The Optimization of A Novel Human-derived Cell-penetrating Peptide Used for Anti-cancer Treatment

doi:10.13523/j.cb.20180706

China Biotechnology

2018, Vol. 38

Issue (7): 40-49 DOI: 10.13523/j.cb.20180706

The Optimization of A Novel Human-derived Cell-penetrating Peptide Used for Anti-cancer Treatment

Si LI¹,Yi-zhou ZHAI¹,Yu-ting LU²,Fu-jun WANG^2,³,Jian ZHAO^1,^**(

)

1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, Chain
2 Zhejiang Reachall Pharmaceutical Co. Ltd, Dongyang 322100,Chain
3 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203,Chain

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Abstract

Cell-penetrating peptides (CPPs) have been widely used in decades for its ability to carry many macromolecular drugs across cell-membrane to exert their effects. Midkine (MK) is a heparin-binding growth factor with a heparin-binding domain (HBD). The HBD in MK that is rich in basic amino acids (MK-S0) was fused with enhanced green fluorescence protein (EGFP) and then it was found that MK-S0 could deliver EGFP into cells, and its transportation capacity is much higher that classical CPPs (such as Tat). After the sequence optimization on MK-S0, MK-Δ4 whose trans-membrane ability was increased about 16-fold than MK-S0 was obtained. The trans-membrane ability of MK-Δ4 was also suitable for a variety of tumor cells. The further investigation of endocytic pathways on MK-Δ4 was shown that MK-Δ4 penetrates cell-membrane through interacting with heparin sulfate on the cell surface and then via macropinocytosis. The results of cell growth inhibition by MTT method showed that MK-Δ4 could enhance the inhibitory effect of a ribosome-inactivating protein-MAP30 about 5.8-fold in HeLa cells which is significantly enhance the anti-tumor activity of MAP30. It was suggested that MK-Δ4 optimized from heparin-binding domain MK is a novel human-derived CPP with high efficiency, and is a new drug vector for anti-tumor therapy.

Key words： Midkine Heparin-binding domain Cell-penetrating peptide Drug delivery Anti-tumor drug

Received: 20 March 2018 Published: 13 August 2018

ZTFLH:

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Corresponding Authors: Jian ZHAO E-mail: zhaojian@ecust.edu.cn

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Cite this article:

Si LI,Yi-zhou ZHAI,Yu-ting LU,Fu-jun WANG,Jian ZHAO. The Optimization of A Novel Human-derived Cell-penetrating Peptide Used for Anti-cancer Treatment. China Biotechnology, 2018, 38(7): 40-49.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180706 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I7/40

Fig.1 SDS-PAGE analysis of fusion protein EGFP-MK-S0 M:Low molecule weight protein marker; 1:Uninduced E.coli (DE3); 2:Induced E.coli (DE3) (EGFP-MK-S0);3:The supernatane of induced E.coli (DE3) (EGFP-MK-S0); 4:The inclusion body of induced E.coli (DE3) (EGFP-MK-S0); 5: Elution; 6: Elution of 20mmol/L imidazole; 7: Elution of 200mmol/L imidazole; 8: Purfied EGFP-MK-S0 for two times dilution; 9:Purfied EGFP-MK-S0

Fig.2 Fluorescence microscopy and flow cytometry among EGFP-MK-S0 and other three CPPs Time-response (a) and concentration-response (b) of fusion protein EGFP-MK-S0; The transporting efficiency was analyzed by FCM among different CPPs (c,d)

Fig.3 Penetrating ability of MK-S0 mutants (a)Fluorescence detection of EGFP-MK-S0 and EGFP-MK-ΔS0 (b)Structural modeling of MK-S0 (c)Fluorescence detection of mutants (d)Flow cytometry of mutants (e),(f)Fluorescence microscopy detection of MK-Δ4

Table 1 Mutant sequence of MK-S0

Fig.4 SDS-PAGE analysis of fusion protein EGFP-MK-Δ4 M:Low molecule weight protein marker; 1:Uninduced E.coli (DE3); 2:Induced E.coli (DE3) (EGFP-MK-Δ4); 3:The supernatane of induced E.coli (DE3) (EGFP-MK-Δ4); 4:The inclusion body of induced E.coli (DE3) (EGFP-MK-Δ4); 5: Elution; 6:Elution of 20mmol/L imidazole; 7:Elution of 200mmol/L imidazole; 8:Elution of 500mmol/L imidazole; 9: Elution of 1mol/L imidazole; 10: Purified EGFP-MK-Δ4; 11:Purified EGFP-MK-S4

Fig.5 Penetrating ability of EGFP-MK-Δ4 into cells of variable origin

Fig.6 Effect of endocyticc inhibitors on penetrating ability of EGFP-MK-Δ4

Fig.7 Growth effect of EGFP,EGFP-MK-Δ4 to HeLa cells

Fig.8 SDS-PAGE analysis of fusion protein MAP30-MK-Δ4 M:Low molecule weight protein marker; 1: Uninduced E.coli (DE3); 2:Induced E.coli (DE3) (MAP30-MK-Δ4); 3:The supernatane of induced E.coli (DE3) (MAP30-MK-Δ4); 4:The inclusion body of induced E.coli (DE3) (MAP30-MK-Δ4); 5: Elution; 6: Elution of 20mmol/L imidazole; 7: Elution of 200mmol/L imidazole; 8: Elution of 500mmol/L imidazole; 9: Elution of 1mol/L imidazole; 10:Purfied MAP30-MK-Δ4 ; 11:Purfied MAP30-MK-Δ4

Fig.9 Effects of fusion protein on tumor cell viability (a)The cytotoxicity of MAP30 and MAP30-MK-Δ4 to HeLa (b)The cytotoxicity of MAP30 and MAP30-MK-Δ4 to SMMC (c)The cytotoxicity of MAP30 and MAP30-MK-Δ4 to MGC803

Table 2 Half maximal inhibitory concentration IC₅₀ of fusion protein to tumor cells


[1]	Chin L, Andersen J N, Futreal P A . Cancer genomics: from discovery science to personalized medicine. Nature Medicine, 2011,17(3):297-303. doi: 10.1038/nm.2323 pmid: 21383744

[2]	Koren E, Torchilin V P . Cell-penetrating peptides: breaking through to the other side. Trends in Molecular Medicine, 2012,18(7):385-393. doi: 10.1016/j.molmed.2012.04.012 pmid: 22682515

[3]	Vivès E, Brodin P, Lebleu B . A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. Journal of Biological Chemistry, 1997,272(25):16010-16017. doi: 10.1074/jbc.272.25.16010

[4]	Morris M C, Gros E, Aldrianherrada G , et al. A non-covalent peptide-based carrier for in vivo delivery of DNA mimics. Nucleic Acids Research, 2007,35(7):e49. doi: 10.1093/nar/gkm053 pmid: 1874649

[5]	Choi Y J, Lee J Y, Park J H , et al. The identification of a heparin binding domain peptide from bone morphogenetic protein-4 and its role on osteogenesis. Biomaterials, 2010,31(28):7226-7238. doi: 10.1016/j.biomaterials.2010.05.022

[6]	Eguchi A, Dowdy S F . siRNA delivery using peptide transduction domains. Trends in Pharmacological Sciences, 2009,30(7):341-345. doi: 10.1016/j.tips.2009.04.009 pmid: 19545914

[7]	Meneghetti M C, Hughes A J, Rudd T R , et al. Heparan sulfate and heparin interactions with proteins. Journal of the Royal Society Interface, 2015,12(110):589. doi: 10.1098/rsif.2015.0589 pmid: 26289657

[8]	Dr I C, Prof R J L . Heparin-protein interactions. Angewandte Chemie International Edition, 2002,41(3):391-412.

[9]	Raman R, Raguram S, Venkataraman G J , et al. Glycomics: an integrated systems approach to structure-function relationships of glycans. Nature Methods, 2005,2(11):817-824. doi: 10.1038/nmeth807 pmid: 16278650

[10]	Mu?oz E M, Linhardt R J . Heparin-binding domains in vascular biology. Arteriosclerosis Thrombosis & Vascular Biology, 2004,24(9):1549-1557. doi: 10.1161/01.ATV.0000137189.22999.3f pmid: 4114236

[11]	Gallagher J T . Heparan sulfate: growth control with a restricted sequence menu. Journal of Clinical Investigation, 2001,108(3):357-361. doi: 10.1172/JCI13713 pmid: 11489926

[12]	Forsten-Williams K, Chu C L, Fannon M , et al. Control of growth factor networks by heparan sulfate Pproteoglycans. Annals of Biomedical Engineering, 2008,36(12):2134-2148. doi: 10.1007/s10439-008-9575-z pmid: 18839312

[13]	Zhao J, Gao P, Xiao W , et al. A novel human derived cell-penetrating peptide in drug delivery. Molecular Biology Reports, 2011,38(4):2649-2656. doi: 10.1007/s11033-010-0406-6 pmid: 21080077

[14]	Xi G, Solum M A, Wai C , et al. The heparin-binding domains of IGFBP-2 mediate its inhibitory effect on preadipocyte differentiation and fat development in male mice. Endocrinology, 2013,154(11):4146-4157. doi: 10.1210/en.2013-1236

[15]	Lee J Y, Seo Y N, Park H J , et al. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo. Biochemical & Biophysical Research Communications, 2012,419(4):597-604.

[16]	Matsuzawa M, Muramatsu T, Yamamori T , et al. Novel neuronal effects of midkine on embryonic cerebellar neurons examined using a defined culture system. Cellular & Molecular Neurobiology, 1999,19(2):209-221.

[17]	Muramatsu T . Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proceedings of the Japan Academy, 2010,86(4):410-425. doi: 10.2183/pjab.86.410 pmid: 3417803

[18]	Kadomatsu K, Tomomura M, Muramatsu T . cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun, 1988,151(3):1312-1318. doi: 10.1016/S0006-291X(88)80505-9

[19]	Kilpelainen I, Kaksonen M, Kinnunen T , et al. Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat. Journal of Biological Chemistry, 2000,275(18):13564-13570. doi: 10.1074/jbc.275.18.13564

[20]	Zhang R, Yang X Z, Wang J W , et al. Evaluating the translocation properties of a new nuclear targeted penetrating peptide using two fluorescent markers. Journal of Drug Targeting, 2015,23(5):444-452. doi: 10.3109/1061186X.2014.1003068

[21]	Hansen M, Kilk K, Langel ü . Predicting cell-penetrating peptides. ScienceDirect, 2008,60(4-5):549-550.

[22]	Goun E A, Pillow T H, Jones L R , et al. Molecular transporters: synthesis of oligoguanidinium transporters and their application to drug delivery and real-time imaging. Chembiochem, 2006,37(52):1497-1515.

[23]	Richard J P, Melikov K, Brooks H , et al. Cellular uptake of unconjugated Tat peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. Journal of Biological Chemistry, 2005,280(15):15300-15306. doi: 10.1074/jbc.M401604200

[24]	Zhang L X, Zhang S X . Mechanism of cell-penetrating peptides-mediated internalization and its application. Chinese Journal of Biochemistry & Molecular Biology, 2008,24(12):1092-1096. doi: 10.1016/S1004-9541(08)60026-9

[25]	Skotland T, Iversen T G, Torgersen M L , et al. Cell-penetrating peptides: possibilities and challenges for drug delivery in vitro and in vivo. Molecules, 2015,20(7):13313-13323. doi: 10.3390/molecules200713313

[26]	Lu Y Z, Li P F, Li Y Z , et al. Enhanced anti-tumor activity of trichosanthin after combination with a human-derived cell-penetrating peptide, and a possible mechanism of activity. Fitoterapia, 2016,112:183-190. doi: 10.1016/j.fitote.2016.03.019

[27]	Lin B, Yang X Z, Cao X W , et al. A novel trichosanthin fusion protein with increased cytotoxicity to tumor cells. Biotechnology Letters, 2017,39(1):71-78. doi: 10.1007/s10529-016-2222-0 pmid: 27714558

[28]	Guidotti G, Brambilla L, Rossi D . Cell-penetrating peptides: from basic research to clinics. Trends in Pharmacological Sciences, 2017,38(4):406-424. doi: 10.1016/j.tips.2017.01.003 pmid: 28209404

[29]	Jobin M L, Alves I D . On the importance of electrosTatic interactions between cell penetrating peptides and membranes: a pathway toward tumor cell selectivity. Biochimie, 2014,107:154-159. doi: 10.1016/j.biochi.2014.07.022

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