Luteolin was purchased from Sigma-Aldrich (EMD Millipore, Billerica, MA, USA); and was dissolved in dimethyl sulphoxide and its concentration was adjusted to 100 mmol/l, as a stock solution. The Cell Counting kit-8 was supplied by Beyotime Institute of Biotechnology (Haimen, China). Annexin V-fluorescein isothiocyanate (FITC) apoptosis and cell cycle detection kits were obtained from BD Biosciences (Franklin Lakes, NJ, USA). A bicinchoninic acid protein assay kit was purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China) and monoclonal antibodies, including rabbit anti-human cell division cycle 2 (CDC2), cyclin-dependent kinase 2 (CDK2), cyclin B1, cyclin A, apoptotic protease activating factor 1 (APAF-1), cytochrome c, caspase-3, mouse anti-human procaspase-9, mouse anti-human caspase-9 and mouse anti-human β-actin, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The human colon cancer cell line, LoVo, was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 1 mmol/l HEPES buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37°C in humidified air containing 5% CO₂ until they reached ~80% confluency, and the cells were used in subsequent experiments.

LoVo cells were trypsinized and plated at 4×10³ cells/well in 96-well plates. Following incubation for 24 h, various concentrations of luteolin (0, 10, 20, 40, 60 and 80 µmol/l) were added and cells were incubated for 12, 24, 48 and 72 h, respectively. Next, 10 µl CCK8 solution (5 g/l) in phosphate-buffered saline (PBS) was added to each well. Following incubation for an additional 3 h, the optical density for each well was measured using a microculture plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm.

A total of 4×10⁵ LoVo cells per well were seeded in six-well plates for 24 h at 37°C. The cells were washed, replaced with fresh medium and subsequently incubated with various doses of luteolin (0, 20, 40 and 60 µmol/l) for 12, 24 and 48 h. The cells were then trypsinized, washed with PBS and stained with 50 µg/ml cold propidium iodide (PI) solution containing 0.1 mg/ml RNase A in PBS (pH 7.4) for 30 min in the dark at room temperature. Thereafter, cell cycle data analysis was performed using a FACSCalibur flow cytometer with CellQuest V.3.3 software (Becton-Dickinson; BD Biosciences, Franklin Lakes, NJ, USA).

Following incubation with 0, 20, 40 and 60 µmol/l luteolin for either 24 or 48 h. A total of 1×10⁵ LoVo cells were harvested, washed and resuspended with PBS. Apoptotic cells were then identified using the FACSCalibur flow cytometer (Becton-Dickinson) according to the manufacturer's protocol. Briefly, the cells were washed and subsequently incubated for 15 min at room temperature in the dark in 100 µl 1X binding buffer containing 5 µl Annexin V-FITC and 5 µl PI. Thereafter, the total apoptosis rate was examined by flow cytometry.

Western blot analysis was performed as described previously (7). Briefly, aliquots of cell lysates containing 25 µg protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, electrophoresed proteins were transferred onto nitrocellulose membranes and detected with specific primary and secondary antibodies. Thereafter, the blots were visualized using an enhanced chemiluminescence system (GE Healthcare Life Sciences, Little Chalfont, UK), and the density of β-actin served as an internal loading control.

Six-week old BALB/C nude mice (18–22 g) were obtained from Shanghai National Center for Laboratory Animals (Shanghai, China). In the present study the nude mice, which were fed with sterilized food and water ad libitum, were maintained at a temperature of 22°C and a humidity environment approximately 40–50% with a light-dark cycle of 12:12 h. All research procedures carried out in the present study were approved by the Medical Ethics Committee of Southeast University (Nanjing, China).

To assess the effect of luteolin on tumorigenicity, BALB/C nude mice were inoculated with LoVo cells for formatting LoVo colon cancer xenografts. In brief, 30 nude mice were inoculated subcutaneously into the flank with 100 ml exponentially growing LoVo cells at a concentration of 5×10⁶ cells/ml, and allowed to proliferate for ~1 week. When the tumor volume of mice reached ~100 mm³, they were randomly divided into five groups with six mice in each group. Three of these groups were administered 10, 20 or 40 mg/kg luteolin intraperitoneally on alternate days for a month. The other groups were administered either normal saline or 15 mg/kg 5-fluorouracil (5-FU) intraperitoneally as controls. During the whole experimental period, the feed intake and motor activity of mice were carefully observed, their body weights were measured, and the tumor volumes were calculated every 5 days using the following formula: Tumor volume (mm³)=(1/2)xaxb², where a is the largest diameter (length) and b is the smallest diameter (width) of the tumor. At the end of the experiments, the mice were sacrificed, the excised primary tumor mass was weighed and the tumor volume was calculated. Thereafter, the relative tumor volume (RTV) was calculated as RTV=V_{day X}/V_{first day}, and the inhibitory rate was calculated using the formula: Inhibitory rate (%)=(1-RTV_{experimental group}/RTV_{control group})×100.

All data are expressed as the means ± standard deviation for experiments performed in triplicate, and the data were analyzed using the Statistical Package for the Social Sciences (SPSS version 18.0; SPSS Inc., Chicago, IL, USA). Comparisons between two groups were performed with unpaired Student's t-test and those between three or more groups were done using one way analysis of variance followed by the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

The growth inhibitory potential of luteolin was determined in cultured LoVo cells by CCK8 assay at various intervals (12, 24, 48 and 72 h) of treatment. As a result, luteolin exhibited a significant growth inhibitory effect against LoVo cells, and the concentration of luteolin required to yield a 50% inhibitory concentration (IC₅₀) of the proliferation, as measured at the 24, 48 and 72 h time points, was 66.70, 41.49 and 30.47 µmol/l, respectively (Fig. 1). Therefore, these results demonstrated that luteolin inhibited the proliferation of LoVo cells significantly in a time- and dose-dependent manner.

The inhibition of cell proliferation may be a result of the induction of apoptosis, which may be mediated by cell cycle arrest. Therefore, the cell cycle distribution in the LoVo cells treated with luteolin was further analyzed for various times. An increased percentage of cells in the G₂/M phase together with a decrease in the S phase was observed to occur in a dose-dependent manner (Fig. 2A and B), while the percentages of G₀/G₁ phase cells remained at almost the same levels. When exposed to 40 µmol/l luteolin for various times, the cell population of LoVo cells in the G₂/M phase was 13.62±2.15% at 12 h, 22.35±2.43% at 24 h and 43.76±3.21% at 48 h, respectively, and there were significant differences compared with the control group (Fig. 2C; 5.07±1.64%; P<0.05).

To investigate the apoptotic mechanisms through which luteolin interferes with cell cycle progression, the expression of cell cycle-associated proteins was measured following treatment with various concentrations of luteolin for 48 h. The measurement of cell cycle-associated protein markers revealed that the protein expression levels of CDC2 and cyclin B, which regulate G₂/M transition in luteolin-treated LoVo cells, were downregulated, whereas those of cyclin A and CDK2 were upregulated in a dose-dependent manner (Fig. 3).

Annexin V/PI analysis was applied to quantify the percentage of cells undergoing apoptosis. Following treatment with luteolin for 24 h, the total percentages of cells undergoing early (Annexin-positive/PI-negative) and late (Annexin-positive/PI-positive) apoptosis were measured, and the results are shown in Fig. 4A and B. These results indicate that luteolin induced apoptosis in the LoVo cells in a dose-dependent manner. Furthermore, when the LoVo cells were incubated with 40 µmol/l luteolin for 12, 24 and 48 h, the apoptotic rate increased significantly with the prolonged duration of the experiment (Fig. 4C; P<0.05).

To explore the molecular mechanisms of luteolin on apoptotic proteins, Western blot analysis was conducted to evaluate the expression of APAF-1, cytochrome c, procaspase-9, caspase-9 and caspase-3 proteins. Following treatment with luteolin for 48 h, a significant decrease of procaspase-9 in LoVo cells was observed in the groups treated with 20, 40 and 60 µmol/l luteolin compared with the control group (P<0.05). By contrast, the protein expression of APAF-1, cytochrome c, caspase-9 and caspase-3 was significantly increased compared with the control group (Fig. 5).

The incidence of subcutaneous tumors derived from LoVo cells was 100%. Luteolin inhibited tumor growth in a dose- and time-dependent manner (Fig. 6A and B). On the final day of the experiment, the excised primary tumor mass was 0.32±0.09 g for 15 mg/kg 5-FU, 0.70±0.20 g for 20 mg/kg luteolin, and 0.60±0.23 g for 40 mg/kg luteolin, which was lower than that of the control group (1.17±0.29 g). Similar results were obtained for the tumor volume, as shown in Fig. 6C, but there was no significant difference between the group receiving a low dose of luteolin (10 mg/kg) and the control group (0.95±0.45 g vs. 1.17±0.29 g, and 1405.8±574.84 mm³ vs. 1081.39±794.58 mm³). The tumor inhibition rate was 72.60% in the 5-FU group, 51.28% in the 20 mg/kg luteolin group and 59.83% in the 40 mg/kg luteolin group, which was higher than that in the 10 mg/kg luteolin group (27.35%; P<0.05). In addition, mice treated with 20 and 40 mg/kg luteolin consumed slightly more food than those in the control group, but there was no mortality or significant change in mice body weight observed throughout the experimental period in the control group or luteolin-treated groups (Fig. 6D). These results suggest that luteolin treatment significantly decreases colon tumor size and tumor weight without having a significant effect on the food intake or total body weight of the mice.