Abstract
Natural compounds of various origins are intensively investigated for their antitumor activity. Potential benefits of antitumor therapy can be achieved when cytotoxic agents kill cancer cells and these dying cancer cells drive adoptive immunity to the tumor. This strategy was successfully demonstrated for chemotherapeutic drugs that induce immunogenic type of cell death (ICD) with release of DAMPs (danger associated molecular patterns) and exposure of “eat me” signals. In this study, we demonstrated that recombinant human milk peptide lactaptin (RL2) induces death of cancer cells with ICD hallmarks in vitro with the release of ATP and high-mobility group box 1 protein (HMGB1) and exposure of calreticulin and HSP70 on the external cell membrane. RL2-treated cancer cells were efficiently engulfed by phagocytic cells. Using the syngeneic mouse model, we demonstrated that RL2-treated MX-7 rhabdomyosarcoma cells confer long-term immune-mediated protection against challenge with live MX-7 cells. We also analyzed the combinatorial antitumor effect of vaccination with RL2-treated cells and the inhibition of indoleamine 2,3-dioxygenase (IDO) with ethyl pyruvate. Compared to solo anti-tumor immunization with RL2-treated cells, additional chemical inhibition of IDO demonstrated better long-term antitumor responses than vaccination alone.
Highlights
Human milk is rich in various bioactive proteins and peptides, which regulate functions of the intestinal tract, vasculature, nervous system, immune system, and endocrine system [1]
Immunogenic cell death has in itself dual advantages for anti-cancer therapy: first, immunogenic type of cell death (ICD) inducers directly kill tumor cells, and second, dying cells can serve as a vaccine material that activate long-term antitumor response
Human adenocarcinoma MDA-MB-231 and MCF-7 cells and murine rhabdomyosarcoma MX-7 cells were used for our experiments
Summary
Human milk is rich in various bioactive proteins and peptides, which regulate functions of the intestinal tract, vasculature, nervous system, immune system, and endocrine system [1]. Resulting from the cytotoxic activity of milk fraction against various cancer cells in vitro, the proteolytic fragment of milk0 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 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]. The N-terminal region, which is essential for proapoptotic activity, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure [9]. RL2 was shown to induce cell death with the hallmarks of various types of programmed cell death: apoptosis, ATP depletion-dependent necrosis, and autophagy [3,10,11]
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