Artemisinin Derivatives Target Topoisomerase 1 and Cause DNA Damage in Silico and in Vitro

Abstract

DNA topoisomerases 1 and 2 are enzymes that maintain DNA topology and play important essential genome functions, including DNA replication and transcription. Aberrant topoisomerases cause genome instability and a wide range of diseases, cancer in particular. Both Topo 1 and 2 are the targets of valuable anticancer drugs, such as camptothecin. It has been previously shown that artemisinin, a sesquiterpene lactone from Artemisia annua L. also known as qinghaosu, possesses anti-cancer effects and one of its derivatives, artesunate inhibits Topo 2. In this study, we evaluated artemisinin and 40 derivatives as potential Topo 1 inhibitors at first by in silico molecular docking analyses. Five compounds that showed comparable binding energies and similar docking poses were selected for in vitro cytotoxicity test and Comet assay for DNA damage. WWLL-013, WWLL-022, and WWLL-1098 showed the lowest binding energy also induced DNA damage in the Comet assay. CMK-0298 and CMK-0398 intercalated into DNA and induced mild DNA damage. All selected compounds, WWLL-013 in particular, were more cytotoxic toward the rat tumor cells than to the normal cells. In conclusion, the artemisinin derivatives such as CMK-0298, CMK-0398, WWLL-013, WWLL-022, and WWLL-1098 can be further developed as Topo 1 inhibitors.

Keywords: DNA damage; artemisinin; cancer; molecular docking; topoisomerase.

Figure 1

Chemical structures of selected artemisinin derivatives (CMK0298, CMK0398, WWLL-013, WWLL-022, and WWLL-1098).

Figure 2

CMK0298 (cyan), CMK0398 (black), WWLL-1098 (violet), WWLL-022 (yellow), WWLL-013 (blue), topotecan (green), hydrolyzed topotecan (red), camptothecin (silver) docking on human Topo 1 (magenta. PDB ID:1K4T).

Figure 3

CMK0298 (cyan), CMK0398 (black), WWLL-1098 (violet), WWLL-022 (yellow), WWLL-013 (blue), topotecan (green), hydrolyzed topotecan (red), camptothecin (silver) docking on N722S mutant human Topo1 (orange. PDB ID 1RRJ).

Figure 4

DNA decatenation assay for CMK-0298 and CMK-0398. Bleomycin was used as control compounds. Lane 1, 1 kb DNA marker; Lane 2, supercoiled pUC18 plasmid DNA; Lane 3, Topo 1-treated pUC18.The other lanes show pUC18 DNA treated with Topo 1 plus test compounds at dosages indicated below: CMK-0298: lanes 4–8: 1, 10, 100, 200, 500 μM; CMK-0398: lanes 4–7: 0.01, 0.1, 1, 2.5 mM; Bleomycin: lanes 4–7: 0.1 mg/mL. 10 ng/mL, 1 ng/mL, 0.1 ng/mL; Ethidium bromide: lanes 4–9: 2, 1.5, 1, 0.75, 0.5, 0.2 μg/ml.

Figure 5

MTT assays of artemisinin derivatives on the normal R6 and transformed R6 (R6T24) cells treated with CMK-0298 (A), CMK-0398 (B), WWLL-013 (C), WWLL-022 (D) and WWLL-1098 (E) respectively.

Figure 6

Comet assays of R6 cells treated with CMK-0298, CMK-0398, WWLL-013, and WWLL-022. Bleomycin was used as the control compound.

Figure 7

DNA melting curves of PCR products incubated in DMSO or test chemicals. The qPCR was conducted according to the “Materials and Methods.” DMSO, 10 μM cisplatin, or 10 μM artemisinin were added to the qPCR reaction.