(D-130) Development of boronic acid-containing artificial alpha-helical cell-penetrating polypeptide-based proteasome inhibitor for cancer immunotherapy.
Introduction:: Proteasomes are intracellular organelles that involved in cellular protein metabolism. Cancer cells have an accelerated rate of protein synthesis for rapid proliferation, making the role of proteasomes crucial. Boronic acid inhibits the function of proteasomes through reversible covalent binding with its catalytic subunits. Inhibition of the proteasomes induces endoplasmic reticulum stress-mediated immunogenic cell death. Alpha-helical polypeptides have both hydrophilicity and cell-penetrating properties, making them easy to deliver them into cells. In this study, we developed an immunogenic cell death-inducing cell-penetrating polypeptide-based proteasome inhibitor, including boronic acid, which has improved solubility, high cellular delivery capacity, and high stability compared to existing boronic acid drugs, for cancer immunotherapy.
Materials and Methods:: We synthesized the boronic acid-containing artificial cell-penetrating polypeptide through organic synthesis. We conducted experiments using B16F10 murine melanoma, CT26 murine colon carcinoma, and NIH-3T3 murine fibroblast, and confirmed cell toxicity, mechanism of action, and expression of damage-associated molecular patterns through various analysis such as cell viability assay, flow cytometry, confocal laser scanning microscopy, and western blotting.
Results, Conclusions, and Discussions:: Results
The boronic acid-containing artificial cell-penetrating polypeptide showed higher cytotoxicity against cancer cells compared to normal cells.
Confocal microscopy and proteasome activity assay confirmed that the peptide interacts and inhibits the function of proteasomes.
As a result, accumulation of ubiquitinated proteins was observed. Accumulated ubiquitinated proteins induced endoplasmic reticulum stress. Reactive oxygen species and intracellular calcium ion levels were increased and endoplasmic stress-related proteins, such as BiP, phospho-eIF2 alpha, ATF-4, and CHOP, were activated. Consequently, apoptosis pathway is activated. Decreased anti-apoptotic protein Bcl-2 and increased pro-apoptotic protein Bax were observed. Also, cleavage of caspase-8 and caspase-3 indicate apoptosis is occurred.
Immunogenic cell death markers such as calreticulin and extracellular ATP levels were increased, which are necessary for recruiting antigen-presenting cells for the activation of anti-tumor immunity. In addition, increased MHC-I expression in cancer cells enables the presentation of tumor antigens to the immune cells.
Conclusions and Discussions
We developed an boronic acid-containing artificial cell-penetrating polypeptide-based proteasome inhibitor for cancer immunotherapy. We validated the high solubility and stability, high toxicity towards cancer cells compared to normal cells, and the ability to induce immunogenic cell death through endoplasmic reticulum stress in cell expreiments. We plan to validate the stability and efficacy in vivo through tumor-bearing animal expreiments.
