ACSL1 Regulates TNFα-Induced GM-CSF Production by Breast Cancer MDA-MB-231 Cells

Abstract

Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in different types of cancer is associated with tumor growth and progression. Tumor necrosis factor-α (TNFα) is involved in the induction of GM-CSF in different cells; however, the underlying molecular mechanism in this production of GM-CSF has not been fully revealed. Recently, it was noted that TNFα mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we investigated the role of ACSL1 in the TNFα mediated production of GM-CSF. Our results showed that MDA-MB-231 cells displayed increased GM-CSF mRNA expression and secretion after incubation with TNFα. Blocking of ACSL1 activity in the cells with triacsin C markedly suppressed the secretion of GM-CSF. However, inhibition of β-oxidation and ceramide biosynthesis were not required for GM-CSF production. By small interfering RNA mediated knockdown, we further demonstrated that TNFα induced GM-CSF production was significantly diminished in ACSL1 deficient cells. TNFα mediated GM-CSF expression was significantly reduced by inhibition of p38 MAPK, ERK1/2 and NF-κB signaling pathways. TNFα induced phosphorylation of p38, ERK1/2, and NF-κB was observed during the secretion of GM-CSF. On the other hand, inhibition of ACSL1 activity attenuates TNFα mediated phosphorylation of p38 MAPK, ERK1/2, and NF-κB in the cells. Importantly, our findings suggest that ACSL1 plays an important role in the regulation of GM-CSF induced by TNFα in MDA-MB-231 cells. Therefore, ACSL1 may be considered as a potential novel therapeutic target for tumor growth.

Keywords: ACSL1; GM-CSF; MDA-MB-231; TNFα.

Figure 1

Effect of tumor necrosis factor-α (TNFα) on granulocyte-macrophage colony-stimulating factor (GM-CSF) production in human MDA-MB-231 cells. MDA-MB-231 cells were cultured in 6-well plates at a concentration of 1 × 10⁶ cells/well. Cells were treated with vehicle and TNFα (2 ng/mL), separately. After 24 h incubation, cells and supernatants were collected. (A) Total cellular RNA was isolated and GM-CSF mRNA expression was determined by real-time PCR. (B) Secreted GM-CSF in culture media was determined by ELISA. (C) MDA-MB-231 cells were treated with vehicle or TNFα for 24 h and then were stained with GM-CSF (red) and DAPI (blue). White arrows indicate typical stained cells. (D) GM-CSF fluorescence intensity is shown. The results obtained from three independent experiments are shown. All data are expressed as mean ± SEM (n ≥ 3). ** p < 0.01, **** p < 0.0001 versus vehicle.

Figure 2

Effect of acyl-CoA synthetase 1 (ACSL1) inhibition on GM-CSF production in MDA-MB-231 cells. MDA-MB-231 cells were pretreated with a long-chain ACSL1 inhibitor (triacsin C, 5 μM), a serine palmitoyltransferase inhibitor (SPT-1) involved in sphingolipid biosynthesis (myriocin, 1 μM), a carnitine palmitoyltransferase 1 (CPT-1) inhibitor (etomoxir, 10 μM), or vehicle for 1 h and then incubated with TNFα for 24 h. (A) GM-CSF mRNA was determined by real-time PCR. (B) Secreted GM-CSF in culture media was determined by ELISA. (C) MDA-MB-231 cells were stained with GM-CSF (green) and DAPI (blue). White arrows indicate typical stained cells. (D) GM-CSF fluorescence intensity was determined. All data are expressed as mean ± SEM (n ≥ 3). * p < 0.05, **** p < 0.001 versus vehicle.

Figure 3

ACSL1 siRNA knockdown suppresses TNFα induced GM-CSF production. MDA-MB-231 cells were transfected with ACSL1 siRNA (targeting the human ACSL1 gene expression or scramble siRNA; a control siRNA). (A) After 36 h, real-time PCR was performed to measure ACSL1 expression to test knocking down efficiency. (B) ACSL1 deficient cells were then treated with vehicle and TNFα for 24 h. GM-CSF mRNA was determined by real-time PCR. (C) Secreted GM-CSF in culture media was determined by ELISA. All data are expressed as mean ± SEM (n ≥ 3). *** p < 0.001, **** p < 0.0001 versus vehicle.

Figure 4

Effect of MAPK and NF-κB pathway inhibitors on TNFα induced GM-CSF production in MDA-MB-231 cells. (A) TNFα activates the MAPK/NF-κB signaling pathway. MDA-MB-231 cells were treated with TNFα for different time points and cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Phosphorylated p38 MAPK, ERK1/2, and NF-κB are depicted in the upper panels and total respective proteins are shown in the lower panels. MDA-MB-231 cells were pretreated with p38 inhibitor (SB203580, 10uM; InvivoGen, San Diego, CA, USA) or ERK1/2 inhibitor (PD98059, 10 µM; InvivoGen, San Diego, CA, USA) or JNK inhibitor (SP600125, 10 µM) or NF-κB inhibitor (resveratrol, 1 µM) for 1 h and then treated with TNFα for 8 h. Cells and supernatants were collected. (B) Cells were used for the isolation of total RNA to assess the GM-CSF gene expression by real-time PCR. (C) Secreted levels of GM-CSF protein were determined in supernatants by ELISA. The results obtained from three independent experiments are shown. All data are expressed as mean ± SEM (n ≥ 3). **** p < 0.001 versus TNFα without respective inhibitors.

Figure 5

Inhibition of ACSL1 affects TNFα-activated MAPK and NF-κB signaling pathways in MDA-MB-231 cells. As shown in Figure 4A, TNFα treatment increases the phosphorylation of p38 MAPK, ERK1/2, and NF-κB in a time-dependent manner. MDA-MB-231 cells were pretreated with p38 inhibitor (SB203580, 10 μM), ERK1/2 inhibitor (PD98059, 10 μM), or NF-kB inhibitor (resveratrol, 1 μM) for 1 h and then treated with TNFα for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Immunoreactive bands were developed using an Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, IL, USA) and visualized by Molecular Imager® VersaDoc^TM MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA). (A) Phosphorylated proteins p38 MAPK, (C) ERK1/2, and (E) NF-κB are depicted in the upper panels and total respective proteins are shown in the lower panels. (B, D, F) Phosphorylation intensity of p38 MAPK, ERK1/2, and NF-κB was quantified using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units. All data are expressed as mean ± SEM (n ≥ 3). **** p < 0.001 versus TNFα without respective inhibitors.

Figure 6

ACSL1 siRNA knockdown reduces the TNFα induced phosphorylation of p38 MAPK, ERK1/2, and NF-κB. MDA-MB-231 cells were transfected with ACSL1 siRNA (targeting the human ACSL1 gene expression) or scramble siRNA (a control siRNA) and incubated for 36 h. ACSL1 deficient cells were then treated with vehicle and TNFα for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Immunoreactive bands were developed using an Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, IL, USA) and visualized by Molecular Imager® VersaDoc^TM MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA). (A) Phosphorylated proteins p38 MAPK, (C) ERK1/2, and (E) NF-κB are depicted in the upper panels and total respective proteins are shown in the lower panels. (B,D,F) Phosphorylation intensity of p38 MAPK, ERK1/2, and NF-κB was quantified using Image Lab Software (Bio-Rad) and presented in arbitrary units. All data are expressed as mean ± SEM. **** p < 0.001 versus TNFα-treated transfected cells with scramble siRNA.