Figure 2

The polyhalogenated monoterpenes RU017 and RU018 inhibit the formation but not development of MCF-7 mammospheres. A) Mammosphere cultures were treated with either the DMSO vehicle control (a), 300 μM of either of the halogenated monoterpene stereoisomers RU017 (b) or RU018 (c) or10 μM fucoxanthin (FXN) (d). The latter concentrations were derived from the IC₅₀ values in MDA-MB-231 or MCF7 breast cancer and MCF12A non-cancerous cells grown in anchorage-dependent conditions in the case of FXN. Images were captured under a light microscope at either 400× or 100× magnification using a Nikon Coolpix 990 camera on Day 1 (i), Day 3 (ii) and Day 6 (ii) after treatment with the relevant compounds upon seeding and on Day 6 for mammospheres treated with compounds four days after seeding (iv). Images were representative of at least three randomly selected fields for each treatment. Analysis of images and calculation of scale was carried out using ImageJ (NIH freeware). Scale bars representative of 0.1 mm. B) Sphere forming efficiency (SFE) and C) mammosphere viability on Day 8 as determined by WST-1 assay. D) Untreated mammospheres were dissociated and seeded as a single cell suspension into regular culture media containing either the vehicle control or 300 μM of RU017 or RU018, cultured under adherent conditions and assessed for viability by means of an MTT assay. The percentage viability in C) and D) was calculated relative to the DMSO vehicle-treated control (taken as 100%) when an equal numbers of cells are seeded and treated for the same period. For B), C) and D) error bars indicate the standard error of the mean where n = 3 (B) or 5 (C and D). Statistical significance was calculated using a Students t-test (**p < 0.01, *p < 0.05).