| Backgrounds and ObjectivesTumor molecular imaging or targeted therapy is a kind of method to achieve the goal of tumor imaging or treatment,which depends on sending the molecular probes or targeted drugs to tumor cells specifically.It has become a hot point in the field of nuclear medicine and cancer research,which brings new hope for the diagnosis and treatment of tumor.Among the factors to success,tumor specific targets selection and molecular probes screening are the two most important elements.VEGF/VEGFR has a high ratio of tumor/non tumor,which plays a key role in the tumor angiogenesis and is closely related to tumor growth and metastasis,so it is an important target in cancer research;molecular probes or targeted drugs based on this target can be used for tumor imaging or tumor treatment.Based on the target VEGFR,our research group has obtained two peptides(QKRKRKKSRKKH,RKRKRKKSRYIVLS)through the bioinformatics screening method.Experiments in vivo and in vitro have confirmed them as tumor targeting inhibitory peptides.As candidate molecular probes or targeted drugs,they may be used in tumors nuclide molecular imaging and tumors targeted therapy.However,the two molecular probes may be degradation in serum,and their function sites are also unclear.So it is important to improve their stability in serum and further explore their function sites,which will complete the mechanism of tumor targeting and the mechanism of inhibiting tumor cell growth.Therefore,this study is aimed to improve the stability of this two tumor molecular probes in serum by protective chemical modification,and further explore their function sites,in order to lay a experimental foundation for their applications in tumor molecular imaging and targeted therapy.Methods1.The stability study of peptides before and after protective chemically modification: the original peptides and the chemical modification peptides(n-terminal acetylation(Ac),c-terminal amide(NH2))were artificially synthesized;3H-TdR and CCK-8 experiments were used to compare their ability of inhibiting the A549 cells proliferation,transwell experiment was used to compare their ability of inhibiting the A549 cells migration before and after modification,evaluating their stability indirectly;high pressure liquid chromatography(HPLC)was used to detect the remaining peptides at different time point in serum and cell environment,comparing their stability directly.2.Prokaryotic expression vectors pGEX4T-1-peptides were constructed,then they were transfected into E.coli BL21(DE3)cells,and induced the expression of GST-peptides fusion proteins;the fusion proteins were purified by Glutathione Sepharose 4B;their expression and purification effects were appraised through protein electrophoresis;the combination of GST-peptides and VEGFR subtypes(VEGFR-1 or VEGFR-2)was identified by Immune coprecipitation(Co-IP)method.3.A549 cells were interacted with peptides,Western Blot experiment was used to detect the phosphorylation level of VEGFR-1 and VEGFR-2.4.RNAi technology silenced VEGF expression,firstly,siRNA was transfected into A549 cells,secondly,Western Blot was used to appraise the level of VEGF proteins expression;CCK-8 experiment was used to investigate whether there exists other ways to inhibit tumor cells proliferation by peptides besides VEGF/VEGFR pathway without endogenous and exogenous VEGF.5.Bioinformatics methods,namely,molecular docking method and dynamic simulation technology were used to predict potential targets of peptides theoretically;and competitive receptor binding assay and Co-IP were used to identified the theoretic potential targets.Results1.After modification,RKRKRKKSRYIVLS peptide's inhibition ratio on A549 cell proliferation was enhanced to(48.50±7.14)% from(2.63±6.19)%(P<0.01),inhibition ratio on cell vitality was changed to(20.80±5.90)% from(1.30±7.90)%(P<0.01),inhibition ratio on A549 cell migration was improved to(38.86 + 7.69)% from(7.02 + 8.64)%(P<0.01)in 10% serum.HPLC showed that the remaining percentage of peptide in serum was increased to(77.90±1.19)% from(70.52±1.55)%(P<0.01)at the point of time 12 h and increased to(64.64±0.30)% from(56.04±1.81)%(P<0.01)at the point of time 24 h after modification.Before and after modification,QKRKRKKSRKKH peptide's inhibition ratio on A549 cell proliferation was(28.38±5.63)% and(34.25±10.90)%(P>0.05)and its inhibition ratio on cell vitality was(8.09±5.84)% and(15.07±8.09)%(P>0.05)in 10% serum;after modification,the inhibition ratio on A549 cell migration was improved to(66.78±4.43)% from(28.27±3.79)%(P<0.01)in 10% serum.HPLC showed that the remaining percentage of peptide in serum was increased to(82.46±0.84)% from(80.14±1.81)%(P<0.05)at the point of time 12 h and increased to(77.43%±1.78)% from(72.72±2.60)%(P<0.05)at the point of time 24 h after modification2.Prokaryotic expression vectors pGEX4T-1-peptides were constructed,GST-peptides fusion proteins were expressed and purified successfully,Co-IP confirmed that the two subtypes of VEGFR(VEGFR-1 and VEGFR-2)were both the function sites of the two peptide probes(QKRKRKKSRKKH and RKRKRKKSRYIVLS).3.Western Blot results showed that peptides(QKRKRKKSRKKH and RKRKRKK SRYIVLS)did not have any obvious influence on the phosphorylation level of VEGFR-1 and VEGFR-2 in A549 cells.4.SiRNA technology silenced VEGF expression successfully,CCK-8 experiment confirmed that both the two peptides could inhibit the proliferation of A549 cells without endogenous and exogenous VEGF.5.Theoretical potential receptors of peptides predicted by bioinformatics were including EGFR,Tie-2R,HGFR,VIPR-2,PLT-3,?v?3;competitive receptor binding assay showed that the peptides could not combine with EGFR.Co-IP showed that the peptides could not combine VIPR-2,but could be combined with Tie-2R,HGFR.Conclusions1.The stability of peptides is improved obviously after protective chemical modification in 10% serum environment,and RKRKRKKSRYIVLS are more prominent.2.GST-peptides fusion proteins are expressed and purified successfully,Co-IP confirms that both the two subtypes of VEGFR,namelyVEGFR-1 and VEGFR-2,are the targets of the two peptides.3.The proliferation inhibition effects of peptides on A549 cells may not act through the way which influence the phosphorylation level of VEGFR-1 and VEGFR-2.The two peptides(QKRKRKKSRKKH and RKRKRKKSRYIVLS)have several targets,together with VEGFR-1 and VEGFR-2,HGFR and TIE-2R are also the targets of them. |
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