Screening And Mechanisms Of Anti-triple Negative Breast Cancer Activity Of Natural Product From Ginkgo Biloba Sarcotestas

Ginkgo biloba is a multi-purpose economic plant including food,medicine and landscape.It has been recoganized as one of the hotspots in the research and development of natural plant active substances.Ginkgo biloba leaves,seeds and sarcotestas all have medicinal values.Ginkgo biloba sarcotestas,which are outside tissues of the ginkgo husk have been discarded as waste substances in the harvest season of fruit,causing waste of resources.In recent years,the application of Ginkgo biloba sarcotesta at home and abroad has focused on bio-pesticide,and there are few studies on its anti-cancer activity.Breast cancer is the common tumor in women around the world and has the high incidence in China.Triple-negative breast cancer has a higher degree of malignancy in breast cancer and has stronger cell proliferation and metastasis characteristics in clinic.At present,the treatment of triple-negative breast cancer is mainly based on the cytotoxicity treatment of chemical drugs.However,there is a lack of effective targeted therapy,and it also brings the risk of recurrence and metastasis as well as serious side effects to the patients.Natural active products have become the major choice for some patients because of their long-term,mild and low-toxic side effects for use.Therefore,searching for natural active substances against triple-negative breast cancer from Ginkgo biloba sarcotesta is of great strategy for the prevention and treatment of breast cancer.In this study,we used the cytotoxicity screening method on triple negative human breast cancer cell line MDA-MB-231 and the murine breast cancer cell line 4T-1,separated the natural active compound:2-hydroxy-6-tridecylbenzoic acid,namely ginkgolic acid C13:0（GA13:0）,and its anti-cancer mechanism was also studied.The main results are as follows:1.A monomer compound was isolated from the sarcotestas of Ginkgo biloba by cytotoxicity guided method.Taking the cytotoxicity as screening indicators,nine components（S₁-S₉）were obtained by silica gel column chromatography from crude ethanol extract of Ginkgo biloba sarcotestas and the fraction of S₄ was further subjected to semi-preparative HPLC as well as the correspondingly MTT assay in MDA-MB-231 and 4T-1 cells.A natural active monomer compound（95.28%purity）,which was isolated from Ginkgo biloba sarcotestas had significant cytotoxicity against MDA-MB-231 and 4T-1 cells.This compound was identified as ginkgolic acid C13:0（GA13:0）by high performance liquid chromatography-mass spectrometry（HPLC-MS）and nuclear magnetic resonance（NMR）.2.GA13:0 can inhibit the proliferation of triple-negative human breast cancer MDA-MB-231 cells and murine breast cancer 4T-1 cells and has similar inhibitory mechanism in vitro.Mechanistic studies indicated that the anti-proliferation of GA13:0 can stimulate the related molecular targets including activation of upstream of AhR and ARNT proteins,and up-regulation of downstream of XRE and CYP1B1 by AhR signaling pathway in MDA-MB-231 and 4T-1 cells.3.We constructed BALB/C mouse model with triple-negative murine breast cancer 4T-1cells to study the inhibitory effect of GA13:0 on the growth of breast tumors in vivo.The results showed that GA13:0 significantly inhibited the growth and lung metastasis of breast tumors compared to the control group,while serum AST,ALT,BUN,CREA and body weight were not significantly changed,indicating that GA13:0 had no adverse effects on the body.4.We investigated the mechanism of GA13:0 for inhibiting the growth and lung metastasis of tumors in vivo.The results showed that the anti-growth of GA13:0 was associated with the up-regulation of the expression of AhR and CYP1B1 proteins through activating AhR signal pathway,and was associated with down-regulation of the expression of Bcl-2 and up-regulation of the expression of Bax and Cleaved caspase-3 proteins through inducing intrinsic mitochondrial apoptosis signal pathway.The anti-metastasis of GA13:0were associated with the up-regulation of the expression of E-cadherin and down-regulation of the expressions of Snail,Vimentin,MMP-2 and MMP-9 through inhibiting the epithelial-mesenchymal transition（EMT）pathway.