The Mechanism Of Xihuang Pill Inhibiting The Proliferation Of Gastric Cancer Cells And The Formation Of Angiogenic Mimicry

Background:China owns a large number of patients with gastric cancer which leads a high mortality.Vasculogenic mimicry(VM)was found recently and serves as a new angiogenesis form and is associated with poor prognosis of gastric cancer.Hypoxia microenvironment could facilitate VM formation and is related to the syndrome of stagnation of blood stasis and toxin.Xihuang Pills(XH)could promote blood circulation and detoxicate,and inhibit kinds of tumors.Based on above,we assume that XH could inhibit gastric cancer by regulating hypoxia microenvironment and reduce VM formation.Methods:(1)Groups were classified as follows:Control,XH,2-ME and combined group.(2)We used MGC-803 cell line,to evaluate proliferation rate,apoptosis rate and cell cycle using CCK-8 kit,AV-PI apoptosis kit,RNAse respectively in vitro.To measure tumor volume and quality in vivo.(3)To build hypoxia model and three-dimensional culture model in vitro,and tumor-bearing model in vivo,counting the mimicry vascular number in both vivo and vitro through PAS-CD31 staining and microscope.(4)To evaluate the expression of VE-Cadherin,EphA2,MMP-2,p-EphA2,HIF-1?,Twist 1 in vivo and vitro using RT-qPCR,Western-blot,IHC methods.(5)Screening the antitumor and hypoxia regulating pathways and genes of XH and verifying them by using RT-qPCR methods.Results:(1)XH could inhibit the proliferation of MGC-803 cells,promote cell apoptosis and induce G0/G1 cell cycle arrest in a concentration and time dependence.The IC50 were 50.424 ?g/ml in 24h,35.129 ?g/ml in 48h and 28.191 ?g/ml in 72h.(2)XH could inhibit the VM formation in vivo and vitro,as same as the effects of 2-ME,the combination group showed the strongest inhibition.(3)XH,2-ME and combined group could down regulate the mRNA expression of VE-Cadherin and MMP-2 and the protein expression of VE-Cadherin,p-EphA2 and MMP-2.The combined group could lead a more significant inhibition of these factors and could down regulate the mRNA expression of EphA2.The protein expression of EphA2 was not affected.Furthermore,XH,2-ME and combined group could decrease the mRNA expression of HIF-la and Twistl and the protein expression of HIF-la in vitro and down regulate the mRNA and protein expressions of HIF-1? and Twist1.(4)XH could down regulate the expression of 628 genes,9 IncRNAs and up regulate the expression of 88 genes,11 lncRNAs and regulate the expression of 54 pathways according to the HTA2.0 gene chip result.We chose the hypoxia and cell cycle pathways to verify.We found that XH could decrease mTOR expression and increase PHD2 and VHL expression.Moreover,XH could down regulate gene expression of cell cycle related proteins(SMC3,CDC25c,CDC6,MCM3).Conclusion:XH could inhibit the progression and VM formation of gastric cancer via the following mechanisms:(1)inhibiting the mTOR-HIF-1?-Twist1 signal pathway;(2)promoting the expression of PHD2 and VHL proteins;(3)inhibiting the expression of VE-Cadherin,MMP-2 and p-EphA2;(4)inhibiting cell cycle related proteins(SMC3,CDC25c,CDC6,MCM3)expressions.