Natural podophyllotoxin analog 4DPG attenuates EMT and colorectal cancer progression via activation of checkpoint kinase 2

Abstract

AbstractEpithelial–mesenchymal transition (EMT) is critical for the metastatic dissemination of cancer cells and contributes to drug resistance. In this study, we observed that epithelial colorectal cancer (CRC) cells transiently exposed to 5-fluorouracil (5-FU) (a chemotherapeutic drug for CRC) as well as 5-FU-resistant cells (5-FU-R) develop EMT characters as evidenced by activation of Vimentin and augmented invasive properties. On the other hand, 4DPG (4′-demethyl-deoxypodophyllotoxin glucoside), a natural podophyllotoxin analog attenuates EMT and invadopodia formation abilities of HCT-116/5-FU-R and SW-620/5-FU-R cells. Treatment with 4DPG restrains Vimentin phosphorylation (Ser38) in 5-FU-R cells, along with downregulation of mesenchymal markers Twist1 and MMP-2 while augmenting the expression of epithelial markers E-cadherin and TIMP-1. Moreover, 4DPG boosts the tumor-suppressor protein, checkpoint kinase 2 (Chk2) via phosphorylation at Thr68 in a dose-dependent manner in 5-FU-R cells. Mechanistically, SiRNA-mediated silencing of Chk2, as well as treatment with Chk2-specific small-molecule inhibitor (PV1019), divulges that 4DPG represses Vimentin activation in a Chk2-dependent manner. Furthermore, immunoprecipitation analysis unveiled that 4DPG prevents complex formation between Vimentin and p53 resulting in the rescue of p53 and its nuclear localization in aggressive 5-FU-R cells. In addition, 4DPG confers suitable pharmacokinetic properties and strongly abrogates tumor growth, polyps formation, and lung metastasis in an orthotopic rat colorectal carcinoma model. In conclusion, our findings demonstrate 4DPG as a targeted antitumor/anti-metastatic pharmacological lead compound to circumvent EMT-associated drug resistance and suggest its clinical benefits for the treatment of aggressive cancers.