The effects of selected therapeutic agents on cell cytotoxicity and Her-2 receptor expression using culturedbreast adenocarcinoma models

Abstract

Introduction: Epidemiological studies suggest that at least 1 in 29 South African women will be diagnosed with breast cancer in their lifetime. Breast cancer is not a single disease. The heterogeneity of breast cancer results in four distinct molecular subtypes including aggressive human epidermal growth factor receptor-2 (Her-2) positive, where Her-2 receptors are overexpressed. Trastuzumab (Herceptin®), is a recombinant, humanized, anti-Her-2 monoclonal antibody that specifically targets subdomain IV of the extracellular domain of the Her-2 receptor and has dramatically altered the prognosis of Her-2 positive breast cancer. Trastuzumab is, however, associated with problems such as primary and acquired resistance, which has prompted investigation into improving its efficacy. Aim: To investigate the ability of selected therapeutic agents to alter in-vitro cell viability, cell cycling, apoptosis and Her-2 expression in models of Her-2-positive and oestrogen receptor positive, Her-2 negative breast adenocarcinoma and bring about an alteration in the efficacy of trastuzumab. Methods: MCF-7 cells which retain the ability to process oestrogen, and SK-Br-3 cells which overexpress Her-2 gene products were used. Cells were exposed to trastuzumab, aspirin, calcipotriol, doxorubicin, epidermal growth factor (EGF-human), geldanamycin, heregulin-β1 and β-oestradiol as single agents and in combination with trastuzumab. Research methodologies included tetrazolium conversion assay for cell viability, AMC-substrate cleavage and annexin-V for apoptosis, propidium iodide staining for cell cycle analysis and anti-Her-2 affibody molecule for relative Her-2 receptor density. Results: Cell survival of 95.39% (±2.69) for MCF-7 cells and 74.17% (±1.60) for SK-Br-3 cells was observed following trastuzumab (100 μg/ml) exposure. Trastuzumab resulted in statistically significant G1 phase accumulation in MCF-7 cells at 72 hours and in SK-Br-3 cells from 24 hours. Furthermore, trastuzumab decreased relative Her-2 receptor density in SK-Br-3 cells by approximately 35% by 24 hours but had no effect in MCF-7 cells. The anti-proliferative effects of trastuzumab were abrogated by EGF, a Her-1 ligand and heregulin-β1, a Her-3 and Her-4 ligand. Most agents altered distribution throughout the phases of cell cycle to a certain degree, with the G1 phase accumulation observed for trastuzumab being potentiated in some combinations. Most of the agents, with the exception of doxorubicin and geldanamycin, did not promote apoptosis and appeared instead to be anti-proliferative. Geldanamycin had the greatest effect on Her-2 receptor density (approximately 80% by 24 hours) followed by EGF, heregulin and trastuzumab, with the biological molecules in combination with trastuzumab producing a further significant reduction. Conclusion: Endogenous Her-receptor ligands (EGF and heregulin) differentially altered the viability parameters for trastuzumab which could play a role in the emergence of clinical resistance to targeted therapy. Doxorubicin with concurrent trastuzumab significantly reduced cell viability compared to each single agent in both cell lines. Furthermore, the cytostatic and cytotoxic abilities of each of the other agents either mimicked trastuzumab alone or the selected agent alone when exposed concurrently.