Cytotoxic and Antitumor Activity of Lactaptin in Combination with Autophagy Inducers and Inhibitors

Abstract

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors—chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)—or inducer—rapamycin (Rap)—can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.

Interplay Between Mitophagy and Apoptosis Defines a Cell Fate Upon Co-treatment of Breast Cancer Cells With a Recombinant Fragment of Human κ-Casein and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

“…However, the recent attention toward the action of RL2 at mitochondria (Richter et al, 2020 ) allowed to suggest in the current study the contribution of mitophagy to RL2-mediated cell death. Moreover, the data presented in this study suggest that the autophagy markers, which were detected previously upon addition of RL2 (Bagamanshina et al, 2019 ), correspond to mitophagy induction. Hence, our current findings seem to be in accordance with previous reports.…”

“…Moreover, the ATP loss induced by mitophagy might lead to the ATP loss mediated necrosis on the single-cell level, which is likely contributing to the overall increase in the number of dying cells upon long-term RL2/TRAIL co-treatment. It has to be mentioned that autophagy induction by RL2 administration has been reported before (Bagamanshina et al, 2019 ). However, the recent attention toward the action of RL2 at mitochondria (Richter et al, 2020 ) allowed to suggest in the current study the contribution of mitophagy to RL2-mediated cell death.…”

“…In particular, this peptide was shown to induce cell death in MDA-MB-231 and MCF-7 cells and suppress tumor growth in mice models (Koval et al, 2012 , 2014 ). RL2 was reported to modulate the expression of apoptotic proteins and induce autophagy in MDA-MB-231 cells (Koval et al, 2014 ; Bagamanshina et al, 2019 ). RL2 acts in monomeric, dimeric, and oligomeric forms; the latter are formed via cysteine bridges (Chinak et al, 2019 ).…”

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

“…Human adenocarcinoma MDA-MB-231 and MCF-7 cells and murine rhabdomyosarcoma MX-7 cells were used for our experiments. Previously, we had already calculated IC50 for lactaptin analogue RL2: for these cells, it was about 250–300 μg/mL [ 10 , 11 ]. This dose of RL2 was used for cell death induction in this study.…”

“…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].…”