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Terpenoids-enriched fraction of Celastrus orbiculatus sensitizes gemcitabine by disrupting Chk1/RAD51-mediated DNA damage response in pancreatic cancer

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Abstract

The stem of Celastrus orbiculatus, a traditional Chinese herbal medicine exhibits prominent anti-inflammatory and anti-tumor activities. In the present study, we prepared terpenoids-enriched fraction termed TTC (Total Terpenoids of C. orbiculatus) and explored the potentials of TTC as a sensitizer of gemcitabine by targeting DNA damage response in treatment of pancreatic cancer. Initially, we characterized the chemical profile of TTC. TTC and constitutional terpenoids showed antitumor effects through prohibiting the proliferation of AsPC-1 and BxPC-3 cells using EdU incorporation assay. Next, we quantified the interaction between TTC and gemcitabine which produced synergistic effects under sub-IC50 concentrations. Moreover, TTC could further block S-phase entry or induce higher extent of apoptosis in the presence of gemcitabine. Of note, we observed that TTC caused DNA breaks in two PC cells assayed by single cell gel electrophoresis and metaphase spreading. We also ruled out the oxidative damage of DNA upon TTC exposure. Mechanically, TTC abrogated Chk1/RAD51, promoted accumulation of γH2AX and reversed replication stress associated DNA damage response triggered by gemcitabine. TTC destabilized nuclear Chk1 and RAD51 loading via proteasomal degradation. Additionally, the ectopic expression of hotspot mutant p53 (R175H, R248W and R273H) in p53-null AsPC-1 cells displayed increased expression of RAD51, which could be compromised by TTC. In an orthotopic mouse model of mCherry-BxPC-3 cells, we confirmed the in vivo anti-metastatic efficacy of TTC in combination with gemcitabine. These data provide insight into how TTC and molecules involved in DNA damage and repair could be effectively multi-targeted and confer chemosensitivity to gemcitabine toward pancreatic cancer.

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Data will be made available upon reasonable request for academic use and within the limitations of the provided informed consent by the corresponding author upon acceptance.

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Funding

This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (no. 81803782, 81903772), the China Postdoctoral Science Foundation (no. 2017M611936) and the Jiangsu Postdoctoral Science Foundation (no. 1701185B).

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Author notes

  1. Yang Zhao, Zhuangzhuang Jiang have contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacy, College of Medicine, Yangzhou University, 136 Jiangyang Avenue, Building 10, Room 111, Yangzhou, 225001, Jiangsu, China

    Yang Zhao, Zhuangzhuang Jiang, Kehui Zhou & Li Tao

  2. The State Administration of Traditional Chinese Medicine Key Laboratory of Toxic Pathogens-Based Therapeutic Approaches of Gastric Cancer, Yangzhou University, 136 Jiangyang Avenue, Building 41, Room 301, Yangzhou, 225009, Jiangsu, China

    Tengyang Ni, Wei Jiang & Yanqing Liu

  3. Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, China

    Yuping Liu

  4. Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, 210028, China

    Yuping Liu

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  1. Yang Zhao
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Contributions

YZ, ZJ performed the experiments and interpretation of data and contributed to the original draft of the manuscript. WJ prepared and offered the purified compounds from TTC. TN, KZ and YL provided methodological and technical support for in vivo experiments. YL supervised the formal investigation and data analysis. LT designed the study, performed the statistical analysis of the data, contributed to the interpretation of the data, reviewed and edited the manuscript. LT and YL have access to and take responsibility for the integrity of the data and for the accuracy of the data analysis.

Corresponding authors

Correspondence to Yanqing Liu or Li Tao.

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Supplementary Information

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42764_2021_55_MOESM1_ESM.tif

Supplementary Figure.1 TTC exhibits certain but lower cytotoxicity in GES-1 cells. (A) Growth inhibition response was determined by MTT assay in GES-1 cells treated with indicated concentrations of TTC (0, 25, 50, 100, 200 and 400 μg/mL) for 48 and 72 h. (B) GES-1 cells were incubated with 0, 25, 50, 100, 200 μg/mL of TTC. After 6 h treatment, whole cell lysates were harvested for immunoblot of cleavage of PARP1. GAPDH was used as an internal control

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Zhao, Y., Jiang, Z., Ni, T. et al. Terpenoids-enriched fraction of Celastrus orbiculatus sensitizes gemcitabine by disrupting Chk1/RAD51-mediated DNA damage response in pancreatic cancer. GENOME INSTAB. DIS. 2, 358–373 (2021). https://doi.org/10.1007/s42764-021-00055-w

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  • DOI: https://doi.org/10.1007/s42764-021-00055-w

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