Abstract 470: Artemisinin derivatives synergize with paclitaxel by targeting FOXM1 through Raf/MEK/MAPK signaling pathway in ovarian cancer

Abstract

Abstract Objective: Artemisinin, the active principle of the Chinese medicinal herb Artemisia annua and its derivatives have the most efficacies against Plasmodium falciparum malaria. Artemisinin contains an endoperoxide bridge that reacts with ferrous iron to generate free radicals which leads to macromolecular damage and cell death. In this study, we aim to determine if artemisinin derivatives dihydroartemisinin (DHA) can improve the efficacy of paclitaxel and cisplatinum in human ovarian cancer cells in vitro and its possible underlying molecular mechanism and novel molecular targets. Methods: Ovarian cancer cell lines SKOV3 and OVCAR3 were treated with paclitaxel/cisplatinum/DHA or a combination for 72 hrs and cytotoxicity was assessed by sulfarodamine B assay. Drug combination effects were determined by calculating the combination index (CI) values using the Chou and Talalay method (CI <0.9 synergy, CI 0.9-1.10 additive and CI >1.10 antagonism). Quantitative RT-PCR and western blot were performed to determine changes in FOXM1 expression and its downstream molecules. In MAPK signaling studies, cells were starved for 24 hrs, stimulated with 20% FBS for 30min with pre-incubation of DHA for 30 min, and then probed with anti-phospho-MAPK for western blot. Results: Application of DHA to ovarian cancer cells induced cytotoxicity with IC50 between 1µM to 2µM. Drug combination studies demonstrated that DHA synergized with paclitaxel (CI value 0.6-0.73) and additive to cisplatinum (CI value 0.98-1.11) in SKOV3. In addition, low dose DHA (0.5µM) significantly synergized to the combination of paclitaxel and cisplatinum in SKOV3 cells (CI 0.40-0.83). To reveal the underlying molecular mechanism, we analyzed the cell cycle upon DHA treatment which proved that DHA alone arrested cells in G2/M phase (30.8%) compare to control (17.3%) after 24 hrs treatment. Furthermore, DHA significantly downregulated oncogenic transcription factor FOXM1 expression and the transcription of downstream molecules such as CCNB1, BIRC5 and STMN1. Therefore, targeting FOXM1 by DHA delay G2/M transition, induce apoptosis and stabilize microtubule dynamics to sensitize the tumor cells to paclitaxel-induced apoptosis, contributing to the synergistic effects. Finally, FOXM1 is phosphorylated for nuclear translocation before entry into G2/M, and one of the major kinase is MAPK. The western blot demonstrated that DHA inhibit serum-induced phosphorylation of MAPK, indicating the inhibitory effects of DHA toward Raf/MEK/MAPK signaling pathway. Conclusion: These results demonstrate that inhibition of Raf/MEK/MAPK signaling by DHA led to suppression of FOXM1 target gene expression and contribute to cellular cytotoxicity and drug synergism in ovarian cancer. In conclusion, we provide the new information in the knowledge of artemisinin derivatives involved signaling pathways and potential molecular targets. Citation Format: Yi Chen, Brian W. Chin, Marcia M. Bieber, Xianjie Tan, Nelson N.h Teng. Artemisinin derivatives synergize with paclitaxel by targeting FOXM1 through Raf/MEK/MAPK signaling pathway in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2014-470