C steroids and their glycosylation products distribute widel

C21 steroids and their glycosylation products distribute widely in Asclepiadaceae plants. Their anticancer activities have been shown in different human cell lines and in vivo animal experiments [[27], [28], [29], [30], [31], [32]]. However, the mechanisms of their anticancer activities are still unclear. In our previous studies, we identified several C21 steroids from Asclepiadaceae plants exhibiting Hh pathway inhibitory activities, which could be new chemotypes of Hh pathway inhibitors [[33], [34], [35], [36]]. In the course of our ongoing effort to search for valuable Hh pathway inhibitors, the present study further screened C21 steroids from the plant Cynanchum bungei Decne, and discovered that a pair of novel epimers, Cynanbungeigenin C (CBC, 1) and D (CBD, 2) (Fig. 1), could block Hh pathway signaling pathway at the level of Gli. In addition, they were demonstrated to significantly suppress the growth of Hh pathway-dependent medulloblastoma both in vitro and in vivo. Furthermore, this pair of epimers exhibited potential for overcoming the primary and acquired resistance to current Smo inhibitors.
Materials and methods
Results
Discussion The dried root of C. bungei (named Bai-Shou-Wu in Chinese) is a traditional Chinese medicine which has been used as a tonic for over 1000 years from Tang Dynasty in China. It is a member of Ascleplaceae family and distributed in Beijing, Jiangsu, Henan, Hebei and Shandong province of China [50]. Previous chemical studies have identified that acetophenones and C21 steroids are the major constituents of C. bungei [51,52]. Pharmacological researches have indicated that the 50% ethanol extract of C. bungei possess gastroprotective activity against indomethacin-induced gastric lesion in rat [53]. The polysaccharide of C. bungei has been found to enhance nonspecific and specific cellular immune function [54]. Cynandione A from C. bungei exhibited anti-ischemic stroke activity [55]. Two acetophenone derivatives, 2,5-dihydroxyacetophenone (2,5-DHAP) and 2,6-DHAP, from C. bungei were identified as tyrosinase inhibitors for the treatment of terbinafine hydrochloride receptor hyperpigmentation [56]. It was also reported that caudatin, a C21 steroid isolated from C. bungei suppressed the growth of several cancer cell lines [57,58]. However, so far, the chemical constituents of C. bungei and their bioactivities, especially their anti-cancer activities are still poorly investigated. Previously, we have identified two C21 steroids from C. bungei as Hh pathway inhibitors [36]. In present studies, by screening chemical constituents and their anti-cancer activities, we discovered two new C21 steroids CBC and CBD, a pair of epimers as two valuable Hh pathway inhibitors in C. bungei. These two newly obtained compounds suppress Hh pathway-dependent medulloblastoma growth by inhibiting Hh signaling at the level of Gli. Ours findings would contribute to develop new drugs for the treatment of malignancy. Though the anticancer activities of plant-derived C21 steroids have been widely reported, the mechanisms of their activities are poorly understood. It has been considered that apoptosis induction, cell cycle arrest, Wnt pathway modulation were involved in the anticancer effects of C21 steroids [59,60]. Our previous [[33], [34], [35], [36]] and current results discovered that more and more plant-derived C21 steroids or their glycosides significantly inhibited Hh pathway, which may shed new lights on the design of new anticancer drugs based on these natural C21 steroids. The chemical structure of our discovered C21 steroids shares the same steroidal skeleton as cholesterol and glucocorticoids. Cholesterol is an endogenous Smo activator and plays a unique role as second messenger in vertebrate Hh signaling, mediating the functional interaction between Ptch1 and Smo. It was reported that the β-OH at C-3 position of cholesterol is essential for its bind to Smo cysteine-rich domain (CRD) to activate Hh pathway [61]. Several glucocorticoid compounds act as specific modulators of Smo ciliary accumulation. Structure-activity relationship analysis suggested that fluorine at C-9, a ketal at C-16 and 17, and protonation at C-11 significantly enhance the potency of this class of compounds in directing Smo accumulation to the primary cilium [62]. However, our data suggested that CBC and CBD inhibited the Hh pathway not through targeting Smo but at the level of Gli. These two compounds suppressed the Hh pathway activation induced either by Smo agonist, or Smo overexpression, or by Sufu knockdown, Gli overexpression. The significant differences in the chemical structure of our C21 steroids to cholesterol and glucocorticoids are that C21 steroids have the additional hydroxy groups at C-8, C-12, C-14, C-17, and C-20, or have the esterified hydroxy groups on C-12 and/or C-20. We conjectured that such structural differences may result in targeting different proteins of these three types of steroid. Meanwhile, we found that stereochemistry configuration of the methylbutyroyl at C-20 did not influence their activities, as both CBC with S configuration and CBD with R configuration showed similar inhibitory activity on Hh pathway as well as in suppressing medulloblastoma growth.