AHVAC-I Inhibits Vasculogenic Mimicry In Triple Negative Breast Cancer Cells

Objective:Triple-negative breast cancer（TNBC）is a type of breast cancer that is negative for estrogen receptor,progesterone receptor and human epidermal growth factor receptor-2 e×pression.As a subtype of breast cancer with poor prognosis,TNBC has attracted much attention.Clinical studies have confirmed that the main causes of death in TNBC patients are recurrence and metastasis.Vasculogenic mimicry（VM）is a tumor microcirculation model,a tubular network structure formed by highly invasive tumor cells that does not require endothelial cells to participate.Clinical studies have confirmed that the positive rate of VM in TNBC tumor tissue is high,and the positive expression rate of VM is related to tumor size and lymphatic metastasis.As an important member of matrix metalloproteinases,matrix metallopeptidase-2（MMP2）has been proved to be not only related to the grading and infiltration of breast cancer,but also found to be closely related to the formation of VM in breast cancer tissue.The study found that the snake venom secreted from the venom glands of the snake head not only has a killing effect on ordinary breast cancer cells,but also has a cytotoxic effect on TNBC cells MAD-MB-231.Although snake venom can inhibit the proliferation of TNBC tumor cells,it has not been studied whether it can reduce the metastasis and recurrence of TNBC by inhibiting the VM function of TNBC tumor cells.The agkistrodon halys venom antitumor component-I（AHVAC-I）is a component extracted from the crude venom of the southern Anhui pit viper venom by the Institute of Snake Venom,Wannan Medical College.In this paper,by studying the effect and mechanism of AHVAC-I on the in vitro vasculogenic mimicry of TNBC cells MDA-MB-231,the potential antitumor mechanism of AHVAC-I in inhibiting the metastasis and recurrence of breast cancer was preliminarily explored.Methods:AHVAC-I was purified from the crude venom of southern Anhui pit viper venom by anion exchanger DE-52 chromatography.MDA-MB-231 cells viability was determined by CCK-8（Cell Counting Kit-8）.Based on the half inhibitory concentration(IC₅₀),the concentrations of AHVAC-I were determined for the following exposure tests.Measured the effect of AHVAC-I on the tubulogenesis of breast cancer cell MDA-MB-231 by Matrigel assay.The level of MMP2,which secreted by MDA-MB-231,was detected by ELISA.The expression level of MMP2 was determined by RT-PCR and Western blot.Results:1.AHVAC-I was successfully isolated and purified from the venom of the southern Anhui pit viper venom.2.MDA-MB-231 cells were treated with varoius concentrations of AHVAC-I（5,10,20,40μg/ml）for 24 hours.The IC₅₀was 32.96μg/ml.Therefore,contrationos of 5,10,and 20μg/ml were chosen for subsequent experients.Matrigel experiment showed that AHVAC-I significantly attenuated formation of capillary-like tube structures in a dose-dependent manner.3.ELISA,RT-PCR and Western blot showed that the level of MMP2 was significantly decreased by AHVAC-I.Matrigel experiment showed that the attenuated vasculogenic mimicry formation was abrogated by treatment with MMP2 protein.Conclusion:AHVAC-I could inhibit the VM of TNBC cell line MDA-MB-231 through down regulating MMP2 production,and is valuable for TNBC therapy.