Figure 2

Synergistic induction of cell death as a function of pitavastatin concentration in combination with the EGFR TKI erlotinib in NSCLC cell lines. (A) Dose response curves of indicated cell lines treated with increasing concentrations of pitavastatin and/or erlotinib (5 µM). Proportions of dead cells were determined via flow cytometry. EC50 values were calculated using Graph Pad Prism (Table 2). Data are given as arithmetic mean ± SD from three independent experiments. Asterisks denote statistical significance as determined via unpaired two-tailed t-test comparing pitavastatin-treated to pitavastatin and erlotinib co-treated cells at matching concentrations: *p < 0.05; **p < 0.01; ***p < 0.001. (B) Bliss independence analysis of drug synergy. Expected fractional effect rate calculated from single drug treatments (grey curve with 95% confidence band) are compared with observed fractional effect rates (black squares and curve). The observed cytotoxicity (black curve) was higher than expected (grey curve) indicating a synergistic rather than additive cytotoxicity of pitavastatin/erlotinib co-treatment in Calu6 and A549 cells compared to H1993 cells. Data are derived from panel A and shown as median and 95% CI. (C) EC50 isobolographic analysis showing the decrease of EC50 of pitavastatin in presence of 5 µM erlotinib [EC50(Pita)]. Data are median and 95% CI. The isobole was generated using data from Table 2. If the effect rate of the combination treatment lies on, above or below the isobole, the drug combination is additive, antagonistic or synergistic, respectively. (D) Representative dot plots of annexin V (AV)/PI labelled cells treated with the indicated drug combinations: untreated (E0P0), pitavastatin 10 µM (E0P10), erlotinib 5 µM (E5P0) and erlotinib 5 µM/pitavastatin 10 µM co-treatment (E5P10). Prominent populations of AV/PI double positive cells appear in presence of pitavastatin (E0P10). This is even more pronounced in combination with erlotinib (E5P10), but absent in untreated (E0P0) or erlotinib-only exposed cells (E5P0). Dot plots were generated using FlowJo vX.0.7.