Tumor-Specific Peptide, Selected from a Phage Peptide Library, Enhances Antitumor Activity of Lactaptin

Abstract

A recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, induces apoptosis in cultured tumor cells. The tumor suppression efficacy of RL2 was shown against mouse hepatoma-1 cells and MDA-MB-231 human breast adenocarcinoma cells. The RL2-based therapeutic drug lactaptin is distributed evenly throughout the organism, which reduces its antitumor efficacy. In the current study, we obtained a genetic construct that allows production of the recombinant fusion protein T3-RL2, consisting of RL2 and T3 peptide (YTYDPWLIFPAN), in E. coli cells. T3 peptide was selected from a phage peptide library as a result of two screenings: in vitro using MDA-MB-231 cell culture and in vivo using a mouse xenograft model of breast cancer MDA-MB-231. It was shown that the displayed peptide T3 provides binding and internalization of phage particles by MDA-MB-231 cells and their specific accumulation in MDA-MB-231 tumor tissue. In addition, based on the nucleotide sequences coding RL2 and the known tumor-targeting peptide iRGD, we obtained genetic constructs that provide synthesis of fusion proteins RL2-iRGD and RL-iRGD-His. We studied the cytotoxic activity of fusion proteins T3-RL2, RL2-iRGD and RL-iRGD-His in vitro using MDA-MB-231 and MCF-7 human adenocarcinoma cells. The in vitro results showed that the fusion proteins inhibit proliferation of both cell cultures, and their cytotoxic activity is higher than that of RL2. In vivo experiments on the study of the antitumor efficacy of the obtained fusion proteins demonstrated that T3-RL2 protein significantly inhibits MDA-MB-231 tumor growth in a xenograft model compared with RL2, while the antitumor effect of RL2-iRGD and RL-iRGD-His proteins is comparable to the effect of RL2.

Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells

“…In Figure 3 shows fluorescence microscopy of cells incubated with bacteriophages 19C, 36C, 92, 26, selected on different phage libraries, in different screening systems. Phage M13, displayed the peptide YTYDPWLIFPAN previously selected for MDA-MB 231 cells, was taken as a negative control [ 5 ]. No significant differences were found in the efficiency of binding to cells of bacteriophages displayed the studied peptides.…”

“…For in vivo screening, we used the previously described methods [ 5 , 18 ], to wit. SCID mice with subcutaneously and orthotopic glioblastoma xenograft U-87 MG were injected into the tail vein with 300 μL of a phage peptide library with a concentration of 2 × 10 11 pfu/mL, diluted in saline.…”

“…U-87 MG cells were incubated on BD Falcon culture slides to 80–90% confluence, washed with PBS twice, and 100μL of the selected phage clone (2 × 10 10 PFU/mL) in PBS-BSA Ca/Mg buffer (0.1% BSA, 1mM CaCl 2 , 10 mM MgCl 2 × 6H 2 O); was added. Cells were incubated with the bacteriophage clone for 2 h at 37 °C with the following treatment according to the previously described technique with slight modifications [ 5 ], namely. After incubation at 37 °C, cells were washed three times with 500 μL buffer (100 mM glycine, 0.5 M NaCl, pH 2.5) at room temperature, fixed with 200 μL cold 4% formaldehyde for 10 min and washed twice with PBS.…”

Design and Modification of Anticancer Peptides

“…More efforts have been made to obtain the targeting properties of lactaptin. Recently, Nemudraya et al [84] reported that two targeting peptides of T3(YTYDPWLIFPAN) and iRGD had the specificity for cancer cell and penetration to targeted cancer cell and tumor tissue. They were used to construct the fusion proteins T3-RL2, RL-iRGD-His and RL2-iRGD and evaluate the activity both in vitro and in vivo.…”

Recombinant Lactaptin Induces Immunogenic Cell Death and Creates an Antitumor Vaccination Effect in Vivo with Enhancement by an IDO Inhibitor

“…Resulting from the cytotoxic activity of milk fraction against various cancer cells in vitro, the proteolytic fragment of milk s k-Casein-lactaptin was purified and identified [2]. Recombinant analogue of lactaptin (RL2), covering the 57-134 amino acid sequence of k-Casein, was also efficient against tumors grafted onto mice [3][4][5][6]. The comparison of RL2 primary structure and the mechanism of its penetration into the cell suggests that it can be assigned to the cell penetrating peptide (supplementary materials) [7,8].…”