G protein-coupled estrogen receptor (GPER) was reported to be a potential target in the breast cancer therapy. This study aimed to illuminate the function of GPER and its mediated PI3K/AKT pathway in cryptotanshinone (CPT) inducing cell apoptosis and antiproliferation effect on GPER positive breast cancer MCF-7 cells. Cell proliferation was tested by MTT assay. Apoptosis rates were tested by Annexin V-FITC/PI double staining and the cell cycle was researched by flow cytometry. Autodock vina was applied to make molecular docking between CPT or estradiol and GPER. siRNA technique and GPER specific agonist G-1 or antagonist G-15 were applied to verify the mediated function of GPER. Apoptosis and cell cycle related proteins, as well as the key proteins on PI3K/AKT signaling pathway were detected by western blot. The results indicated that CPT could exert antiproliferation effects by arresting cell cycle in G2/M phase and downregulating the expression of cyclin D, cyclin B and cyclin A. Besides, apoptosis induced by CPT was observed. CPT might be a novel GPER binding compounds. Significantly, suppression of PI3K/AKT signal transduction by CPT was further increased by G-1 and decreased by G-15. The study revealed that the effect of antiproliferation and apoptosis treating with CPT on MCF-7 cells might be through the downregulation of PI3K/AKT pathway mediated by activated GPER.
Due to the lack of classic estrogen receptors, there has been a shortage of targeted therapy for triple-negative breast cancer (TNBC), resulting in a poor prognosis. However, the newly discovered G protein-coupled estrogen receptor (GPER) has been found to be expressed in TNBC cells. Salvia miltiorrhiza (Danshen) is an essential Chinese medicine for gynecological disorders, and its component tanshinone IIA (Tan IIA) exerts an anticancer effect. Therefore, this study attempted to investigate whether GPER is involved in the inhibitory effect of Tan IIA on TNBC. We applied various databases and GO pathway analysis to predict the possible mechanism of Tan IIA. We identified 39 overlapping targets, including c-Jun, c-Fos, and caspase-3, and enriched cell cycle-related pathways. Next, we demonstrated the strong binding ability of Tan IIA to GPER by molecular docking assay. In the subsequent validation tests, Cell Counting Kit-8 (CCK8) assay showed that Tan IIA inhibited proliferation of MDA-MB-231 cells time and dose dependently without affecting normal cells. Using Transwell plate, flow cytometry, and Western blot assays, we showed that Tan IIA inhibited migration and induced apoptosis of MDA-MB-231 dose dependently. Importantly, protein expressions of GPER, epidermal growth factor receptor (EGFR), extracellular regulated protein kinases (ERK), c-Fos, and c-Jun were all decreased by Tan IIA dose dependently. Administration of GPER inhibitor partly abolished these effects. Furthermore, nuclear translocation of c-Fos and c-Jun as well as cell cycle-related proteins was downregulated by Tan IIA dose dependently. In summary, Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce apoptosis, and the possible mechanism may be the regulation of GPER-mediated pathways, suggesting that GPER could be a therapeutic target for TNBC.
Background Bone protective effect of Si-Wu-Tang (SWT), a classical prescription of traditional Chinese medicine, is verified in clinical for thousand years. However, its mechanisms were still unclear. This study aims to investigate the molecular mechanism in ApoE -/- mice fed a high-fat diet combining network pharmacology and in-vivo experiments. Methods Femurs were collected from 6 ~ 8-week-old female ApoE-/- C57BL/6J mice (n = 12, 18–22 g) and their age-matched wild-type (WT) littermates C57BL/6J mice (n = 6, 18–20 g). They were divided into 3 groups: the control, SWT and model groups. Serum levels of high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by the serum biochemical index. HE staining and immunohistochemistry analysis were performed to observe the pathological tissue structure and the location and expression level of targets from the pathway screened out by the network pharmacology method. Western blot (WB) and RT-PCR analyses were performed to detect the expression levels of target proteins and mRNAs, respectively. Results The results of the network pharmacology analysis showed that the mechanism of SWT in treating osteopenia was closely related to the oestrogen receptor (ER) signalling pathway. In vivo experiments indicated that, compared with control group, the distribution of bone trabeculae was sparse, and the bone density decreased. The levels of HDL-C and LDL- C in the serum of the model group increased significantly (p < 0.01). The expression of GPER, PI3K, AKT and BCL-2 in the bone tissue of the model group decreased, and P53, BAX, ERα and ERβ were upregulated. Compared with the model group, the body mass of the SWT group increased slowly. The bone density and the distributions of bone trabeculae both increased. The expression of ERα, ERβ, GPER, PI3K, AKT and BCL-2 increased. The decreased expression of apoptotic genes, including P53 and BAX, was observed. Conclusion SWT significantly reduced bone loss in ApoE -/- mice fed a high-fat diet. An important mechanism might be that SWT could activate the PI3K/AKT signalling pathway mediated by ER and then inhibit apoptosis-related proteins to exert bone protective effects.
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