gms | German Medical Science

Objective: Roscovitine, a purine analogue and cyclin-dependent kinase (CDK) inhibitor, shows strong antiproliferative and proapoptotic effects in solid and hematologic cancers, such as non small-cell lung cancer and various lymphomas. It targets preferentially CDK2, 7 and 9, which are also overexpressed in glioblastoma. Previous studies have shown that the combined therapy with roscovitine influenced survival of glioma patients. We therefore investigated the molecular effects of roscovitine in glioblastoma cell lines in vitro.

Method: Glioblastoma cell lines A172 and G28 were incubated with serial concentrations of roscovitine for 24-120 hours. Proliferation was measured using the xCELLigence Real Time Cell Analyzer, an impedance-based cell viability system. Cell cycle distribution was assessed using flow cytometry and gene expression quantified using real-time PCR.

Results: xCELLigence showed the real time growth and death of the cells after 24h in A172 and proliferation stop after 48 h in A172 by concentration of 100 µM. Roscovitine targets CDK 2, 7 and 9 were reduced on mRNA levels in a dose-dependent manner to values of 0.3, 0.02 and 0.04-fold in A172 and 0.1, 0.3 and 0.1-fold in G28 cells, respectively at 100 µM roscovitine for 72 hours. Cycline A and E were both reduced to values of 0.01-fold in A172 and to a value of 0.01 and 0.06-fold in G28. In addition, we observed a reduced p53 expression to 0.3-fold in A172 and 1.9-fold in G28. p21 showed a differential change in expression, increasing 4.63-fold in A172 and of decreasing 0.05-fold in G28 cells. Cell cycle analysis by flow cytometry showed a dose-dependent increase of the G2/M phase to 37.2 at 24 hours already and to 51.5% after 72 hours. Apoptosis increased to 15.8% after 72 hours in A172 cells vs. 2.5% in untreated controls and in 20.2% vs.0.9% in G28 cells.

Conclusions: Roscovitine suppresses CDK2, 7, and 9 on the mRNA level and has antiproliferative effects in concentrations of 100 µM. Its effects on cell cycle are represented by an G2/M arrest which result in reduced proliferation. In addition, it induces apoptosis and could therefore be a promising therapeutic agent in glioblastoma.