| Background:Tumor vascular targeting therapy is currently a hot field in the research of cancer treatment.Compared to traditional chemotherapy,anti-angiogenesis therapy has a role in a broad spectrum,less drug resistance advantages.Currently there are two groups of vascular targeting agents,angiogenesis inhibitors(AIs)and vascular disrupting agents(VDAs).AIs have weak anti-tumor effect,the use of combination chemotherapy is often required;VDAs have some anti-tumor effect,mainly affect the relatively mature tumor vadculature,but no influence on neovascularization.Single AIs or VDAs can not completey destroy tumor vascular network,so we combined AIs and VDAs,to achieve stonger alti-tumor effect.Endostatin is a tumor angiogenesis inhibitor,with a strong inhibitive effect of angiogenesis,which can selectively interfere with microvascular endothelial cells.Recombinant human endostatin(Endostar),is synthesized by adding 9 amino acids at the amino terminus of endostatin.This conformational change has not diminished biological effect of endostatin,while its activity and stability have been significantly improved.Combretastatin A4(CA4)is a polyhydroxy cis-stilbene natural material,isolated from a kind of bush Combretum caffrum,whose structure is very similar with colchicine,but less toxicity.CA4 can combine on tubulin at colchicine binding site,inhibiting tubulin polymerization,changing the structure of the endothelial cells' cytoskeleton,interferencing vascular function,resulting in tumor tissue necrosis.CA4P is a phosphate precursor of CA4,with water soluble ability,which in vivo can be transformed via a non-specific phosphatase to CA4,and have improved anti-tumor activity.This study was designed to assess the anti-vascular effects by combination of Endostar and CA4P in vivo and in vitro experiments,to explore the theoretical foundation for the combination of AIs and VDAs.Methods and Results:1.We combined use Endostar and CA4P in human umbilical vein endothelial cells(HUVEC)in a short time interval,MTT cell viability assay found low concentration of endostatin(0.05-50?g/ml)or CA4P(0.1-100nM)with no significant cytotoxicity.Scratch and Transwell experiments found single Endostar(10?g/ml)or CA4P(10nM)inhibited endothelial cell migration,the migration ability after treated by two agents combination was stronger than monotherapy group;the results of tube formation is similar to Scratch and Transwel experiment.2.We used confocal microscope to detect F-actin generation in endothelial cells after treatment of Endostar or CA4P,the results suggested the two drugs alone may increase the F-actin formation,combined use two agents in a short time interval decreased production of F-actin.Then we used pull-down assays with western blot to detect RhoGTPases family activation of RhoA,Racl and Cdc42,which found relative expression of active Rael/Cdc42 were increased by endostar,with inhibititon of RhoA activation,thereby interfering with the the biological effects of CA4P.3.We extend the Endostar and CA4P adminstation interval to 12h in scratch test and Transwell experiments,and interference disappeared.Then we constructed HUVEC cells and MNNG/HOS osteosarcoma cells xenograft model,when endostar and CA4P administration interval was 24h,the anti-tumor effect were the strongest and tumor microvessel density was minimum.Conclusions:1.We firstly found when the adminstation interval is short Endostar and CA4P can interfere with each other;2.Competitive activation RhoGTPases family members may play a key role in the interfere between Endostar and CA4P;3.Extending Endostar and CA4P administration time interval can eliminate interference effects between the two drugs;... |
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