ACCA/acetyl-CoA carboxylase-α; PPARalpha; apoptosis

TOFA (5-tetradecyloxy-2-furoic acid) exhibits cytotoxicity against NCI-H460 lung cancer cells as well as HCT-8 and HCT-15 colon cancer cells, with IC50 values of around 5.0, 5.0, and 4.5 μg/mL, in that order. In a dose-dependent manner, TOFA at 1.0–20.0 μg/mL efficiently inhibits the synthesis of fatty acids and causes cell death [1]. The IC50 values of ~26.1 and 11.6 μg/mL for TOFA indicated that it was cytotoxic to COC1 and COC1/DDP cells, respectively. TOFA stops the cell cycle in the G0/G1 phase, triggers apoptosis, and inhibits the growth of cancer cells in a time- and dose-dependent way [2]. Acetyl-CoA-carboxylase-α, or ACCA, is an essential enzyme that controls the production of fatty acids. In most PCa cell lines, TOFA-induced ACCA inhibition results in decreased fatty acid production, caspase activation, and cell death [3].

---

TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.[1]

---

The aim of the present study was to investigate the effects of 5‑tetradecyloxy‑2‑furoic acid (TOFA), an allosteric inhibitor of ACC, on the proliferation and cell cycle progression of the ovarian cancer cell lines COC1 and COC1/DDP. TOFA was found to be cytotoxic to COC1 and COC1/DDP cells with a 50% inhibitory concentration (IC50) of ~26.1 and 11.6 µg/ml, respectively. TOFA inhibited the proliferation of the cancer cells examined in a time‑ and dose‑dependent manner, arrested the cells in the G0/G1 cell cycle phase and induced apoptosis. The expression of the cell cycle regulating proteins cyclin D1 and cyclin-dependent kinase (CDK) 4, as well as the expression of the apoptosis‑related proteins caspase‑3 and Bcl‑2, were detected by western blot analysis. Cyclin D1, CDK4 and Bcl‑2 protein expression was inhibited by TOFA, while caspase‑3 was cleaved and activated. [2]

---

Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. [3]

COC1/DDP cell growth in ovarian tumor mouse xenografts is inhibited by TOFA. The tumor growth rate was significantly inhibited by TOFA in comparison to control mice treated with DMSO (1649±356.3 vs. 5128±390.4 mm)3. The tissues of the lung, kidney, spleen, heart, and intestines did not exhibit any toxicity. TOFA may develop into a promising small molecule medication for the treatment of ovarian cancer by blocking ACC [2].

---

TOFA inhibits growth of ovarian cancer xenografts in mice[2]
To investigate whether TOFA suppresses tumor growth in vivo, the effects of TOFA were investigated using a human ovarian cancer mouse xenograft model. COC1/DDP cells were injected into female nude mice, and tumor sizes were measured following TOFA treatment. The tumor growth rate was significantly inhibited by TOFA compared with the DMSO-treated control mice (1649±356.3 vs. 5128±390.4 mm3; Fig. 4A). To test for TOFA toxicity, the effect of TOFA was examined on multiple mouse organs with H&E staining (Fig. 4B). No toxicity was observed in the heart, liver, spleen, lung, kidney and intestinal tissues (data not shown).[2]

Apoptosis[1]
Apoptosis was evaluated by PARP cleavage, DNA fragmentation, and annexin-V staining and FACScan analysis. PARP cleavage was detected by Western blot as described above; DNA fragmentation was examined as addressed previously; and annexin-V staining and FACScan was performed as previously described.

---

Fatty acid synthesis[1]
Cells were pulsed with 1 μCi 2-14C-labeled acetate (53 mCi/mmol) per well of 12-well plates for 4 hours at 37°C, 5% CO2, followed by lipid synthesis analysis as previously described.

Cell proliferation assay[2]
TOFA was dissolved in dimethylsulphoxide (DMSO) to make a 50 mg/ml stock solution. The cells (1×104 cells/well) were seeded in 96-well plates and incubated with TOFA at various concentrations (0, 1, 5, 10, 20 and 50 μg/ml). Cell proliferation was assessed 24, 48 and 72 h following TOFA treatment by measuring the reduction of the tetrazolium salt WST-8 to formazan using the Cell Counting kit-8 (CCK-8) purchased from Dojindo according to the manufacturer’s instructions. At each time point, 10 μl of the CCK-8 solution was added to each well and cultured at 37°C for 2 h. The supernatant from each plate was collected and the absorbance was measured at 450 nm. The inhibition rate of cell proliferation and the 50% inhibitory concentration (IC50; calculated by nonlinear regression) was determined according to the following equation: Inhibition rate (%) = [(Ac−Ae)/(Ac−Ab)] × 100%, where Ae, absorbance of the culture media in experimental wells; Ac, absorbance of the culture medium in control wells and Ab, absorbance of the culture medium in blank wells.

---

Cell cycle analysis[2]
COC1 and COC1/DDP cells were seeded in 6-well plates at a density of 2×105 cells/well and treated with or without TOFA (5 or 10 μg/ml) for 24, 48 and 72 h. For cell cycle analysis, the cells were centrifuged at 466 × g for 5 min and washed twice with cold phosphate-buffered saline (PBS). The cells were fixed with 70% ice-cold ethanol and stored at −20°C. The cells were washed with PBS 24 h later, treated with RNase A and incubated at 37°C for 30 min according to the manufacturer’s protocol. Propidium iodide (PI) was added (400 μl of a 20-μg/ml solution), the cells were incubated in the dark for 30 min and the absorbance was measured at 488 nm.

---

Apoptosis assay[2]
Ovarian cancer cells COC1 and COC1/DDP (2×105 cells/well) were treated with TOFA (0, 1, 5, 10, 20 and 50 μg/ml) for 24, 48 and 72 h. Subsequently, an apoptosis detection kit was used according to the manufacturer’s instructions. The cells were collected by centrifugation at 466 × g for 5 min, washed twice with cold PBS and suspended in 100 μl binding buffer. Annexin V-FITC (5 μl) and PI (5 μl) were added to the culture media of cells and the cells were incubated for 15 min at room temperature in the dark. Binding buffer (400 μl) was added and fluorescent intensities were determined by flow cytometry at 488 nm

---

TOFA suppresses ovarian cancer cell proliferation[2]
To investigate the effect of TOFA on ovarian cancer cells, COC1 and COC1/DDP cell lines were treated with various concentrations of TOFA (1–50 μg/ml) for 24–72 h, and cell proliferation was assessed. The various concentrations of TOFA used were found to inhibit COC1 and COC1/DDP cell growth in a concentration- and time-dependent manner (Fig. 1). TOFA was also shown to be highly cytotoxic to ovarian cancer cells. The IC50 values of COC1 and COC1/DDP cells for TOFA were ~26.1 and ~11.2 μg/ml, respectively, after 48 h of treatment. These data indicate that fatty acid synthesis plays an important role in the proliferation of the ovarian cancer cell lines COC1 and COC1/DDP.

---

TOFA induces cell cycle arrest[2]
To investigate whether TOFA induced cell cycle arrest in ovarian cancer cells, COC1 and COC1/DDP cells were cultured with TOFA for 24, 48 and 72 h, followed by cell counting using FACScan analysis. COC1 cells treated with TOFA were arrested in the G0/G1 phase. Particularly, treatment with 5 μg/ml TOFA for 48 and 72 h increased the percentage of COC1 cells in this phase to 50.2 and 51.1%, compared with the percentage of the untreated control groups, 28.1 and 34.4% (P<0.01), respectively (Fig. 2B and C). The treatment of COC1/DDP cells with TOFA (10 μg/ml) for 72 h increased the percentage of cells in the G0/G1 phase from 38.3 (control) to 43.0% (P<0.05; Fig. 2G). These results indicate that cell cycle progression from the G1 to the S phase is associated with fatty acid synthesis. To investigate the mechanism underlying cell cycle arrest, the protein levels of cyclin D1 and cyclin-dependent kinase (CDK) 4, two proteins that regulate progression from the G1 to the S phase, were examined using western blot analysis (Fig. 2D and H). TOFA treatment of COC1 and COC1/DDP cells decreased cyclin D1 protein levels in a dose-dependent manner. However, CDK4 protein levels were increased in TOFA-treated COC1 cells (1–20 μg/ml), followed by a subsequent decrease when 50 μg/ml TOFA was used (Fig. 2D).

---

NCI-H460, human lung cancer cells, and HCT-8 and HCT-15 cells (5,000/well) are seeded in 96-well plates overnight and then exposed to TOFA at indicated concentrations (0, 1, 5, 10, 20, 50 µg/mL) for 72 hours. Viable cells are detected using MTT assay.

COC1/DDP tumor xenografts in mice[2]
Female athymic BALB/c nude mice, 4–5 weeks old, weighing 17–20 g were used. COC1/DDP cells were collected by centrifugation at 300 × g for 5 min and suspended in RPMI-1640 medium at a density of 2×106 cells/100 μl. The cells were subcutaneously injected into both right and left flanks of each mouse. Twenty days later, mice were randomly allocated into one of the 2 groups (n=5 mice/group): i) mice treated with 50 μl DMSO (control group) or ii) mice treated with TOFA (50 mg/kg). The drugs were injected intraperitoneally daily for two weeks. Tumor volumes were recorded every two days by measuring dimensions (length and width) with Vernier calipers. The mice were sacrificed four weeks after the final treatment. Tumor weights were measured by a scale. The formula used to determine tumor sizes was A × B2 × 0.5236 (A, length; B, width; all measured in mm). For histopathological examination, tumors and multiple organs were fixed in 10% neutral-buffered formalin and wax embedded. The tissues were cut into 4-mm sections and stained with hematoxylin and eosin (H&E).

---

50 mg/kg; i.p.

Mice

[1]. Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis. Biochem Biophys Res Commun. 2009 Jul 31;385(3):302-6.

[2]. TOFA suppresses ovarian cancer cell growth in vitro and in vivo. Mol Med Rep. 2013 Aug;8(2):373-8.

[3]. TOFA (5-tetradecyl-oxy-2-furoic acid) reduces fatty acid synthesis, inhibits expression of AR, neuropilin-1 and Mcl-1 and kills prostate cancer cells independent of p53 status. Cancer Biol Ther. 2011 Jul 1;12(1):80-5.

5-(tetradecyloxy)-2-furoic acid is a member of the class of furans that is 2-furoic acid in which the hydrogen at position 5 is replaced by a tetradecyloxy group. It has a role as an EC 6.4.1.2 (acetyl-CoA carboxylase) inhibitor, a PPARalpha agonist, an antineoplastic agent and an apoptosis inducer. It is a furoic acid and an aromatic ether. It is functionally related to a 2-furoic acid.

C19H32O4

324.45

324.23

C, 70.33; H, 9.94; O, 19.72

54857-86-2

115175

White to off-white solid powder

1.0±0.1 g/cm3

441.7±25.0 °C at 760 mmHg

112-115ºC

220.9±23.2 °C

0.0±1.1 mmHg at 25°C

1.483

7.82

1

4

15

23

293

0

CZRCFAOMWRAFIC-UHFFFAOYSA-N

InChI=1S/C19H32O4/c1-2-3-4-5-6-7-8-9-10-11-12-13-16-22-18-15-14-17(23-18)19(20)21/h14-15H,2-13,16H2,1H3,(H,20,21)

5-tetradecoxyfuran-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~4 mg/mL ( 12.32 mM）
Water: Insoluble
Ethanol: Insoluble

Solubility in Formulation 1: ≥ 1 mg/mL (3.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 2: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O : 3mg/ml (9.25mM)

---

 (Please use freshly prepared in vivo formulations for optimal results.)