1, 8-cineole (eucalyptol) was obtained from Sigma (St. Louis, MO, USA). For cell culture experiment, 1, 8-cineole was dissolved in ethanol and then in water at a concentration of 0.1 mg/ml. In an in vitro experiment, 5–50 mM of 1, 8-cineole was added to the medium and cell viability assay and western blot experiments were performed 24 h later.

The human CRC cell lines HCT116 and RKO were used and tested for mycoplasma-free cell lines. The cancer cell lines were subdivided into 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 1, 8-cineole against these cancer cells, 3×10³ cells were plated per well onto 96-well plates. Following overnight culture, 1, 8-cineole and oxaliplatin were added at specified concentrations. After 24 h 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 as per the manufacturer's instructions. Plates were read at A450 on a spectrometer.

To measure the cell proliferation activity of 1, 8-cineole against HCT116 and RKO cancer cells, 3×10³ cells were plated per well onto 96-well plates. Following overnight culture, 1, 8-cineole was 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.

In order to evaluate the activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) released from the cytosol when cells were damaged under stress, HCT116 and RKO cells were seeded (1×10⁵ cells/ml) on 96-well plates. The LDH activity was determined using a commercial kit (Takara Bio, Tokyo, Japan). Absorbance values were then correlated with the number of viable cells to predict the cytotoxic activity. Triton X-100 (1%) (Sigma) was used as a positive control.

The In Situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland) was used for the demonstration of apoptotic cell death of cell culture and liver tissue. 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 1, 8-cineole treatment and separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). The following antibodies were used as primary antibodies: cleaved caspase-3 (9661), cleaved poly(ADP-ribose) polymerase (PARP) (5625p), survivin (2808p), phosphoserine 473 Akt (9271), phospho p38 (4511p) and GAPDH (2118) (Cell Signaling Technology, Beverly, MA, USA). Secondary goat anti-rabbit antibody conjugated with horseradish peroxidase was purchased from Cell Signaling Technology.

Seven-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 two groups as follows: control group, mice without any treatment (n=10); and 1, 8-cineole group, mice with 1, 8-cineole treatment (n=9). 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 jurisdication of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Cells (2×10⁶) of RKO 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, 50 mg/kg of 1, 8-cineole was injected subcutaneously every 3 days. Twenty-one days after inoculation, mice were sacrificed and tumors were removed for examination.

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

We initially determined whether 1, 8-cineole treatment led to the inhibition of human CRC cell proliferation. CRC cells were treated with various doses of 1, 8-cineole 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 1, 8-cineole increased from 5 to 50 mM, cell growth inhibition increased in a dose-dependent fashion in CRC cell lines HCT116 and RKO. 1, 8-cineole-induced growth inhibition was found to be statistically significant (p<0.01) (one-way ANOVA) in 5–50 mM of 1, 8-cineole compared to 0 mM in WST-8 assay and 5–25 mM in BrdU assay.

Fig. 2 shows the cytoplasmic LDH released of HCT116 and RKO cells treated with each sample at 5–50 mM of 1, 8-cineole. Compared to culture medium (negative control), no statically significant increase in LDH released by these cells was observed. However, all samples showed a statistically significant difference (p<0.01) in LDH released when compared to 1% Triton X-100 (positive control). Damaged cells or those under stress can release cytoplasmic LDH and other substances into the medium due to disruption of the cytoplasmic membrane and cell necrosis (13). Therefore, it is possible that the cytotoxic activity of the samples was not based on the mechanism of cell death by necrosis. Consequently, these results indicate that the cytotoxic activity of the samples can involve pathways inducing cell death by apoptosis.

In subsequent experiments, we determined the mechanism of the observed suppressive effect of 1, 8-cineole by WST-8 and BrdU assays. The overexpression of survivin has been observed to cooperate with survival pathways, including the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Suppression of survivin and constitutive activated Akt and activation of p38 was observed in 12.5–50 mM of 1, 8-cineole. These molecules induced increments of cleaved PARP and caspase-3 in 1, 8-cineole treatment (Fig. 3A). TUNEL staining of RKO cells showed apoptotic cells in 50 mM of 1, 8-cineole (Fig. 3B).

We evaluated the potential effectiveness of 1, 8-cineole in a xenograft model of RKO, which was subcutaneously injected into the right flank of each mouse. Fig. 4A shows representative mice of each group. No skin damage was observed in the 1, 8-cineole group. Fig. 4B shows tumors removed from the representative control and mice with 1, 8-cineole groups. Fig. 4C indicates tumor growth curve. Fig. 4D indicates tumor weight in the 2 groups. Tumor growth was significantly inhibited in the 1, 8-cineole group, compared to the control group.