Cell lines, reagents and antibodies
RKO and SW480 human colon carcinoma cell lines and CCD-18Co colon fibroblasts were obtained from American Type Culture Collection (Manassas, VA). Cells were initially grown and multiple aliquots were frozen and stored at -80°C for future use. Cells were purchased more than 6 months ago and were not further tested or authenticated by the authors. Cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) with phenol red supplemented with 10% FBS, and 10 mL/L of 100X antibiotic/antimycotic solution (Sigma-Aldrich Co., St. Louis, MO). Cells were cultured in 150-cm2 plates in an air/CO2 (95:5) atmosphere at 37°C. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) except c-MET and survivin (Cell Signaling Technology, Danvers, MA), NFκB-p50 and NFκB-p65 (Abcam Inc., Cambridge, MA), Sp1 (Millipore, Billerica, MA), and FAS (Sigma-Aldrich Co., St. Louis, MO). Glutathione, 98% (γ-L-glutamyl-L-cysteinyl-glycine, GSH) and lactacystin (proteasome inhibitor) were purchased from Sigma-Aldrich. Carboxy-H2DCFDA was purchased from Invitrogen (Carlsbad, CA). Curcumin (98% pure) was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ), and curcumin analogs were synthesized as described
 and RL197 synthesis is outlined below.
Melting points were determined on a Mettler Toledo FP62 melting block and were uncorrected. High resolution mass spectrometry was recorded using a VG70-250S double focusing magnetic sector mass spectrometer. NMR spectra, at 25°C, were recorded at 500 MHz for 1H and 125 MHz for 13C on Varian INOVA-500 spectrometer. Chemical shifts are given in ppm on the δ scale referenced to the solvent peaks CHCl3 at 7.26 and CDCl3 at 77.00. 1-Boc-4-piperidone, and 2,5-dimethoxybenzaldehyde were purchased from the Sigma-Aldrich Company. (3E,5E)-3,5-Bis(2,5-dimethoxybenzylidene)-1-t-butoxycarbonylpiperidin-4-one (RL197). To a mixture of 1-Boc-4-piperidone (0.70 g, 3.5 mmol) and 2,5-dimethoxybenzaldehyde (1.20 g, 7.4 mmol) in methanol (50 mL) was added sodium methoxide (5M, 0.75 ml) and the mixture was stirred for 18 hr at room temperature. The resulting precipitate was removed by filtration, then washed with cold methanol and purified by recrystallisation from ethanol to give RL197 as a yellow solid (1.20 g, 69%); mp 167.7°C. Found: C, 67.79; H, 6.79; N, 2.73. C28H33NO7 requires: C, 67.86; H, 6.71; N, 2.83. 1H-NMR (CDCl3) δ: 1.26 (s, 9H), 3.79 (s, 6H), 3.82 (s, 6H), 4.59 (bs, 4H), 6.80 (bs, 2H), 6.85 (d, J = 9Hz, 2H), 6.89 (dd, J = 2, 9 Hz, 2H), 7.95 (bs, 2H); 13C-NMR (CDCl3) δ: 187.76, 154.35, 153.05, 152.74, 133.30 (br), 124.83, 116.13, 115.46 (br), 111.80, 80.20, 56.04, 55.84, 45.05, 28.05: (HRMS (+ve ESI) calc for C28H33NaNO7: 518.2149 m/z [MNa+], found: 518.2115 m/z.
Cell proliferation assay and annexin V staining
RKO and SW480 cancer cells were seeded in DMEM High Glucose with 10% FBS on 12-well plates and allowed to attach for 24 hr. The medium was then changed to DMEM High Glucose containing 2.5% charcoal-stripped FBS and cells were treated with either the vehicle (DMSO) or the indicated compounds for 24 hr. Cells were trypsinized and counted using a Coulter Z1 particle counter. For Annexin V staining, cells were seeded in 6-well plates, allowed to attach overnight, and treated with curcumin or RL197 as indicated. Annexin V and propidium iodide staining was determined using the Vybrant apoptosis assay kit #2 (Molecular Probes, Grand Island, NY) and images were captured at 20X magnification using IN cell analyzer 6000 (GE Healthcare Biosciences, Piscataway, NJ).
RKO and SW480 cancer cells were seeded in DMEM High Glucose with 10% FBS on 6-well plates and allowed to attach for 24 hr. The medium was then changed to DMEM High Glucose containing 2.5% charcoal-stripped FBS and treated with either the vehicle (DMSO) or the indicated compounds and analyzed by western blots as described
Cellular ROS levels were evaluated with the cell permeant probe carboxy-H2DCFDA (5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate) from Invitrogen. Following treatment, cells seeded on 6-well plates were loaded with 10 mM of carboxy-H2DCFDA for 1 hr, washed once with serum-free medium, and analyzed for ROS levels using BD Accuri C6 Flow Cytometer using the FL1 channel. Analysis of data was determined with BD Accuri CFlow software (set at 480 nm and 525 nm excitation and emission wavelengths, respectively). Each experiment was carried out in triplicate and results are expressed as means ± S.E. for each treatment group.
Measurement of mitochondrial membrane potential (MMP)
MMP was measured with Mitochondrial Membrane Potential Detection Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol using the JC-1 dye; mitochondrial membrane potential was measured using BD Accuri C6 Flow Cytometer and data were analyzed using the BD Accuri CFlow software. J-aggregates are detected as red fluorescence and J-monomers are detected as green fluorescence. Each experiment was determined in triplicate, and results are expressed as means ± S.E. for each treatment group.
Quantitative real time PCR of mRNA and miRNAs
The mirVana miRNA Isolation Kit (Applied Biosystems, Carlsbad, CA) was used for miRNA extraction and, miRNAs (RNU6B, miRNA-27a, miRNA-20a, and miRNA 17-5p) were quantitated by real time PCR using the Taqman miRNA assay (Applied Biosystems) according to the manufacturer’s protocol. U6 small nuclear RNA (RNU6B) was used as a control to determine relative miRNA expression. mRNA was extracted using the RNeasy Protect Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol, and cDNA was prepared by reverse transcription using Reverse Transcription Kit (Promega, Madison, WI) according to the manufacturer’s protocol. Each PCR was carried out in triplicate using SYBR Green PCR Master Mix (Invitrogen) at one cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min on MyIQ2 Real Time PCR Detection System (BioRad, Hercules, CA) with 1 μmol/L of each primer and 1 μL cDNA template in each 20 μL reaction. TATA binding protein (TBP) was used as an endogenous control to compare the relative mRNA levels. Comparative CT methods were used for relative quantitation of samples and the following primers, purchased from Integrated DNA Technologies (Coralville, IA ), were used:
Sp1 (Forward): 5′-TCA CCT GCG GGC ACA CTT-3′
Sp1 (Reverse): 5′-CCG AAC GTG TGA AGC GTT-3′
TBP (Forward): 5′-TGCACAGGAGCCAAGAGTGAA-3′
TBP (Reverse): 5′-CACATCACAGCTCCCCACCA-3′
ZBTB10 (Forward): 5′-GCTGGATAGTAGTTATGTTGC-3′
ZBTB10 (Reverse): 5′-CTGAGTGGTTTGATGGACAGA-3′
ZBTB4 (Forward): 5′-ACCTGTGCAGGAATTTCCAC-3′
ZBTB4 (Reverse): 5′-GAGCGGCCAAGTTACTGAAG-3′
Primers for Sp3 and Sp4 were purchased from Qiagen.
Statistical significance of differences between the treatment groups was determined using the Student’s t test, and levels of probability were noted. IC50 values were calculated using linear regression analysis and expressed in micromolar (μM) concentrations at 95% confidence intervals.