Suppression of macrophages- Induced inflammation via targeting RAS and PAR-4 signaling in breast cancer cell lines

Highlights: • Tumor associated macrophages (TAMs) stimulate breast cancer progression. • TAMs upregulate Ras, Cox-2, PGE₂, IL-1β, IL-6, VEGF-A and MMP-9. • TAMs inhibit apoptosis via upregulation of Bcl-2 and downregulation of Par-4. • Both NSAIDs suppress the stimulatory effect of TAMs and induce apoptosis. -- Abstract: Tumor associated macrophages (TAMs) have a crucial role in cancer progression, metastasis and drug response. Piroxicam and sulindac sulfide are non-steroidal anti-inflammatory drugs (NSAID) that decrease the incidence and progression of several types of cancer. However, their role in suppressing the interactions between TAMs and cancer cells remain unclear. Herein, we studied the impact of human monocytes conditioned media (CM) on cellular proliferation of ER-dependent MCF-7 and ER-independent MDA-MB-231 cells, and the effects of piroxicam and sulindac sulfide on the expression levels of RAS, COX-2, IL-6, IL-1β and PAR-4 (qRT-PCR), BCL-2 and BAX (western blot), Caspase-3, VEGF-a and PGE₂ (ELISA), MMP-2 and -9 (zymography) in the stimulated cells. Our results showed that CM caused a significant increase in cells survival through significant increase in RAS expression which resulted in upregulation of COX-2, PGE₂, BCL-2, IL-6, IL-1β, VEGF-A and MMP-9 and down regulation of PAR-4. Treatment with one of the NSAIDs used in this study produced a time and concentration dependent growth inhibition of stimulated cells by inhibiting RAS expression. Suppression of RAS was accompanied by downregulation of its downstream signaling of IL-1β, IL-6, COX-2 and PGE₂, activation of apoptotic machinery through upregulation of PAR-4 and caspase-3, as well as, inhibition of BCL-2, VEGF-A, MMP-2 and MMP-9. In conclusion, our data support the role of piroxicam and sulindac sulfide in suppressing inflammation-driven breast cancer progression and identifies promising novel target in RAS and PAR-4 signaling.