Effect And Its Related Mechanism Of Brucine On Vasculogenic Mimicry In Human Triple-Negative Breast Cancer Cell Line MDA-MB-231

Background and Objectives Vasculogenic mimicry(VM)with the pattern of endothelial independent tubular structure formation lined by aggressive tumor cells mimics regular tumor blood vessels to ensure robust blood supply and correlates with the proliferation,invasion,metastasis and poor prognosis of malignant tumors,which was demonstrated to be a major obstacle for resistance to anti-angiogenesis therapy.Therefore,it’s urgent to discover methods to abrogate the VM formation of tumors,which possesses important practical significance for improving tumor therapy.Brucine is a traditional medicinal herb extracted from seeds of Strychnos nux-vomica L.(Loganiaceae)exhibiting antitumor activity in a variety of cancer models.Previous research groups have demonstrated its potential anti-tumor angiogenesis.This study was to investigate the role and mechanism of brucine in the formation of VM in triple negative breast cancer cell line MDA-MB-231.Methods The inhibitory effect of different concentrations of brucine on the growth and proliferation of human breast cancer cell line MDA-MB-231 was determined by MTT assay.Application of Annexin V-FITC/PI double staining flow cytometry to detect apoptosis of human breast cancer cell apoptosis cells treated with different concentrations of brucine.Detection of death of human breast cancer cells by Hoechst33342/propidium iodide(HO/PI)staining.Detection of the effects of strychnine on migration and invasion of breast cancer cells by scratch test and cell invasion assay.Effects of brucine on cytoskeletal microfilaments and microtubules by immunofluorescence staining under laser confocal microscopy.Then,the human breast cancer cell MDA-MB-231 VM model was constructed in vitro.Matrigel collagen was used as a growth scaffold to culture human breast cancer cells.PAS staining is used to confirm whether human breast cancer cells have the ability to form VM in vitro.And then,we further explored the effects of different doses of brucine on VM.We finally used western blot to detect the expression of apoptosis-related protein caspase-3 and VM-related proteins Eph A2,MMP-2 and MMP-9 after treatment with different concentrations of brucine.Results1.In the triple negative breast cancer cell line MDA-MB-231,we found that brucine induced dose-dependent cell death with obvious increase at the higher concentrations after treatment with brucine.Western blot assay also showed that brucine induced cell apoptosis indicated by increased cleaved caspase-3 only at the higher concentrations.2.Through scratch experiments and cell invasion experiments,we found that brucine inhibited cell migration and invasion in a dose-dependent manner.3.Our results for the first time indicated that brucine could disrupt F-Actin cytoskeleton and microtubule structure,thereby impairing hallmarks of aggressive tumors,like migration,invasion,and holding a possibility of suppressing VM.4.On Matrigel Matrigel,human breast cancer cell MDA-MB-231 has the ability to form VM.Hence,the inhibitory effect of brucine on VM was further verified.The results illustraed that brucine significantly suppressed VM tube formation with a dose dependent effect indicated by the change of the number of tubules,intersections and mean length of tubules.5.The in-depth molecular mechanism of VM suppression induced by brucine was finally suggested.It was demonstrated that brucine inhibited VM mightly through the downregulation of erythropoietin?producing hepatocellular carcinoma?A2,and matrix metalloproteinase?2,9.Conclusion Taken together,our results demonstrate that brucine inhibits the migration,invasion and VM in human triple-negative breast cancer cell line MDA-MB-231,might through the disruption of cytoskeleton and down-regulation of Eph A2,MMP-9 and MMP-2.