Network pharmacology predicted HDAC6 as a potential target of flavones from Daphne giraldii on hepatocellular carcinoma

Abstract Daphne giraldii Nitsche, belongs to Daphne genus, has been reported to exert anti-tumor activities. Our previous study suggested that flavones from Daphne giraldii have significant inhibitory effects on hepatocellular carcinoma (HCC) cells. However, the potential target of this type flavone was still unknown. In this study, 74 flavonoids compounds of Daphne giraldii and 41 potential targets of HCC were analyzed by the network, the most potential target was histone deacetylase 6 (HDAC6). Considering the cytotoxicity, compound 70 (Daphnegiravone D, DGD) was chosen for further confirmation. Molecular docking study revealed that DGD formed high binding affinity with HDAC6. Concomitantly, pharmacological studies indicated that DGD could inhibit the expression of HDAC6 in vitro and in vivo. In this study, network pharmacology along with experimental validation predicted and verified HDAC6 as one of potential targets of flavones, these investigations provide a new insight for further study of Daphne giraldii on HCC treatment. Graphical Abstract


Introduction
HCC is one of the most prevalent malignancies and one of the most common causes of cancer-related deaths worldwide. Although there are some medical strategies, the worse prognosis and disease recurrence still affect the life quality of patients (He et al. 2018). Therefore, it is urgent to search for the effective drugs for the therapy of HCC. Natural products have been recognized as a novel strategy with less toxicity and higher efficacy for the therapy of cancer (Yuan et al. 2017).
The plants of Thymelaeaceae family were reported to exhibit anti-tumor activities, which were attribute to the compounds flavone (Sun et al. 2016), terpenoids (Xu et al. 2011;Sun et al. 2018), phorbol (Venditti et al. 2017;Venditti et al. 2019) and their derivatives in it. Daphne giraldii, belonging to the genus Daphne of Thymelaeaceae family (Su et al. 2008), several reports suggested that a series of flavone derivatives isolated from it displayed marked cytotoxic effects on HCC cells (Wang et al. 2017;Shang et al. 2018;Yao et al. 2018). However, the effective target of compounds from D. giraldii is still unknown. Network Pharmacology as a novel field based on the general ideas of systems biology has been considered to be an efficient method to identify the potential targets for drug discovery (Zhang et al. 2019). The systematized strategy with largely existing databases allows us to search for the potential target and mechanism of the ingredients in natural products. To better understand the potential target of the flavone derivatives of D. giraldii on HCC, network pharmacology prediction along with the experimental verification in vitro and in vivo was implemented in this study.

Results and discussion
There were 74 candidate flavone derivatives (Supplementary material, Table S1) from D. giraldii identified through literatures and 41 HCC-related genes (Supplementary material, Table S2) obtained from the GeneCards and Malacards databases. The resulting compound-target network based on molecular docking was constructed by Cytoscape 3.2.1 software illustrated in Figure S1 (Supplementary material). Degree and betweenness centrality were set as the standards to screen the most potential target 5G0G (HDAC6). HDAC6, known as a microtubule-associated deacetylase, is a member of histone deacetylases family. The overexpression of HDAC6 could promote cell proliferation, migration and invasion activity of HCC (Chu et al. 2019). Recently, some HDAC6 selective inhibitors have been investigated in tumor therapy with few side effects .
Previous studies showed that flavone derivatives isolated from D. giraldii had obvious cytotoxic activities (Wang et al. 2017;Yao et al. 2018), the results (illustrated in Supplementary material, Figure S2) showed that DGD had most potent inhibitory effect against HCC cells. The finding was consistent with the network result, DGD The protein level of HDAC6 was detected in Hep3B cells and ImageJ software was applied to quantify the band density of protein; (E, F) HDAC6 expression in vivo was determined by using immunoblotting analysis. The data were presented as the mean ± SD from three independent experiments. Ã p < 0.05, ÃÃ p < 0.01, vs control group. ranked first among this type of compounds. Therefore, DGD was selected as the representative active component of D. giraldii for further pharmacology experiments. Besides, the top 10 compounds in the network pharmacology mostly have prenyl chains on the flavonoid skeleton, implying that prenylation might be critical for the combination of flavonoids and HDAC6.
Thereafter, computational molecular docking study was employed to analyze the molecular interaction between DGD and HDAC6, and the result (Supplementary material, Figure S3) revealed that DGD bound tightly to a highly hydrophobic pocket of HDAC6 through hydrophobic interactions with 8 residues, including CYS-203, . DGD was also predicted to form Hbonds with LYS-330 (distance: 2.11 Å). Our previous research reported that DGD could inhibit the growth of HCC cells in vitro and in vivo (Wang et al. 2017;Shang et al. 2018), the MTT assay depicted in Figure 1A also showed that DGD displayed a stronger inhibitory effect against Hep3B cells (IC 50 ¼ 1.69 ± 0.11 lM) than the positive control sorafenib (IC 50 ¼ 6.58 ± 0.37 lM). To provide further evidence that the potential molecular target HDAC6 is modulated by DGD, we conducted PCR and Western blotting assays in Hep3B cells. As shown in Figure 1B and D, the mRNA and protein expression levels of HDAC6 were obviously reduced by DGD in a concentration-dependent manner. In addition, immunoblotting analysis was also applied to confirm whether HDAC6 was regulated by DGD in vivo, the result from Figure 1E and F demonstrated that the level of HDAC6 was dose-dependently decreased. Together, DGD, a prenylated flavone isolated from D. giraldii, was likely to suppress the growth of HCC cells by targeting HDAC6.

Conclusion
The network pharmacology was used to predict that HDAC6 was a potential target of flavones from D. giraldii on HCC cells. Additionally, experimental validation confirmed that DGD markedly suppressed the level of HDAC6 in vitro and in vivo. This investigation provided an important research basis for further understanding of the targets and underlying mechanism of D. giraldii in the HCC treatment.