Materials
Emodin [1,3,8-trihydroxy-6-methylanthracene-9,10-dione; purity ≥ 95] was procured from LKT Laboratories (Cat#:518-82-1) (St. Paul, MN, USA) and DMSO was purchased from Corning Cellgro (Cat#:MT-25950CQC) (Manassas, VA, USA). For western blot analyses, rabbit derived antibodies specific for Bad (Cat#9239T), Bak (Cat#12105), Bax (Cat#5023), Bim (Cat#2933), total (Cat#9662) and cleaved-Caspase-3 (Asp175) (Cat#9664), cleaved-Caspase-9(Asp315) (Cat#20750), Cytochrome C (Cat#11940), phosphorylated (Thr202/Tyr204) (Cat#4370) and total (Cat#9102) p44/p42 MAPK ERK1/2, p53 (Cat#2527), PARP (Cat#9532), PDK1 (Cat#5662), p-PTEN(Ser380) (Cat#9551), PUMA (Cat#12450), Ras (Cat#3339), STAT-1 (Cat#9172), STAT-3 (Cat#30835S), and anti-rabbit IgG HRP-linked secondary antibody was purchased from CST (Denvers, MA, USA). Mouse derived antibodies specific for Bid (Cat#8762), cleaved (Asp387) (Cat#8592) and total (Cat#9746) Caspase-8, total Caspase-9 (Cat#9508), p-NF-κβ (S380) (Cat#6956), and anti-mouse IgG HRP-linked secondary antibody was purchased from CST (Danvers, MA, USA). β-actin mouse (C4) (Cat#sc-47778) mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Bcl-2 mouse (Bcl-2-100) (Cat#13-8800) mAb was purchased from ThermoFisher Scientific (Waltham, MA, USA).
Cell lines and culture conditions
Human CoCa cells (DLD-1 and COLO-20) and normal colon epithelial cells (CCD 841 CoN) were purchased from the ATCC (Rockville, MD, USA). Cells were cultured in RPMI-1640 medium (Cat#MT10040CM) (Corning Cellgro, Manassas, VA, USA) supplemented with 10% FBS, amphotericin B (250 μg/mL), HEPES Buffer (238.3 μg/mL), streptomycin (100 μg/mL) and penicillin (100 U/mL) (Corning Cellgro, Manassas, VA, USA). All cells were cultured at 37 °C with 5% CO2 atmosphere. Prior to each experiment, cells were cultured in 2% FBS containing media for 24 h.
Cell viability
Normal colon epithelial and CoCa cells were seeded (1.5 × 104 cells/well) in triplicate in a 96-well plate for 24 h. Cells were treated with increasing concentrations of Emodin (0,10, 20, 40, and 80 μM), dissolved in DMSO (0.01% final concentration), or vehicle control (0.01% DMSO) for 24, 48, and 72 h. At the conclusion of each time point, 20 μL of MTT reagent (5 mg/mL in PBS) was added to each well and incubated for 2 h to allow formazan crystallization. Media was then discarded and formazan crystals were solubilized in DMSO. Absorbance was measured at 570 nm using a SpectraMax M5 spectrophotometer (Molecular Devices, San Jose, CA, USA). The half maximal effective concentration (EC50) of each cell line as determined by a linear regression using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA) was used for subsequent experiments.
Apoptosis assay
Cells were seeded (106 cells/well) in duplicate in 6-well plates for 24 h and treated with EC50 of Emodin or vehicle control (0.01% DMSO) for 24, 48, or 72 h. Cells were harvested, washed twice with ice-cold cell staining buffer and equal number of cells were then stained with FITC-Annexin V and 7-AAD using the Apoptosis Detection Kit (Cat#640922) (BioLegend, San Diego, CA, USA), according to manufacturer’s instructions. The fluorescence of the stained cells was acquired (30,000 events/sample) using a Guava flow cytometer (Millipore, Billerica, MA, USA) and analyzed using FlowJo 10.0.06 software (Treestar Inc., Ashland, OR, USA).
Antibody microarray
An apoptosis associated antibody microarray (Cat#APP069) (Full Moon BioSystems, Sunnyvale, CA, USA) consisting of 73 highly specific and well characterized antibodies against apoptosis and related survival signaling was used to evaluate effect of Emodin treatment on CoCa cells. Cells were treated with EC50 of Emodin or DMSO for 48 h. Protein extracts from all samples were collected and biotinylated (75μg protein/reaction) using the Antibody Array Assay Kit (Cat#KAS02) (Full Moon BioSystems). Samples were coupled with array slides and labeled with 0.5 mg/mL Cy3-Streptavidin as directed in the manufacturer’s instructions. The arrays were scanned for signal quantification by Full Moon BioSystems using an Axon GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA). Each antibody was replicated six times and β-actin and GAPDH were used as internal controls. Average signal intensity of replicate spots was normalized first to the median signal of the slide, then to that internal control. Ratio of intensities of Emodin treated versus untreated samples was used as fold changes in protein expression. Heat map was generated using the CIMminer platform (https://discover.nci.nih.gov/cimminer/oneMatrix.do) developed by the Genomics and Bioinformatics Group at the NCI.
Mitochondrial membrane potential assay
Cells were seeded (106 cells/well) in 6-well plates for 24 h followed by treatment with Emodin, or vehicle (0.01% DMSO) for 24, 48, or 72 h. Cells were harvested, washed twice with ice cold PBS, resuspended in JC-1 containing assay buffer (Cat#30001) (Biotium Inc., Fremont, CA, USA), and stained as per the manufacturer’s instruction. Cells were then incubated for 15 min at 37 °C with 5% CO2, washed and resuspended in PBS. Fluorescence was acquired and analyzed as mentioned before.
Cytosolic and mitochondrial protein isolation
Cytosolic and mitochondrial protein fractions were extracted from vehicle-treated or Emodin treated CoCa cells using Mitochondria/Cytosol Fractionation Kit (Cat#K256-25) (BioVision Inc, Milpitas, CA, USA). Briefly, 4 × 106 cells were harvested, washed and centrifuged at 600g/5 min/4 °C. Cells were resuspended in cytosol extraction buffer mix, incubated on ice for 10 min, and homogenized on ice using Omni TH homogenizer (Omni Inc., Kennesaw, GA, USA). The homogenate was centrifuged at 700g/10 min/4 °C. Supernatant was collected and centrifuged at 10,000g/30 min/4 °C. That supernatant was used as the cytosolic fraction. The pellet was resuspended in mitochondrial extraction buffer mix, vortexed for 10 s, and used as the mitochondrial fraction.
Western blot analyses
Total protein from Emodin or vehicle-treated cells was isolated using RIPA buffer containing protease (Thermo Fisher Scientific, Waltham, MA, USA) and phosphatase inhibitors (Millipore Sigma, St. Louis, MO, USA). Protein was quantified by BCA method (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (50 µg/lane) were loaded, resolved on 12% SDS-PAGE, and transferred to PVDF membranes. Membranes were incubated in blocking buffer (5% nonfat milk in PBST i.e. PBS/0.1% Tween-20) for 1 h at RT. Membranes were incubated with specific primary antibodies overnight at 4 °C followed by three washes with PBST, then incubated with respective secondary antibody for 2 h. Primary and secondary antibodies were diluted 1:1000 and 1: 2000, respectively in 3% non-fat milk dissolved in PBST. West Pico or Trident Femto Chemiluminescent substrate (GeneTex Inc., Irvine, CA, USA) was used to develop the bands, and Image J software (http://www.rsbweb.nih.gov/ij) was used to quantify the results.
Statistical analyses
The statistical significance between different groups was determined either by Student’s t test or by two-way ANOVA followed by Holm-Sidak’s post hoc test for multiple comparisons using GraphPad Prism version 6.0 (GraphPad Software Inc., La Jolla, CA, USA). The graphs presented are representative of three independent experiments. Statistical significance was established at p < 0.05 and results are shown as mean ± SEM.