Synergistic effects of PARP inhibitors and ionizing radiation on growth and survival of rhabdomyosarcoma cells

Background: The poly(ADP-ribose) polymerase inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma in children and adolescents. Our study evaluated the effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthetized PARP1/2/3 inhibitor, in RMS cells both as single-agent and in combination with ionizing radiation (IR). Methods: Cell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was investigated by clonogenic assays. Results: Olaparib (1.5 and 5 μM) or AZD2461 (5 and 10 μM) treatments dose-dependently reduced RH30 and RD cell growth by arresting cell cycle at G2/M phase. A significant modulation in the expression/activation and subcellular localization of specific cell cycle regulators, such as cyclin B1, cyclin D1, cdc2, cdc25C and p21, was observed in both Olaparib- and AZD2461-treated cells in comparison to mocked controls. Phosphorylation of H2AX (γH2AX), a specific marker of DNA damage, was significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to cellular death as assessed by BCL2 down-regulation, caspase-3 activation and cytoplasmic vacuolization. PARPi treatment was also able to induce autophagy in addition to apoptosis, as confirmed by the concurrent LC3-II cleavage and p62 reduction. Interestingly, both PARP inhibitors strongly enhanced the effects of IR by blocking DNA damage repair, increasing G2/M arrest and drastically reducing the colony formation capacity of RMS-cotreated cells. Conclusions: Our findings suggest that combined exposure to PARP inhibitors and IR might have therapeutic benefits in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.