Tumor Biology

, Volume 37, Issue 8, pp 10643–10652 | Cite as

iRGD-targeted delivery of a pro-apoptotic peptide activated by cathepsin B inhibits tumor growth and metastasis in mice

  • Wang Qifan
  • Ning Fen
  • Xue Ying
  • Feng Xinwei
  • Du Jun
  • Zhang Ge
Original Article


The use of cytolytic peptides with potential therapeutic properties is a promising approach to cancer therapy due to their convenient automated synthesis and their capacity for modifications. However, the use of cytolytic peptides is limited due to their nonspecific cytolytic activity. In this study, we designed a tumor-targeting proapoptotic system based on an amphipathic D-amino acid-modified apoptotic peptide, KLA, a variant of (KLAKLAK)2, which is fused with a linear tumor-penetrating homing peptide iRGD through specific cathepsin B (CTSB) cleavage sequences that are overexpressed in many types of tumor tissues. Our data show that the procytotoxic peptide D(KLAKLAKKLAKLA)K-GG-iRGD (m(KLA)-iRGD) is internalized into cultured tumor cells through a neuropilin-1 (NRP1)-activated pathway by iRGD delivery. Once inside the cells, the peptide triggers rapid apoptosis through both the mitochondrial-induced apoptotic pathway and the death receptor pathway in NRP1+/αvβ3/CTSB+ tumor cells. Furthermore, m(KLA)-iRGD spread extensively within the tumor tissue when it was injected into 4T1 tumor-bearing mice. The m(KLA)-iRGD peptide inhibited tumor growth to a certain degree, resulting in a significant reduction in tumor volume (P < 0.05) and the total inhibition of metastasis at the end of the treatment. These results suggest that m(KLA)-iRGD has the potential for development as a new antitumor drug.


iRGD Cathepsin B Tumor growth Metastasis 



This work was supported by the National Natural Science Foundation of China (No. 81072670) and the Fundamental Research Funds for the Central Universities.

Compliance with ethical standards

Ethics statement

All of the animal procedures and experiments were approved by the Institutional Ethical Committee for Animal Research at Sun Yat-sen University. Twelve female BABL/c mice were used in the study.

Conflicts of interest


Supplementary material

13277_2016_4961_MOESM1_ESM.tif (349 kb)
Suppl. Fig 1 The chemical structure of m(KLA)-iRGD. The chemical structure of m(KLA)-iRGD (a) and control peptide D(KLA)-iRGD (b). m(KLA)-iRGD consists of a membrane-disrupting domain, a tumor-homing domain and a GG linker. The yellow domain represents the L-enantiomeric domain, and the blue domain represents the D-enantiomeric domain. The C-terminus of the yellow Lys is the cleavage site of CTSB. (TIF 348 kb)
13277_2016_4961_Fig7_ESM.gif (37 kb)

(GIF 37 kb)

13277_2016_4961_MOESM2_ESM.tif (5.4 mb)
Suppl. Fig 2 MS and HPLC analysis of m(KLA)-iRGD. a The molecular weight of m(KLA)- iRGD is 2570 Da. b The purity of m(KLA)-iRGD is approximately 95 %. (TIF 5573 kb)
13277_2016_4961_Fig8_ESM.gif (51 kb)

(GIF 50 kb)

13277_2016_4961_MOESM3_ESM.tif (504 kb)
Suppl. Fig 3 Expression levels of CTSB and NRP1 in different tumor cells. a The relative level of CTSB mRNA to GAPDH in different tumor cells measured by RT-PCR. CTSB mRNA shows higher expression in most tumor cell lines, except for SKBR3. b The expression levels of CTSB, NRP1, αv, β3 in MDA-MB-231, 4 T1, SKBR3 and B16 cell lines measured by Western blotting. (TIF 503 kb)
13277_2016_4961_Fig9_ESM.gif (60 kb)

(GIF 60 kb)

13277_2016_4961_MOESM4_ESM.tif (2 mb)
Suppl. Fig 4 Tumor tissues of the m(KLA)-iRGD group and the PBS group were removed and measured at the end of the experiments. a and b Tumor volumes of the m(KLA)-iRGD-treated mice were much smaller than those of the PBS control. (TIF 2084 kb)
13277_2016_4961_Fig10_ESM.gif (97 kb)

(GIF 97 kb)


  1. 1.
    Hait WN. Targeted cancer therapeutics. Cancer Res. 2009;69:1263–7.CrossRefPubMedGoogle Scholar
  2. 2.
    Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer. 2006;6:583–92.CrossRefPubMedGoogle Scholar
  3. 3.
    Svensen N, Walton JG, Bradley M. Peptides for cell-selective drug delivery. Trends Pharmacol Sci. 2012;33:186–92.CrossRefPubMedGoogle Scholar
  4. 4.
    Chen R, Braun GB, Luo X, Sugahara KN, Teesalu T, Ruoslahti E. Application of a proapoptotic peptide to intratumorally spreading cancer therapy. Cancer Res. 2013;73:1352–61.CrossRefPubMedGoogle Scholar
  5. 5.
    Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Del Rio G, et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med. 1999;5:1032–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Cieslewicz M, Tang J, Jonathan LY, Cao H, Zavaljevski M, Motoyama K, et al. Targeted delivery of proapoptotic peptides to tumor-associated macrophages improves survival. Proc Natl Acad Sci. 2013;110:15919–24.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Smolarczyk R, Cichoń T, Graja K, Hucz J, Sochanik A, Szala S. Antitumor effect of RGD-4C-GG-D (KLAKLAK) 2 peptide in mouse B16 (F10) melanoma model. Acta Biochim Pol. 2005;53:801–5.Google Scholar
  8. 8.
    Lemeshko VV. Potential-dependent membrane permeabilization and mitochondrial aggregation caused by anticancer polyarginine-KLA peptides. Arch Biochem Biophys. 2010;493:213–20.CrossRefPubMedGoogle Scholar
  9. 9.
    Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, Greenwald DR, et al. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science. 2010;328:1031–5.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Sugahara KN, Braun GB, de Mendoza TH, Kotamraju VR, French RP, Lowy AM, et al. Tumor-penetrating iRGD peptide inhibits metastasis. Mol Cancer Ther. 2015;14:120–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Ruoslahti E, Bhatia SN, Sailor MJ. Targeting of drugs and nanoparticles to tumors. J Cell Biol. 2010;188:759–68.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lao X, Liu M, Chen J, Zheng H. A tumor-penetrating peptide modification enhances the antitumor activity of thymosin alpha 1. PLoS One. 2013;8(8):e72242.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Yang X-Z, Du X, Wang J-W, Zhang R, Zhao J, Wang F-J, et al. Enhancing tumor-specific intracellular delivering efficiency of cell-penetrating peptide by fusion with a peptide targeting to EGFR. Amino Acids. 2015;47:997–1006.CrossRefPubMedGoogle Scholar
  14. 14.
    D’Onofrio N, Caraglia M, Grimaldi A, Marfella R, Servillo L, Paolisso G, et al. Vascular-homing peptides for targeted drug delivery and molecular imaging: meeting the clinical challenges. Biochim Biophys Acta Rev Cancer. 2014;1846:1–12.CrossRefGoogle Scholar
  15. 15.
    Beer AJ, Schwaiger M. Imaging of integrin αvβ3 expression. Cancer Metastasis Rev. 2008;27:631–44.CrossRefPubMedGoogle Scholar
  16. 16.
    Jubb AM, Strickland LA, Liu SD, Mak J, Schmidt M, Koeppen H. Neuropilin-1 expression in cancer and development. J Pathol. 2012;226:50–60.CrossRefPubMedGoogle Scholar
  17. 17.
    Dubowchik GM, Firestone RA, Padilla L, Willner D, Hofstead SJ, Mosure K, et al. Cathepsin b-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: Model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug Chem. 2002;13:855–69.CrossRefPubMedGoogle Scholar
  18. 18.
    Sinha AA, Morgan JL, Betre K, Wilson MJ, Le C, Marks LS. Cathepsin B expression in prostate cancer of native Japanese and Japanese-American patients: an immunohistochemical study. Anticancer Res. 2008;28:2271–7.PubMedGoogle Scholar
  19. 19.
    Chan AT, Baba Y, Shima K, Nosho K, Chung DC, Hung KE, et al. Cathepsin B expression and survival in colon cancer: implications for molecular detection of neoplasia. Cancer Epidemiol Biomark Prev. 2010;19:2777–85.CrossRefGoogle Scholar
  20. 20.
    Gong F, Peng X, Luo C, Shen G, Zhao C, Zou L, et al. Cathepsin b as a potential prognostic and therapeutic marker for human lung squamous cell carcinoma. Mol Cancer. 2013;12:10.1186.Google Scholar
  21. 21.
    Blondelle SE, Houghten RA. Design of model amphipathic peptides having potent antimicrobial activities. Biochemistry. 1992;31:12688–94.CrossRefPubMedGoogle Scholar
  22. 22.
    Dufort S, Sancey L, Hurbin A, Foillard S, Boturyn D, Dumy P, et al. Targeted delivery of a proapoptotic peptide to tumors in vivo. J Drug Target. 2011;19:582–8.CrossRefPubMedGoogle Scholar
  23. 23.
    Karjalainen K, Jaalouk DE, Bueso-Ramos CE, Zurita AJ, Kuniyasu A, Eckhardt BL, et al. Targeting neuropilin-1 in human leukemia and lymphoma. Blood. 2011;117:920–7.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Alves ID, Carré M, Montero MP, Castano S, Lecomte S, Marquant R, et al. A proapoptotic peptide conjugated to penetratin selectively inhibits tumor cell growth. Biochim Biophys Acta. 1838;2014:2087–98.Google Scholar
  25. 25.
    Kim HY, Kim S, Youn H, Chung JK, Dong HS, Lee K. The cell penetrating ability of the proapoptotic peptide, KLAKLAKKLAKLAK fused to the N-terminal protein transduction domain of translationally controlled tumor protein, MIIYRDLISH. Biomaterials. 2011;32:5262–8.CrossRefPubMedGoogle Scholar
  26. 26.
    Lemeshko VV. Electrical potentiation of the membrane permeabilization by new peptides with anticancer properties. Biochim Biophys Acta Biomembr. 2013;1828:1047–56.CrossRefGoogle Scholar
  27. 27.
    Dubowchik GM, Mosure K, Knipe JO, Firestone RA. Cathepsin B-sensitive dipeptide prodrugs. 2. Models of anticancer drugs paclitaxel (Taxol®), mitomycin C and doxorubicin. Bioorg Med Chem Lett. 1998;8:3347–52.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhong Y-J, Shao L-H, Li Y. Cathepsin B-cleavable doxorubicin prodrugs for targeted cancer therapy (review). Int J Oncol. 2013;42:373–83.PubMedGoogle Scholar
  29. 29.
    Bröker LE, Kruyt FA, Giaccone G. Cell death independent of caspases: a review. Clin Cancer Res. 2005;11:3155–62.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Wang Qifan
    • 1
  • Ning Fen
    • 2
  • Xue Ying
    • 1
  • Feng Xinwei
    • 1
  • Du Jun
    • 1
  • Zhang Ge
    • 1
  1. 1.Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical SciencesSun Yat-sen UniversityGuangzhouChina
  2. 2.Guangzhou Institute of Pediatrics, Department of ObstetricsGuangzhou Women and Children’s Medical CenterGuangzhouChina

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