Cardenolides from Calotropis gigantea as potent inhibitors of hypoxia-inducible factor-1 transcriptional activity

Ethnopharmacological relevance Calotropis gigantea (L.) Dryand (Apocynaceae) is a medicinal plant native to southern China, India and Southeast Asia. It has been traditionally used for the treatment of several diseases including cancers in these countries. Aim of the study This study aimed to isolate bioactive cardenolides from C. gigantea , to screen their hypoxia-inducible factor (HIF-) 1 inhibitory activity, and to analyze the structure-activity relationship (SAR). Materials and methods Isolation and purification of cardenolides from the latex and the fruits of C. gigantea were performed by using a series of separation techniques. Their structures were fully characterized by elucidating their NMR and HRMS data. The HIF-1 inhibitory activities of cardenolides were evaluated by using a T47D cell-based dual-luciferase reporter assay. The potent cardenolides were selected to further evaluate their dose-response manner. Cytotoxic effects of selected cardenolides were also examined against breast cancer cell line (MCF-7) and normal mammary epithelial cell line (MCF-10A) by MTT assay. Results Among twenty isolated cardenolides, compounds 1 , 3 , 4 , 6–8 , 14 and 17 exhibited stronger HIF-1 inhibitory activities than that of digoxin, a well-known HIF-1 inhibitor ( P <0.001). These eight cardenolides inhibited HIF-1 transcriptional activity in a dose-dependent manner with IC 50 values in nanomolar potency (21.8–64.9   nM). An analysis of SAR revealed the great contributions of a β -configuration of the substituents at positions of C-2′ and C-3′, an aldehydic moiety on C-19, and the dioxane moiety between the aglycone and sugar parts of cardenolides to the HIF-1 inhibitory activity. In contrast, a hydroxyl group at any positions of C-15, C-16 and C-4′ of cardenolides showed negative effects on suppressing HIF-1 transcriptional activity. In addition, these eight cardenolides also exhibited potent cytotoxic effects against human breast cancer cell MCF-7 (IC 50 values ranged from 30.5 to 68.8   nM), but less toxic effects to human normal mammary epithelial cell MCF-10A (IC 50 values >20   µM). Conclusions This is the first report of a comprehensive study of SAR on cardenolides from C. gigantea as HIF-1 inhibitors. Eight cardenolides ( 1 , 3 , 4 , 6–8 , 14 and 17 ) showed both potent HIF-1 inhibitory activity and strong cytotoxic effect against MCF-7 cancer cells in nanomolar level. The findings of these cardenolides provided important insights into the development of these potent HIF-1 inhibitors as anticancer drug .