Proteasome is a large protein complex responsible for intracellular proteolysis. Hematopoietic cells assemble a specialized form of proteasomes, known as the immunoproteasomes, in which the constitutive catalytic sites ß1, ß2, ß5 are replaced by the inducible homologs ß1i, ß2i and ß5i. As proteasome is a well-known target in haematological malignancies, we sought to investigate whether the level of catalytic subunits of proteasomes affects the susceptibility of breast cancer cells to the cytotoxic effects of proteasome inhibitors. Breast cancer cell lines with different metastatic potential were treated with IFN-γ that up-regulates the immunoproteasome expression in time- and dose-dependent matter. By western blotting and activity profiling with irreversible fluorescent probes, we determined that exposure of cell lines to a 150 IU/mL concentration of IFN-γ stimulates the expression of β1i, β2i and β5i immunoproteasome subunits which is most noticeable after 48 hours. Subsequently, the cytotoxic effect of proteasome inhibitor treatment (bortezomib, carfilzomib, KZR-504, LMP7-IN-1) was determined by a metabolic activity assay. We determined that presence or absence of IFN-γ has no significant impact on cytotoxic profiles of used proteasome inhibitors We next used co-cultures of platelets and breast cancer cells, as such in vitro models better mimic the in vivo conditions in terms of the immunosuppressive microenvironment that enables the formation of metastases. Breast cancer cells were pre-treated with IFN-γ or proteasome inhibitors and later co-cultured with isolated platelets. By flow cytometry levels of selected chemokines (IL-8, CCL2, CCL5, MIG, CXCL9, CXCL10) were assessed. Analysis of the supernatants showed large differences in the levels of CCL5 and IL-8 secretion compared to the basal secretion from the cells. We also observed significant changes in the levels of secreted CXCL10 and MIG after treatment with proteasome inhibitors, which indicates that the degranulation process can be influenced by treating cells with selective proteasome inhibitors.
These results indicate the potential role of proteasome in shaping the tumour microenvironment. However, further studies are required to ascertain whether proteasome could serve as an efective therapeutic against metastatic breast cancer.
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