According to the result, the cell morphology showed little influence under 1

According to the result, the cell morphology showed little influence under 1.8M ICJ treatment (equivalent to 1g/ml,) in both MDA-MB-231(named MDA-231 for short) and 4T1 high-invasive breast cancer cell lines (Determine ?(Figure1D).1D). have obtained Caldaret a promising extract named ESC and further isolated a Caldaret chemical compound Chamaejasmenin B (named ICJ) from it, which had little record of its anti-tumor activities. With minimal toxic effects, we have for the first time identified ICJ as the potent metastatic inhibitor in breast cancer by specifically targeting TRII: ITGB3: FAK: p38, the central pathway for non-canonical TGF signaling. Notably, different from other TGF pan-antagonists, ICJ was not the universal blocker for TGF-beta. In contrast, the cytostatic effect of TGF-beta can be significantly activated after ICJ treatment, and as such, ICJ re-balanced the functional output of TGF Paradox in tumor microenvironment. Our study broke the limit of traditional toxic efficacy of SCL and provided a novel and promising candidate for clinical metastatic intervention. RESULTS Drug efficacies screening and identification of chamaejasmenin B from SCL As described in the introduction, TGF-beta is the pivotal oncotarget for controlling of metastasis. Leading by this, we have established the natural products screening platform targeting tumor motility and TGF regulation. During this study, the extracts from L(SCL) greatly attracted our attention. Through efficacy screening, among ten tested extracts, we clearly exhibited that ESC (named T6) efficiently inhibited breast cancer cell migration at the low dose (Physique ?(Figure1A).1A). Indicated by this, we further isolated a highly-content compound Chamaejasmenin B (ICJ) from ESC, which had little record of its bioactivity against cancers. Firstly, chemical structure analysis identified that ICJ was a flavonoid with molecular formula of C32H26O10. Its relative molecular mass was 570. The chemical structure of ICJ was showed in Figure ?Physique1B1B and the purity of prepared (+)-chamaejasmenin B was 99.4%, which was determined by the area normalization method using a HPLC equipped with a photodiode array detector (Determine ?(Physique1C).1C). The purity of the product met the requirement of further pharmacological study. Open in a separate window Physique 1 Efficacy screening for SCL extracts and identification of ICJ isolated from ESCA. Extracts efficacy screening from SCL targeting tumor cell motility. In this platform, ten different extracts (T1 to T10) with the concentrations of 1g/ml were prepared by newly established extraction protocol and they were utilized to treated 4T1 for 24 Caldaret hours. Then the cell motility changes were measured by Transwell assay. As indicated by the black arrow in the physique, one of the extract, T6 (named ESC), possessed the Klf2 strongest activity against breast cancer migration. B. The chemical structure of Chamaejasmenin B (ICJ). The absolute configuration of ICJ was decided through NMR and CD. C. Purity detection of prepared ICJ using a WATERS-2695 HPLC equipped with a photodiode array detector (UV wavelength was 295 nm). D. The morphological observation of MDA-MB-231 cells (shown as MDA-231 for short) and 4T1 cells treated with 1.8M (1g/ml) low-dose ICJ. Images were collected on 24 hours after drug treatment. E. MTT assay in breast cancer cells treated with a serial doses of ICJ (from 2.8 to 45M) for 72 hours. F. Transwell assay in ICJ or ESC treated 4T1 cells. Results were further quantified through the cell counting in randomly selected 5 microscopic fields. G. Matrigel cell invasion assay in 4T1 cells. The result was quantified through the same method in Physique ?Figure1F1F. Next, the dose-toxicity test was performed. According to the result, the cell morphology showed little influence under 1.8M ICJ treatment (equivalent to 1g/ml,) in both MDA-MB-231(named MDA-231 for short) and 4T1 high-invasive breast cancer cell lines (Determine ?(Figure1D).1D). Additionally, the cell proliferation intensity was further quantified by MTT assay. With the same initial cell confluency, after culturing for 72 hours, result showed no significant difference of cell proliferation rate in low-dose ICJ treated group comparing to that in unfavorable Caldaret control (Physique ?(Figure1E).1E). From the above data, we could clearly conclude that less than 22.4M of low-dose ICJ was optimal for drug efficacy studies with little cytotoxicity. Based on the above results, we next investigated if ICJ, at the nontoxic dose interval, possessed the same efficacy as ESC. As expected, in transwell assay and Matrigel invasion assay, under 1g/ml ICJ treatment (equivalent to 1.8M), the transmembrane cells were 179 and 6 respectively, which were more than 3 and 14 times lower than it in unfavorable control. Moreover, ICJ showed stronger activities against cell migration and invasion than ESC, indicating that, in high-invasive breast cancer model, low-dose ICJ had the.