Abstract
PurposePARP inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent childhood soft-tissue sarcoma. The cellular and molecular effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthesized PARP1/2/3 inhibitor, were assessed in alveolar and embryonal RMS cells both as single-agent and in combination with ionizing radiation (IR).MethodsCell 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 assessed by using clonogenic assays.ResultsOlaparib and AZD2461 dose-dependently reduced growth of both RH30 and RD cells by arresting growth at G2/M phase and by modulating the expression, activation and subcellular localization of specific cell cycle regulators. Downregulation of phospho-AKT levels and accumulation of γH2AX, a specific marker of DNA damage, were significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to apoptosis-related cell death. Both PARPi significantly enhanced the effects of IR by accumulating DNA damage, increasing G2 arrest and drastically reducing the clonogenic capacity of RMS-cotreated cells.ConclusionsThis study suggests that the combined exposure to PARPi and IR might display a role in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.
Highlights
Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, representing approximately 50% of all sarcomas in children aged 0–14 years (McDowell 2003; Francesca Megiorni and Carlo Dominici contributed .Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Extended author information available on the last page of the articleO’Neill et al 2013)
Poly(ADP-ribose) polymerases (PARPs) expression was analysed in two ARMS (RH4 and RH30) and three ERMS (RD, RD18 and TE671) cell lines, and higher levels of PARP1, PARP2 and PARP3 transcripts were demonstrated in comparison to proliferating myoblasts (HFMs), with the only exception of PARP3 in RH4 cell line (Fig. 1b)
PARP inhibitors (PARPi)-treated cells showed a significant dose-dependent reduction in their number (Fig. 2b): direct counting for living cells using the trypan blue dye exclusion test showed that Olaparib exposure is able to inhibit cell growth by about 35% at 1.5 μM in both RMS cell lines, whilst higher concentrations (5 μM) reduced proliferation by 70% in RH30 cells and by 60% in RD compared to the untreated samples (Fig. 2b); AZD2461 led to about 35% decreased cell growth at 5 μM and to 60% at 10 μM in both RMS cell lines compared to mocked controls (Fig. 2b)
Summary
Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, representing approximately 50% of all sarcomas in children aged 0–14 years Extended author information available on the last page of the article. Alveolar RMS (ARMS) and embryonal RMS (ERMS), the two most common histological subtypes in childhood, have distinct clinicopathological features and outcomes (Coffin 1997; Parham and Barr 2013). ARMSs and ERMSs are both characterised by distinctive genetic alterations that are likely to play a decisive role in their pathogenesis
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