Cell lines and reagents
All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to ATCC protocols. The human medulloblastoma cell line DAOY was cultured in MEM supplemented with 10% fetal bovine serum, glutamine and penicillin/streptomycin in a humidified, 5% CO2 atmosphere at 37°C.
Antibodies against α-tubulin, acetylated α-tubulin, cleaved caspase3, cleaved PARP, GAPDH, cyclin A, and cyclin D1 and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA), APC2, APC7, and APC8 from Biolegend (San Diego, CA) and Cdc27, Cdc20, BubRI, and β-actin from BD Transduction Laboratories (Franklin Lakes, NJ). Antibody against cyclin B1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and securin from Abcam (Cambridge, MA). Cdh1 and cyclin E antibodies, curcumin and half-curcumin (dehydrozingerone, DHZ, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) were purchased from Sigma-Aldrich (St. Louis, MO).
Lactate dehydrogenase (LDH) levels as a measure of cell death were determined using the Non-radioactive Cytotoxicity kit (Promega, Madison, WI) according to manufacturer's instructions. LDH release was determined from curcumin-treated and untreated control cells grown on 24-well plates by collecting growth medium. Cell debris was removed by centrifugation. Viable cell LDH was collected from cells lysed by freezing for 15 min at -70°C followed by thawing at 37°C. The medium was collected and cleared from cell debris by centrifugation. The relative release of LDH was determined as the ratio of released LDH versus total LDH from viable cells.
Immunoblotting, immunoprecipitations, and λ-phosphatase treatment
Cell lysates were prepared in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride and 5 μg/ml of antipapain, leupeptin and pepstatin (protease inhibitor cocktail). Protein concentrations were determined by the Dc protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose. The membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Primary antibodies diluted in 5% bovine serum albumin/TBST were incubated overnight at 4°C and HRP-conjugated secondary antibodies in 5% non-fat milk/TBST for 2 h at room temperature. Protein bands were visualized by Enhanced Chemiluminescene Plus (GE Healthcare, Piscataway, NJ).
For immunoprecipitation, cells were lysed at 4°C for 30 min in a buffer of 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM Na3VO4, 1 mM aprotinin, 1 mM leupeptin and 1 mM PMSF. Equal amounts of protein (from 0.5 to 2 mg) were incubated with Cdc27 antibody for 4 h at 4°C followed by protein G-sepharose (GE Healthcare) for 2 h, washed extensively, and analyzed by immunoblotting with indicated antibodies. For λ-phosphatase treatment, Cdc27 was immunoprecipitated as above except that phosphatase and protease inhibitors were omitted and then incubated with λ - phosphatase according to the manufacturer's protocol (New England Biolabs, Ipswich, MA).
Cell cycle analysis
Interphase DAOY cells were treated with curcumin for indicated times, trypsinized, and fixed in cold 70% ethanol. DNA was stained with 100 μg/ml propidium iodide (PI) in hypotonic citrate buffer with 20 μg/ml ribonuclease A. Stained nuclei were analyzed for DNA-PI fluorescence using an Accuri C6 flow cytometer (Accuri Cytometers Inc., Ann Arbor, MI). Resulting DNA distributions for sub G0/G1, G0/G1, S and G2/M phase of the cell cycle were analyzed with CFlow plus software (Accuri Cytometers Inc).
For analysis of cell cycle profiles after mitotic block, cells were synchronized with 2 mM thymidine for 24 h. The block was released for 3 h and cells were arrested in prometaphase with 100 nM nocodazole for 12 h, resulting in approximately 70% of the cells arrested in G2/M. For G1/S arrest, cells were synchronized for 18 h with 2 mM thymidine, released for 9 h, followed by a second thymidine arrest for 18 h, resulting in a G1/S block in about 50% of the cells. The block was then released in the presence of DMSO or curcumin as indicated and the cells were processed as described above.
In vitro APC assay
In vitro APC assays were performed as described  using an in vitro transcribed and translated N-terminal fragment of cyclin B1 (cyclin B1-N1-102) as substrate. 35S-methionine labeled cyclin B1-N1-102 was obtained using the TNT quick-coupled Transcription/Translation system (Promega, Madison, WI). Cell pellets of control and curcumin-treated DAOY cells were snap frozen in liquid nitrogen. The cell pellets were resuspended in an ice-cold hypotonic buffer (20 mM Hepes pH 7.6, 20 mM NaF, 1.5 mM MgCl2, 1 mM DTT, 5 mM KCl, 20 mM β-glycerophosphate, 250 μM NaVO3, 1 mM PMSF, and EDTA-free protease inhibitors) and incubated for 30 min on ice. The lysates were briefly homogenized and cleared by a 1 h centrifugation at 13,000 rpm in a micro centrifuge. For the assay, 30 μg of total protein were added to reaction buffer containing 20 mM Tris pH 7.5, 20 mM NaCl, 5 mM MgCl2, 5 mM ATP-γ-S, 20 μg/ml MG-132, 0.5 μg UbcH10, 20 μM ubiquitin, 1 μm ubiquitin aldehyde, protease inhibitors, and 2 μl of in vitro translated35S-cyclin B1-N1-102 and incubated at 37°C for 60 min. The reactions were stopped by adding sample buffer and proteins were separated by SDS-PAGE on a 4-15% gradient gel. To visualize the bands, the gel was incubated and enhanced with salicylate, dried, and then subjected to autoradiography.
Immobilization of curcumin on epoxy-activated Sepharose 6B
Curcumin was coupled to epoxy-activated Sepharose 6B as previously described . Briefly, 20 mM curcumin dissolved in coupling buffer (50% dimethylformamide/0.1 M Na2CO3/10 mM NaOH) was incubated with swollen epoxy-activated Sepharose 6B beads overnight at 30°C. After washing, unoccupied binding sites were blocked with 1 M ethanolamine by overnight incubation. Low (0.1 M acetate buffer, pH 4) and high (0.1 M Tris-HCl, pH 8, 0.5 M NaCl) pH buffers were used each three times to wash and equilibrate the beads. Control beads were prepared in parallel with curcumin-coupled beads but curcumin was omitted. DAOY cell lysates were prepared in a lysis buffer of 100 mM HEPES, pH 7.6, 300 mM NaCl, 0.1% Triton X-100, 2 mM EDTA, 2 mM EGTA supplemented with phosphatase and protease inhibitors. 500 μg of protein was mixed with 20 μl of curcumin-coupled Sepharose beads and incubated for 3 h at 4°C. After washing bound proteins were eluted with 1× SDS-PAGE sample buffer and processed for immunoblotting.
Data are presented as mean ± SD unless otherwise indicated. The differences between means of two groups were analyzed by a two-tailed unpaired Student's t-test. When required, P values are stated in the figure legends.