87 glioma cell line A. Immunohistochemistry for H&E, PCNA and Aurora A. Data are representative of four mice per group. B. Quantification of tumor cell nuclear density (H&E staining); *p < 0.05, **p < 0.001 vs. control group, ***p < 0.001 vs. all other groups (one-way ANOVA+ Newman-Keuls test, n = 4. C. Quantification of the PCNA positive tumor cell nuclei staining; one-way ANOVA revealed significant differences between all groups (p < 0.001). Data presented as mean values ± SD

Fig. 4

Morphological evidence of the tumor invasiveness derived from U87 glioma cell line. a. Representative example of immunohistochemistry staining for H&E and GFAP. Staining by H&E showing infiltration of the brain parenchyma by tumor cells detached from the main tumor mass, arrows indicate the infiltrative cells (left column). Staining for GFAP showing the astrocytes cells around the tumor borders, arrowheads indicate the GFAP positive astrocytes (right column) b. Quantification of the number of astrocytes as indicated by positive GFAP staining,; ***p < 0.001 vs. all other groups, One-way ANOVA+ Newman-Keuls test, n = 4

Fig. 5

Survival rate in CD1 nude mice-bearing intracranial U87 MG cells. Kaplan-Meier survival of CD1 nude mice treated with 0.3 M NaCl 50 ml/day (n = 13), OxAc 2.14 mg/kg/day/hGOT 2.14 mg/kg on the first day followed by a maintenance dose of 0. 214 mg hGOT/kg/day (n = 13), TMZ 5 mg/kg for 4 days (n = 13) and a combined TMZ/ OxAc/hGOT treatment with the same doses as the rest groups (n = 13). Animals were monitored up to 115 days after the tumor cell implantation. P-Values obtained using a Long-rank test show P < 0.0001