The effects of various combinations of different classes of anticancer drugs and tyrosine kinase inhibitors on the human MCF-7 and triple-negative MDA-MB 231 breast carcinoma cell lines

Abstract

Globally, breast cancer is the most common cancer affecting women and it is predicted that in 2030 about 12 million deaths will occur with approximately 21.7 million new cases. Genetic risk factors as well as race and ethnicity, account for about 5-10% of all breast cancer occurrences. Triple negative breast cancer (TNBC), tumors that tested negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), contribute to 10-20% of all breast carcinomas and is known to be a more aggressive type of cancer with varying degree of response to chemotherapeutic and radiation therapy. The epidermal growth factor receptor (EGFR) is abnormally activated in many cancers and its signal transduction pathways play an important role in regulating cell proliferation and survival. It is often overexpressed in TNBC. P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for active transport of substances across the cell membrane out of a cell. It functions as an efflux pump and is overexpressed in many cancer cells. The altered expression of both EGFR and P-gp parallels an aggressive clinical course and the development of resistance to anticancer and adjuvant endocrine therapies. Anti-EGFR compounds that specifically target the intracellular and extracellular domains of the EGFR include tyrosine kinase inhibitors (TKIs), e.g., gefitinib and erlotinib and monoclonal antibodies, e.g., cetuximab and trastuzamab, that are amongst the most effective agents that are currently used together with chemotherapeutic agents in clinical practice. However, the emergence of multidrug resistance (MDR), a mechanism that is not well understood, has, over time, eclipsed the progress that has been made in drug therapeutics. In this study we examined the effects of doxorubicin (DOX), cisplatin (CDDP) and one investigational TKI (EGFR inhibitor I, hereinafter referred to as EGFRi), individually and in combination, on human MCF-7 breast carcinoma cells and a triple negative breast cancer (TNBC) cell line (MDA-MB-231), with specific focus on P-gp and EGFR inhibition. Analyses of MCF-7 and MDA-MB-231 TNB carcinoma cells exposure to DOX, CDDP and EGFRi included growth and dose-response curves (cytotoxicity assays), caspase3/7 assays for apoptosis detection, P-glycoprotein function (intracellular Calcein-AM accumulation) and EGFR and P-glycoprotein (ABCB1 gene) mRNA expression, using quantitative Real-Time PCR. When used as an individual treatment, EGFRi demonstrated a higher growth inhibition in both MCF-7 and MDA-MB 231 cell lines, compared to DOX and CDDP. Combination treatment of DOX with CDDP at equimolar log dose concentrations, synergistically inhibited cell growth that was significantly different to the compounds used individually. The pairwise combinations of EGFRi with both DOX and CDDP also demonstrated synergistic and antagonistic drug interactions consistent with the Bliss independence and Loewe additivity synergy models. Apoptosis was confirmed in both MCF-7 and MDA-MB-231 TNBC cells after 24-hour drug treatments. Intracellular calcein accumulation that is indicative of P-glycoprotein inhibition, was determined using the Calcein-AM assay. Drug treatments significantly increased the intracellular accumulation of calcein inside the cells, but only at very high concentrations in MCF-7 cells. No significant levels of calcein accumulation were detected in MDA-MB-231 cells, following individual treatment with EGFRi, DOX and CDDP. The pairwise combination drug treatments of EGFRi + DOX demonstrated significant accumulation of calcein only in MCF-7 cells. RT-qPCR was utilized to quantify the gene expression levels of EGFR and ABCB1 in both MCF-7 and MDA-MB-231 TNBC cell lines following drug treatment. The expression levels of EGFR gene were significantly reduced in both cells following drug treatment, which correlates with the cellular growth inhibition results. The expression levels of ABCB1 gene could not be quantitated following optimization of expression due to undesirable CT values that was outside the normal acceptable ranges. This is a due to the undetectably low expression levels of ABCB1 gene in our samples. Our scientific findings confirmed our hypothesis of the EGFRi’s ability to successfully reduce the efflux function of P-glycoprotein as well as demonstrating its combinatorial potential with DOX and CDDP, as compounds from different classes are more effective in enhancing growth inhibition efficacy in the two human breast carcinoma cell lines.