Effect Of The Combination Of Disulfiram And Copper On Tumor Angiogenesis,invasion And Metastasis

Abnormal vessel growth and metastasis are hallmarks of cancer and contribute to the progression of solid malignancies.Because angiogenesis together with metastasis is essential for tumor growth beyond minimal size,anti-angiogenesis and anti-metastasis have become a potential therapeutic strategy for treating cancer.Disulfiram(DSF)has been reported to regulate tumor growth through the induction of apoptosis,blockage of drug resistance,inhibition of invasion and angiogenesis and suppression of stem cell-like properties.Moreover,we have found that DSF showed high toxicity to cancer cells in a copper(Cu),Zinc(Zn),cadmium(Cd)dependent manner.Previous studies revealed that DSF inhibited angiogenesis in nude mice,and inhibited chick embryo chorioallantoic membrane(CAM)angiogenesis.It is also reported that DSF inhibited invasion in both tumor and endothelial cells at non-toxic concentrations through the inhibition of MMP-2 and MMP-9 activity.However,neither the anti-angiogenic and antimetastatic activity nor the molecular mechanisms has been reported.Therefore,preclinical evaluation of the anti-angiogenic anti-metastatic capacity of DSF/Cu in in vitro and in vivo models is urgently required to determine whether Cu can improve the inhibition of angiogenesis and metastasis by DSF.1.Firstly,we verified for the first time that a combination of Cu and DSF resulted in improved inhibition of endothelial cell proliferation,migration,invasion,adhesion and tube formation in vitro.DSF/Cu also inhibited the growth of new microvessels in the rat aortic ring assay,the Matrigel plug assay and the glioblastoma xenograft model.We also explored the antimetastasis effects and molecular mechanisms of DSF/Cu on HCC cells in vitro.DSF inhibits the proliferation,migration and invasion of HCC cells.Cu improved the anti-metastatic activity of DSF.2.Secondly,We demonstrated for the first time that Cu improves the anti-angiogenic activity of DSF in endothelial cells by targeting the EGFR/Src/VEGF signaling pathway.These findings suggest that DSF/Cu may be a better angiogenesis inhibitor in clinical applications than DSF alone.DSF/Cu inhibited the TGF-? and NF-?B signaling,including Smad4 and the nuclear translocation of NF-?B subunit.These two pathways lead to down-regulation of Slug,which contributed to EMT.Therefore,the metastasis of tumor cells was weakened by DSF/Cu.3.Finally,U87 tumor xenografts and U87 orthotopic mouse model were established.DSF/Cu reduced expression of VEGF and microvessel density(MVD),which in turn the growth of the U87-derived tumors.Becides,in the comparision of DSF,DSF/Cu exerted increased anti-tumor effects on subcutaneous and intracerebral U87 xenograft models by reducing microvessel density(MVD)and VEGF expression.These results indicate that Cu improves the anti-angiogenic activity of DSF by targeting the EGFR/Src/VEGF signaling pathway.We also determined the effect of DSF/Cu on metastasis in vivo.It turned out that DSF/Cu showed improved anti-metastatic activity not only in a subcutaneous tumor model but also in an orthotopic liver metastasis assay by inhibiting phenotype epithelial-mesenchymal transition.In summary,we demonstrated for the first time that Cu improves the anti-angiogenic activity of DSF in vitro and in vivo by targeting the EGFR/Src/VEGF signaling pathway.DSF/Cu exerted effects on EMT corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo.Smad signaling together with NF-?B was found to be key players in the suppression of EMT caused by DSF/Cu.These preclinical findings may provide a plausible scientific basis for DSF/Cu to be used in clinical research targeting tumor angiogenesis and invasion.