| Ribosome inactivating proteins(RIPs)can inactivate ribosomes through N-glycosidase activity,thereby exerting antitumor effect by inhibiting protein synthesis.Gelonin is a protein derived from the Euphorbiaceae plant Gelonium multiflorum with a molecular weight of 29 k D.It belongs to type I RIPs and has N-glycosidase activity,which can cleave 28 S r RNA to inactivate ribosomes and cause cell death,and is a highly effective antitumor protein.However,Gelonin lacks the cell-binding domain and is difficult to internalize into tumor cells,which extremely limits its application.To solve this problem,based on the characteristics of acidic microenvironment and overexpression of MMP-2/9 in tumors,a functional protein of plant toxin gelonin Trx-PVGLIG-pHLIP-Gelonin(TPpG)was designed in this study.Trx can improve the water solubility of the fusion protein;PVGLIG peptide can be specifically cleaved by MMP-2/9(highly expressed in various tumor tissues)to expose pHLIP;pHLIP can respond to the weakly acidic tumor microenvironment and transport gelonin to tumor cells;and gelonin can exert potent antitumor activity.In this study,TPpG protein was successfully obtained by prokaryotic expression,and its cellular uptake and in vitro antitumor effect were investigated.This paper contains the following three parts:(1)We successfully constructed the recombinant plasmid p ET32a-PVGLIG-pHLIPGelonin through full-plasmid PCR,the fusion protein TPpG was obtained successfully by prokaryotic expression system and purified by nickel affinity chromatography coloum.TPpG was characterized by SDS-PAGE,Western Blot and MALDI-TOF mass spectrometry,which proved that TPpG was successfully expressed.The secondary structure and thermal stability of TPpG protein were determined by circular dichroism,and the serum stability of TPpG protein was also determined.The results showed that TPpG exhibited good thermal stability and excellent pH and serum stability.(2)In order to study the cellular internalization behavior of functional proteins,TPpG and Gelonin were labeled with FITC.The UV-visible spectrophotometer confirmed that we successfully obtained FITC-TPpG and FITC-Gelonin.The cellular internalization behavior of TPpG was determined by confocal laser scanning microscopy,flow cytometry and Western Blot.These results indicated that TPpG could enter HT1080 cells with high MMP-2 expression more significantly under weak acidic conditions than MCF-7 cells with low MMP-2 expression.(3)MCF-7 cells with low expression of MMP-2 and HT1080 cells with high expression of MMP-2 were utilized to investigate the in vitro antitumor activity of TPpG.Firstly,the inhibitory effect of TPpG on tumor cell proliferation was monitored by MTT,SRB,crystal violet staining and CFDA-SE staining.Then the apoptosis induction effect of TPpG on tumor cells is determined by Annexin V-FITC/PI double staining and Caspase-3activity detection kit.Finally,the total protein level in tumor cells was measured by BCA assay.These results demonstrated that TPpG could effectively inhibit the proliferation of tumor cells,significantly induce tumor cell apoptosis,markedly elevated the relative activity of Caspase-3 and obviously inhibited intracellular protein synthesis under weak acidic conditions.The above-mentioned phenomenon is much more evident in HT1080 cells with high expression of MMP-2.In summary,we firstly designed and prokaryotically expressed a plant toxin Gelonin functional protein TPpG with MMP and tumor acidity responsive property,and systematically investigated its in vitro antitumor activity.TPpG could mediate the effective internalization of Gelonin into cells in a MMP and pH dual responsive manner,inhibit tumor cell proliferation,induce cell apoptosis and suppress intracellular protein synthesis,thereby efficiently killing cancer cells.This study can lay a new theoretical foundation for the biological application of Gelonin,and provide a general and efficient strategy for the targeted delivery of macromolecular agents such as proteins. |
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