Materials and methods

Materials   All chemicals and materials for cell culture (unless otherwise indicated) were obtained from Sigma (Milan, Italy). Lactate dehydrogenase (LDH) assay kit was purchased from Sclavo Diagnostics (Siena, Italy). Rottlerin was dissolved in dimethyl sulfoxide (DMSO). Carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) was dissolved in 95% ethanol.

Cells and culture conditions   MCF-7 cells, purchased by Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, Italy, were grown in a humidified atmosphere (95% air/5% CO₂) at 37°C in MEM containing 10% FBS, Na-pyruvate (1 mM), and antibiotics.

Primary human microvascular endothelial cells (HMVEC), purchased by LONZA (Milano, Italy), were cultured in EBM-2 medium supplemented with EGM-2 Single Quots (LONZA), and experiments were performed on cultures from passages 3 to 9.

After reaching subconfluence, cells were incubated in serum-free medium for 24 h and then subjected to treatments in 2.5% serum.

Cell counting   The number of cells, cultured in 25-cm² culture flasks (Falcon, Perugia, Italy) was evaluated by detaching with trypsin solution (0.05% trypsin–0.02% sodium EDTA) and counting using a Bürker chamber and trypan blue solution (0.2% w/v final dye concentration). Viable MCF-7 cell number counts were obtained at 1-, 2-, 4-, 15-, and 24-h incubation in the presence or absence of Rottlerin 5 and 20 μM.

Viable HMVEC cell number counts were obtained at 24-h incubation in the presence or absence of 20 μM Rottlerin.

Three replicate counts were determined by the same operator at each time point. The data were presented as proportional viability (%) by comparing the Rottlerin-treated with the vehicle-treated cells, whose viability was assumed to be 100%.

MTT assay   Cell viability was assessed using the MTT colorimetric assay. MTT is taken up into cells by endocytosis or protein-facilitated mechanism and reduced, mainly by mitochondrial enzymes, to yield a purple formazan product which is largely impermeable to cell membranes, thus resulting in its accumulation within living cells. Solubilization of the cells results in the liberation of the purple product which can be detected using a colorimetric measurement. The ability of cells to reduce MTT provides an indication of the mitochondrial integrity and activity which, in turn, may be interpreted as a measure of cell number/proliferation/viability/survival/toxicity.

Operatively, 100 μl of cell suspension was inoculated to each well of 96-well plates at the density of 2 × 10⁴ cells/well (the area of each well was 0.32 cm²). After 24 h of culture, the medium was removed by aspiration and replaced with 100 μl of experimental medium. The treatments were performed with 5 and 20 μM Rottlerin for 1, 2, 4, 15, and 24 h. After incubation, cells were observed under a contrast phase microscope before adding MTT solution, prepared fresh as 5 mg/ml in H₂O, filtered through a 0.22-μm filter, and kept for 5 min at 37°C. MTT solution (10 µl) was added to each well, and the plates were incubated in the dark for 4 h at 37°C.

To check for the direct effect of Rottlerin on the formazan production, a parallel set of experiments was carried out in cell-free plates. The followed procedure was the same as described above with some modifications: (a) The Rottlerin doses were 5, 20, 50, and 100 μM; (b) the incubation time with MTT was prolonged from 4 up to 24 h; (c) different media (DMEM, MEM, and RPMI 1640), enriched or not with 10% serum or 20 μM NAD or NADH, were tested.

In another set of experiments MCF-7 cells were treated for 30 min with equal doses (5 and 20 μM) of Rottlerin or the chemical uncoupler FCCP, before incubation with 10 µl of MTT for 1 h at 37°C.

HMVEC were treated for 24 h with 20 μM Rottlerin before incubation with MTT for 4 h.

At the end of each experiment, the medium was removed by inverting and tapping the plates and 100 μl solution of 4% HCl 1 N in isopropanol was added to immediately dissolve the formazan crystals. Absorbance at 570 nm was read on a Multiwell scanning spectrophotometer (Sclavo, Siena, Italy), and the results were expressed as a percentage (%) of the control (vehicle alone).

The medium from the plates without cells was collected and both read directly and centrifuged before solubilization of (eventual invisible) formazan crystals. In this experiment, the reducing agent dithiothreitol was used as a positive control.