The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells

Abstract

Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2–TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.

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

“…RL2 treatment alone induces cell viability loss of breast cancer cells ( Figure 1A ), whereas TRAIL treatment alone causes the cell death of TRAIL-sensitive cells (Sprick et al, 2000 ; von Karstedt et al, 2017 ; Richter et al, 2020 ). To analyze the effects of RL2/TRAIL coadministration on cell viability, the treatment of MDA-MB-231 breast carcinoma cells with RL2, TRAIL, or their combination was carried out ( Figure 1B ).…”

“…RL2 acts in monomeric, dimeric, and oligomeric forms; the latter are formed via cysteine bridges (Chinak et al, 2019 ). Moreover, recently, it has been shown that RL2 mediates cell death, which was accompanied by the loss of mitochondrial membrane potential and intracellular adenosine triphosphate (ATP) loss (Richter et al, 2020 ). Mass spectrometry-based screening of RL2 interactome identified the mitochondrial import protein TOM70 as an interaction partner of RL2.…”

“…Mass spectrometry-based screening of RL2 interactome identified the mitochondrial import protein TOM70 as an interaction partner of RL2. Further, it was unveiled that RL2 is targeted to the mitochondria after internalization into the cells (Richter et al, 2020 ). The requirement for TOM70/RL2 interaction in RL2-induced reduction of intracellular ATP levels was validated by downregulation of TOM70, resulting in partial rescue of the intracellular ATP yield.…”

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].…”