Targeting glioma cells by antineoplastic activity of reversine

Abstract

Gliomas are the most common type of primary central nervous system tumors and despite great advances in understanding the molecular basis of the disease very few new therapies have been developed. Reversine, a synthetic purine analog, is a multikinase inhibitor that targets aurora kinase A (AURKA) and aurora kinase B (AURKB). In gliomas, a high expression of AURKA or AURKB is associated with a malignant phenotype and a poor prognosis. The present study investigated reversine-related cellular and molecular antiglioma effects in HOG, T98G and U251MG cell lines. Gene and protein expression were assessed by reverse transcription-quantitative PCR and western blotting, respectively. For functional assays, human glioma cell lines (HOG, T98G and U251MG) were exposed to increasing concentrations of reversine (0.4-50 µM) and subjected to various cellular and molecular assays. Reversine reduced the viability and clonogenicity in a dose- and/or time-dependent manner in all glioma cells, with HOG (high AURKB-expression) and T98G (high AURKA-expression) cells being more sensitive compared with U251MG cells (low AURKA- and AURKB-expression). Notably, HOG cells presented higher levels of polyploidy, while T98G presented multiple mitotic spindles, which is consistent with the main regulatory functions of AURKB and AURKA, respectively. In molecular assays, reversine reduced AURKA and/or AURKB expression/activity and increased DNA damage and apoptosis markers, but autophagy-related proteins were not modulated. In conclusion, reversine potently induced mitotic catastrophe and apoptosis in glioma cells and higher basal levels of aurora kinases and genes responsive to DNA damage and may predict improved antiglioma responses to the drug. Reversine may be a potential novel drug in the antineoplastic arsenal against gliomas.

Keywords: apoptosis; aurora kinases; glioblastoma; gliomas; reversine.

Figure 1.

Expression of AURKA and AURKB in glioma cell lines. (A) RT-qPCR analysis of AURKA and AURKB mRNA expression in U87MG, HOG, T98G and U251MG cells. Bar graphs represent the mean ± SD of at least 3 independent samples. AURKA expression analysis: ***P<0.001, HOG vs. U87MG or U251MG; **P<0.05, T98G vs. U87MG or U251MG. AURKB expression analysis: ***P<0.001, HOG vs. other cell lines; **P<0.05, T98G vs. U87MG. (B) Western blot analysis for AURKA, AURKB and p-histone H3^S10 in total cell extracts from U87MG, HOG, T98G, and U251MG; membranes were reprobed with the antibody for the detection of β-actin. (C) Bar graphs represent the mean ± SD of 3 independent experiments quantifying band intensities of indicated proteins. AURKA protein levels: ***P<0.01, T98G vs. other cell lines; **P<0.01, U87MG vs. other cell lines. AURKB protein levels: ***P<0.001, HOG vs. other cell lines; **P<0.01, T98G vs. U87MG cell line. Phosphorylated-histone H3^S10 levels: ***P<0.01, T98G vs. other cell lines. ANOVA test and Bonferroni post-test. AURKA, aurora kinase A; AURKB, aurora kinase B; p, phosphorylated; RT-q, reverse transcription-quantitative.

Figure 2.

Reduction of cell viability and clonogenicity by reversine in glioma cells. (A) Dose- and time-response cytotoxicity analyzed by the sulforhodamine B (SRB) assay for HOG, T98G and U251MG cells treated with vehicle (DMSO) or graded concentrations of reversine (0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25 and 50 µM) for 24, 48 and 72 h. Values are expressed as the percentage of viable cells for each condition relative to vehicle-treated cells. Results are shown as mean ± SD of at least 3 independent experiments. (B) Colony formation after reversine exposure for 24 h and placement in drug-free media for an additional 10–15 days. Representative images of colony formation after vehicle or reversine (0.2, 0.4, 0.8, and 1.6 µM) treatment are illustrated. (C) Bar graph represents mean ± SD of relative number of colonies (% of control). *P<0.05, **P< 0.01 and ***P<0.001. IC, inhibitory concentration.

Figure 3.

Reversine induced mitotic aberrations and polyploidy in glioma cells. (A) Cell cycle progression was determined by PI staining and flow cytometry in HOG, T98G and U251MG cells treated with vehicle (DMSO) or 0.8 and 1.6 µM of reversine for 24 h. A representative histogram for each condition is illustrated. Bar graphs represent the mean ± SD of the percent of cells in _subG₁, G₀/G₁, S, G₂/M and >4N cells for conditions from at least three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. (B) Immunofluorescence analysis of HOG, T98G and U251MG cells treated with 1.6 µM reversine or vehicle for 24 h, displaying α-tubulin (green) and DAPI (blue) staining. The zoomed images highlight mitotic aberrations and/or polyploidy observed upon reversine exposure. Scale bar, 100 µm. PI, propidium iodide.

Figure 4.

Reversine triggered apoptosis and DNA damage markers in glioma cells. Western blot analysis for AURKA, AURKB, p-histone H3^S10, γH2AX, PARP1 (total and cleaved), LC3BI/II and SQSTM1/p62 in total cell extracts from HOG, T98G and U251MG cells treated with (A) vehicle (DMSO) or graded concentrations of reversine (vehicle, 1.6, 3.2 or 6.4 µM) for 24 h or (B) graded time of exposure (24, 48 and 72 h) to reversine at 1.6 µM. Membranes were reprobed with the antibody for the detection of α-tubulin. (C) Heatmap illustrates the RT-qPCR analysis of BCL2, BCL2L1, BIRC5, BNIP3, BNIP3L, BAD, BAX, BBC3, PMAIP1, CDKN1A, CDKN1B and GADD45A gene expression in HOG cells upon treatment with reversine (1.6 µM; mean, n=4) for 24 h. The data are represented as the fold-change of vehicle-treated HOG cells and down and upregulated genes are shown by blue and red, respectively. Bar graph represents mean ± SD of the fold-change of vehicle-treated cells (dotted line) for PMAIP1, CDKN1A and GADD45A in HOG, T98G, and U251MG cell lines upon reversine exposure for 24 h. **P<0.1 and ***P<0.001. (D) Trypan blue exclusion dye assay in HOG, T98G, and U251MG cells upon vehicle or 1.6 µM reversine treatment for 72 h. Bar graph represents relative mean ± SD of viable (gray) and non-viable (blue) cells. *P<0.05 and **P<0.01. p, phosphorylated; RT-q, reverse transcription-quantitative; AURKA, aurora kinase A; AURKB, aurora kinase B; γH2AX, phosphorylated histone 2AX; PARP1, poly(ADP-ribose) polymerase 1; SQSTM1/p62, sequestosome 1; LC3BII, microtubule associated protein 1 light chain 3 beta, BCL2, BCL2 apoptosis regulator; BCL2L1, BCL2 like 1; BIRC5, baculoviral IAP repeat containing 5; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BAD, BCL2 associated agonist of cell death; BAX, BCL2 associated X, apoptosis regulator; BBC3, BCL2 binding component 3; PMAIP1, phorbol-12-myristate-13-acetate-induced protein 1; CDKN1A, cyclin dependent kinase inhibitor 1A; CDKN1B, cyclin dependent kinase inhibitor 1B; GADD45A, growth arrest and DNA damage inducible gene 45 alpha.