Chemicals and cell lines
All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Tocotrienols (α, β, γ, and δ) and α- and δ-tocopherol were obtained from Davos Life Ltd (Helios, Singapore). Gemcitabine-HCl was purchased from Eli Lilly and Company (Indianapolis, IN). L-glutamine, penicillin, streptomycin, and HEPES buffer were purchased from Mediatech (Herndon, VA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT). Dulbecco’s modified minimal essential medium (DMEM), RPMI 1640, keratinocyte serum-free medium, sodium pyruvate, trypsin-EDTA, and phosphate-buffered saline (PBS) were purchased from Invitrogen (Carlsbad, CA). Ethanol (100%) was purchased from Aaper Alcohol and Chemical (Shelbyville, KY). Pancreatic cancer cell lines MiaPaCa-2 and AsPc-1 were obtained from ATCC (Manassas, VA). Human pancreatic ductal epithelial cells (HPDE6 C7) and HPDE6 C7-KRas cells were gifts from Dr. Tsao, Ontario Cancer Institute, University of Toronto. These cells were authenticated by morphologic, cell proliferation, and Mycoplasma tests, as recommended in ATCC Technical Bulletin No. 8 (2007).
Cell culture and growth
HPDE6 C7 and HPDE6 C7-KRas cells were grown in keratinocyte growth media (Invitrogen) supplemented with human EGF and bovine pituitary extract. Mia PaCa-2 and AsPc-1 cells were grown in monolayers with DMEM and RPMI media, respectively, supplemented with 10% FBS, 2 mM L-glutamine, penicillin (50 IU/mL), and streptomycin (50 mg/mL). RPMI medium was also supplemented with 10−2 M HEPES buffer and 10−3 M sodium pyruvate. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2-95% O2.
Cell proliferation MTT assay
Cells were seeded in 96-well plates at a density of 3,000 cells per well and allowed to attach overnight. Cells were then incubated for 72 hours with various concentrations of α, β, γ, and δ-tocotrienol and α-tocopherol (10−5 to 10−4 M). In other experiments, cells were incubated for 72 hours with gemcitabine and δ-tocotrienol (10−5 to 10−4 M) and their combination or ethanol (<5%) in PBS as vehicle control. Media were aspirated and replaced with 20 μL of 1 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and incubated for 2–4 hours at 37°C in a humidified atmosphere of 5% CO2. Media were aspirated, 200 μL of DMSO were added to each well, plates were incubated for 5 minutes with shaking, and absorbance was read at 540 nm.
Gemcitabine and δ-tocotrienol were combined at ratios of 1:1 and 1:2 of their IC50
values to plot the isobologram using fraction effects and combination index (CI) through ClacuSyn software (Biosoft, Cambridge, UK). Constant ratio (IC50
ratio) of the two-drug combination is used. The dose range (different fraction of the IC50
values) and effects (proliferation) are entered in ClacuSyn, which displays the parameters (Dm
, m, and r) as well as isobologram using CI, where Dm
is the median-effect dose signifying the potency, m is measurement of the sigmoidicity (shape) of the dose-effect curve, and r is linear correlation coefficient of the median-effect plot. The software analyzes the quantitative measure of the degree of drug interactions in terms of additive effect (CI = 1), synergism (CI <1), or antagonism (CI >1), based on a published method (18
Trypan blue dye exclusion assay
Treated cells were trypsinized and washed with PBS, and cell suspension (50 μL) was mixed with 50 μL of Trypan blue dye and incubated for 2 minutes at room temperature. A 10-μL volume was loaded onto a hemacytometer, and cells were scored as live or dead based on Trypan blue exclusion.
Cell death assay
Cytoplasmic DNA fragments and histone were detected in cells using Cell Death Detection ELISA kit from Roche Diagnostic (Indianapolis, IN).
Colonogenic survival (anchorage-independent) growth assay
The standard soft agar colony formation assay was performed in MiaPaca-2 cells. Cells were seeded at a density of 5,000/well in 12-well plates in 0.3% agar over a 0.6% bottom agar layer. Colonies were fed with growth media and α, β, γ, and δ-tocotrienol or α/δ-tocopherol (5× 10−5 M), and colony formation growth was observed for 14 days. In other experiments, colonies were fed with growth media and gemcitabine (2× 10−5 M) and δ-tocotrienol (5× 10−5 M) and their combination, and colony formation growth was observed for 14 days. Colonies were photographed after overnight incubation with 1 mg/mL MTT in the wells. Colonies were counted under stereomicroscope and compared with controls. Experiments were done at least twice, each in triplicate.
NF-κB (p65) binding activity assay
NF-κB binding activities in nuclear and cytosolic fractions of MiaPaca-2 cells were determined using the Trans AM ELISA kit from Active Motif (Carlsbad, CA), according to manufacturer’s instructions.
Caspase enzyme activity assay
Treated cells were lysed with lysis buffer (0.5 mM Tris/pH 8.0, 5 mM EDTA/pH 8, 0.15 mM NaCl, 0.5% NP-40). The enzymatic activities of caspase 3, 8, and 9 were determined using specific fluorogenic substrates. The liberated fluorescent 7-amido-4-methyl-coumarin groups were quantified using a multi-well plate VersaFluorTM fluorometer at 355-nm excitation and 460-nm emission wavelengths (Bio-Rad).
Cell lysis and protein determination
Cells were washed three times in cold PBS (pH 7) and then lysed in mammalian-protein extraction reagent lysis buffer (Pierce, Rockford, IL) containing EDTA and protease inhibitor cocktail. Protein concentration was determined using BCA reagents (Pierce) according to manufacturer’s instructions.
Western blot analysis
Extracted proteins from cells and tumor tissues (40 μg) were resolved on 12.5% SDS-PAGE running gel and 5% stacking gel. Proteins were then electrotransferred onto nitrocellulose membranes. After blocking in 5% nonfat powdered milk for 1 hour, membranes were washed and treated with antibodies to CK18, PARP-1, NF-κB subunits (p65/p50), IκBα, p-IκBα, Bcl-XL, XIAP, survivin, and β-actin (1:1,000 and 1:5,000) overnight at 4°C (Axxora, San Diego, CA; Santa Cruz Biotechnology, Santa Cruz, CA; Cell Signaling, Danvers, MA). After they were washed, blots were incubated with horseradish peroxidase-conjugated secondary antibody IgG (1:5,000 and 1:10,000) for 1 hour at room temperature. Washed blots were then treated with SuperSignal West Pico chemiluminescent substrate (Pierce) for positive antibody reaction. Membranes were exposed to X-ray film (Kodak) for visualization and densitometric quantization of protein bands using AlphaEaseFC software (Alpha Innotech).
Animals and treatments
Female NIH SCID nude mice (4–5 weeks old, 20–25 g) were obtained from Charles River (Wilmington, MA) and kept in our Center’s animal facility for 1 week for quarantine. Mice (n = 25) were injected with AsPc-1 cells (one million) with Matrigel (100 μL) to both flanks. When tumor volume reached 100 mm3, mice were randomly divided into 5 treatment groups: 1) normal controls (100 μL oral gavage of ethanol extracted olive oil (vehicle) twice daily for 4 weeks, 2) α-tocotrienol treated, 3) β-tocotrienol treated, 4) γ-tocotrienol treated, and 5) δ-tocotrienol treated. Tocotrienols were administered at 200 mg/kg oral gavage twice daily for 4 weeks. In other experiments, mice (n = 20) were injected with AsPc-1 cells (one million) with Matrigel (100 μL) to both flanks. When tumor volume reached 100 mm3, mice were randomly divided into 4 treatment groups: 1) normal controls (100 μL oral gavage of ethanol extracted olive oil (vehicle) twice daily for 4 weeks, 2) δ-tocotrienol treated (200 mg/kg oral gavage twice daily for 4 weeks), 3) gemcitabine treated (100 mg/kg IP twice a week for 4 weeks), and 4) δ-tocotrienol + gemcitabine treated (200 mg/kg oral gavage daily plus gemcitabine at 100 mg/kg IP twice a week for 4 weeks). Tumor volumes were measured every other day. Animal studies were approved by our Institutional Laboratory Animal Care and Use Committee and were conducted per the guidelines of the National Institutes of Health.
Data are expressed as means ± SEM and were analyzed statistically using unpaired t-tests or one-way analysis of variance (ANOVA) where appropriate. ANOVA was followed by Duncan’s multiple range tests using SAS statistical software for comparison between different treatment groups. Significance was set at P < 0.05.