Several essential oils possess pharmacological effects. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects such as anti-bacterial and anti-inflammatory effects. The effect of 1, 8-cineole on human colorectal cancer cells, however, has not reported previously. In this study, we have investigated the anti-proliferative effect of 1, 8-cineole on human colon cancer cell lines HCT116 and RKO by WST-8 and BrdU assays. The cytotoxicity of 1, 8-cineole was investigated by LDH activity and TUNEL staining. The mechanism of apoptosis by 1, 8-cineole was determined by western blot analyses. In
The use of plant-derived natural products for medical benefits is playing an important role globally. Several anticancer drugs available on the market today such as Taxol, Oncovin, Navelbine and Vumon trace their origins to plants (
Several essential oils from plants possess medical benefits. Among the various constituents of essential oils, 1, 8-cineole has been shown to possess pharmacological effects. The content of 1, 8-cineole in the essential oils varies in the different Eucalyptus species, from 25 to 90% (
In this study, we found that 1, 8-cineole exerts antitumor activity on human colon cancer cell lines HCT116 and RKO. We investigated whether 1, 8-cineole induces apoptosis in two human colorectal cancer cell lines and in a xenograft model.
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
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
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 (
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 (
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.
Essential oils and their components have been investigated regarding their effect against a variety of human cancer cell lines. Lemon balm was verified to show cytotoxic activity against some human cancer cell lines and a mouse cell line (
Among the various constituents of essential oils extracted from Eucalyptus species, 1, 8-cineole is one of the main constituent in the essential oils from Eucalyptus species (
The mechanism of cell killing by 1, 8-cineole is not fully understood. Suppression of growth by 1, 8-cineole in the leukemia cell lines was reported to the induction of apoptosis (
In a xenotransplant mouse model, 1, 8-cineole therapy showed tumor shrinkage comparable to the control group. Throughout the experiments, the animals had no weight loss in 1, 8-cineole and control groups (data not shown). Moreover, we could not observe any skin damage in the 1, 8-cineole group, which was injected locally.
In conclusion, these findings demonstrate that 1, 8-cineole might exert its antitumor activity by triggering apoptosis in human colorectal cancer cells
The authors thank Satoko Nakabayashi for technical assistance. This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
Effect of 1, 8-cineole on the WST-8 assay and BrdU assay of human CRC cell lines. CRC cell lines were treated with 0–50 mM 1, 8-cineole for 24 h. (A) RKO; (B) HCT116. *p<0.01 compared to 0 mM 1, 8-cineole. The values indicate ratio compared to 0 mM 1, 8-cineole as 100% control. In the BrdU assay of human CRC cell lines, cell lines were treated with 0–25 mM 1, 8-cineole for 24 h. (C) RKO; (D) HCT116. *p<0.01 compared to 0 mM 1, 8-cineole. The values indicate ratio compared to 0 mM 1, 8-cineole as 100% control.
Effect of 1, 8-cineole on the LDH activity assay of human CRC cell lines. CRC cell lines were treated with 0–50 mM 1, 8-cineole for 24 h. (A) RKO; (B) HCT116. *p<0.01 compared to 1% Triton X-100. The values indicate ratio compared to 1% Triton X-100 as 100% control.
(A) RKO cells were treated with 0–50 mM of 1, 8-cineole for 24 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to Immobilon membrane and immunoblotted with antibodies against survivin, p-Akt, p-p38, cleaved caspase-3, cleaved PARP and GAPDH as indicated. 1, 8-cineole treatment caused suppression of survivin, dephosphorylation of constitutive phosphorylation of Akt, p38 activation and cleavage of PARP and caspase-3. (B) TUNEL staining of RKO cells, which were treated with 0, 5 and 50 mM of 1, 8-cineole. Stained cells indicate TUNEL positive apoptotic cells.
1, 8-cineole therapy significantly inhibits tumor growth of the xenograft RKO tumors in SCID mice. RKO cells were injected into the right flank subcutaneously. Seven days later, 50 mg/kg of 1, 8-cineole was injected subcutaneously 4 times, with 3-day intervals. Control animals were prepared as no treatment. (A) Representative mice of the 2 groups. Arrows indicated tumors. No skin damage was observed in the 2 groups. (B) Representative xenograft tumors of the 2 groups. (C) Tumor volume of the 2 groups. Columns, mean; bars, SD. White bar, control group. Black bar, 1, 8-cineole group. *p<0.05 versus control group. (D) Tumor weight of the 2 groups. **p<0.01 versus control group.