The proteasome is a large multi-subunit enzyme complex with a crucial role in protein regulation. In immune system cells, a specialized form known as the immunoproteasome replaces the standard catalytic subunits β1c, β2c, and β5c with β1i, β2i, and β5i. The immunoproteasome influences tumor cell growth, inflammatory cytokine production, and T-cell differentiation and proliferation. It is also expressed in platelets, which can aid tumor growth by shielding cancer cells from the immune system. This makes the immunoproteasome a potential pharmacological target to reduce the immunosuppressive environment created by platelets. In our study, we explored the effects of various inhibitors on immunoproteasome function. Platelets were isolated from healthy donors and exposed to the non-selective proteasome (carfilzomib) and the selective inhibitors of β1i (KZR-504) and β5i (M3258). Using western blotting, we assessed the expression and activity of proteasome catalytic subunits and potential changes in the selected signaling pathways. We found that all proteasome subunits are expressed in healthy donors' platelets and that their activity is reduced when exposed to inhibitors. We also demonstrated that the inhibitors alter the investigated signaling pathways. Further, we established co-cultures of platelets with breast cancer cell lines (MDA-MB-231, SK-BR-3 or MCF-7) to mimic the tumor microenvironment. Through co-cultivation, we aimed to study whether modulation of the proteasome activity affects the immunosuppressive environment. We monitored the secretion levels of selected cytokines and chemokines. An increase or decrease in release of IL-6, IL-8 and TGF-β was noted in the case of MDA-MB-231/platelet co-cultures.
Our findings suggest variations in the function of (immuno)proteasome subunits after inhibitor use and their role in tumor microenvironment formation. Further studies are needed to explore the impact of proteasomes and their inhibitors on anti-tumor activity.
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