Cytotoxicity and Radiosensitizing Activity of the Fatty Acid Synthase Inhibitor C75 Is Enhanced by Blocking Fatty Acid Uptake in Prostate Cancer Cells

Abstract

Prostate cancers, like many other types of cancer, express elevated levels of fatty acid synthase (FASN) to make more fatty acids, which are required for energy, signaling, and proliferation. Because inhibition of FASN has been shown to sensitize tumors to chemotherapy and radiation, we studied the effect of C75, a radiosensitizing FASN inhibitor, and compared its single agent and radiosensitizing activities in 2 prostate cancer cell lines, PC3 and LNCaP, with alternative FASN inhibitors that have progressed into clinical trials. We also investigated the effect of serum and fatty acid supplementation on responses to FASN inhibitors, probing expression of key proteins related to fatty acid uptake in response to FASN inhibition, irradiation, and serum lipid concentration and how this may be modulated to increase the potency of C75. We demonstrated that C75 was the only FASN inhibitor to sensitize cells to ionizing radiation; no sensitization was apparent with FASN inhibitors TVB-3166 or Orlistat. The prostate cancer cell lines were able to take up fatty acids from the culture medium, and the availability of fatty acids affected sensitivity of these cells to C75 but not the other FASN inhibitors tested. C75 also increased expression of fatty acid transporter proteins FATP1 and CD36. Furthermore, blocking CD36 with antibody increased the sensitivity of cells to C75. We suggest that the potency of C75 is affected by fatty acid availability and that the effectiveness of FASN inhibitors in combination with ionizing radiation can be further enhanced by regulating fatty acid uptake.

Figure 1

Cytotoxic effect of inhibitors of fatty acid synthase. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of (A) PC3 cells and (B) LNCaP cells after 24-hour exposure to C75, TVB-3166, or Orlistat. Data are means ± standard error of the mean (SEM), n = 3. (C) Representative images of PC3 and LNCaP cells after 24-hour treatment with control dimethyl sulfoxide (DMSO) or fatty acid synthase (FASN) inhibitors (50 μM drug) in medium containing 10% serum. Bars represent 200 μm.

Figure 2

Combination of drugs with ionising radiation. (A) Clonogenic assay of PC3 cells 24 hours after simultaneous administration of x-rays and C75 (30 μM). (B) Clonogenic assay of PC3 (prostate cancer), MCF7 (breast cancer), UVW (glioma), and SK-N-BE(2c) (neuroblastoma) cells 24 hours after simultaneous administration of x-rays (2 Gy) and C75 (35 μM). Data are means ± standard error of the mean (SEM), n = 3. ^∗P < .05 and ^†P < .01 compared with single agent treatments. Clonogenic assay of PC3 cells 24 h after simultaneous administration of x-rays and (C) TVB-3166 or (D) Orlistat.

Figure 3

Serum concentration affects sensitivity to C75. (A) Clonogenic assay of PC3 cells after 24-hour culture in serum containing low (1%) or normal (10%) serum. (B) Surviving fraction of PC3 cells in clonogenic assay carried out 24 hours after exposure to x-rays (1 or 2 Gy). No significant difference was observed between treatments in 1% or 10% serum. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of (C) PC3 cells and (D) LNCaP cells 24 hours after treatment with C75 administered as either the racemic mixture (±) or the (-) enantiomer in medium containing 1% or 10% serum. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of (E) PC3 and (F) LNCaP cells was also carried out in 1% serum-containing medium with the addition of oleic acid-bovine serum albumin solution (100 μM oleic acid) and compared with the effect of C75 (50 μM) in 10% serum-containing medium. (G) Clonogenic assay of PC3 cells 24 h after culture in medium containing 1% or 10% serum or 1% serum with oleic acid (100 μM) with and without C75 (35 μM). Data are expressed compared with vehicle treated controls and are means ± standard error of the mean (SEM), n = 3, except (G) n = 4. ^∗P < .05 and ^†P < .01 compared with C75 in 1% serum-containing medium.

Figure 4

Serum concentration does not affect sensitivity to TVB-3166 or Orlistat. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of (A) PC3 and (B) LNCaP cells 24 h after administration of TVB-3166 in either 1% or 10% serum medium. (C) PC3 or (D) LNCaP cells were cultured in 1% or 10% serum medium with TVB-3166 (50 μM) and oleic acid (100 μM) before MTT assay. (E) MTT assay of PC3 and LNCaP cells 24 h after administration of Orlistat in 1% or 10% serum medium. Data are expressed compared with vehicle treated controls and are means ± standard error of the mean (SEM), n = 3-5.

Figure 5

Lipid accumulation and fatty acid uptake in cells. (A) Protein expression of fatty acid transporter 1 (FATP1) and CD36 in PC3 and LNCaP cells was decreased by culture for 24 h in increased concentrations of serum in medium. (B) FATP1 and CD36 expression was increased by x-rays (2 Gy) and C75 (35 μM) in PC3 and LNCaP cells cultured in medium containing 10% serum. Representative blots are shown. Loading control was β-actin. (C) Representative images of lipid droplets in PC3 and LNCaP cells visualized by Oil Red O staining 24 h after culture in medium containing 1% serum with or without oleic acid (100 μM). Bars represent 500 μm. (D) Absorbance of PC3 cells and LNCaP cells exposed to oleic acid for 24 h then stained with Oil Red O. Stain was solubilized with isopropanol. n = 3. (E) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of PC3 cells every 24 h after culture in medium containing 1% serum and 100 to 500 μM oleic acid. Data are expressed compared with vehicle treated controls and are means ± standard error of the mean (SEM), n = 4.

Figure 6

CD36 blocking antibody enhanced effect of C75. (A) Clonogenic assay of cells cultured for 24 h in medium containing 1% or 10% serum and treated with x-rays (2 Gy), C75 (35 μM), or a combination of both. ^∗P < .05 between different serum concentrations. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of (B) PC3 and (C) LNCaP cells in 10% serum medium after 24 h treatment with C75 (50 μM) in the absence or presence of CD36 antibody (10 μg/mL). (D) Clonogenic assay of PC3 cells treated in 10% serum containing medium for 24 h with x-rays (2 Gy), C75 (35 μM), or combination treatment in the absence or presence of CD36 antibody (10 μg/mL). Data are expressed compared with vehicle treated controls and are means ± standard error of the mean (SEM), n = 3. ^∗P < .05 between presence and absence of antibody.