Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway

Abstract

Along with surgery and chemotherapy, radiation therapy (RT) is an important modality in cancer treatment, and the development of radiosensitizers is a current key challenge in radiobiology to maximize RT efficiency. In this study, the radiosensitizing effect of a natural compound from the withanolide family, withanolide D (WD), was assessed. Clonogenic assays showed that a 1 h WD pretreatment (0.7 µM) before irradiation decreased the surviving fraction of several cancer cell lines. To determine the mechanisms by which WD achieved 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 were higher between 1 and 24 h after 2 Gy-irradiation in WD-treated cells compared to vehicle-treated cells, suggesting that WD induced the persistence of radiation-induced DNA damages. Immunoblotting was then performed to investigate protein expression involved in DNA repair pathways. Interestingly, DNA-PKc, ATM, and their phosphorylated forms appeared to be inhibited 24 h post-irradiation in WD-treated samples. XRCC4 expression was also down-regulated while RAD51 expression did not change compared to vehicle-treated cells suggesting that only non-homologous end joining (NHEJ) pathways was inhibited by WD. Mitotic catastrophe (MC) was then investigated in SKOV3, a p53-deficient cell line, to assess the consequence of such inhibition. MC was induced after irradiation and was predominant in WD-treated samples as shown by the few numbers of cells pursuing into anaphase and the increased amount of bipolar metaphasic cells. Together, these data demonstrated that WD could be a promising radiosensitizer candidate for RT by inhibiting NHEJ pathway and promoting MC. Additional studies are required to better understand its efficiency and mechanism of action in more relevant clinical models.

Natural Compounds That Target DNA Repair Pathways and Their Therapeutic Potential to Counteract Cancer Cells

“…In lung squamous carcinoma cells, luteolin caused an increase in the levels of MHT1, OGG1, and AP-1 (106). Withanolide D, a compound obtained from Withania somnifera, was shown to improve the radiosensitivity of different cancer cell lines by inhibiting DNA damage via non-homologous end joining repair pathway (107). Isoorientin is a flavonoid extracted from many plant species, such as flax straw, watery leaf, Gypsophila elegans, Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum.…”

Use of a healthy volunteer imaging program to optimize clinical implementation of stereotactic MR-guided adaptive radiotherapy

“…Future work should compare imaging and simulation parameters with more quantitative measures such as signal to noise ratio. Additional work is also needed to aggregate results from experiences across centers and across vendors [26] , which will better inform the most optimal parameters for SMART.…”

“…Although contouring atlases for a limited number of sites are available [22] , [23] , [24] , these atlases do not provide instructions or consensus for contouring across MR field strengths and MR sequences and disease sites. Gaps exist in both training for SMART [25] , and consensus regarding its delivery [19] , [26] . In this study, a healthy volunteer imaging program was developed and implemented to establish optimal simulation parameters for SMART prior to clinical implementation and to provide a simulated clinical environment in which to develop and practice multi-disciplinary skills in the safe and effective implementation of SMART.…”

Plant-Based Scaffolds Modify Cellular Response to Drug and Radiation Exposure Compared to Standard Cell Culture Models

“…For the drug response assay, withaferin A (WFA) from the aerial parts of aeroponically grown W. somnifera was used and characterized as previously described ( Lacombe et al, 2019 ). WFA was added at concentrations ranging from 0.156 to 40 μM.…”