Construction Of Dual Antagonistic Proteins Of VEGF And FGF-2 And Their Antitumor Mechanism In Rectal Cancer

Colorectal cancer(CRC)is one of the most common gastrointestinal tumors.It is the third most common malignancy in the world,which seriously threatens human health.The growth and metastasis of tumor cells are closely related to the angiogenesis.Vascular endothelial growth factor(VEGF)is the most important regulator in the regulation of angiogenesis.Therefore,the application of therapeutic drugs targeted to VEGF has become the important treatment of CRC in recent years.However,tumor angiogenesis is regulated simultaneously by various factors,such as fibroblast growth factor-2(FGF-2).Single targeted drugs may easy to cause drug resistance and limit clinical efficacy.Studies have reported that a variety of targeted drugs combination therapy could make up for the shortcoming of drug resistance produced by single targeted drugs,and achieved better clinical efficacy,but the medicines were high cost and had significant side effects.In our study,a protein drug(VR-FR-Fc)which targeted VEGF and FGF-2 at the same time was designed,constructed and successfully expressed.And the inhibitory effects of VR-FR-Fc on proliferation,migration,invasion and angiogenesis of rectal cancer(RC)cells were verified in follow-up cytological experiments.Part?Quantitative study of expression levels of VEGF and FGF-2 in rectal tumor tissuesBackground:CRC is one of the most common gastrointestinal tumors.It is the third most common malignancy in the world,which seriously threatens human health.The growth and metastasis of tumor cells are closely related to the angiogenesis.VEGF and FGF-2 are important regulators in the regulation of angiogenesis.In this part of experiment,we detected the expression levels of VEGF and FGF-2 in the RC tissue samples,to determining the relationship between the expressions of VEGF and FGF-2 and tumors.This will provide a solid theoretical basis for the subsequent development of multi-targeted drug with VEGF and FGF-2 as two targets.Material and Methods:The RC tissue samples were collected from Department of colorectal surgery,the First Affiliated Hospital of Zhejiang University Medical College.To exclude the influence of different tumor sites on the experimental results,the 30 samples of this experiment were all patients with rectal cancer.15 tissue samples were taken from the primary lesions as experimental group.While 15 tissue samples taken from the corresponding paracancerous tissue as control group.RT-PCR,Western Blot and immunohistochemistry technology were used to detect the gene and protein expression levels of VEGF and FGF-2 in the 30 samples in the two groups,respectively,which to investigate the expression changes of VEGF and FGF-2 in tumor tissue.Results:As the RT-PCR results showed,the mRNA expressions of VEGF and FGF-2 in the experimental group were significantly higher than those in the control group(P<0.01).Similarly,the protein levels of VEGF and FGF-2 were also obviously higher than those in the control group.The results were consistent and confirmed that VEGF and FGF-2 were highly expressed in tumor tissues(P<0.01).Conclusion:This study examined the mRNA and protein expression levels of VEGF and FGF-2 in different tissue samples of rectal cancer,and revealed the correlation between the expression of VEGF and FGF and tumors,indicated the direction for the subsequent development of multi-target drugs.Part ? Construction of dual antagonistic proteins of VEGF and FGF-2 and their inhibitory effect on RC cellsBackground:At present,targeted therapy is undisputed as a major means of cancer treatment.However,the occurrence of cancer is due to a variety of regulatory factors.A single targeted drug can only block one signal pathway,and cause drug resistance,then lead to poor prognosis.The emergence of the research ideas of multi-targeted drugs and fusion protein technology maybe improve the above problems.In this part of experiment,the protein drug(VR-FR-Fc)which targeted VEGF and FGF-2 were obtained through the secretory expression of eukaryotic cells.And the inhibitory effects of VR-FR-Fc on RC cells were verified in the follow-up cytological experiments.Methods:The extracellular domain of VEGFR1 and FGFR1,and the cDNA sequence of IgG1 Fc segment were retrieved in NCBI.The relevant fragments were fused according to the fusion protein sequence and constructed into expression vector.The recombinant plasmids were transfected into 293T cells,and the cells were cultured with serum-free medium after transfection.3 days after culture,culture supernatant was collected.The protein concentrations of SP-VR-FR-Fc,SP-FR-Fc and SP-VR-Fc in the culture supernatant were determined by dual quantitative of SDS-PAGE and BCA.96-well plates was coated with recombinant human VEGF or FGF-2 respectively,recombinant human VEGF or FGF-2 was marked by Protein A/G agarose beads respectively,the affinity of each fusion protein to recombinant human VEGF and FGF-2 was detected by Enzyme linked immunosorbent assay(ELISA)and Co-Immunoprecipitation(Co-IP),the differences were compared between groups after treated with SP-VR-Fc protein(VR group),SP-FR-Fc protein(FR group),SP-VR-FR-Fc fusion protein(VR-FR group)and placebo(Control group).The above-mentioned proteins were added to the culture systems of SW837 and SW1463,respectively.The differences were compared between groups after treated with SP-VR-Fc protein(VR group),SP-FR-Fc protein(FR group),SP-VR-Fc and SP-FR-Fc proteins(VR+FR group),SP-VR-FR-Fc fusion protein(VR-FR group)and placebo(Control group).ELISA were used to detect the contents of free VEGF and FGF-2 in the culture supernatant,CCK8 was used to measure cell viability,transwell assay was used to detect cell migration and invasion ability,and western blot was used to detect AKT and ERK1/2 pathway related proteins expression.Results:Three plasmids were successfully constructed.As the result of dual quantitative of SDS and BCA showed,the protein concentrations of SP-VR-FR-Fc,SP-FR-Fc and SP-VR-Fc were all 0.1 ?g/?L.A same trend was found between the two strains of cells.The ELISA and Co-IP result showed that the affinity of fusion protein SP-VR-Fc and SP-VR-FR-Fc to VEGF was significantly higher than that of the blank control and the SP-FR-Fc(P<0.01),while the affinity of fusion protein SP-FR-Fc and SP-VR-FR-Fc to FGF-2 was significantly higher than that of the blank control and the SP-VR-Fc(P<0.01).The ELISA result showed that the content of free VEGF in the culture supernatant decreased significantly when adding SP-VR-Fc to the cell culture fluid(P<0.01),while no significant change in the content of free FGF-2(SW837 P=0.9017,SW1463 P=0.9812).The content of free FGF-2 in the culture supernatant decreased significantly when adding SP-FR-Fc to the cell culture fluid(P<0.01),while no significant change in the content of free VEGF(SW837 P=0.6978,SW1463 P=0.6928).After treated with SP-VR-FR-Fc,SP-FR-Fc and SP-VR-Fc,the ability of cell proliferation,migration and invasion significantly reduced(P<0.01).The western blot result showed that,the protein levels of p-AKT and p-ERKl/2 both distinctly decreased after the addition of protein antagonism(P<0.01).Moreover,the effect of double antagonism was better than the single antagonism.However,there was no significant difference in the effect of fusion plasmid and plasmid mixed by VR and FR(P>0.05).Conclusions:The protein drug(VR-FR-Fc)which targeted VEGF and FGF-2 was designed,constructed and successfully expressed in our study.And through further experiments,we found that VR-FR-Fc has high affinity to recombinant human VEGF and FGF-2 and VEGF and FGF-2 secreted by RC cell lines SW837 and SW1463 at the same time.In addition,it could simultaneously inhibit the activation of PI3K/AKT signaling pathway and ERK signaling pathway mediated by VEGF and FGF-2,thereby inhibiting the ability of proliferation,migration and invasion of RC cells.These findings suggest that VR-FR-Fc may have a better therapeutic effect on RC,and it could be based on the development of dual-targeted protein drugs for RC.Part ? Study on inhibitory effects of VEGF and FGF dual antagonistic proteins on angiogenesis in RC cells Background:Angiogenesis refers to the growth of new blood vessels on the basis of the original blood vessels,it is crucial for tumor growth and metastasis.Angiogenesis is mainly regulated by proangiogenic factor and angiogenic factor.In tumor cells,when the expressions of proangiogenic factor and angiogenic factor were out of balance,endothelial cells will be stimulated to proliferate,which promotes the proliferation and metastasis of tumor cells.So in this part of experiment,we verified the effects of VR-FR-Fc on the proliferation,migration and angiogenesis of endothelial cells in the extracellular milieu of tumors by RC cells and human umbilical vein endothelial cell(HUVEC)co-culture models.Methods:HUVEC cells were cultured respectively in normal medium(Control group)and the SW1463 cell culture fluid(RC conditioned media group,RCCM group).The RCCM were added SP-VR-FR-Fc(VR-FR group),SP-VR-Fc(VR group),SP-FR-Fc(FR group)and SP-VR-Fc and SP-FR-Fc mixed proteins(VR+FR group)when cultured.CCK8 was used to measure cell viability and transwell assay was used to detect the cell migration ability of each group.Finally,the ability of angiogenesis was detected.Results:The viability,migration ability and angiogenesis ability of HUVEC cells were obviously enhanced after cultured with RCCM(P<0.01).While the viability,migration ability and angiogenesis ability of that slightly lower when cultured with RCCM that contained VR or FR protein(P<0.05).Compared with the VR group or FR group,the viability,migration ability and angiogenesis ability of cells treated with VR+FR or VR-FR protein were significantly lower(P<0.01).The results also showed that the dual antagonistic effect was better than that of the single antagonism(P<0.01),but there was no significant change in the mixture of VR and FR plasmid and the fusion plasmid(P>0.05).Conclusions:In present research,we used RC conditioned medium to culture HUVEC cells to construct a co-culture model of tumor cells and endothelial cells.Using this model,we found that VR-FR-Fc could inhibit HUVECs proliferation,migration and in vitro angiogenic ability,and its inhibitory effect is significantly better than that of single-target protein.The results suggest that VR-FR-Fc could inhibit the activity of vascular endothelial cells in the tumor microenvironment,thereby inhibiting angiogenesis and playing an important role in inhibiting tumor development.