Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

Abstract

Malignant gliomas are characterized by an intrinsic ability to invade diffusely throughout the normal brain tissue. This feature contributes mainly to the failure of existing therapies. Deregulation of small GTPases signaling, in particular Rac1 activity, plays a key role in the invasive phenotype of gliomas. Here we report the effect of ZINC69391, a specific Rac1 inhibitor developed by our group, on human glioma cell lines LN229 and U-87 MG. ZINC69391 is able to interfere with the interaction of Rac1 with Dock180, a relevant Rac1 activator in glioma invasion, and to reduce Rac1-GTP levels. The kinase Pak1, a downstream effector of Dock180-Rac1 signaling, was also downregulated upon ZINC69391 treatment. ZINC69391 reduced cell proliferation, arrested cells in G1 phase, and triggered apoptosis in glioma cells. Importantly, ZINC69391 dramatically affected cell migration and invasion in vitro, interfering with actin cytoskeleton dynamics. We also evaluated the effect of analog 1A-116, a compound derived from ZINC69391 structure. 1A-116 showed an improved antiproliferative and antiinvasive activity on glioma cells. These findings encourage further preclinical testing in clinically relevant animal models.

Keywords: Dock180; GTPases. invasion; small molecule.

Figure 1

Chemical structures of Rac1 inhibitors. Notes: (A) Chemical structure of ZINC69391 (C14H15F3N5; molecular weight, 310.303). (B) Chemical structure of 1A-116 analog (C16H16F3N3; molecular weight, 307.31).

Figure 2

ZINC69391 inhibited Rac1 activation on LN229 glioma cells. Notes: (A) Blockade of Rac1–Dock180 interaction. Dock180 was affinity-precipitated with bacterially expressed Rac1 immobilized in glutathione agarose beads in the presence of varying concentrations of ZINC69391. Western blot analysis was carried out with anti-Dock180 antibody. The experiment was repeated three times. Densitometric values are shown below (arbitrary units). (B) Concentration-dependent Rac1 inhibition by ZINC69391 in glioma cells. Serum-starved LN229 cells were treated for 1 hour with different ZINC69391 concentrations and stimulated with epidermal growth factor (EGF) (100 ng/mL) for 15 minutes. Densitometric values are shown below (arbitrary units). (C) Concentration-dependent inhibition of Pak1 phosphorylation using the same experimental procedure as before. Densitometric values are shown below (arbitrary units).

Figure 3

ZINC69391 affected cell proliferation and cell cycle progression of glioma cells. Notes: (A) ZINC69391 inhibited cell proliferation. LN229 and U-87 MG cells were treated for 72 hours with different concentrations of ZINC69391. Cell viability was measured using MTT assay. (B) ZINC69391 arrested cell cycle progression in G1 phase. LN229 cells were synchronized and treated for 48 hours with ZINC69391 with different concentrations. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry to estimate the percentage of cells in sub-G0 phase, G1 phase, S phase, and G2/M phase. Bars, standard error of the mean. *P<0.05, **P<0.01 determined by analysis of variance contrasted. Dunnett’s multiple comparison test versus control in each phase.

Figure 4

ZINC69391 triggered apoptosis on malignant glioma cells. Notes: (A) Apoptosis of LN229 cells after 10 μM and 50 μM treatment for 6 hours using annexin V staining. Representative micrographs taken at 200×. (B) Late apoptosis was evaluated at 10 μM and 50 μM for 6 hours using TUNEL assay. Representative micrographs taken at 100×. (C) Quantification of apoptotic cells per field using TUNEL assay. Bars, standard error of the mean, *P<0.05, **P<0.01 determined by analysis of variance cont. Dunnett’s multiple comparison test versus control.

Figure 5

ZINC69391 inhibited glioma cell migration and invasion modulating actin cytoskeleton reorganization. Notes: (A) LN229 cells were seeded in uncoated transwell chambers with or without ZINC69391, incubated for 18 hours, and quantified. Bars, standard error of the mean. *P<0.05; ***P<0.001 determined by analysis of variance cont. Dunnett’s multiple comparison test. (B) LN229 cells were seeded in Matrigel-coated transwell chambers with or without ZINC69391, incubated for 48 hours, and quantified. Bars, standard error of the mean. *P<0.05; **P<0.01; ***P<0.001 determined by analysis of variance cont. Dunnett’s multiple comparison test. (C) Representative micrographs taken at 1,000× showing inhibition of epidermal growth factor (EGF)-induced actin reorganization by ZINC69391 in LN229 cells. Cells were grown on cover slips, serum-starved for 16 hours (untreated panel), and treated for 1 hour with ZINC69391. After 15 minutes stimulation with EGF (100 ng/mL) (EGF control), cells were fixed and actin filaments were visualized with AlexaFluor555-phalloidin.

Figure 6

1A-116 analog is a more potent Rac1 inhibitor. Notes: (A) LN229 and U-87 MG cells were treated for 72 hours with different concentrations of ZINC69391 and 1A-116. Cell viability was measured using MTT assay. (B) LN229 were transiently transfected with Rac1 small interfering (si)RNA or control siRNA. Cells were treated for 72 hours with different concentrations of 1A-116 analog and cell viability was measured using MTT assay. (C) LN229 cells were seeded in Matrigel-coated transwell chambers with or without ZINC69391, incubated for 48 hours, and quantified. **P<0.01, ***P<0.001. Determined by analysis of variance contrasted with Dunnett’s multiple comparison test.