Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide

Abstract

Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl-CoA synthetase (ACS), which converts long-chain fatty acids to acyl-CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c-resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax-induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well-known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non-toxic dose enhanced the antitumor efficacy of a low-dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide.

Figure 1

Acyl‐CoA synthetase (ACS) catalytic activity requirement for glioma cell survival. (a) Effect of 48‐h Triacsin c treatment (3 µM) on ACS activity in mock‐, ACSL5‐, and ACSL5‐MT–transduced SF268 cells. ACS activity was measured as described in ‘Materials and Methods’. Inset, The expressions of ACSL5 and ACSL5‐MT were examined by western blot with anti‐FLAG M2 antibody. The expression of α‐tubulin was analyzed as a loading control. (b) Apoptosis induction after Triacsin c treatment. Cells were treated as in (a). The cell nuclei were stained with Hoechst 33342. Induction of apoptosis in each cell was evaluated using the characteristic nuclear features of apoptosis, such as chromatin condensation and nuclear fragmentation. Arrows indicate apoptotic cells. (c) Caspase activation after Triacsin c treatment. Cells were left untreated or treated with 3‐µM Triacsin c in the absence or the presence of 50‐µM caspase inhibitor, Z‐Val‐Ala‐Asp(OMe)‐CH2F (Z‐VAD), for 30 h. Caspase activity was measured as described in ‘Materials and Methods’. (d) Cell number after Triacsin c treatment. Cells were treated as in (c) for 48 h and relative cell number was measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) method, as described in ‘Materials and Methods’. Data are mean values of three independent experiments. Error bars show standard deviations.

Figure 2

Organelle‐localized acyl‐CoA synthetase (ACS) is critical for cancer cell survival. (a) Localization of human ACSL5 in SF268 cells. ACSL5 (FLAG) and the mitochondria marker cytochrome c (Cyto c) were detected by indirect immunofluorescence stain of ACSL5‐transduced SF268 cells with anti‐FLAG M2 (red) and anti‐cytochrome c (green) antibodies, respectively. DAPI staining of DNA is shown in blue. (b) Subcellular fractionation of ACSL5 and its N‐terminal deletion mutant (delta L) in SF268 cells. Cytoplasmic (C), organelle/membrane (O/M), and nuclear (N) fractions were prepared, and subjected to western blot analysis with the indicated primary antibodies. Blots with anti‐FLAG M2 antibody (FLAG) indicate the expression of ACLS5. Blots with anti‐Heat shock protein (Hsp)‐90, voltage‐dependent anion channel (VDAC), epidermal growth factor receptor (EGFR), and poly(ADP‐ribose) polymerase (PARP) antibodies demonstrate the purity of their respective fractions. (c) Caspase activation in SF268/ACSL5 and SF268/delta L cells after Triacsin c treatment. Mock‐, ACSL5‐, and delta L–transduced SF268 cells were left untreated or treated with 3‐µM Triacsin c for 30 h. Caspase activity was measured as described in ‘Materials and Methods’. (d) Cell number after Triacsin c treatment. Cells were treated as in (c) for 48 h and relative cell number was measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) method. In (c) and (d), data are mean values of three independent experiments, and error bars show standard deviations.

Figure 3

Potentiating the mitochondrial apoptosis pathway by acyl‐CoA synthetase (ACS) inhibition. (a) To estimate Bax‐induced caspase activation, mock‐ and ACSL5‐transduced SF268 cells were seeded in six‐well plates and transiently transfected with pCGBL‐HA‐Bax (0, 0.1, and 0.2 µg/well) and pGVC, a luciferase‐expressing construct (0.4 µg/well). At 6 h after transfection, cells were left untreated or were treated with 1 µM Triacsin c for an additional 24 h. Each cell lysate was prepared and caspase activity measured as described in ‘Materials and Methods’. The expression of Bax (HA) was examined by western blot. The expression of α‐tubulin was analyzed as a loading control. (b) Cells were transiently transfected with the Bax plasmid vector and then were treated with Triacsin c as in (a). Cytochrome c release from the mitochondria to cytoplasm was monitored by western blot analysis. (c) Mock‐ and ACSL5‐transduced SF268 cells were left untreated or treated with 1 µM Triacsin c for 24 h. Cytosolic extracts were prepared and incubated with 10 µM cytochrome c and 1 mM dATP for 0–40 min. After the incubation, caspase activity was measured, as described in ‘Materials and Methods’. In (a) and (c), data are mean values of three independent experiments. Error bars show standard deviations.

Figure 4

Potentiating etoposide‐induced cell death by acyl‐CoA synthetase (ACS) inhibition. (a) Enhanced activation of caspase by etoposide in combination with Triacsin c. Mock‐ and ACSL5‐transduced SF268 cells were left untreated or were treated with 0.3 µg/mL of etoposide, 1 µM Triacsin c or 0.3 µg/mL of etoposide and 1 µM Triacsin c for 48 h. Caspase activity was measured as described in ‘Materials and Methods’. (b) Potentiation of apoptosis by etoposide in combination with Triacsin c. Mock‐ and ACSL5‐transduced SF268 cells were treated as in (a), and apoptotic cells were evaluated and counted. (c) Effect of ACS inhibition on etoposide‐induced cytotoxicity. Mock‐ and ACSL5‐transduced SF268 cells were left untreated or were treated with the indicated concentrations of etoposide in the absence or presence of 1 µM Triacsin c for 48 h. Cell viability was measured, using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) method. Data are mean values of three independent experiments. Error bars show standard deviations.

Figure 5

Triacsin c enhances the efficacy of etoposide in vivo. Therapeutic experiments (five mice per group) were started (day 0) when U251 tumors reached 90–170 mm³. Etoposide (12 mg/kg/day) was administrated i.v. on days 0, 1, and 2. Triacsin c (4 mg/kg/day) was administrated by intratumoral injection in 40 µL of saline on days 0, 1, and 2. Control mice received the same volume of saline. Relative tumor volumes and body weight changes of the mice are shown in (a) and (b), respectively. Data are mean values for five mice, and error bars show standard deviations. Statistical evaluations were performed as described in ‘Materials and Methods’. **P < 0.01.