Figure 2.
The FAO inhibitor perhexiline amplifies the antitumor activity of gemcitabine by decreasing cell viability and increasing apoptosis in primary pancreatic cancer cells
(A) Dose-response experiments with etomoxir and perhexiline treatments in selected primary PDAC cells. Live cells were quantified using the crystal violet assay after 72 h of treatment. (Top) Curves are representative of three independent experiments performed in triplicates, and data are the mean ± SD. (Bottom) Percentage of live PDAC cells treated for 72 h with etomoxir (62.5 μM) or perhexiline (7 μM), compared with non-treated controls. The cells showing a percentage of live cells below the mean (75% for etomoxir and 72% for perhexiline [dotted lines]) are considered as responding efficiently to etomoxir and perhexiline (“high responders”). Two other groups of cells show moderate response with values close to the mean (“intermediate responders”) and low response (“low responders”) with a percentage of live cells above the mean. Data are means of triplicates ±SEM and are representative of three independent experiments.
(B) Representative cell death assays in four PDAC cells treated with perhexiline (7 μM) for 72 h compared with controls (vehicle DMSO). Cell survival was measured using an Annexin V apoptosis assay with PI, and the number below each panel is the percentage of viable cells (Annexin V-/PI- cells). The corresponding quantification (right) indicates the percentage of viable cells. Data are presented as mean ± SEM of three independent experiments performed in duplicates.
(C) Dose-response combination assays were performed in four selected PDAC cells according to the sensitivity to Perhexiline. Cells were treated for 72 h with increasing doses of gemcitabine, perhexiline (5 μM), or the combination, and cell viability was measured by crystal violet assay (the curves are shown in Figure S2D). Graphs show the percentage of live cells of each treatment: gemcitabine at one dose (1 nM), perhexiline (5 μM), or the combination. To determine the synergistic effect in the combination treatments, we calculated the predicted values by multiplying the percentage of live cells in the gemcitabine and perhexiline groups, according to the method previously described.28 Then, when the observed value for the combination is lower than the predicted value, we consider it as a synergistic effect. Data are the mean ± SEM of two independent assays performed in triplicates.
(D) Representative cell death assay of PDAC084T cells treated for 24 h with gemcitabine (1 μM), perhexiline (7 μM), or the combination. The corresponding cell survival quantification (%) is shown below. Data are mean ± SEM of two independent assays performed in duplicates. p values from Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.