Screening And Characterization Of A Novel High-efficiency Tumor-targeting Cell-penetrating Peptide From The Buffalo Antimicrobial Peptides

Targeted delivery of anticancer drugs can reduce toxic side effects and is especially important for the treatment of cancer patients.Cell-penetrating peptids(CPPs)are short peptides consisting of 5-30 amino acids that are rich in basic amino acids.CPPs can transport wide range of different bioactive macromolecules into living cells.Currently,CPPs have become effective drug delivery vehicles.However,most CPPs lack cell specificity,such as TAT,a classical CPP derived from HIV virus,which limits their clinical application as drug delivery vehicles.In many organisms,there are numerous naturally occurring small peptides called antimicrobial peptides(AMPs)that have bactericidal activity against a variety of microorganisms.AMPs have similar physicochemical properties as CPPs,such as amphipathicity,hydrophobicity,isoelectric point and cationicity.The overlapping structural features of AMPs and CPPs make naturally occurring AMPs an important source for the identification of novel CPPs.Furthermore,it has been reported that some AMPs can selectively penetrate tumor cells.Buffalo CATHL4(buCATHL4)is a recently discovered family of buffalo-derived AMPs that are rich in arginine and aromatic amino acid residues,and their antibacterial mechanism is mainly dependent on membrane disruption,leading to membrane instability.There is currently no research reporting whether such AMPs have CPP-like functions.In this study,several buCATHL4 members were randomly selected as candidates for CPP screening among 12 newly identified buCATHL4s.These AMP sequences were fused to the enhanced green fluorescent protein(EGFP)gene and expressed in Escherichia coli BL21(DE3)to investigate their cell-penetrating activity by fluorescence microscopy and flow cytometry analysis.The results showed that all these AMPs had cell-penetrating activity on HeLa cells,and an AMP with the highest cell-penetrating activity(named CAT)was screened,which cellpenetrating activity was 2-8 times higher than that of the other AMPs.CAT showed an approximately 3-6-fold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides,including the well-known classical CPP TAT.Moreover,the cell penetrating ability of CAT in various tumor cell lines(HeLa,SMMC7721,A549,H460)and normal cell lines(LO-2,HEK293,293T,MRC-5)was further observed.The delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells,especially for the human hepatoma cell line SMMC-7721,for which delivery was 7.37times more efficient than the normal human embryonic lung cell line MRC-5,according to fluorescent labeling experiment results.MAP30 is a Momordica charantia-derived type-I ribosome-inactivating protein with antitumor properties.To further evaluate whether CAT can be used as an excellent vector for targeted delivery of antitumor drugs to tumor cells,MAP30 gene was fused with CAT and expressed in an E.coli prokaryotic expression system.The cytotoxicity of the MAP3 0-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared to that of the unconjugated MAP30.The IC50 value of MAP30-CAT was approximately 83 times lower than the IC50 value of the original MAP30.Interestingly,the IC50 value of MAP30 alone for MRC-5 was approximately 2-fold higher than the value for SMMC-7721,showing a small difference.However,when MAP30 was conjugated to CAT,the difference in IC50 values between the two cell lines was significantly increased by 3 8-fold.The same results were observed in HeLa and 293T cells.These results indicated that the introduction of CAT greatly increased the ability of MAP30 to target tumor cells rather than normal cells.The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein.These results suggest that CAT,as a novel tumor-targeting CPP,has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.