TY - JOUR
T1 - Withanolide D Enhances Radiosensitivity of Human Cancer Cells by Inhibiting DNA Damage Non-homologous End Joining Repair Pathway
AU - Lacombe, Jerome
AU - Cretignier, Titouan
AU - Meli, Laetitia
AU - Wijeratne, E. M.Kithsiri
AU - Veuthey, Jean Luc
AU - Cuendet, Muriel
AU - Gunatilaka, A. A.Leslie
AU - Zenhausern, Frederic
N1 - Funding Information:
Authors would like to greatly thank Colleen's Dream Foundation for its financial support. We also thank Dr. Kurt Gustin from the Biomedical Imaging Core at the UA College of Medicine—Phoenix for providing epifluorescence imaging service. Funding. Colleen's Dream Foundation funded part of this work. The funders had no role in the study design, data collection, data analysis, decision to publish, or manuscript preparation.
Publisher Copyright:
© Copyright © 2020 Lacombe, Cretignier, Meli, Wijeratne, Veuthey, Cuendet, Gunatilaka and Zenhausern.
PY - 2020/1/8
Y1 - 2020/1/8
N2 - 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.
AB - 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.
KW - DNA damage repair
KW - cancer
KW - mitotic catastrophe
KW - radiation
KW - radiosensitizer
KW - withanolide D
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U2 - 10.3389/fonc.2019.01468
DO - 10.3389/fonc.2019.01468
M3 - Article
AN - SCOPUS:85078255587
SN - 2234-943X
VL - 9
JO - Frontiers in Oncology
JF - Frontiers in Oncology
M1 - 1468
ER -