Pharmacological blockade of group II metabotropic glutamate receptors reduces the growth of glioma cells in vivo

Abstract

U87MG human glioma cells in cultures expressed metabotropic glutamate (mGlu) receptors mGlu2 and mGlu3. Addition of the mGlu2/3 receptor antagonist LY341495 to the cultures reduced cell growth, expression of cyclin D1/2, and activation of the MAP kinase and phosphatidylinositol-3-kinase pathways. This is in line with the evidence that activation of mGlu2/3 receptors sustains glioma cell proliferation. U87MG cells were either implanted under the skin (1x10(6) cells/0.5 ml) or infused into the caudate nucleus (0.5x10(6) cells/5 microl) of nude mice. Animals were treated for 28 days with mGlu receptor antagonists by means of subcutaneous osmotic minipumps. Treatments with LY341495 or (2S)-alpha-ethylglutamate (both infused at a rate of 1 mg/kg per day) reduced the size of tumors growing under the skin. Infusion of LY341495 (10 mg/kg per day) also reduced the growth of brain tumors, as assessed by magnetic resonance imaging analysis carried out every seven days. The effect of drug treatment was particularly evident during the exponential phase of tumor growth, that is, between the third and the fourth week following cell implantation. Immunohistochemical analysis showed that U87MG cells retained the expression of mGlu2/3 receptors when implanted into the brain of nude mice. These data suggest that mGlu2/3 receptor antagonists are of potential use in the experimental treatment of malignant gliomas.

Fig. 1

Response of U87MG cells to treatment with group II mGlu receptor ligands. A. Reverse transcriptase polymerase chain reaction analysis of mGlu1, 2, 3, 4, 5, and 7 receptor RNA in U87MG cells. The adult male rat cerebral cortex (CTX) is shown as a positive control. The absence of genomic DNA contamination was proved in parallel samples incubated without the reverse transcriptase (not shown). B. Western blotting of mGlu2/3 receptors in U87MG cells. Thirty micrograms of proteins were loaded for each lane. CTX is shown as a positive control. The blot was repeated three times with similar results. C. Exposure to the mGlu2/3 receptor antagonists LY341495 (1 μM), MTPG (100 μM), or Eglu (100 μM) reduced U87MG cell growth in culture. Drugs were applied to the cultures every 24 h for four days. Values are means ± the standard error of the mean (SEM) of three individual determinations. *P < 0.05 (one-way ANOVA + Student-Newman-Keuls test to isolate the differences) versus the respective controls. D–F. Expression of phosphoERK1/2 (panel D), cyclin D1/D2 (panel E), and phospho-Akt (panel F) in cultured U87MG stimulated with EGF and treated with LY341495 (1 μM) and/or LY379268 (1 μM). Densitometric values are means + SEM of three to four determinations. *P < 0.05 (one-way ANOVA + Student-Newman-Keuls test) as compared with all other groups treated with EGF.

Fig. 2

Western blot analysis of mGlu2/3 receptors in cultured U87MG cells treated for one to four days with LY341495. Densito-metric values are means + SEM (n = 3).

Fig. 3

Effects of continuous systemic administration of mGlu2/3 receptor ligands on the growth of U87MG cells implanted under the skin of nude mice. Animals were treated for 28 days with saline, LY341495 (1 mg/kg per day), Eglu (1 mg/kg per day), LY379268 (1 mg/kg per day), or LY341495 + LY379268, all by means of subcutaneous osmotic minipumps. Two independent experiments are shown. A. n = 10. B. n = 8 to 10. Values are means + SEM; *P < 0.05 (one-way ANOVA + Student-Newman-Keuls test) versus mice treated with saline.

Fig. 4

Growth to day 28 of a brain tumor initiated with U87MG cells. A. MRI analysis of a brain tumor originating from unilateral intra-striatal infusion of U87MG cells in a control nude mouse. The T2-weighted (T2-W) image shows the absence of brain edema at seven days following cell implantation. Sequential contrast-enhanced T1-W images (from 7 to 28 days following cell implantation) show the progressive expansion of the brain tumor. B–E. Hematoxylineosin staining after 28 days shows (B) the sharp border between the tumor and the surrounding brain tissue (objective = 10×), (C) tumor cells at higher (40×) magnification, (D) the mGlu2/3 receptor immunoreactivity at 10×, and (E) the immunoreactivity at 40× magnification. Arrowheads in panels B and D show the borders between tumors and normal brain.

Fig. 5

Tumor growth over 28 days in LY341495-treated and control mice. A–B. Sequential frontal contrast-enhanced T1-W images of brain tumor originated from unilateral intrastriatal infusion of U87MG cells in mice. A. Representative control mouse. B. Mouse treated with LY341495 (10 mg/kg/day). The treatment was started 7 days following cell implantation. C–D. Data (means + SEM) from two independent experiments. C. Ten mice were treated with saline or LY341495 (10 mg/kg per day). Four mice treated with LY341495 were withdrawn from the drug at day 21 (black circle and dashed line in the graph). D. Twelve mice were treated with saline, and six were treated with LY341495. *P < 0.05 (one-way ANOVA + Student-Newman-Keuls test in panel C; and Student t test in panel D) versus the corresponding values obtained in mice treated with saline (C and D) or in mice withdrawn from LY341495 (C).

Fig. 6

Ki-67 immunostaining of U87MG cells implanted in the mouse brain. A–B. A representative immunostaining of tumor developed in mice treated with saline (A) or LY341495 (B) released subcutaneously by osmotic minipumps (objective = 20×). C. Quantification of immunostaining, where data are normalized per square centimeter of microscopic field and represent the means ± SEM of cell counts obtained from five mice per group (four random microscopic fields for each mouse). *P < 0.05 (Student t test), if compared with mice treated with saline.