Development of stomach cancer-targeted and cytotoxic hybrid peptides

SUMMARY Development of targeting drug to reduce side effect of cancer chemotherapy is important for improving patient compliance and convenience. Previously we screened stomach cancer cell binding peptide (named gastric tumor-binding peptide-1, GT1) by phage display. We prepared hybrid peptides that contain GT1 and apoptosis inducing peptide (Bak-BH3). This hybrid peptide showed specific apoptosisinducing effect on stomach cancer cell lines. Also, we designed the neutravidin nanocarriers containing biotin-labeled GT1, Bak-BH3, and endosome escape peptide. It showed more efficient apoptosis-inducing effect than free form of GT1-Bak-BH3 hybrid peptide. These results suggest that GT1-mediated delivery of cytotoxic peptide is a promising tool for targeting stomach cancer. INTRODUCTION One of the major problems in cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their non-selectivity for tumor versus normal cells [1]. To increase the therapeutic index, various drug delivery systems are being developed [2]. With phage display technology, we identified a stomach cancer binding peptide and named it gastric tumor-binding peptide-1 (GT1). In order to develop stomach-cancer targeted therapeutics, we synthesized a hybrid peptide that contains GT1, a stomach-cancer targeting moiety, and Bak-BH3, an apoptosis-inducing peptide, has been developed. Furthermore, we designed a cassette type of nanocarrierdisplayed hybrid peptide, which is composed of neutravidin (NA) complexed with biotin-labeled GT1, Bak-BH3, and HGP, an endosomal escape peptide from HIV gp41 (NA:biotin-peptides = 1:4). In this study, we compared the cytotoxic activity of free form of hybrid peptide versus nanocarrier-displayed hybrid peptide on stomach tumor cells. EXPERIMENTAL METHODS To measure cell viability, the Cell Count Kit-8 (Dojindo, Japan) was used. Human stomach cancer cell line, SNU-16 cells, was seeded into 96-well plates at 5×10 cells per well, and treated with peptides. After incubation, CCK-8 solution was added. After two hours, color formation was detected by measuring absorbance at 450 nm. Apoptotic cells were stained with fluorescence labeled Annexin-V (Invitrogen, Carlsbad, CA). Annexin V detect the phosphatidylserine that exposed on cell membrane surface in apoptosis condition. Cells were seeded into 6-well plates at 1×10 cells per well and treated with peptides. After incubation, cells were harvested and stained with Annexin V. The binding of Annexin V was analyzed with flow cytometry (BD Bioscience, San jose, CA, USA). NA/biotin-peptide complex was prepared as previously described at room temperature for 1 hour [3,4]. RESULTS AND DISCUSSION The reduction of cell viability was already detected in the GT1-Bak-BH3 treated cells at 6 h and gradually increased to 24 h (Fig. 1A). On the other hand, treatment of GT1 or Bak-BH3 alone, or mixture of them did not show significant cytotoxicity (Fig. 1A). The induction of apoptosis measured by annexin V staining was remarkable at 24 h after treatment, which was similar to that of octarginine (R8)-Bak-BH3 (Fig. 1B). The induction of apoptosis by GT1-Bak-BH3 was not observed at MDA-MB-231 breast cancer cells that little bind to GT1, while R8-Bak-BH3 significantly induced apoptosis (Fig. 1C). It was certified that GT1 peptide can specifically target stomach cancer cells and GT1-Bak-BH3 hybrid peptide can be a specific apoptosis-inducing agent against stomach cancer cells. Fig. 1. Cytotoxic effects of stomach cancertargeted cytotoxic hybrid peptide versus untargeted cytotoxic peptide. NA complexed with GT1 and Bak-BH3 induced apoptosis higher levels than that with Bak-BH3 only (Fig. 2, p < 0.05). The apoptosisinducing effect NA with GT1/Bak-BH3 was enhanced by adding HGP endosomal escape peptide, while it was less cytotoxic than NA with R8/Bak-BH3 (Fig. 2, p < 0.05). NA armed with GT1 and Bak-BH3 induced apoptosis higher levels than GT1-Bak-BH3 hybrid peptide containing the same concentration of Bak-BH3 (Fig. 2, p < 0.01). These results represent the benefit of using multimeric nanocarriers. Nanocarriers could increase the cytotoxic drug effect on cancer cell compared to free peptide. Also, the results of this study show that such a cassette type of nanocarrier can be easily conjugated with different types of biotinylated peptides or drugs to achieve maximum therapeutic effect. Fig. 2. Apoptosis induction of stomach cancer cells by multimeric NA/biotin nanocarriers versus free form of hybrid peptides CONCLUSION This study demonstrates that GT1 peptide a useful targeting moiety against s cells and GT1-Bak BH3 hybrid apoptosis inducing effect against stomach cancer cells. In addition, multimeric nanocarriers composed of GT1, Bak HGP can improve apoptosis-inducing effect of free form of peptide. REFERENCES 1. Langer, M. , Kratz, F. , Rothen B. , Wunderli-Allenspach, H. , & Beck Sickinger, A. G. (2001). "Novel Peptide Conjugates for Tumor Chemotherapy §". Journal chemistry, 44(9), 1341-1348. 2. Chen, X. , Plasencia, C. , Hou, Y. , & Neamati, N. (2005). "Synthesis and biological evaluation of dimeric RGD peptide paclitaxel conjugate as a model for integrin targeted drug delivery". medicinal chemistry, 48(4), 1098 3. Pähler, A. , Hendrickson, W. A. , Kolks, M. A. , Argarana, C. E. , & Cantor, C. R. (1987). "Characterization and crystallization of core streptavidin". Journal of Biological Chemistry, 262(29), 13933-13937. 4. Wang, Y. , Liu, X. , Nakamura, K Rusckowski, M. , & Hnatowich, D. J. (2009). "In vivo delivery of antisense MORF oligomer by MORF/carrier streptavidin nanoparticles". Cancer Biotherapy and Radiopharmaceuticals, 24(5), 573 -peptide . is tomach cancer has a specific