Parhira Supawadee, Zhu Guo-Yuan, Chen Ming, Bai Li-Ping, Jiang Zhi-Hong
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR, People's Republic of China; Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Muang, Phitsanulok 65000, Thailand.
State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR, People's Republic of China.
J Ethnopharmacol. 2016 Dec 24;194:930-936. doi: 10.1016/j.jep.2016.10.070. Epub 2016 Oct 26.
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.
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).
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.
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 values in nanomolar potency (21.8-64.9nM). 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 values ranged from 30.5 to 68.8nM), but less toxic effects to human normal mammary epithelial cell MCF-10A (IC values >20µM).
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.
牛角瓜（萝摩科）是一种原产于中国南部、印度和东南亚的药用植物。在这些国家，它传统上被用于治疗多种疾病，包括癌症。
本研究旨在从牛角瓜中分离生物活性强心苷，筛选其对缺氧诱导因子（HIF-）1的抑制活性，并分析构效关系（SAR）。
采用一系列分离技术从牛角瓜的乳胶和果实中分离纯化强心苷。通过解析其核磁共振（NMR）和高分辨质谱（HRMS）数据对其结构进行全面表征。采用基于T47D细胞的双荧光素酶报告基因检测法评估强心苷对HIF-1的抑制活性。选择活性较强的强心苷进一步评估其剂量反应方式。通过MTT法检测所选强心苷对人乳腺癌细胞系（MCF-7）和正常乳腺上皮细胞系（MCF-10A）的细胞毒性作用。
在分离得到的20种强心苷中，化合物1、3、4、6 - 8、14和17表现出比已知的HIF-1抑制剂地高辛更强的HIF-1抑制活性（P<0.001）。这8种强心苷以剂量依赖方式抑制HIF-1转录活性，IC值在纳摩尔水平（21.8 - 64.9nM）。构效关系分析表明，C-2'和C-3'位取代基的β构型、C-19位的醛基部分以及强心苷苷元与糖部分之间的二氧六环部分对HIF-1抑制活性有重要贡献。相反，强心苷C-15、C-16和C-4'位的任何位置上的羟基对抑制HIF-1转录活性均有负面影响。此外，这8种强心苷对人乳腺癌细胞MCF-7也表现出较强的细胞毒性作用（IC值范围为30.5至68.8nM），但对人正常乳腺上皮细胞MCF-10A的毒性作用较小（IC值>20µM）。
这是首次对牛角瓜强心苷作为HIF-1抑制剂进行构效关系的全面研究报告。8种强心苷（1、3、4、6 - 8、14和17）在纳摩尔水平上既表现出较强的HIF-1抑制活性，又对MCF-7癌细胞有较强的细胞毒性作用。这些强心苷的研究结果为开发这些强效HIF-1抑制剂作为抗癌药物提供了重要的见解。
