Cardiac glycosides from Digitalis lanata and their cytotoxic activities

Cardiac glycosides (CGs) are good candidates as drug leads in the treatment of cancer because of their structural diversities and potent biological activities. In this study, fifteen CGs including three new ones (1–3) were isolated from Digitalis lanata Ehrh. Their structures were elucidated by HRESIMS, NMR spectroscopic methods, including homonuclear and heteronuclear coupling constant analysis, and acid-catalyzed hydrolysis and derivatization analysis of the sugar chain. The cytotoxic activities of these CGs were evaluated against three human cancer cell lines (A549, HeLa and MCF-7 cell lines), and all of them showed strong activities at nanomolar scale. The flow cytometric analysis indicated that compound 1 induced cell cycle arrest in the G2/M phase. Transcriptome analysis revealed a panel of possible targets for compound 1. RT-PCR and western blot experiments showed that 1 significantly inhibited the expression of vasohibin-2 (VASH2). Moreover, compound 1 restrained angiogenesis in a concentration-dependent manner in the chick embryo chorioallantoic membrane (CAM) model.


Introduction
Natural products are an important source for drug discovery. Drugs directly obtained or indirectly derived from natural products play a signicant role in cancer chemotherapy. 1 Cardiac Glycosides (CGs) are a class of steroid-like compounds from a diverse group of plants, including Strophanthus, Digitalis, Nerium, and animals. 2,3 CGs have traditionally been used to treat congestive heart failure and arrhythmias, for their specic inhibitory effect on the Na + /K + -ATPase pump, an ion pump present on almost all eukaryotic cell membranes. 4 Recently, the remarkable anticancer effects of CGs have drawn academic attention to their applications in cancer chemotherapy. 5 An increasing number of research studies revealed that CGs exhibited selective anti-proliferative and pro-apoptosis effects against various human cancer cell lines, such as cervical, lymphoma, lung, colon, breast, prostate, melanoma, pancreas, and liver cancer cell lines. 6,7 In addition, several CGs have been evaluated in clinical trials as anticancer drugs. [8][9][10][11] The anticancer activity mechanisms of CGs have been studied to include apoptosis, 12 autophagy, 13 anoikis, 14 immunogenic cell death 15 and cell cycle arrest. 16 These ndings provided insights into developing CGs as novel anticancer therapeutics.
In our ongoing research to discover more bioactive CG molecules as lead compounds, 16 a series of CGs including three new ones were isolated from Digitalis lanata Ehrh., an herb of Digitalis genus (Plantaginaceae). 17 Their structures were unambiguously elucidated by extensive spectral data and chemical transformation. The cytotoxic activities and structure-activity relationship (SAR) were evaluated. Flow cytometric analysis indicated that compound 1 caused signicant cell cycle arrest. The possible molecular target of 1 was investigated by transcriptomic analysis, RT-PCR, and western blot. Furthermore, the concentration-dependent antiangiogenic activity of compound 1 was also evaluated on the CAM model. These ndings provide new insights for the development of CGs into anti-tumor lead compounds.
The cis-, trans-and cis-fused A/B, B/C and C/D tetracyclic skeleton was determined by the NOESY correlations of  the signals in aglycone of 1 as those corresponding to digitoxigenin. 19 All of the four sugar moieties were b-glycosides, which were supported by the relatively large homonuclear 3 The molecular formula, C 55 H 86 O 24 of compound 2 was unequivocally deduced by the HRESIMS sodium adduct ion peak at m/z 1153.5343 [M + Na] + (calcd for C 55 H 86 O 24 Na 1153.5401) and the 13 C NMR data (  Detailed 2D NMR analysis suggested the sugar moieties of 2 were identical to those of 9 except for one more b-D-glucopyranosyl, which was determined to be 1 00000 / 4 0000 connection based on the HMBC correlations of H-1 00000 /C-4 0000 and downeld signal of C-4 00 (69.7 in 9 and 80.8 in 2). The structure of compound 2 was thereby established as digitoxigenin Compound 3 was isolated as a white solid.  (Table 3)       1 000 -H-1 000 ), and 162.6 Hz (C-1 0000 -H-1 0000 ) (see the ESI Fig. S20 †). These glycosyl were linked in the same order as 9, which was conrmed by the HMBC correlations from H-1 00 to C-4 0 , H-1 000 to C-4 00 , and 1 0000 to C-4 000 and validated the linkages of 4 0 / 1 00 , 4 000 / 1 000 , and 4 000 / 1 0000 .   (14), 28 and diginatin (15) 29 were elucidated by comparing their spectroscopic data with those in literature.
The cytotoxic activities of the obtained CGs 1-15 were measured on A549 (non-small lung carcinoma) cells by the MTT method in preliminary assays. The proliferation inhibition rates of all the isolated CGs were greater than 70% aer 24 hours of exposure (see the ESI Table S27 †). Based on these results, IC 50 values of these CGs were then measured against A549, HeLa (human cervical cancer cells), MCF-7 (breast cancer), and BEAS-2B (normal human lung epithelial cells) cell lines. Paclitaxel was used as a positive control ( Table 4). Most of the CGs presented potent cytotoxic activities on all four cancer cell lines on the nanomolar scale (IC 50 < 1 mM). In general, these CGs were more active against A549 and HeLa cells than against MCF-7. The cytotoxic selectivities of these CGs of cancer cell lines over normal cell lines were evaluated by comparing their IC 50 values on cancer cell lines with those on BEAS-2B cell lines. Most of the isolated CGs exhibited two to eight-fold higher IC 50 values on BEAS-2B cell lines than those on cancer cell lines, indicating that they were relatively selective against cancer cell lines.
It has been shown in literature that subtle changes in the core structure of CGs can inuence the biological activity signicantly. 30 To obtain a preliminary knowledge above the structure-activity relationship (SAR) of the isolated CGs, we examined the impact of structural variations on their antiproliferative activities against A549 cells. In general, CGs bearing saccharide moieties at C-3 showed greater activity than the aglycone, as compounds without saccharide units exhibited a dramatic decrease in the activity, as shown by 7 (0.15 mM), 8 (0.15 mM) and 9 (0.19 mM) vs. aglycone 6 (1.28 mM). These data suggested that C-3 saccharide moieties were benecial to the activity. Similar results have been reported previously. 31 In addition, except for 2, compounds 1, 4 and 5 (0.04 # IC 50 # 0.09 mM), showed greater activity than 7-9 (0.15 # IC 50 # 0.19 mM) and 10-13 (0.14 # IC 50 # 0.57 mM), suggesting that hydroxylation at C-12 or C-16 was unfavorable to the activity. Meanwhile, O-acetylation was thought to be responsible for the reduced activity of the compound 2. Compounds 14 (0.67 mM) and 15 (0.36 mM) displayed lower antiproliferative activity, indicating that simultaneous hydroxylation at C-12 and C-16 was detrimental to the activity. Moreover, the transformation of aglycone led to a dramatic decrease of the activity, as shown by 3 (0.33 mM), which could be due to the acetylation. Noteworthy, compounds 1, 4, and 5, displayed extremely low IC 50 values comparable to the positive control paclitaxel. Compound 1 exhibited statistically signicant selectivity against cancer cell lines (Fig. 5). Based on these data, compound 1 was chosen as a representative compound to export further. Based on literature reports, 12,32 we rst hypothesized that the cytotoxic activity of compound 1 was attributed to its apoptosisinducing effect. To test our hypothesis, we rst monitored the morphological change induced by compound 1. A549 cells showed substantial apoptotic morphological change aer treatment with 1 for 24 h (see the ESI Fig. S27 †). Subsequent Annexin V-FITC/PI assay revealed that accumulated over time, the apoptotic cell fraction increased with the treatment of 1 (Fig. 6). Interestingly, the apoptotic fraction of A549 cells was less than 20% even aer being exposed to 80 nM of 1 for 48 h, indicating that alternative mechanisms of action other than apoptosis also contribute to the cytotoxic activity. The ow cytometric analysis also showed a surprising result that no signicant apoptosis-induced effects on BEAS-2B cells were observed under the same experimental settings. This result further suggested that 1 showed selectivity against cancer cells over normal cells.
The so agar assay measures the proliferation ability of cells in simulated matrices and provides an ideal way to rigorously test the inhibitory activity of compounds on a longer time scale. 33 compound 1 can suppress the long-term proliferative capacity of A549 cells and the colony ability of surviving cells was signicantly reduced aer compound 1 treatment (Fig. 7), which is in accordance with the MTT assay results. The ow cytometric analysis was also performed to investigate the impact of 1 on cell cycle distribution. A549 cells were treated with increasing concentration (40,80, 120 nM) of compound 1 for 24 h and DNA content was measured. As illustrated in Fig. 8, a signicantly increased cell accumulation in the G2/M phase with a dose-dependence. The proportion of cells in G2/M phase increased from 21.68% to 24.02%, 35.32% and 40.99% respectively, with the increase of concentration of 1. According to the above results, compound 1 can inhibit cancer cell proliferation via arresting cell cycle progression in G2/M phase.   The application of transcriptomics can offer new insights into locating drug targets and pinpointing relevant pathways in tumor progression. 34 To nd an alternative mechanism of 1 for its anti-proliferative activities, a transcriptome analysis was performed. The expression of mRNA with a padj < 0.05 and jlog 2 (fold change)j > 0 were considered for differential expression between the control groups and compound 1 treatment groups. The result showed that 6460 genes were up-regulated and 6317 were down-regulated in all 12 777 differentially expressed genes (DEGs) as visually shown in volcano plot (Fig. S28A †). A heatmap (Fig. S28B †) was also used to illustrate the asymmetry of the relative up/down regulation of genes. Analysis of the top 40 cancer associated DEGs revealed that three tumor suppressor genes KLF15 (Kruppel Like Factor 15), FOSB (fosb proto-oncogene), and FTCD (formimidoyltransferase cyclodeaminase) were up-regulated and two carcinogenic genes vasohibin 2 (VASH2) and tryptophan 2,3-dioxygenase (TDO2) were down-regulated aer the treatment with 1. The differential expression of the ve candidate genes were further validated using RT-PCR (Fig. 9A), which showed consistent results with transcriptomic analysis otherwise FOSB. The most signicantly downregulated gene VASH2, an angiogenesis regulatory factor, was proposed to participate in the anti-proliferative activities of 1. The inhibited effect of 1 on protein level of VASH2 was also examined with a western blot experiment. As shown in Fig. 9B and C, compound 1 caused  signicantly concentration-dependent down-regulation of VASH2. Together, VASH2 was proposed to be the potential molecular target of compound 1.
VASH2, a member of the vasohibin family, is abnormally expressed in various cancer cells and is strongly associated with tumor progression and chemoresistance. 35,36 In addition, VASH2 was shown to promote angiogenesis in hepatic, ovarian and endometrial cancers, and was proposed to be a potential drug target. 37 To better understand the molecular interactions between 1 and VASH2, a molecular docking analysis was performed. 3D interaction represented in Fig. 10 showed that 1 can t in a binding pocket of VASH2 by forming eleven hydrogen bonds at Tyr239, Gln53, Asn189, Ile133, Met136, Arg212, Lys157, and His241 with a glide score of À14.943. The molecular docking result indicated that 1 was likely to show a good binding affinity with its potential molecular target VASH2. As VASH2 has been shown to be a potential molecular target of 1, and that VASH2 is a regulatory factor of angiogenesis, we sought to study the anti-angiogenesis activities of 1. Angiogenesis is a complicated pathological process, which is essential for cancer progression and metastasis by providing oxygen and nutrients. 38 Antiangiogenic therapy, a way of blocking tumor neovascularization before the angiogenic switch or causing tumor vessel regression, has been showing to be a prospective treatment for cancer. [39][40][41][42] To assess whether 1 can suppress tumor migration, the mobility of A549 cells was measured by wound healing and transwell assay. As shown in Fig. 11A and B, the width of the wound became smaller in control groups aer 48 h. Nevertheless, the migration of A549 cells in 1-treated groups were signicantly inhibited at designated time points. Transwell assay simultaneously showed that the quantity of migrated A549 cells obviously decreased with the increase in the concentrations of 1 (Fig. 11C and D), indicating the inhibitory effect of 1 on carcinoma cell metastasis. We further conrmed the inhibitory effect of 1 on angiogenesis in vivo by implementing compound 1 on CAM model, which has been an  awesome tool in studying of tumor angiogenesis and metastasis. 43 Chick embryos were treated with 0.3 or 0.6 mg of 1 or DMSO (control) within pre-placed polyethylene resin rings, and then were photographed at 0 h and 48 h (Fig. 12A). Experimental results showed that the density and extension of the CAM blood vessel plexuses were strongly reduced by 1 (Fig. 12B), which illustrates that compound 1 possessed antiangiogenesis potentiality.

Conclusions
Collectively, 15 naturally occurring CGs were isolated and iden-tied from the leaves of D. lanata. All the isolated CGs exhibited excellent anti-proliferation activity against cancer cell lines on nanomole scale. Compounds 1, 4, and 5 exhibited promising inhibitory activities with IC 50 values of 40 nM, 90 nM and 80 nM respectively against A549 cell line. The mechanism studies indicated that the antiproliferative activity of 1 was associated with the cell cycle arrest at G2/M phase. Furthermore, 1 can signicantly inhibit the expression of VASH2 and restrain angiogenesis on the CAM model in a concentration-dependent manner. To the best of our knowledge, this is the rst attempt to repurpose CGs as potential inhibitors of tumor angiogenesis. Our study suggested that compound 1 is a potential lead compound for developing novel therapeutics for the treatment of cancer.

Author contributions
Kun Gao and Jian-Jun Chen performed conceptualization and reviewed the nal manuscript. Hong-Ying Yang performed the experiments and wrote the preliminary manuscript. Ya-Xiong Chen provide signicant guidance in the biological experimental procedure. Shangwen Luo modied the syntax. Yi-Lin He, Wei-Jiao Feng and Yue Sun carried out the spectral analysis.

Conflicts of interest
There are no conicts to declare.