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PARP inhibitor olaparib sensitizes cholangiocarcinoma cells to radiation.

ABSTRACT: Cholangiocarcinoma (CCA) is a highly malignant tumor with resistance to radiotherapy alone. Olaparib, a highly potent poly(ADP-ribose) polymerase (PARP) inhibitor, has been shown to sensitize many types of tumor to radiotherapy. However, the effect of olaparib, either as monotherapy or as combination therapy with radiotherapy, on CCA is not known, and our study aimed to explore this. To assess radiosensitization in three CCA cell lines (QBC939, HuH28 and TFK-1), viability and clonogenic assays were conducted. The absorbed radiation doses were 0 Gy, 2 Gy, 4 Gy, and 6 Gy; olaparib concentrations were 0 nmol/L, 1 nmol/L, 10 nmol/L, 100 nmol/L, 1000 nmol/L, 2500 nmol/L, 5000 nmol/L and 10 000 nmol/L. The mechanism of olaparib radiosensitization was explored by Western blotting. Immunofluorescence staining and flow cytometry were conducted to explore DNA damage and apoptosis. The radiosensitivity of CCA cells was enhanced by olaparib, which alone had little effect on the CCA cell lines without BRCA mutations. The degree of radiosensitization increased with increasing doses of olaparib by viability and clonogenic assays in vitro. Olaparib was able to enhance the effect of radiation by inhibiting PARP1, inducing DNA lesions and apoptosis. These findings emphasize the role of olaparib in the radiosensitization of CCA cells.

SUBMITTER: Mao Y

PROVIDER: S-EPMC5911590 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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PARP inhibitor olaparib sensitizes cholangiocarcinoma cells to radiation.

Mao Yize Y Huang Xin X Shuang Zeyu Z Lin Guohe G Wang Jun J Duan Fangting F Chen Jianlin J Li Shengping S

Cancer medicine 20180226 4

Cholangiocarcinoma (CCA) is a highly malignant tumor with resistance to radiotherapy alone. Olaparib, a highly potent poly(ADP-ribose) polymerase (PARP) inhibitor, has been shown to sensitize many types of tumor to radiotherapy. However, the effect of olaparib, either as monotherapy or as combination therapy with radiotherapy, on CCA is not known, and our study aimed to explore this. To assess radiosensitization in three CCA cell lines (QBC939, HuH28 and TFK-1), viability and clonogenic assays w ...[more]

PMID: 29479816

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Project description:The use of polyADPribose polymerase inhibitors in cancer treatment provides a unique opportunity to target DNA repair processes in cancer cells while leaving normal tissue intact. The PARP-1 enzyme repairs DNA single strand breaks (SSB). Therefore PARP-1 inhibition in BRCA1 negative cancers results in the formation of cytotoxic DNA double strand breaks (DSB) causing synthetic lethality. The use of PARP1 inhibitors is gaining momentum in the treatment of a variety of tumours with BRCA1 involvement including breast, ovarian, pancreatic and prostate cancer. Our previous work showed that the PARP-1 inhibitor Olaparib causes both hypersensitivity of BRCA1+/- cells following exposure to gamma radiation due to the persistence of DNA strand breaks in cells, measured by the DNA damage biomarker ?-H2AX. Therefore dual treatment of cancers with radiotherapy and PARP1 inhibition may lead to cases of increased normal tissue toxicity in cancer patients. In this study we exposed two normal lymphoblastoid cell lines and three heterozygous BRCA1 lymphoblastoid cell lines to the PARP-1 inhibitor Olaparib and gamma radiation and after measured BRCA1 protein expression and apoptosis levels following treatment. BRCA1 protein foci analysis was performed on cells exposed to 2 Gy radiation in the presence or absence of 5 ?M Olaparib. Using immunofluorescence and imaging flow cytometry, foci were measured in untreated cells and at 0.5, 3, 5 and 24 hours post-irradiation. Exposing normal and BRCA1+/- cells to Olaparib followed by gamma radiation results in a dramatic change in BRCA1 protein foci expression, with a significant reduction in BRCA1 protein expression observed in the heterozygote cells, together with an increase in apoptosis levels in these cells. In conclusion, combining PARP1 inhibitors with radiotherapy in treating of BRCA1-related cancers has clinical relevance, however this study and our previous publications serve to highlight the potential problems of increased side effects in these scenarios.

Project description:Objective: Osteosarcomas are derived from bone-forming mesenchymal cells that are insensitive to radiation. This study aimed to investigate the radiosensitization of osteosarcoma cells (U2OS and K7M2) using the PARP inhibitor olaparib combined with X-rays or carbon ions (C-ions). Methods: The effect of olaparib on the proliferation of osteosarcoma cells after irradiation was assessed using CCK-8 and clone formation assays. Cells were treated with olaparib and/or radiation and the effects of olaparib on the cell cycle and apoptosis were analysed by flow cytometry after 48h. Immunofluorescence was used to stain the nuclei, γ-H2AX, 53BP1, and Rad51 proteins, and the number of γ-H2AX, 53BP1, and Rad51 foci was observed under a fluorescence microscope. The effect of olaparib combined with radiation on double-stranded DNA breaks in osteosarcoma cells was evaluated. Results: At the same radiation dose, olaparib reduced the proliferation and colony formation ability of irradiated osteosarcoma cells (P < 0.05). Olaparib monotherapy induced minimal apoptotic effects and G2/M phase arrest in osteosarcoma cells and irradiation alone induced moderate apoptosis and G2/M phase arrest. However, radiation combined with olaparib significantly increased the percentage of apoptotic cells and G2/M phase arrest in osteosarcoma cells (P < 0.05). Immunofluorescence experiments showed that compared to the radiation group, the formation of γ-H2AX and 53BP1 foci was significantly increased in the combined group (P < 0.05). The expression levels of Rad51 foci in the irradiated group were higher than those in the control group (P < 0.05). However, the number of Rad51 foci in the combined group was significantly decreased (P < 0.05). Conclusion: The PARP inhibitor olaparib combined with irradiation (X-rays or C-ions) enhanced the radiosensitivity of osteosarcoma cell lines (U2OS and K7M2). Our findings provide a potential theoretical basis for the clinical application of olaparib in overcoming radiation resistance in osteosarcoma.

Project description:PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines.The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and ?H2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN.The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student's t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and ?H2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN?+ cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN?+ cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN.Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer.

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PARP inhibitor olaparib sensitizes cholangiocarcinoma cells to radiation.

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PARP inhibitor olaparib sensitizes cholangiocarcinoma cells to radiation.

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