Effects And Mechanisms Of Small Molecular Compound DMBT Against Breast Cancer Vasculogenic Mimicry

Breast cancer is one of the most serious malignant tumors that harm to women health. Invasion and metastasis is the most important biological characteristics of cancer and is also the main reason for the clinical treatment failure. In the clinical diagnosis of malignant cancer patients, more than 50% patients have been suffering metastasis. Tumor invasion and metastasis is a complex multi-steps process, in the process, tumor growth and metastasis is closely related to the new blood vessels formation. The formation of new blood vessels not only provides ongoing nutrition and oxygen for tumor growth and take away metabolites at the same time, but also transport tumor cells to target organs, which provides a necessary condition for tumor metastasis. Traditional point of view considers that tumor blood supply patterns mainly include vasculogenesis and angiogenesis, and their common characteristic is endothelial cells dependent. Prior anti-tumor angiogenesis therapy just focuses on vascular endothelial cells, and the malignant tumor itself can form angiogenesis mimicry structure was not taken into consideration, which is called vasculogenic mimicry (VM). VM is a brand new pattern of the tumor blood supply which doesn’t depend on the endothelial cells and is formed by the high invasive tumor cells. These high invasive tumor cells can mimic the function of endothelial cells to form a pipeline structure which convey plasma and red blood cells for tumor cells. VM is often located inside of tumor. VM is obviously negatively related to the tumor prognosis. But at present the research on the VM is still at an early stage, the molecular mechanisms of VM formation is not very clear. The growth and proliferation of tumor cells depends on sufficient oxygen and energy supply. When the tumor cell growth and proliferation sharply, consume a large amount of nutrients and oxygen. Because the tumor tissue has yet to establish effective network of new blood vessels in time, or the structure and function of new blood vessels is in abnormal state, the oxygen supply is far less than the demand of oxygen, which results to the tumor cells in an acute or chronic hypoxia state. While in the hypoxia condition, many molecules are involved in the formation of VM, including HIF-la, VE-Cadherin, MMPs, VEGF, etc. HIF-1 a may start a series of signal pathway to promote the happening of the VM. This is also a bottleneck of breast cancer therapy. At present, clinical commonly used anti-angiogenesis drugs mainly target the endothelial cells. However, the tumor therapy of these molecular-targeted inhibitor is not ideal in the clinical. Therefore, the research and development of new therapy for breast cancer VM is the problem demanding prompt solutions.Trehalose derivatives Brartemicin is isolated from Actinomycete metabolites by Japanese scientists Igarashi. Which structure is 6,6’-double-2,4-dihydroxy-6-methyl benzoate alpha, alpha-D-trehalose. Brartemicin has stronger anti-invasion and metastasis ability on Colon 26-L5 cells and have low toxicity. Professor Zhao-peng Liu from our Institute of Pharmaceutical Chemistry optimize the Brartemicin molecular structure in order to obtain better resistance to the invasion and metastasis of tumor, build the structural diversity of trehalose derivatives. After screening and activity comparison, found that the compound 6,6’-bis (2,3-dimethoxybenzoyl)-a, a - D-trehalose (DMBT) have stronger anti-tumor activity of invasion and metastasis. But whether the anti-invasion and metastasis ability have something related to VM is not clear.This study is aimed at the effects of compound DMBT on human breast cancer VM in vivo and in vitro and discuss its mechanisms and make the foundation for the development of high-efficiency and low toxicity anti-invasion and metastasis of breast cancer drugs in our country.Methods:In this study, we investigated the effects of DMBT on VM of human breast cancer cells MDA-MB-231 and MCF-7. MTT assay, colony forming method and western blotting were used to choose the proper concentration of sodium hydrosulfite to make the hypoxia environment in vitro; Wound healing assay and Transwell experiments to detect tumor invasion and metastasis ability in normoxia, hypoxia and hypoxia with DMBT respectively. VM formation assay was used to detect the VM forming ability in 3D matrigel in vitro. The expression of VM related proteins such as HIF-la, VE-cadherin, MMP-9, Racl, Cdc42, and EGFR, p-Akt, p-mTOR were detected by using western blotting. PAS/CD34 double stain were used to investigate the anti-VM ability of DMBT in vivo.Results:We chose sodium hydrosulfite lmM and 0.5mM to make the hypoxia environment on MDA-MB-231 and MCF-7 cells through MTT assay and colony forming method. The results of Western blotting showed that the expression of HIF-la, VE-cadherin, MMP-9, MMP-2, and VEGF were increased as time goes on in hypoxic environment in MDA-MB-231 cells. While in MCF-7 cells, the expression of VM related proteins were increased after 4h then decreased after 6h in hypoxic environment. Wound healing assay and Transwell experiments showed that hypoxia could promote tumor cells invasion and migration and DMBT could reversed these effects; 3D culture in vitro showed that hypoxia enhanced tumor cells VM forming ability and DMBT could reversed this ability. PAS/CD34 double stain showed that DMBT 1mg/kg and 10mg/kg inhibit VM forming in vivo. When tumor cells exposed to hypoxic environment, the expression of HIF-1a, VE-cadherin, MMP-9, MMP-2, Rac1, and Cdc42 were increased in hypoxia but decreased when exposed to hypoxia medium with DMBT in MDA-MB-231 cells. In MCF-7 cells, DMBT has little effects on the expression of VE-cadherin, MMP-2 and VEGF but can inhibit the expression of HIF-la, Beclinl, Rac1, and Cdc42 and at the same time increase mTOR expression. These results suggested that DMBT could serve as a therapeutic agent to inhibit VM formation in human breast cancer.Conclusion:Hypoxic microenvironment can promote breast cancer MDA-MB-231, MCF-7 cells invasion and metastasis and VM formation while DMBT can reverse this phenomenon in vitro. DMBT suppress the formation of VM by inhibiting HIF-1 a/VE-cadherin/MMPS signaling pathway in MDA-MB-231 cells. In MCF-7 cells, DMBT can inhibit autophagy through inhibiting Beclinl and promoting the expression of mTOR, thereby inhibiting the VM formation.