Fig. 2. The effect of drug combinations on cell viability, caspase 3/7 activity and PARP cleavage.

a Ovarian or breast cancer cells were exposed to paclitaxel (9.5 nM), CMVA (100 μM), DCBC (100 μM) or combinations of these drugs as indicated for 48 h and cell viability was assessed by staining with trypan blue. The effect of the drug combination if the results were additive was estimated using the Bliss independence criterion which is indicted with the horizontal bar. The effect of the combination (mean ± S.D., n = 3) was significantly different from the expected additive effect where shown (*P < 0.05; **P < 0.005; 1; paired t test). b Ovarian or breast cancer cells were exposed to the indicated drugs as described in a and caspase-3/7 activity determined after 48 h. To control for effects of the drug on proliferation, parallel samples were also exposed to the same drug combinations and relative cell number estimated by staining with SRB. The caspase-3/7 activity was then normalised to relative cell number. The results are expressed as a fraction of the caspase activity measured in cells exposed to paclitaxel alone (mean ± SD, n = 3) and are significantly different to those obtained with cells exposed to paclitaxel alone where indicated (*P < 0.05 paired t test). c Expression of BCKDK was repressed with the indicated siRNA and the cells treated with paclitaxel. After 48 h, caspase-3/7 activity was assessed. The results were normalised to total protein and expressed (mean ± SD, n = 3) as proportion of that measured in cells transfected with a non-targetting siRNA (NT-1). The results are significantly different to the caspase activity measured in cells transfected with NT-1 and treated with paclitaxel where shown (*P < 0.05; **P < 0.01; ***P < 0.005; paired t test). d OVCAR-4 and MCF-7 cells were exposed to the indicated drugs as described in a and PARP cleavage was assessed by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The result is representative of three independent experiments. e OVCAR-4 cells were exposed to combinations of paclitaxel as described above, in the absence or presence of additional BCAA (1 mM) and PARP cleavage assessed. The result is representative of three independent experiments. f MCF-7 cells were grown in the bespoke media prepared to contain BCAA at a fraction of that normally found in DMEM (0.8 mM). The potency of paclitaxel (IC₅₀ ± S.D., n = 3) was determined in cell growth assays and was significantly different from that measured in the normal medium.