Targeting HSP70 chaperones by rhein sensitizes liver cancer to artemisinin derivatives

Liver cancer is one of common types of cancer with poor prognosis and limited therapies. Heat shock proteins (HSP) are molecular chaperones that have important roles in tumorigenesis, and emerging as therapeutic targets. Artemisinin and rhein are natural agents from Artemisia annua L. and Rheum undulatum L., respectively. Both rhein and artemisinin have anticancer effects; however, the molecular targets of rhein remain to be identified. It is also unclear whether rhein can synergize with artemisinin derivatives to inhibit liver cancer.We aim to identify the targets of rhein in the treatment of hepatocarcinoma and determine the effects of combining rhein and artemisinin derivatives on liver cancer cells.The targets of rhein were detected by mass spectrometry and validated by rhein-proteins interaction assays. The effects of rhein on the chaperone activity of HSP72/HSC70/GRP78 were determined by luciferase refolding assays. Cell viability and apoptosis were determined by CCK8 and flow cytometry assays. For in vivo study, xenograft tumor models were established and treated with rhein and artesunate. Tumor growth was monitored regularly.Mass spectrometry analysis of rhein-binding proteins in HepG2 cells revealed that HSP72, HSC70 and GRP78 were more profoundly pulled down by rhein-crosslinked sepharose 4B beads compared to the control beads. Further experiments demonstrated that rhein directly interacted with HSP72/HSC70/GRP78 proteins, and inhibit their activity of refolding denatured luciferase. Meanwhile, rhein induced proteasomal degradation of HIF1α and β-catenin. Artesunate or dihydroartemisinin in combination with knockdown of both HSP72 and HSC70 significantly inhibited cell viability. The HSP70/HSC70/GRP78 inhibitors VER-155,008 and rhein phenocopied HSP72/HSC70 knockdown, synergizing with artesunate or dihydroartemisinin to inhibit hepatocarcinoma cell viability. Combinatorial treatment with rhein and artemisinin derivatives significantly induced hepatocarcinoma cell apoptosis, and inhibited tumor growth in vivo.The current study demonstrates that rhein is a novel HSP72/HSC70/GRP78 inhibitor that suppresses the chaperone activity of HSP70s. Dual inhibition of HSP72 and HSC70 can enhance the sensitivity of hepatocarcinoma cells to artemisinin derivatives. Combined treatment with artemisinin derivative and rhein significantly inhibits hepatocarcinoma. Artemisinin derivatives in combination with dual inhibition of HSP72 and HSC70 represents a new approach to improve cancer therapy.