Kwon et al. BMC Cancer 2010, 10:392 http://www.biomedcentral.com/1471-2407/10/392 pathway is an attractive and effective way for develop- ment of anti-cancer agents. NF B and AP1 constitu- tively active in many kinds of cancers and play critical roles in tumor development and progression through modulation of their target genes involved in angiogen- esis, metastasis and cell survival [19-21]. Recently we have reported that anti-cancer effect of cinnamon extracts is associated with modulation of angiogenesis and effector function of CD8+ T cells [22]. In this study we further identified that anti-tumor effect of cinnamon extracts is also linked with their enhanced pro-apoptotic activity by inhibiting the activities of NF B and AP1 in mouse melanoma model. Methods Animals C57BL/6 mice (6~8 weeks, male) were purchased from SLC (Japan) and maintained under specific pathogen- free conditions in an animal facility at the Gwangju Institute of Science and Technology (GIST). All of the animal experiments were approved by the GIST Animal Care and Use Committee. Preparation of cinnamon extract Dried Cinnamomum cassia bark (Hwajin Distribution Co., Seoul, Korea) was pulverized and extracted for three hours in a hot water extractor. The extract was filtered and the supernatant was concentrated with a rotary evaporator. The extract was then freeze dried resulting in a powder extract. The powder extract was suspended in sterilized distilled water at appropriate concentrations. As we reported in our previous work [22], HPLC analysis was performed by comparing the levels of trans-cinnamic acid (Sigma, USA) and cinnamic aldehyde (kindly provided by Dr. Ehren., Germany) as known standards makers for the quality control of composition of cinnamon extract in each experiment. Chromatography was carried out using 1% acetic acid (H20)-MeOH (50: 50 v/v) at room tempera- ture on a Phenomenex Luna 5u C18, 100 A pore size, 250 × 4.60 mm I.D. column. The flow rate of the mobile phase was 2 ml/min. The amount of trans-cinnamic acid and cinnamic aldehyde was about 2.9 (mg/g extract) and 7.9 (mg/g extract) in each extract [22]. Cell lines B16F10 and Clone M3 (mouse melanoma cell), Hela (human cervical carcinoma cell) and Caco2 (human epithelial colorectal adenocarcinoma cell) were obtained from the Korean Cell Line Bank (Seoul National Univer- sity, Korea) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, USA), 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin Page 2 of 10 (Sigma). To check effects of cinnamon extract in normal cells, primary mouse lymphocytes were isolated and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, nonessential amino acids, sodium pyruvate, vitamins, HEPES and 2-mercaptoethanol. Cell viability analysis Cell viability and proliferation were determined with EZ-Cytox Cell Viability Assay Kit (Daeil Labservice, Korea) based on the cleavage of the tetrazolium salt to water-soluble formazan by succinate-tetrazolium reduc- tase. Briefly, cells were treated with cinnamon extract (0.5 mg/ml) or Doxorubicin (Sigma) for indicated time points in 6 well plates. After treatment, cells were trans- ferred into 96 well plates in 100 μl of medium and incu- bated with 10 μl of Ez-CyTox solution for 5 hours in the 37°C incubator. Then absorbance were measured using the Easy Reader EAR 400 (SLT-Lab Instruments, Austria) at 420~480nm. Data was presented by relative growth inhibition to PBS treated cells. Cell cycle analysis The effect on cell division by cinnamon treatment was determined by assessing cellular DNA content using propidium iodide (PI) staining [23]. Briefly, cells were treated with 0.5 mg/ml of cinnamon extract for indi- cated time periods and then each sample was harvested and fixed in 70% ethanol for 10 hours. After fixation, cells were washed with PBS, treated with 0.5 μg/ml of DNase-free RNase (Sigma) for 20 mins at room tem- perature and stained with 100 μg/ml of PI in 0.1 M sodium citrate buffer (pH 7.4) for 30 mins at 4°C. Flow cytometric analysis (FACS) was performed with EPICS XL Cytometer (Beckman Coulter) and cell cycle distri- bution was determined with Expo32 program (Beckman Coulter) Apoptosis analysis Cells (1 × 106) were treated with cinnamon extract (0.5 mg/ml) for indicated time periods and then resus- pended in 1ml of 1× Annexin V binding buffer (BD bioscience). After incubating for 15 mins with 5 μl of Annexin V-PE and 7-ADD, at 25°C in the dark, 400 μl of 1× binding buffer was added to each tube and imme- diately analyzed by FACS. Cells stained with isotype matched normal IgG used as a control and showed less than 0.2% positive population (data not shown). Luciferase assay B16F10 cells were transfected with AP1- or NF B- dependent reporter construct that contains repeated copies of NF B or AP1 response elements. After