Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines

The prognosis of malignant glioma, the most common brain tumor, is still poor, thus underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell cycle control, there is a strong rationale to examine the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact or deleted or mutated p53 or PTEN or p14ARF, or EGFR amplification status. Dinaciclib was found to inhibit cell proliferation and induce cell cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72 h MTS assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitor ABT-263 or ABT-737 with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c, smac/DIABLO and apoptosis-inducing factor (AIF), phosphatidylserine exposure on the plasma membrane surface and activation of caspases and PARP. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1. These observations may have important clinical implications for the design of experimental treatment protocols for malignant human glioma. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. as described in the Materials and Methods. Nuclei were stained with DAPI. Control cells received DMSO. Morphological and nuclear changes in response to inhibitor treatment were evaluated by microscopic inspection. C , U87, U87-EGFRviii, LNZ308, U373, LN229, LN18 and A172 cells were seeded at 60% confluence, allowed to attach overnight, and treated with dinaciclib (2.5µmol/L) for 24 h. Cell cycle analysis using propidium iodide (PI) staining was performed as described in the Materials and Methods Section. The results represents the mean of 3 three independent experiments. C ) for 24 h. The integrity of the mitochondrial membranes of the cells was examined by DiOC6 staining and flow cytometry. D , U87, U87-EGFRviii, LNZ308, LN229, LN18 and T98G cells were treated with dinaciclib (50 nmol/L) or ABT-737 (50 nmol/L) or the combination of both for indicated durations. Cytosolic extract was prepared, and equal amounts of protein were separated by SDS-PAGE and subjected to Western blotting analysis with the indicated antibodies. E , U87, U87-EGFRviii, LNZ308, LN229, LN18 and T98G cells were treated with dinaciclib (50 nmol/L) or ABT-737 (50 nmol/L) or the combination of both for the indicated duration and lysed with 1% CHAPS buffer. An equal amount of protein (500 µg) was immunoprecipitated (IP) with monoclonal anti-Bax (6A7; Sigma-Aldrich) antibody and then subjected to Western blot analysis with polyclonal anti-Bax antibody (Cell Signaling Technology). F , U87, U87-EGFRviii, LNZ308, LN229, LN18 and T98G cells were treated with dinaciclib (50 nmol/L) or ABT-737 (50 nmol/L) or the combination of both for 12 h. Control cells received equivalent amounts of DMSO. Membrane fractions were obtained as described under Materials and Methods, and proportional amounts corresponding to total protein were analyzed for Bax oligomerization by Western blotting under non-reducing conditions. Slow-moving Bax oligomers in DSP cross-linked cells were derived from Bax monomers, and the molecular masses of oligomers containing Bax were calculated by plotting their migrations against migrations of molecular mass standards (left panel, Mol. Wt. Marker). The results of a representative study are shown; two additional experiments similar Mcl-1 Forty-eight cells were ABT-737 of ABT) for and viability was by Annexin V/PI apoptosis assay (lower In parallel, cell lysates were collected and protein was subjected to Western blot analysis using Mcl-1 antibody. Immunoblots and reprobed with