Along with surgery and chemotherapy, radiation therapy (RT) is an important modality in cancer treatment, with about half of cancer patients receiving RT during their treatment. However, there are still a lot of challenges to improve its efficiency by minimizing normal tissue toxicity while maximizing tumor control. In this perspective, radiosensitizers are a promising approach in order to create a better tumor response to ionizing radiation (IR) while allowing for better dose modulation. Withanolides are natural steroidal lactones found in the plant Withania somnifera and already known for their numerous biological effects, in particular an antitumor activity due to induction of ROS production, cell cycle arrest or cytoskeleton destabilization. Hence, we tested in this study our hypothesis that withanolide D (WD), a compound with an important antitumor effect, could also act as a radiosensitizer. Clonogenic assays showed that 1-hour WD pretreatment (0.7µM) before IR decreased surviving fraction of several cancer cell lines. To determine the mechanisms by which WD achieves its radiosensitizing effect, we then assessed whether WD could promote radiation-induced DNA damages and inhibit double-strand breaks (DSBs) repair in SKOV3 cells. Comet and γH2AX/53BP1 foci formation assays confirmed that DSBs are higher from 1 hour up to 24 hours after 2Gy-irradiation in WD-treated cells compared to vehicle-treated cells, suggesting that WD induces the persistence of radiation-induced DNA damages. We then performed immunoblotting to investigate protein expression involved in DNA repair pathways. Interestingly, DNA-PK, ATM and their phosphorylated forms appeared to be inhibited at 24 hours post-irradiation in WD-treated samples. XRCC4 expression is also decreased while RAD51 expression does not change compared to vehicle-treated cells suggesting that only non-homologous end joining pathways is altered by WD. In order to assess the consequence of such inhibition, we then investigated cell death, and especially delayed death, referred to as mitotic catastrophe (MC), which was described as the main form of epithelial cell death induced by IR. MC is induced after IR and but is predominant in WD-treated samples as showed by the few numbers of cells pursing into anaphase. Interestingly, our results showed that cells preferentially undergo necrosis-like death after MC instead of apoptosis. This can be explained by the p53 mutated profile of SKOV3 in addition to the WD-induced ATM inhibition. Together, these data demonstrate that WD is a promising radiosensitizer candidate for RT and additional studies are required to investigate its effect in more relevant clinical models.

Citation Format: Jerome Lacombe, Titouan Cretignier, Laetitia Meli, Jean-Luc Veuthey, Leslie Gunatilaka, Muriel Cuendet, Frederic Zenhausern. Withanolide D enhances radiosensitivity of human cancer cells to X-rays radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2932.