Figure 5.
AYNE eliminates the leukemic population and hinders clonogenic growth by targeting long chain FAO at the site of VLCAD. (A) Death of leukemia cell lines TEX (left panel) and AML2 (right panel) following 72-hour treatment with AYNE. (B) Clonogenic growth of primary AML cells (#9-#13) and normal MNCs (n = 5) following a 2-week treatment with solvent vehicle or 10 µM AYNE. (C) Intact leukemia cell basal respiration (left panel) and maximal respiration (right panel) following 1-hour treatment with a solvent vehicle or 10 µM AYNE or 100 µM ETO. (D) Rates of complete FAO, using radiolabeled palmitate, in AML cell lines following 3-hour treatment with a solvent vehicle, AYNE (10 or 50 µM), ETO (100 µM; positive control for FAO inhibition), or cytarabine (Ara-C; 1 µM, negative control for FAO inhibition). (E) Following 12-hour treatment with a solvent vehicle or AYNE (10 or 25 µM), ACAD activity in AML2 cells was directly quantified with a fluorescence-based assay and palmitoyl-CoA (C16-CoA) or octanoyl-CoA (C8-CoA) to assess VLCAD or MCAD activity, respectively. (F) Following 1-hour treatment with a solvent vehicle or AYNE (10 or 25 µM), ACAD activity in AML2 cells was indirectly quantified via ETF respiration with palmitoyl-carnitine or octanoyl-carnitine to assess VLCAD or MCAD activity, respectively. (G) AC profiling after 96 hours in spent media of human nontransformed fibroblasts treated with a solvent vehicle or AYNE (25 or 50 µM). Viability of TEX cells (H) and AML2 cells (I) following 72-hour treatment with sodium heptanoate (C7) and AYNE. In (A,H-I), the 7-aminoactinomycin D exclusion assay was used for all viability experiments in which cells were treated for 72 hours. Data in (B-G) are mean ± standard deviation (SD). *P ≤ .05, **P ≤ .002, ***P ≤ .001, 2-tailed paired Student t test (B); 1-way ANOVA with Tukey’s post hoc test (C-G). In (H-I), 50% inhibitory concentrations were calculated using the dose response–inhibition equation using GraphPad Prism 7.0 and then compared as mean ± SD using a 2-tailed unpaired Student t test.

AYNE eliminates the leukemic population and hinders clonogenic growth by targeting long chain FAO at the site of VLCAD. (A) Death of leukemia cell lines TEX (left panel) and AML2 (right panel) following 72-hour treatment with AYNE. (B) Clonogenic growth of primary AML cells (#9-#13) and normal MNCs (n = 5) following a 2-week treatment with solvent vehicle or 10 µM AYNE. (C) Intact leukemia cell basal respiration (left panel) and maximal respiration (right panel) following 1-hour treatment with a solvent vehicle or 10 µM AYNE or 100 µM ETO. (D) Rates of complete FAO, using radiolabeled palmitate, in AML cell lines following 3-hour treatment with a solvent vehicle, AYNE (10 or 50 µM), ETO (100 µM; positive control for FAO inhibition), or cytarabine (Ara-C; 1 µM, negative control for FAO inhibition). (E) Following 12-hour treatment with a solvent vehicle or AYNE (10 or 25 µM), ACAD activity in AML2 cells was directly quantified with a fluorescence-based assay and palmitoyl-CoA (C16-CoA) or octanoyl-CoA (C8-CoA) to assess VLCAD or MCAD activity, respectively. (F) Following 1-hour treatment with a solvent vehicle or AYNE (10 or 25 µM), ACAD activity in AML2 cells was indirectly quantified via ETF respiration with palmitoyl-carnitine or octanoyl-carnitine to assess VLCAD or MCAD activity, respectively. (G) AC profiling after 96 hours in spent media of human nontransformed fibroblasts treated with a solvent vehicle or AYNE (25 or 50 µM). Viability of TEX cells (H) and AML2 cells (I) following 72-hour treatment with sodium heptanoate (C7) and AYNE. In (A,H-I), the 7-aminoactinomycin D exclusion assay was used for all viability experiments in which cells were treated for 72 hours. Data in (B-G) are mean ± standard deviation (SD). *P ≤ .05, **P ≤ .002, ***P ≤ .001, 2-tailed paired Student t test (B); 1-way ANOVA with Tukey’s post hoc test (C-G). In (H-I), 50% inhibitory concentrations were calculated using the dose response–inhibition equation using GraphPad Prism 7.0 and then compared as mean ± SD using a 2-tailed unpaired Student t test.

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