| ObjectiveTo observe the effect of antiangiogenic drug(E109 and Endostar)on the biological behavior of the HUVEC under hypoxia and hypoxia/starvation condition.And to study the change of markers in HUVEC when E109 and Endostar acts on cell under hypoxia condition.On the basis of our previous research,we compare the effect of antiangiogenic drugs(E109,Endostar and bevacizumab)under same condition and identify the similarities and differences between them.Method:The in vitro hypoxia model(1%O2+5%CO2+94%N2)was established using Billups-rothenberg.then on this basis,cell was cultured in Serum free medium to mimic the hypoxia/starvation condition.MTT was used to detect the cell proliferation,and the ability of HUVEC migration and tube formation was tested by cell tubule formation assay and migration assay separately.The concentration of drug in cell tubule formation assay and migration assay was accroding to the result of MTT.The expression of endoglin(CD105)and response gene to complement-32(RGC-32)was detected by western-blot.Result:1.The results determined by MTT methods: In E109 group,after processing 24 h,the inhibition rate of hypoxia ± E109(1,4,6μmol/L)were 9.54%,26.67%,51.89%,64.42% respectively.And afte processing 48 h,the inhibition rate of them increased to 12.49%,33.61%,54.24%,65.46% respectively.After processing 24 h,the inhibition rate of hypoxia/starvation condition ± E109(1,4,6 μmol/L)were 10.88%,21.62%,59.87%,66.70% respectively.And after processing 48 h,the inhibition rate of them increased to 15.82%,30.99%,65.55%,78.49% respectively.In endostar group,after processing 24 h,the inhibition rate of hypoxia ± endostar(10μg/ml,200μg/ml,400μg/ml,800μg/ml)were 8.40%,15.32%,18.02%,21.2%,21.74% respectively.And after processing 48 h,the inhibition rate of them increased to 12.50%,12.99%,16.17%,24.37%,24.81% respectively.After processing 24 h,the inhibition rate of hypoxia/starvation condition ± endostar(10μg/ml,200μg/ml,400μg/ml,800μg/ml)were 10.19%,17.08%,19.58%,21.92%,23.10% respectively.And after processing 48 h,the inhibition rate of them increased to 14.25%,24.03%,29.22%,32.25%,37.59% respectively.In E109 group,the concentration of 1,4,6mol/L caused the inhibition rate rised from low to high.In endostar group,the 10,200,400,800μg/ml endostar cover the concentration from low to high.2.The changes of HUVEC morphology: Compared with the cells cultured in normoxia,cells cultured in hypoxia,starvation,hypoxia/starvation condition showed special changes,including cell displayed long fusiform shape,and generate filopodia.E109 promoted HUVEC death under hypoxia and hypoxia/starvation condition,reduced and broke the filopodia in survival cells.Endostar did not induce HUVEC death observably under forementioned condition,the filopodia did not change greatly as the drug concentration rose.3.The result of transwell migration experiment: Compared with the cells cultured in normoxia,the ability of cell migration was enhanced after HUVEC was cultured in hypoxia,starvation condition for both 24 and 48 hours,also in hypoxia/starvation condition for 24 hours.E109(1μmol/L,4μmol/L,6μmol/L for 24 h,48h)obviously inhibited the migration ability of HUVEC under hypoxia condition and hypoxia/starvation condition.Endostar(10μg/ml,200μg/ml,400μg/ml,800μg/ml for 24h)inhibited the migration ability of HUVEC under hypoxia condition and hypoxia/starvation condition.But there was no difference between group H+EN10 and group H+EN200.or between group H/S+EN10 and group H/S+EN200.4.Three-dimensional training results: The ability of cell tube formation did not change significantly in hypoxia group,starvation group and hypoxia/starvation guoup for 24 hours compared with the normoxia group.But the ability of cell tube formation was inhibited after processing 48 h under the environment described.E109(1μmol/L,4μmol/L,6 μmol/L for 24 h,48h)obviously inhibited the tube formation ability of HUVEC under hypoxia condition and hypoxia/starvation condition.Endostar(10μg/ml,200μg/ml,400μg/ml,800μg/ml for 24h)inhibited the tube formation of HUVEC under hypoxia and hypoxia/starvation condition.But there were no significant difference between group H+EN10 and group H+EN200.5.Western blot detecting the expression of protein:Compared with the normoxia group,hypoxia(24h)group did not influence the expression of CD105,but it up-regulate the expression of RGC-32 obviously.E109(1μmol/L,4μmol/L,6 μmol/L for 24h)can down-regulate the expression of CD105 and RGC-32 obviously.Endostar(10μg/ml,200μg/ml,400μg/ml,800μg/ml for 24h)can down-regulate the expression of CD105 and RGC-32.But for CD105,there was no significant difference between group H+EN10 and group H+EN200,also between the group H+EN400 and group H+EN800.For RGC-32,there were no significant difference between group hypoxia and group H+EN10,and the change among H+EN200,H+EN 400,H+EN 800 groups had no statistical significance.Conclusion:1.Hypoxia and hypoxia/starvation conditon promoted the abilities of migration of HUVEC,but suppress the ability of tube formation and proliferation.Both E109 and Endostar can suppress the biological behavior of HUVEC under hypoxia and hypoxia/starvation condition.As the concentration increased,the efficacy of endostar on biological behavior of HUVEC did not enhance greatly.2.The expression of RGC-32 can induced by hypoxia.E109 and Endostar can down-regulate the expression of RGC-32 and CD105 under hypoxia condition.The degree of down-regulation induced by E109 was related to the drug level,which was not obvious when increase the concentration of endostar in certain range.3.The effect of antiangiogenic drugs(E109,Endostar and bevacizumab)on HUVEC under hypoxia condition were different.E109 and Endostar dose not promote the malignant biological behavior or expression of CD105 but bevacizumab do.Since CD105 may induce endothelial-to-mesenchymal transition in HUVEC,we think that E109 and Endostar has advantage in inhibiting the malignant biological behavior of HUVEC under hypoxia condition compare to bevacizumab.The down-regulation of RGC-32 is related to the enhancement of biological behavior in HUVEC.But the effects of durg(E109 and Endostar)on inhibiting the malignant biological behavior of HUVEC is powerful,they inhibited the RGC-32 and the malignant biological behavior of HUVEC under hypoxia condition in the same time. |
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