Figure 1.
Cerulenin and orlistat reduce cell viability and induce apoptosis in the melan-a cell line.
Melan-a cells were treated with increasing concentrations of cerulenin or orlistat for 24 or 48 h, respectively; cell viability was determined using trypan blue (A and B) or MTT assays (C and D), and apoptosis was determined by flow cytometry after Annexin V staining (E and F). The values represent the mean ± s.e.m of at least three independent experiments. *Significantly different from the respective control at p<0.05.
Figure 2.
FASN inhibitors blocked cell cycle progression in non-tumorigenic cells.
Melan-a cells were treated with 22 µM cerulenin or 30 µM orlistat for 24 or 36 h, respectively. Then, the percentage of cells in each phase of the cell cycle was determined by flow cytometry after PI staining (A). Western blot analysis of protein extracts prepared from cerulenin- and orlistat-treated melan-a cells revealed the accumulation of the p21WAF1/Cip1 tumor suppressor protein (B). The data were normalized using beta-actin as a loading control. The values represent the mean ± s.e.m of at least five independent experiments. *Significantly different from the respective control at p<0.05.
Figure 3.
Treatment of melan-a cells with FASN inhibitors leads to the release of mitochondrial cytochrome c and the activation of caspases-3 and -9 but not -8.
Melan-a cells were treated with 22 µM cerulenin or 30 µM orlistat for 12 or 24 h, respectively; then, the release of cytochrome c was determined by flow cytometry (A). The cells were also treated with cerulenin or orlistat under the same conditions, and the activation of caspase-3 was estimated using FITC-DEVD-FMK (B). The activities of caspase-9 and -8 (C and D) were determined as described in Material and Methods. The values represent the mean ± s.e.m of at least three independent experiments. *Significantly different from the respective control at p<0.05.
Figure 4.
FASN inhibitors result in decreased ΔΨm and increased superoxide production in melan-a cells.
Melan-a cells were treated with 22 µM cerulenin or 30 µM orlistat for 6 or 24 h, respectively; then, approximately 2×106 viable cells/ml were permeabilized with 15 µM digitonin. ΔΨm was estimated by Safranin fluorescence. The arrows indicate the addition of 15 µM digitonin, 100 µM ADP, 5 µM carboxyatractyloside (CAT) and 1 µM CCCP (A and B, representative of at least three independent experiments). Melan-a cells were also treated with 22 µM cerulenin or 30 µM orlistat for 24 or 48 h, respectively and also incubated in the presence or absent of NAC; the cells were then washed and probed with 5 µM MitoSOX (C). The values represent the mean ± s.e.m of four independent experiments. *Significantly different from the respective control at p<0.05. #Significantly different from the respective condition in the absence of NAC at p<0.05.
Figure 5.
Treatment with FASN inhibitors promotes the inhibition of respiration in melan-a cells.
Oxygen consumption by the melan-a cells was measured after treatment for 24 h with 22 µM cerulenin (A) or for 48 h with 30 µM orlistat (B) using high-resolution respirometry (Oroboros) in a closed chamber equipped with a magnetic stirrer and temperature control set to 37°C. Approximately 2×106 viable cells/ml were permeabilized with 15 µM of digitonin and then were added to 2 ml of reaction medium (described in Materials and Methods). Analyses of oxidative phosphorylation and respiratory activity of the mitochondria were made by sequential additions of 300 µM ADP, 2 µg/ml oligomycin, 100 nM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), 5 mM succinate, 0.5 µM antimycin and 200 µM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) with 2 mM ascorbate (A–D). The activity of citrate synthase was measured in melan-a cells after 24 or 48 h of treatment with 22 µM cerulenin or 30 µM orlistat (E). The values represent the mean ± s.e.m of at least four independent experiments. *Significantly different from the respective control at p<0.05.
Figure 6.
NAC pre-incubation protects melan-a cells from cerulenin or orlistat-induced apoptosis.
Melan-a cells were pre-incubated with 5 mM NAC for 1 h, followed by treatment with 22 µM cerulenin for an additional 24 h (A) or 30 µM orlistat for 48 h (B). NAC was also present during the incubations with cerulenin or orlistat. Apoptosis was then determined by flow cytometry after Annexin V staining. The values represent the mean ± s.e.m of six independent experiments. *Significantly different from the respective control at p<0.05. #Significantly different from the respective condition in the absence of NAC at p<0.05.
Figure 7.
FASN silencing does not induce apoptosis in melan-a cells.
Melan-a cells were either transfected with specific siRNAs (siRNA), treated with the transfection reagent alone (mock) or maintained in culture with equimolar concentrations of a nonspecific control oligo (control). Cells were incubated for 48 h. FASN protein content was determined by Western blot analysis (A), and apoptosis was estimated by flow cytometry after Annexin V staining (B). The values represent the mean ± s.e.m of three independent experiments.