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 (H2
0)-MeOH (50: 50 v/v) at room temperature 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
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 University, 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 (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 reductase. 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 transferred into 96 well plates in 100 μl of medium and incubated 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 indicated 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 temperature 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 distribution was determined with Expo32 program (Beckman Coulter)
Cells (1 × 106) were treated with cinnamon extract (0.5 mg/ml) for indicated time periods and then resuspended 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 immediately 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).
B16F10 cells were transfected with AP1- or NFκB-dependent reporter construct that contains repeated copies of NFκB or AP1 response elements. After 18 hours culture in complete media, cells were stimulated with PMA (phorbol 12-myristate 13-acetate) and ionomycin (P+I) for 4 hours in the presence or absence several dose of cinnamon extract from 0.1 mg/ml to 0.5 mg/ml. Luciferase activity measured by dual luciferase assay system (Promega) is expressed relative to expression of the cotransfected Renilla luciferase promoter (phRL-null; Promega) to control for transfection efficiency.
RNA isolation, cDNA synthesis, quantitative RT-PCR and standard RT-PCR
Total RNA was prepared using TRI Reagent (Molecular Research Center) according to the manufacturer's protocol. For reverse transcription, cDNA was generated using 1 μg of total RNA, oligo (dT) primer (Promega) and Improm-II Reverse Transcriptase (Promega) in a total volume of 20 μl. One μl of cDNA was amplified using the following RT-PCR primer sets: L32 (5'-GAGGACCAAGAAGTTCATCAG-3' and 5'-GCACAGTAAGATTTGTTGCAC-3'), BcL-xL (5'-GACAAGGAGATGCAGGTATTGG-3' and 5'-TCCCGTAGAGATCCACAAAAGT-3'), Bcl-2 (5'-ATGCCTTTGTGGAACTATATGGC-3'); Bak (5'-GTGACCTGCTTTTTGGCTGAT-3' and 5'-GGTCTCTACGCAAATTCAGGG-3'); Bax (5'-TGAAGACAGGGGCCTTTTTG-3' and 5'-AATTCGCCGGAGACACTCG-3'); Bim (5'-CCCGGAGATACGGATTGCAC-3' and 5'-GCCTCGCGGTAATCATTTGC-3'); Bad (5'-AAGTCCGATCCCGGAATCC-3' and 5'-GCTCACTCGGCTCAAACTCT-3') and 5'-GGTATGCACCCAGAGTGATGC-3'), and Survivin (5'-CTACCGAGAACGAGCCTGATT-3' and 5'- AGCCTTCCAATTCCTTAAAGCAG-3').
Preparation of nuclear extracts
Cell lines or cells isolated from tumor tissues were washed twice with ice cold PBS and incubated in 1ml of lysis buffer (10 mM Tris/HCl, 3 mM CaCl2, 2 mM MgCl2) containing a protease inhibitor cocktail (Roche) for 10 mins on ice. Then the cells were vortexed gently and incubated in 1ml of NP-40 buffer (10 mM Tris/HCl, 3 mM CaCl2, 2 mM MgCl2, 1% NP-40) for 5 mins at 4°C, and the suspension was centrifuged at 3000 rpm for 10 mins at 4°C. Nuclei was washed with 1ml of Buffer A (20 mM Hepes-KOH, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF), and 100 μl of Buffer C (20 mM Hepes-KOH, 25% Glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, 1% Triton X-100) was added to the pellet and vortexed vigorously at 4°C for 10 mins. Nuclear debris was removed by centrifugation at 13000 g for 5 mins. Protein concentrations were determined by the Bradford Assay (Bio-Rad). The nuclear extract was confirmed by immunoblotting with anti-Lamin B and anti-Tubulin beta. For single cell suspension of tumor tissues, tumor tissues from each group was homogeized with homogenizer (Fluko).
Proteins were resolved by 10% (for NFκB and AP1) or 15% (for caspase-3, Bcl2, Bcl-xL, Bad, Bax, Bak, Bim and Sruvivin) SDS-PAGE gels, transferred onto a PVDF membrane (Bio-RAD) and subjected to Western blot analysis using anti-NFκB (Abcam), anti-pc-JUN (SantaCruz), anti-caspases-3 (Abcam), anti-Bcl-2 (Abcam), anti-Bcl-xL (Cell signaling), anti-Survivin (Cell signaling), anti-Bad (Abcam), anti-Bax (Abcam), anti-Bak (Abcam), anti-Bim (Abcam) and peroxidase-conjugated secondary antibodies (DAKO). Proteins were visualized with a chemiluminescence kit (Amersham Bioscience). The levels of Tubulin (anti-tubulin; Santa Cruz), beta-actin (anti-beta-actin; Abcam) and Lamin B (anti-lamin B; SantaCruz) detected by relevant antibodies were monitored as a loading control.
Melanoma induction and anti-tumor assay
Mouse melanoma B16F10 (1 × 106 cells/0.1ml) cells were injected subcutaneously (s.c) into the flanks of C57BL/6 mice (6 weeks old male). One week after the injection, mice were divided into two groups (10 mice/each group) and orally treated with either 10 mg/dose (400 μg/g mouse weight) of cinnamon extract in 100 μl of PBS or same volume of PBS alone as a sham control for 30 days. During the treatment period, the tumor size was measured with vernier calipers every 2 days, and tumor volumes were calculated using the standard formula: width2 × length × 0.52. Mice were sacrificed for further analysis after 30 days of treatment.
DNA fragmentation assay
Genomic DNA isolation was performed with gDNA purification kit (Solgent, Korea). Briefly, mouse tumor tissues from the differentially treated group were collected, pooled, and 5 mg of tumor tissues from each group were transferred. They were dissolved in 300ml of cell lysis buffer with 25 mg of proteinase K for 4 hours at 55°C, and then mixed with 100ml of protein precipitation solution. Then solution was centrifugated at 14000 rpm for 3 mins. After centrifugation, DNAs was precipitated, washed with isopropanol and 70% ethanol. DNA pellets were dissolved in 100 μl of DNA hydration solution. Finally, fragmented DNAs (10 ml) were visualized in 2% agarose gels.
Cells seeded on the glass in 12 well plate were incubated with cinnamon extract for 72 hours, washed with PBS and fixed with 4% paraformaldehyde for 15 mins at RT. Fixed cells were incubated in PBS (pH 7.4) containing 200mg of DNase-free RNase (Sigma) for 30 mins at 37°C and stained with 2 mg/ml of Hoechst for 10 mins at 37°C. Nuclear morphology of the cells was observed under fluorescence microscope.
A two-tailed Student's t-test was employed where P < 0.05 was considered to be statistically significant (*p < 0.05, **p < 0.005, and ***p < 0.001).