The radiosensitising effect of olomoucine derived synthetic cyclin-dependent kinase inhibitors

Bohemine and roscovitine are the most important representatives of the group of compounds structurally derived from olomoucine. Biologically they function as inhibitors of cyclin-dependent kinases (CDKs), the key regulators of cell cycle, which is often disrupted in cancer cells resulting in uncontrollable proliferation. Bohemine and roscovitine have demonstrated their cytostatic and cytotoxic in vitro and also in vivo effects. Currently the phase II clinical trials for roscovitine are underway. The aim of the study was to evaluate the potential in vitro radiosensitising effect of bohemine (BOH) and roscovitine (ROS). Clonogenic survival assay and human lung adenocarcinoma cell line A549 were used. Tested schedules were: A-pretreatment, B-concomitant application and C-posttreatment. Concentrations corresponded to IC10, IC25 and IC50 for BOH/ROS (0.1-30 mu M). The radiation doses were 1, 2 and 3 Gy. Flow cytometry and western blot analysis were used to characterize cell cycle distribution, BrdU incorporation and DNA repair processes. The highest in vitro radiosensitising effect of BOH/ROS was observed for Schedule A in all tested concentrations (SER(37%) 1.46-3.20). Cell cycle analysis showed an inclination towards G0/G1 delay 48 hours posttreatment and unaltered level of apoptosis. Changes in the DNA repair processes were observed - inhibition of DNA-PK kinase, inhibition of BrdU incorporation, strong and enduring induction of p21 protein and long-lasting phosphorylation of gamma H2AX(Ser139). Certain low concentration activities of BOH/ROS in monotherapy were detected, mainly the activation of DNA-PK kinase. The results demonstrated strong in vitro radiosensitising effect of BOH/ROS that is concentration and especially schedule dependent. The strong cytostatic effect of the pretreatment schedule is mediated through the inhibition/rearrangements of DNA repair processes.