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Objective To investigate the inhibitory effects of Tum5 on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and alkali-induced corneal neovascularization.Methods HUVECs in logarithmic growth phase were divided into 4 groups,cells with untreated as normal control group,cells with the infection of rAdGFP virus as rAd-GFP group,cells with the infection of rAd-Tum5 virus as rAd-Tum5 group,and cells with the infection of rAd-Tum5 virus followed by VEGF treatment as rAd-Tum5 + VEGF group.Then cell proliferation,migration,and tube formation of HUVECs were examined by CCK-8,Transwell and Matrigel assays,respectively.Sixty-four healthy male SD rats were randomly divided into 4 groups (n =16) by using random number table,and they were normal control group,alkali-burn group,alkali-burn + rAd-GFP group,and alkali-burn + rAd-Tum5 group.The alkali-burn rat model was then established except normal control group,and the normal control group received no treatment,whereas the alkali-burn,alkali-burn + rAd-GFP,and alkali-burn + rAd-Tum5 groups received subconjunctival injection of equal volumes of sterilized saline,rAd-GFP virus,and rAd-Tum5 virus,respectively following the alkaline burn.The relative area of corneal neovascularization and the number of infiltrating inflammatory cells were recorded on day 1,7,and 14 after injection.Results The CCK-8 assay showed that the proliferative rate of rAd-Tum5 group was lower than that of the normal control group and rAd-GFP group (both P ＜0.01),while rAd-Tum5 + VEGF group exhibited a significantly greater cell proliferative capability than rAd-Tum5 group (P =0.004).There were no statistical differences between rAd-Tum5 + VEGF group,normal control group and rAdGFP group (all P ＞ 0.05).Transwell assay showed the significantly lower number of migrating cells in the rAd-Tum5 group than those in the normal control group and rAdGFP group (both P ＜ 0.01).The number of migrating cells in rAd-Tum5 + VEGF group was higher than those in rAd-Tum5 group (P =0.000);however,the migration capacity had not been restored to normal level,and rAd-Tum5 + VEGF group had significant difference with normal control group and rAd-Tum5 group (both P ＜0.05).Matrigel assay showed that the number of meshes in rAd-Tum5 group was lower than that in the normal control group and rAd-GFP group (both P ＜0.01);while the number of meshes in rAd-Tum5 + VEGF group was significantly increased compared with rAd-Tum5 group(P =0.001).The density and number of corneal neovascularization increased gradually from day 1 to day 14 after alkali burn,while the relative neovascularization area in the alkali-burn + rAd-Tum5 group was significantly reduced as compared to those in the alkali-burn group and alkali-burn + rAd-GFP group on day 7 and day 14,suggesting that Tum5 could reduce the growth rate and density of corneal neovascularization,so as to inhibit corneal neovascularization induced by alkali burn.On day 7 and 14 after alkali burn,in the normal control group,the corneas were intact,no infiltrating inflammatory cells and cells were arranged in an orderly manner.On day 7 after alkali burn,there were disordered epithelial structure,corneal edema and infiltrating inflammatory cells in alkali-burn group and alkali-burn + rAd-GFP group.The number of infiltrating inflammatory cells in alkaliburn + rAd-Tum5 group was significantly lower than that in alkali-burn group and alkali-burn + rAd-GFP group (both P ＜0.01).On day 14 after alkali burn,the number of infiltrating inflammatory cells in alkali-bum,alkali-burn + rAd-GFP and alkali-burn + rAd-Tum5 group were significantly lower than those on day 7,while the corneal epitheliums were intact and dropsy was alleviated,while the number of inflammatory cells was significantly lower than that in alkali burn group and alkali-burn + rAd-GFP group,with significant difference (both P ＜ 0.01).Conelusion Tum5 can inhibit the angiogenic capability of HUVECs by VEGF pathway,as well as suppress the alkali-burn-induced corneal neovascularization and inflammatory cell infiltration in rats.
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Objective: To explore the inhibitory effect of Tum-5 gene on the tumor growth and its influence in the angiogenesis of hepatocellular carcinoma (HCC), and to illustrate its mechanism involved in antitumorigenesis. Methods: The human HepG2 cells were selected in in vitro experiment and treated with different multiplicity of infection (MOD (0, 1, 5, 10, 25 and 50) of pLXSN and pLXSN-Tum-5 virus particles for 72 h. The proliferation rates of cells in various groups were detected by MTT method. In vivo, the H22 tumor-bearing mouse models were established. The mice were divided into saline group, pLXSN group and pLXSN-Tum-5 group (n=5). The mice in saline group were intratumorally injected with normal saline, and the mice in pLXSN group and pLXSN-Tum-5 group were intratumorally injected with virus particles (MOI =5), 5 times every other day. The volumes and weights of transplanted tumor and the weights of mice in various groups were measured. The CD31 expressions in transplanted tumor tissue were detected by immunohistochemical method and the microvessel density (MVD) was calculated. Results: Compared with pLXSN group, the proliferation rates of cells in pLXSN-Tum-5 group after infected with different MOI of virus particles were not significantly different (P> 0. 05). The volumes and weights of transplanted tumor of the mice in pLXSN-Tum-5 group were significantly smaller than those in pLXSN group and saline group after intratumoral injection (P
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Objective:To explore the inhibitory effect of Tum-5 gene on the tumor growth and its influence in the angiogenesis of hepatocellular carcinoma (HCC),and to illustrate its mechanism involved in antitumorigenesis.Methods:The human HepG2 cells were selected in in vitro experiment and treated with different multiplicity of infection (MOI) (0,1,5,10,25 and 50) of pLXSN and pLXSN-Tum-5 virus particles for 72 h.The proliferation rates of cells in various groups were detected by MTT method.In vivo,the H22 tumor-bearing mouse models were established.The mice were divided into saline group,pLXSN group and pLXSN-Tum-5 group (n=5).The mice in saline group were intratumorally injected with normal saline,and the mice in pLXSN group and pLXSN-Tum-5 group were intratumorally injected with virus particles (MOI =5),5 times every other day.The volumes and weights of transplanted tumor and the weights of mice in various groups were measured.The CD31 expressions in transplanted tumor tissue were detected by immunohistochemical method and the microvessel density (MVD) was calculated.Results:Compared with pLXSN group,the proliferation rates of cells in pLXSN-Tum-5 group after infected with different MOI of virus particles were not significantly different (P＞ 0.05).The volumes and weights of transplanted tumor of the mice in pLXSN-Tum-5 group were significantly smaller than thosein pLXSN group and saline group after intratumoral injection (P＜ 0.05 or P＜ 0.01).The immunohistochemical results showed that there was irregular angiogenesis in each group.Compared with saline group and pLXSN group,the mean value of MVD of the transplanted tumor tissue of the mice in pLXSN-Tum-5 group was significantly decreased (P ＜ 0.05).Conclusion:Tum-5 can exert its antitumor activity by inhibiting the formation of neovascularization in HCC.
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Background Tumstatin is the most active endogenous angiogenesis inhibitor,which has a marked inhibitory effect on pathological neovascularization,and Tum5 is an angiogenesis inhibitors fragment of fulllength tumstatin.Objective This study was to investigate the effects of adenovirus-mediated overexpression of recombinant Tum5 gene on the proliferation,migration and tube formation of human umbilical vein endothelial cells (HUVECs) in physiological status.Methods The empty adenoviral vector expressing green fluorescent protein (rAd-GFP) and the viral vector expressing recombinant Tum5 gene were constructed.The HUVECs cultured in RPMI1640 medium were divided into normal control group,empty vector group (rAd-GFP group) and Tum5 gene infection group (rAd-GFP-Tum5 group).The rAd-GFP and rAd-GFP-Tum5 adenoviral particles at the density of 1 × 1010/ml were added into the medium to infect the cells for 48 hours.The proliferation of the cells was assayed at 24,48 and 72 hours by cell counting kit-8 (CCK-8) to evaluate the proliferative rate;the migration number of the cells was detected at 48 hours after infection by Transwell chamber;the tube formation number of the cells were detected by Matrigel method.The concentration of vascular endothelial growth factor (VEGF) in cell supernatants was assayed by ELISA at 24,48,and 72 hours following adenoviral infection.Results The cultured cells showed green fluorescence in the rAd-GFP group and rAd-GFP-Tum5 group under the inverted fluorescence microscope,and the infection efficiency of rAd-GFP and rAd-GFP-Tum5 was 55.13％ and 50.31％,respectively.No significant difference was found in cell proliferative rate among normal control group,rAd-GFP group and rAd-GFP-Tum5 group both at 24 and 48 hours after infection (both at P＞0.05),and the cell proliferative rate was significantly lower in the rAd-GFP-Tum5 group than that in the normal control group and rAd-GFP group at 72 hours after infection (both at P＜0.01).The migration number of the cells at 48 hours after infection was 2 260.25-±930.44,2 370.00±441.06 and 723.75± 363.80 in the normal control group,rAd-GFP group and rAd-GFP-Tum5 group,showing a significant difference among the groups (F =8.524,P =0.008),and the migrated cells were evidently decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group and the normal control group (both at P＜ 0.01).The tube number at 48 hours after infection was 95.67±5.86,88.00±4.58 and 20.67±3.51 in the normal control group,rAd-GFP group and rAdGFP-Tum5 group,showing a significant difference among the groups (F=226.498,P＜0.01),and the tube number in the rAd-GFP-Tum5 group was significantly reduced in comparison with the normal control group and rAd-GFP group (both at P＜ 0.01).The considerably differences in VEGF concentration in the cell supernatants were found in different groups and various time points (Fgroup =73.260,P＜0.01;Ftime =73.477,P＜0.01),and VEGF concentration in the cell supernatants was significantly decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group at both 48 hours and 72 hours (both at P＜0.01).Conclusions The overexpression of the recombinant Tum5 can inhibit the proliferation,migration and tube formation of the HUVECs in physiological status,which may be associated with Tum-5-mediated down-regulation of VEGF protein in the cell supernatant.
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Growth of tumor need new blood vessel to meet the demand for adequate oxygen and nutri -ents.Inhibiting the formation of blood vessel can inhibit growth of tumor .In all of the endogenous angiogenesis in-hibitors deriving from the type IV collagen .Study of tumstatin is the most widely .Tumstatin can inhibit endothelial proliferation and induce the apoptosis of endothelial cell .This article reviews the research status of the tumstatin .
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Objective To observe the effect of human telomerase reverse transcriptase-thymidine kinase/ganciclovir (phTERT-TK/GCV) system combined human telomerase reverse transcriptase-tumstatin (phTERT-tumstatin) system on apoptosis of human HepG2 and mRNA expression and protein content of AFP,RhoC related with cancer.Methods Fluorescence microscopy was used to observe expression of EGFP and MCHERRY in transfected HepG2 and L-02.Real-time PCR and Western blot were used to detect AFP and RhoC mRNA and protein content.Flow cytometry was used to detect the apoptosis of transfected HepG2.Results phTERT-tumstatin and phTERT-TK/GCV genes expressed in transfected HepG2.Real-time PCR showed that AFP and RhoC mRNA expression in different group were 0.76±0.09 and 0.80±0.04 (TK/GCV group),0.62±0.09 and 0.40±0.02 (TM group),0.49±0.07 and 0.54±0.03 (MK group).The differences were significant (P ＜ 0.01) except TK/GCV group compared with empty plasmid group.Western blot test results showed that protein content of AFP and RhoC were higher in TK/GCV group (0.97±0.02/1.17± 0.01),TM group (0.83±0.02/0.99±0.02),MK group (0.69±0.01/0.77±0.02) than in empty plasmid group (1.19±0.03/1.32±0.05) and non-transfected group (1.15±0.05/1.29±0.30) (P ＜ 0.01).Additionally,protein content of AFP and RhoC in MK group were significant difference with TK/GCV group and TM group (P ＜ 0.01).Flow cytometry showed that phTERT-tumstatin,phTERT-TK/GCV co-transfected HepG2 cells apoptosis rate was significantly higher than both individually transected group.Cells apoptosis rate of alone and co-transfected groups was significant difference compared with empty vector group and untransfected group.Conclusions The effect of phTERT-TK/GCV and phTERT-tumstatin on pro-apoptotic of HepG2 cells is significant.phTERT-TK/GCV combined with phTERT-tumstatin has strong therapeutic function.
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OBJECTIVE: To study the effect of biological activity of tumstatin 7 peptide (CNYYSNS) on the cell proliferation and apoptosis of B16 melanoma cell. METHODS: The inhibitory effect of tumstatin 7 peptide on the proliferation of B16 cell was observed by MTT and cell growth curves. The influence of tumstatin 7 peptide on morphology of B16 cell was perceived by TUNEL, HE staining and the transmission electron microscope(TEM). Human umbilical vein endothelial cell (ECV304) as control cell was detected that tumstatin 7 peptide affected the proliferation of non-tumor cells. RESULTS: Tumstatin 7 peptide can significantly inhibit the proliferation of B16 cell in dose-and time-dependent manner. Its IC50 was 8.53 × 10-5 mol·L-1. The mophology of B16 cell was obviously changed by means of TUNEL assay, HE staining and TEM. They appeared karyopyknosis and apoptotic bodies. The apoptosis rate of B16 cell was 68.45%. The effect of 7peptide on human endothelial cell was weak, its IC50 was 5.78 × 10-4mol·L-1. CONCLUSION: Tumstatin 7 peptide can inhibit the proliferation of B16 cell and promote B16 cell apoptosis. It has little effect on endothelial cell, which revealed 7 peptide having a certain specificity of anti-tumor. It will be of great potential value to melanoma treatment.
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Objective: To express Tumstatin183-230-TRAIL fusion protein and to observe its biological functions. Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin183-230 and TNF-related apoptosis-inducing ligand (TRAIL114-281). An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c2; the vector was used to transfect E. coli BL21 (DE3) and expression of MBP-Tu-T fusion protein was induced by IPTG. Amylose Resin columns were employed to purify the fusion protein. The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation, standard tumor cell cytotoxic assay, in vitro tube formation inhibition, and electron microscopic observation (apoptosis). Results: The expression rate of MBP-Tu-T fusion protein in E. coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation (IC50 12.5 μg/ml), induced apoptosis of pancreatic cancer cells, and inhibited tube formation. Conclusion: Constructed MBP-Tu-T fusion protein is bifunctional, which lays a solid foundation for further investigation of antitumor effect of Tumstatin183-230-TRAIL in vivo.
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Aim: To construct a prokaryotic expression vector carrying NuBCP-9-tumstatin(74-98) (abbreviated as NT) gene and to obtain the fusion peptide with antitumor activity. Methods: Nucleotide sequences of antitumor peptides, NuBCP-9 and Tumstatin( 74-98), were connected via a linker(G_4S)_3 based on biased codons of E. coli the fused NT gene was reconstructed using SOE PCR, and inserted into pET32a(+) vector, and transformed in E. coli BL21(DE3). After expression, the novel fusion peptide was purified through nickel-affinity chromatogra-phy, Factor Xa digestion and ultrafiltration. Biological activity of the fusion peptide on ECV304 and A549 cells was evaluated by MTT assay. Results: A prokaryotic expression system with NT gene was successfully constructed. The soluble fusion peptide was accounted for approximately 25% when induced by 0. 5 mmol/L IPTG at 30 ℃ for 4 h. The purified fusion peptide could inhibit cell growth of ECV304 and A549 with inhibition rates of 60. 8% and 65. 2% at 20 μmol/L, respectively. Conclusion: A novel fusion peptide with antitumor activity was cloned, expressed and purified.
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Objective:To express Tumstatin_(183-230)-TRAIL fusion protein and to observe its biological functions.Methods: SOE-ing PCR was employed to amplify the recombinant sequence of Tumstatin_(183-230)and TNF-related apoptosis-inducing ligand (TRAIL_(114-281)).An expression vector pMAL-Tu-T was constructed by inserting Tu-T sequence into pMAL-c_2;the vector was used to transfect E.coli BL21(DE3)and expression of MBP-Tu-T fusion protein was induced by IPTG.Amylose Resin columns were employed to purify the fusion protein.The biological functions of MBP-Tu-T protein was examined by inhibitory test of endothelial cell proliferation,standard tumor cell cytotoxic assay,in vitro tube formation inhibition,and electron microscopic observation(apoptosis).Results:The expression rate of MBP-Tu-T fusion protein in E.coli was about 20%. Purified recombinant protein obviously inhibited endothelial cell proliferation(IC_(50)12.5?g/ml),induced apoptosis of pancreatic cancer cells,and inhibited tube formation.Conclusion:Constructed MBP-Tu-T fusion protein is bifunctional,which lays a solid foundation for further investigation of antitumor effect of Tumstatin_(183-230)-TRAIL in vivo.