Figure 3.

TP4O induces ROS in human CRC cell lines. (A) ESR measurement. HCT116 and RKO cells were treated with 0 or 1,000 µM of TP4O for 15 min. ESR was measured in a respiration buffer containing 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trapping agent. The production of the hydroxyl radical was detected with four spikes in ESR. Spikes derived from the hydroxyl radical (indicated with arrows) became apparent in cells exposed to 1,000 µM TP4O. (B) Quantitative measurements of cellular populations undergoing oxidative stress, based on the detection of ROS. HCT116 and RKO cells were untreated or treated with 1,000 µM TP4O for 24 h. The distribution of cells according to levels of intracellular ROS was represented as a histogram. Cells were divided into two groups: The blue area (M1) represented the cells with low ROS, and the red area (M2) represented the cells with high ROS. The administration of TP4O led to an increase in the number of cells in the red area. (C) The mean fluorescence intensity of each group is presented. The values were compared with those obtained with 0 µM TP4O. The data are presented as the mean ± SD. *P<0.05. The results were obtained from four independent experiments. TP4O, terpinen-4-ol; CRC, colorectal cancer; IC₅₀, half maximal inhibitory concentration; SD, standard deviation; ROS, reactive oxygen species; DHE, dihydroethidium; ESR, electron spin resonance.