Screening of a Gelonin Fusion Protein with High Cell-penetrating Efficiency and Its Anti-tumor Activity and Apoptosis Pathway

doi:10.13560/j.cnki.biotech.bull.1985.2017-1119

Abstract

Abstract: By fusing gelonin with a variety of cell penetrating peptides（CPP），a recombinant protein gelonin-HBP with the highest anti-tumor activity was obtained and its molecular mechanism of inhibiting tumor cell growth was also studied. The recombinant protein of gelonin fused with four different CPPs（R9，TAT，HBD，and HBP）was expressed in Escherichia coli BL21（DE3）and then purified by NI-NTA affinity chromatography. Superhelical cleavage experiment and in vitro translation inhibition assay were used to detect the bioactivity of gelonin fusion protein. The translocation efficiency of gelonin recombinant protein was observed by laser scanning confocal microscopy. The cell growth inhibition effect of gelonin fusion protein on tumor cells was detected by MTT assay and the molecular mechanism of cell apoptosis was observed by flow cytometry and Western blot. The results showed that four gelonin recombinant proteins that retained the original biological activity of gelonin were obtained，and the fusion with a CPP promoted the cell penetration of gelonin，improved the inhibition effect of gelonin on tumor cells，and accelerated the apoptosis of these tumor cells. Compared to gelonin alone，the gelonin-HBP fusion protein increased its potency in 4 tumor cells of HeLa，B16，MCF-7 and HepG2 by 16.4 times，9.6 times，14.0 times and 24.6 times，respectively. Western blotting analysis demonstrated that gelonin-HBP induced an enhancement in the activity of Caspas 3，Caspas 8 and Caspas 9 in tumor cells，thus the expression of Bax significantly increased，while the expression of Bcl-2 significantly decreased. Conclusively，CPP may enhance the anti-tumor effect and the apoptosis of gelonin on tumor cells；among them，HBP，a CPP derived from human heparin-binding epidermal growth factor-like growth factor，makes the gelonin-HBP have the strongest anti-tumor effect. The mechanism of inducing tumor cell apoptosis by this recombinant protein is related to mitochondrial and death receptor-mediated apoptosis signaling pathway.

Key words: gelonin, CPPs, anti-tumor, cell apoptosis

ZHAI Yi-zhou ,LU Mei-ya ,ZHAO Jian ,WANG Fu-jun. Screening of a Gelonin Fusion Protein with High Cell-penetrating Efficiency and Its Anti-tumor Activity and Apoptosis Pathway[J]. Biotechnology Bulletin, 2018, 34(6): 204-212.

References

[1] Lu YZ, Li PF, Li YZ, et al. Enhanced anti-tumor activity of trichosanthin after combination with a human-derived cell-penetrating peptide, and a possible mechanism of activity[J] . Fitoterapia, 2016, 112：183-190.
[2] Puri M, Kaur I, Kanwar RK, et al. Ribosome inactivating proteins（RIPs）from Momordica charantia for anti viral therapy[J] . Current Molecular Medicine, 2009, 9（9）：1080-1094.
[3] Antignani A, Fitzgerald D. Immunotoxins：The role of the toxin[J] . Toxins, 2013, 5（8）：1486-1502.
[4] Puri M, Kaur I, Perugini MA, et al. Ribosome-inactivating proteins：current status and biomedical applications[J] . Drug Discovery Today, 2012, 17（13-14）：774-783.
[5] Stirpe F, Olsnes S, Pihl A. Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A[J] . Journal of Biological Chemistry, 1980, 255（14）：6947-6953.
[6] Nimmanapalli R, Lyu MA, Du M, et al. The growth factor fusion construct containing B-lymphocyte stimulator（BLyS）and the toxin rGel induces apoptosis specifically in BAFF-R-positive CLL cells[J] . Blood, 2007, 109（6）：2557-2564.
[7] Atkinson SF, Bettinger T, Seymour LW, et al. Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells[J] . Journal of Biological Chemistry, 2001, 276（30）：27930-27935.
[8] Shin MC, Zhang J, David AE, et al. Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity[J] . Journal of Controlled Release, 2013, 172（1）：169-178.
[9] Snyder EL, Dowdy SF. Cell penetrating peptides in drug delivery [J] . Pharmaceutical Research, 2004, 21（3）：389-393.
[10] J?rver P, Langel U. The use of cell-penetrating peptides as a tool for gene regulation[J] . Drug Discovery Today, 2004, 9（9）：395-402.
[11] Koren E, Torchilin VP. Cell-penetrating peptides：breaking through to the other side[J] . Trends in Molecular Medicine, 2012, 18（7）：385-393.
[12] Cheung CHA, Sun X, Kanwar JR, et al. A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy[J] . Cancer Cell International, 2010, 10（1）：36-47.
[13] Luo Z, Cao XW, Li C, et al. The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery[J] . Journal of Peptide Science, 2016, 22（11-12）：689-699.
[14] Lv Q, Yang XZ, Fu LY, et al. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity[J] . Protein Expression and Purification, 2015, 111：9-17.
[15] Creagh EM. Caspase crosstalk：integration of apoptotic and innate immune signalling pathways[J] . Trends in Immunology, 2014, 35（12）：631-640.
[16] Danial NN, Korsmeyer S J. Cell death：critical control points[J] . Cell, 2004, 116（2）：205-219.
[17] Green DR, Guido K. The pathophysiology of mitochondrial cell death[J] . Science, 2004, 305（5684）：626-629.