Cerulenin and oxaliplatin were obtained from Sigma (St. Louis, MO, USA). For cell culture and i.p. injections, cerulenin was dissolved in acetone at a concentration of 20 mg/ml and stored at −20°C. Oxaliplatin was dissolved in sterile water. In in vitro experiments, 12.5–100 μM of cerulenin and 0.5–2.5 μM of oxaliplatin were added to the medium. Cell viability assay and western blot experiments were performed 24 h later after adding cerulenin and oxaliplatin. In in vivo experiments, treatment with cerulenin at 15 and 30 mg/kg were given i.p. at days 7, 10, 14 and 17 after tumor inoculation. In in vivo treatment with oxaliplatin, 2.5 and 5 mg/kg of oxaliplatin were given i.p. at the same schedule as cerulenin.

The human CRC cell lines HCT116 and RKO were used and tested for mycoplasm-free cell lines. These cancer cells were subdivided in multiple tubes for stock in liquid nitrogen immediately after possession. All cell lines were subjected to the present experiment within 6 months of resuscitation. Stock cultures were grown in high-glucose DMEM containing 10% FBS and 1% antibiotics. The cells were grown in growth medium at 37°C in a 95% air, 5% CO₂-humidified incubator.

To measure the cytotoxicity of cerulenin against HCT116 and RKO cells, 3×10³ cells were plated per well onto 96-well plates. Following overnight culture, cerulenin and oxaliplatin were added at specified concentrations. After 24 h of incubation, cell viability was measured by the mitochondrial activity in reducing 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to formazan using a Cell Counting kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cells were incubated with a reagent according to the manufacturer’s instructions. Plates were read at A450 on a spectrometer.

To measure the cell proliferation activity of cerulenin and oxaliplatin against HCT116 and RKO cancer cells, 3×10³ cells were plated per well onto 96-well plates. Following overnight culture, cerulenin and oxaliplatin were added at specified concentrations. After 24 h of incubation, cell proliferation was measured with a BrdU assay kit (Roche Diagnostics, Penzberg, Germany). Cells were incubated with a reagent as per the manufacturer’s instructions. Plates were read at A450 on a spectrometer.

The In situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland) was used for the demonstration of apoptotic cell death of cell culture. Cells (3×10⁴) were plated per well onto Lab-Tek II Chamber Slides (Nalge Nunc International, Tokyo, Japan) and were incubated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction mixture according to the manufacturer’s recommendations.

For western blot analysis, total protein extracts of HCT116 cells were obtained 24 h after cerulenin and oxaliplatin treatment and separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). The following antibodies were used as primary antibodies: total Akt (9272), phosphoserine 473 Akt (9271), cleaved caspase-3 (9661), phospho-p38 (4511p), phosphoserine 15 p53 (9284p), p21waf1 (2947p) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118) (Cell Signaling Technology, Beverly, MA, USA). Purified mouse anti-FAS antibody (610962) was purchased from BD Biosciences (San Jose, CA, USA). Secondary goat anti-rabbit and goat anti-mouse antibodies conjugated with horseradish peroxidase were purchased from Cell Signaling Technology. Immunoblots were analyzed by enhanced chemiluminescence.

Eight-week-old male severe combined immunodeficiency (SCID) mice (Clea, Tokyo, Japan), weighing 24–28 g were utilized. The mice were kept in a temperature-controlled room on a 12-h light-dark cycle. They had free access to water and standard chow throughout the experiment. After an acclimation period of ≥7 days, the mice were separated into four groups as follows: control group, mice without any treatment (n=12); cerulenin group, mice with cerulenin treatment 15 (n=5) and 30 mg/kg (n=5); oxaliplatin group, mice with oxaliplatin treatment 2.5 (n=8) and 5 mg/kg (n=5); and combination group, mice with 15 mg/kg of cerulenin and 2.5 mg/kg of oxaliplatin treatment (n=10). All animal experiments were carried out in a humane manner after receiving approval from the Institutional Animal Committee of Teikyo University and in accordance with the Regulation for Animal Experiments of the University and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Cells (2×10⁶) of HCT116 were injected subcutaneously into the right flank of each mouse with a 27-gauge needle. Tumors were detected by palpation and measured periodically with calipers. Seven days after tumor injection, cerulenin and oxaliplatin were injected intraperitoneally every 3 days. Twenty-one days after inoculation, the mice were sacrificed and tumors were removed for examination. Tumor tissue, fixed in 10% buffered formalin, was used for histological analyses.

All data are expressed as the mean ± SD of samples. Comparisons between various points were made using one-way ANOVA. Significant data were examined by the Bonferroni-Dunn multiple comparisons post hoc test. In all cases, P<0.05 was considered significant.

We initially determined whether cerulenin treatment led to the inhibition of human CRC cell proliferation. CRC cells were treated with various doses of cerulenin for 24 h and cell viability was assayed using WST-8 assay (Fig. 1A and B) and BrdU assay (Fig. 1C and D). Fig. 1 shows that as the dose of cerulenin increased from 12.5 to 100 μM, cell growth inhibition increased in a dose-dependent manner in CRC cell lines HCT116 and RKO. Cerulenin-induced growth inhibition was found to be statistically significant (p<0.01) (one-way ANOVA) in 12.5–100 μM of cerulenin compared to 0 μM.

Next, we determined whether oxaliplatin treatment led to the inhibition of human CRC cell proliferation. CRC cells were treated with various doses of oxaliplatin for 24 h and cell viability was assayed using WST-8 assay (Fig. 2A and B) and BrdU assay (Fig. 2C and D). Fig. 2 shows that as the dose of oxaliplatin increased from 0.5 to 2.5 μM, cell growth inhibition increased in a dose-dependent manner in CRC cell lines HCT116 and RKO. Oxaliplatin-induced growth inhibition was found to be statistically significant (p<0.01) (one-way ANOVA) in 0.5–2.5 μM of oxaliplatin compared to 0 μM.

Next we determined whether a synergistic antitumor effect exists between cerulenin and oxaliplatin. The cerulenin effect under 0.5 μM of oxaliplatin was evaluated using WST-8 assay (Fig. 3A and B) and BrdU assay (Fig. 3C and D). The oxaliplatin effect under 25 μM of cerulenin was evaluated using WST-8 assay (Fig. 4A and B) and BrdU assay (Fig. 4C and D). The results indicated that cerulenin and oxaliplatin have synergic antitumor effects.

In subsequent experiments, we determined the mechanism of the observed suppressive effect of combination therapy by WST-8 and BrdU assays. The overexpression of FASN has been observed to cooperate with survival pathways, including the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. HCT116 cells expressed FASN and p-Akt constitutively and treatment of cerulenin suppressed FAS expression in 100 μM of cerulenin as previously reported by us (data not shown). Dephosphorylated constitutive activated Akt, activation of p38 and increased cleaved caspase-3 in cerulenin treatment (Fig. 5A). Oxaliplatin induced p53–p21 pathway and p38, but did not increase cleaved caspase-3 (Fig. 5B). In combination therapy, p53–p21 pathway and p38 activation occurred in a lower dose and induced caspase-3 cleavage (Fig. 5C).

TUNEL staining of HCT116 cells shows apoptotic cells in 25 μM of cerulenin and 0.5 μM of oxaliplatin. Combination with cerulenin and oxaliplatin induced apoptosis significantly (Fig. 6).

We evaluated the potential effectiveness of cerulenin and oxaliplatin combination for a xenograft model of HCT116, subcutaneously injected into the right flank of each mouse. Fig. 7A shows the weight of animals in control, cerulenin 15 mg/kg (cer 15) and 30 mg/kg (cer 30), oxaliplatin 2.5 mg/kg (ox 2.5) and 5 mg/kg (ox 5) and combination group, with 15 mg/kg of cerulenin and 2.5 mg/kg of oxaliplatin (cer 15 ox 2.5). In control, cer 15, ox 2.5 and ox 5 groups, significant body weight loss was observed during treatment. However, in cer 15 ox 2.5 group, significant weight loss was not observed. Fig. 7B shows tumors removed from the representative control, cer 15, cer 30, ox 2.5, ox 5 and cer 15 ox 2.5 groups. Tumor growth was significantly inhibited in the cer 30 and ox 5 group compared to the control group. In cer 15 ox 2.5 group, the tumor growth was inhibited at the same level compared to cer 30 and ox 5. Fig. 7C indicates tumor weight in the 6 groups. Tumor growth was significantly reduced in cer 30, ox 5 and cer 15 ox 2.5 groups compared to control group.