Integrated Strategy of UHPLC-Q-TOF-MS and Molecular Networking for Identification of Diterpenoids from Euphorbia fischeriana Steud. and Prediction of the Anti-Breast-Cancer Mechanism by the Network Pharmacological Method

Breast cancer is one of the most common malignancies in women worldwide. Traditional Chinese medicine has been used as adjunctive or complementary therapy for breast cancer. Diterpenoids from Euphorbia fischeriana Steud. have been demonstrated to possess anti-breast-cancer activity. This research was aimed to systematically explore the diterpenoids from E. fischeriana and study the multiple mechanisms on breast cancer. The structures of diterpenoids were identified by the integrated strategy of UHPLC-Q-TOF-MS and molecular networking. A total of 177 diterpenoids belonging to 13 types were collected. In silico ADME analysis was performed on these compounds. It indicated that 130 of 177 diterpenoids completely adjusted to Lipinski’s rule. The targets of compounds were obtained from PharmMapper. The targets of breast cancer were collected from GeneCards. Then, 197 compounds-related targets and 544 breast cancer-related targets were identified. After the intersection process, 58 overlapping targets between compounds-related targets and breast cancer-related targets were acquired. The STRING database was applied to predict the protein-protein interactions. The GO and KEGG pathway enrichment analysis were performed by using the KOBAS database. It indicated that these predicted pathways were closely related to breast cancer. The treatment effect of E. fischeriana on breast cancer might be performed through signaling pathways, such as IL-17 signaling pathway, MAPK signaling pathway, and PI3K-Akt signaling pathway. The predicted top genes such as EGFR, ESR, MAPK, SRC, CASP3, CDK2, and KDR were involved in cell proliferation, gene transcription, apoptosis, signal transduction, DNA damage and repair, tumor differentiation, metastasis, and cell cycle, which indicated that E. fischeriana might treat breast cancer comprehensively. A compounds-KEGG pathways-related targets network was built by using cytoHubba to analyze the hub compounds and targets. It concluded that E. fischeriana treated breast cancer not only by the main components but also by the microconstituents, which reflected the overall regulatory role of multicomponents treating breast cancer. To estimate the binding affinities, binding sites, and binding postures, molecular docking simulations between 177 diterpenoids and top 19 targets were carried out. The results are basically in line with expectations. In conclusion, these results can serve as references for researchers studying potential targets of diterpenoids from E. fischeriana on breast cancer in the future.

The Molecular Mechanism of ESZM Extract on Treatment ER(-) Breast Cancer in Vivo: Application of “Combat Poison with Poison” Theory

Background: Extract of Euphorbia fischeriana S. and Ziziphus jujuba M. (ESZM) is a formula preparation, was composed of Euphorbia fischeriana S. (E. fischeriana) and Ziziphus jujuba M. (Z. jujuba). The aim of the current research was to examine for the first time the pharmacological effects and the underlying molecule mechanism of ESZM extract on the growth inhibition and apoptosis in human ER(-) or ER(+) breast cancer in vivo, and enrich and innovate the theory of traditional Chinese medicine of combating poison with poison. Methods: Growth inhibition, cell cycle arrest and apoptosis in tumor tissues were determined by tumor inhibition rates, cycle analysis kit, Annexin V-FITC/PI staining, Hoechst 33342/PI staining, TUNEL test and TEM observation. Related genes and proteins expression levels were identified by qRT-PCR assay, Western Blotting method, immunohistochemistry and immunofluorescence means. Drug toxicity was analysised by serum biochemistry detection and H&E staining.Results: We found that ESZM extract can inhibite growth, block cell cycle at G2/M phase and induce apoptosis of subcutaneously transplanted tumor, especially ER(-) breast cancer transplanted tumor. Furthermore, we demonstrated that the polyjuice can down-regulate or up-regulate the expression of Bcl-2 family and PI3k/Akt pathway -related signaling molecules proteins and genes in vivo. ESZM extract or E. fischeriana extract increased the levels of ALT, AST, Cr and BUN, and increased liver and kidney toxicity in tumor-bearing mice by serum biochemistry detection and H&E staining.Conclusion: The pharmacological detection suggested that ESZM extract had significant anti-breast cancer effect, especially ER(-) breast cancer xenograft, and this process may be implement through the mitochondrion dependent pathway and the PI3k/Akt signaling pathway. Drug toxicity detection proved that ESZM extract because of compatibility with Z. jujuba was lower toxicity than E. fischeriana extract, was no significant difference in hepatorenal toxicity between ER(-) or ER(+) tumor-bearing mice.