Effects of cPLA2α inhibition in the MDAMB-468 cell model system for basal-like breast cancer and/or triple negative breast cancer
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Basal-like breast cancer (BLBC) is a highly aggressive breast cancer sub-type and represents ~15% of all breast cancers. BLBC are often associated with early age of onset, early relapse and poor overall survival in contrast to other types of breast cancers. Moreover, patients diagnosed with BLBC do not benefit from currently available targeting treatments on the marked, such as anti-oestrogen, anti-progesterone and antibody against the HER2-receptor, and therefore, BLBC are also referred to as triple negative breast cancer (TNBC). In the last years, many researches have focused on studying the molecular characteristic of BLBC/TNBC to develop molecular targets against BLBC/TNBC and several molecular targets are currently being investigated and some are already proposed. BLBC/TNBC has shown overexpression and/or amplification of various receptors and proteins involved in cell survival, proliferation and apoptosis. Thus, targeting these pathways have shown increasing interests for researchers who want to develop such inhibitors for the treatment of BLBC/TNBC. It has previously been shown that BLBC/TNBC overexpress the gene PLA2G4A that encode the cPLA2α-enzyme, which is responsible for regulating the production of eicosanoids by liberating arachidonic acid (AA). The eicosanoids are biologically active lipid mediators, which are involved in regulating many responses in the cells, such as inflammation, proliferation, apoptosis, metastasis and angiogenesis. To our knowledge, the exact role of cPLA2α in cell proliferation in BLBC/TNBC is not fully identified. In this master thesis, we investigate the cellular effects of cPLA2α inhibition by using the MDA-MB-468 cell line as a model for BLBC/TNBC. More specifically, cell proliferation and/or viability, apoptosis and cell cycle distribution of these cells was investigated. Another goal of this master project was to evaluate the possible synergistic/additive effects of combining cPLA2α-inhibitors with selective mTOR/PI3K inhibitors, which represent a promising treatment strategy for BLBC/TNBC. By using the resazurin viability assay, the effects of different compounds/inhibitors/comparative drugs on the viability of the MDA-MB-468 cells were investigated. A doseresponse experiment was conducted for each compound/inhibitor/comparative drug in order to select a sub-optimal concentration to further experiments. Caspase activity of cells treated with selective cPLA2α inhibitors alone and in combination with pan-PI3K inhibitor buparlisib/BKM120 were measured by caspase GLO®. Flow cytometry was utilized to measure cell cycle phase distribution of the MDA-MB-468 cells treated with AVX002 separately, buparlisib/BKM120 separately and when combined with AVX002. Doxorubicin was used as apoptosis control. Rezasurin viability assay, revealed positive results and reduced the viability of the MDAMB468 cells in a dose-dependent manner for all the tested compounds. Combination treatment of selective cPLA2α-inhibitors with buparlisib/BKM120 revealed additive effects in the MDA-MB-468 cells. Caspase assay showed increased caspase activity of cells treated with different cPLA2α-inhibitors and combing these inhibitors with buparlisib/BKM120 increased these effects considerably. Flow cytometry analyses showed cell cycle arrest at G1 phase in cells treated with AVX002. Cells treated with buparlisb/BKM120 showed increased number of cells at the G2+M phase of the cell cycle. The use of selective cPLA2α inhibitors could offer a novel targeted therapy for BLBC/TNBC which otherwise have low survival prognosis. Also, combining selective cPLA2α inhibitors with selective PI3K inhibitors could potentially balance the side effect profile of each inhibitor and reduce resistance problems. Further studies need to be conducted in order to confirm or reject these observations.