Anticancer effect of linalool via cancer-specific hydroxyl radical generation in human colon cancer

Figure 1 Anticancer effect of linalool. A: HCT 116 cells in the control group showing rapid proliferation; B: HCT 116 cells treated with 250 μmol/L linalool for 24 h showed shrinkage of cells and tightly packed organelles; C: A dose-dependent increase in the cytotoxic effect of linalool was observed in all cancer cell lines. Values are expressed as mean ± SD (n = 3).

Figure 2 Anticancer mechanism of linalool. A: TUNEL staining of HCT 116 cells in the control group; B: Staining of HCT 116 cells treated with 100 μmol/L linalool; C: Staining of HCT 116 cells treated with 50 nmol/L staurosporine. The corresponding percentage of TUNEL-positive cells (white arrow) was 0.7% ± 0.7% (A), 10.5% ± 7.7% (B), and 25.1% ± 11.2% (C); D: The percentage of apoptotic cells in each group (mean ± SD, n = 10). ^aP < 0.05 vs control group; ^bP < 0.05 vs 100 μmol/L linalool group; E: Flow cytometry using Annexin V for the detection of live cells, cells showing early and late apoptosis, and cell death; F: Percentages of early and late apoptosis fraction. ^cP < 0.05 vs control group; ^dP < 0.01 vs control group; ^eP < 0.05 vs control group; one-way ANOVA followed by Dunnett’s test, n = 3.

Figure 3 Changes in mouse tumor size and weight after drug administration. A: The pictures show the size of the tumors in each group. Upper: control group. Middle: 100 mg/kg linalool group. Lower: 200 mg/kg linalool group. As concentration of linalool increased, the xenografted tumor size decreased macroscopically; B: Tumor weight after removal showed a significant decrease in the group of 200 mg/kg administration, compared to that of control group [expressed as geometric mean, n = 5 (control), 5 (100 mg/kg linalool), 6 (200 mg/kg linalool)], ^aP < 0.05 vs control group; one-way ANOVA followed by Dunnett’s test); C: No significant change was detected in serum AST and ALT level in each group; values are expressed as mean ± SD, n = 5 (control), 5 (100 mg/kg linalool), 6 (200 mg/kg linalool).

Figure 4 Oxidative stress assay in vitro. A: Electron spin resonance (ESR) spectroscopy performed 30 min after linalool treatment. The signal intensity of 1:2:2:1 suggests generation of hydroxyl radical, the strongest reactive oxygen species (ROS); B: The signal of HCT 116 without linalool at the same time period; C: Diphenyl-1-pyrenylphosphine (DPPP) assay performed to detect lipid peroxidation showed that linalool induced cell membrane lipid peroxidation as green fluorescent on the cell surface(white arrow); D: The cell membrane lipid peroxidation was not observed in without linalool treatment group.

Figure 5 Oxidative stress assay in vivo. Although accumulation of 4-hydroxynonenal (4-HNE) was observed in the tumors from the high-dose linalool group, it was not observed in stomach or liver tissues, except for the physiological accumulation (black arrow) of 4-HNE near the basal membrane of the stomach.