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

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.

Funding

Young Scientists Fund(81803782)

Postdoctoral Research Foundation of China(2017M611936)

Postdoctoral Science Foundation of Jiangsu Province(1701185B)