Breast cancer is the most commonly diagnosed type of cancer and leading cause of cancer-related death among women worldwide. Although there are several breakthroughs in the treatment of breast cancer on the past few decades, high incidence of relapse and progression after conventional therapies is deeply concerning, indicating a great need for developing new therapeutics for breast cancer. Strategies including utilization of combined treatments in order to enhance the efficacy of cancer treatment have been widely developed in recent years, targeting different signaling pathways to inhibit proliferation or induce apoptosis. In the present study, the anti-cancer effects of combinational therapy of Letrozole (Aromatase inhibitor) or Olaparib (PARP inhibitor) with Aspirin, non-steroidal anti- inflammatory drugs (NSAIDs), were examined on well differentiated ER-positive and poorly differentiated triple negative breast cancer cell lines, T-47D and MDA-MB-231, respectively. In addition, The effect of combined treatment was also examined by detecting cell survival, cell cycle analysis, and alterations in signaling pathway that play a role in breast cancer progression such as β -catenin, STAT3, cyclin D1, BAX, c-PARP, wild and Mutant p53. In addition the effect of combined treatments were examined on gene expression levels of Cytochromes; CYP1A1, CYP1B1, Aurora kinases; Aurora-A and Aurora-B were determined by RT-PCR.

Our data showed that combined treatments of 50 nm Letrozole or 250 nm Olaparib with 5mm, 2.5mm Aspirin for 48 & 24 h respectively promoted apoptosis induction through enhanced histone release and caspase-3 activation compared to control and cells treated with each drug alone. Furthermore, apoptotic induction was associated with upregulation of Bax and downregulation of cyclin D1, β-catenin, and STAT3 signaling pathways in both tested cell lines compared to control, suggesting that Aspirin may enhance the sensitivity of tested cells to Letrozole or Olaparib in both (ER) positive as well as in triple negative breast cancer cell lines. In addition, cell cycle analysis showed a cell arrest at G0/G1 phase in both cell lines.

Interestingly, in T-47D cells, combined treatment of Letrozole with Aspirin significantly upregulated wild p53 at protein level and downregulated CYP1A1, CYP1B1, Aurora-A, and Aurora-B expression at gene level compared to control, suggesting that combined treatment may inhibit breast cancer cell proliferation via down-regulation of Aurora-A and Aurora-B. In MDA- MB-231 cells, combined treatment of Olaparib with Aspirin showed a significant downregulation of Mutant p53 at protein level and CYP1A1, CYP1B1, Aurora-B and upregulation of Aurora-A at gene level compared to control, suggesting that there is a crosstalk between Olaparib/ Aspirin and P53. In conclusion, combined treatment of Letrozole or, Olaparib with Aspirin might be a promising approach to inhibit both ER (+) and triple negative breast cancer cell lines survival and proliferation.