Background
Pancreatic ductal adenocarcinoma represents a major healthcare problem, with a high fatality rate and increasing incidence, and is likely to increase from the fourth- to the second-leading cause of cancer deaths in the US by 2030 [
1‐
4]. Escape from apoptosis (programed cell death) is a characteristic of pancreatic cancer cells, contributing to their resistance to currently available interventions [
5,
6]. Therefore, targeting apoptosis resistance is an important strategy to improve pancreatic cancer outcomes.
Apoptosis is controlled by 2 major signaling pathways: the extrinsic pathway, initiated by death receptors, and the intrinsic pathway, initiated via the mitochondria [
7,
8]. Crosstalk between these 2 pathways is mediated by signaling involving the proapoptotic protein Bid [
9]. The activation of the extrinsic death receptor-mediated apoptosis pathway relies on a death ligand (e.g., tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) binding to its corresponding death receptors [
10]. Activation of the extrinsic death receptor-mediated apoptotic pathway has received attention as a potential strategy for cancer treatment because its activation preferentially induces apoptosis in transformed or malignant cells but not in most normal cells. Recombinant human TRAIL and the agonist antibodies against death receptor 4 (DR4) and death receptor 5 (DR5), which directly activate extrinsic apoptotic pathways, are being tested in early-phase clinical trials. However, recent studies have shown that many types of cancer cells, including pancreatic ductal adenocarcinoma cells, are resistant to the apoptotic effects of TRAIL [
11‐
13], suggesting that treatment with TRAIL alone may not be sufficient for treating pancreatic cancer. Therefore, sensitizers capable of overcoming TRAIL resistance in pancreatic cancer cells are needed to establish more effective TRAIL-based pancreatic cancer therapies.
Tocotrienols (α-, β-, δ-, and γ-) are 1 of 2 groups of compounds that constitute the vitamin E family [
14]. The β-, δ -, and γ-tocotrienols have shown promising efficacy in preventing and treating human cancers, including pancreatic cancer [
15‐
18]. Vitamin E delta-tocotrienol (VEDT) is undergoing early-phase human clinical trials for pancreatic cancer intervention. VEDT exhibits pleiotropic pancreatic anticancer effects, including induction of cell cycle arrest and apoptosis [
16,
19] and prevention of angiogenesis and invasion [
20‐
22], demonstrating VEDT’s potential utility as a pancreatic anticancer drug. VEDT also augments gemcitabine activity in pancreatic cancer, suggesting its potentiality as an adjunct in combination therapy. Multiple targets, including p27, K-ras, p53, NF-kB, and VEGF, have been proposed to explain the anticancer effects of VEDT [
16,
19‐
22], but the underlying molecular mechanisms have not yet been fully elucidated.
Here, we show for the first time that VEDT effectively sensitizes pancreatic cancer cells but not human pancreatic ductal epithelial cells to TRAIL-induced apoptosis, suggesting that this combined treatment may provide a safe and effective therapeutic strategy against pancreatic cancer. Furthermore, we provide novel evidence that the prominent sensitizing effect of VEDT on TRAIL-induced apoptosis is primarily through inducing cellular FLICE inhibitory protein (c-FLIP) degradation. Thus our findings highlight a novel mechanism by which VEDT modulates apoptosis in human cancer cells.
Methods and materials
Ethics statement
All experiments were carried out in accordance with guidelines set by the Animal Experimental Ethics Committee.
Chemicals and reagents
Vitamin E analogs α-, β-, γ-, and δ-tocopherols and tocotrienols were kindly gifted by Davos Life Sciences (Helios, Singapore). Ethanol was purchased from Aaper Alcohol & Chemical (Shelbyville, KY). Recombinant human TRAIL was purchased from Invitrogen (Carlsbad, CA). FLICE inhibitory protein (FLIP) antibody (NF6) was purchased from Alexis Biochemicals (Enzo Life Sciences, Farmingdale, NY). DR5, DR4, DCR1, DCR2, and DCR3 antibodies were purchased from Abcam (Cambridge, MA). Caspase-3 (#9662), caspase-8 (#9746), PARP (#9542), cleaved caspase 3 (#9661S), cleaved caspase 8 (#9496), cleaved PARP (#5625), and green fluorescence protein (GFP) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Hemagglutinin- (HA-) probe antibody, protein A/G PLUS-Agarose, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All cDNA plasmids were purchased from Origene (Rockville, MD). The sulforhodamine B- (SRB-) based In Vitro Toxicology Assay Kit and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.
Cell lines and culture conditions
AsPc-1, BxPc-3, MiaPaCa-2, PANC-1, and SW1990 human pancreatic cancer cell lines (American Type Culture Collection, Manassas, VA) were cultured in complete 1 × Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin–streptomycin (Mediatech, Herndon, VA) at a final concentration of 50 IU/mL (penicillin), 50 mg/mL (streptomycin), and 2 mM l-glutamine (Mediatech). AsPc-1 and BxPc-3 cells were cultured in complete RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum, penicillin–streptomycin, 10 mM HEPES buffer (Mediatech), 1 mM sodium pyruvate (Invitrogen), and 2 mM l-glutamine (Mediatech). HPDE6-C7 human pancreatic ductal epithelial cells (gift from G. Springett, Moffitt Cancer Center) were cultured in keratinocyte serum-free medium (Invitrogen) containing the provided epidermal growth factor and bovine pituitary extract supplements. Immortalized human pancreatic normal epithelial (HPNE) cells (gift from P. Campbell, Moffitt Cancer Center) were cultured in a mixture of DMEM and M3:F media (INCELL, San Antonio, TX) at a ratio of 3 parts DMEM and 1 part M3:F media and were supplemented with 5% fetal bovine serum. All cell lines were maintained at 37 °C in a humidified incubator with 5% CO2. Cells were passaged regularly with .05% trypsin-ethylenediaminetetraacetic acid (Invitrogen) to maintain logarithmic-phase growth. For all experiments, cells were gently detached with Accutase enzyme cell detachment medium (eBioscience, San Diego, CA) in accordance with the provided protocol, with Trypan Blue and hemacytometer used to determine viable cell number.
Cell viability and cell survival assay
Cells were seeded in 96-well plates at a density of 3000 cells/well and allowed to attach overnight. After cells were incubated for 72 h with various concentrations of drugs (10
−5 to 10
−4 M) or ethanol (< 5%) 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 to 4 h at 37 °C in a humidified atmosphere of 5% CO
2. Media were aspirated, 200 µL of dimethyl sulfoxide were added to each well, plates were incubated for 5 min with shaking, and absorbance was read at 540 nm. Cell survival was determined using the SRB colorimetric assay for cytotoxicity in accordance with manufacturer’s instructions. The SRB assay is an established surrogate for cell survival [
23]. Absorbance was recorded using a microplate reader at 565 nm.
Determination of apoptosis
Cells (1 × 106) were seeded in 100-mm tissue culture dishes and allowed to adhere overnight. Cells were treated the following day at varying doses with δ-tocotrienol and collected at specific time points to assess apoptosis by either terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) or annexin V staining. Cells were washed with phosphate-buffered saline (PBS), pelleted, and counted using a hemacytometer. For all TUNEL experiments, ~ 1 × 105 cells were fixed onto a glass slide using a Cytospin III centrifuge (Thermo Shandon Inc., Pittsburgh, PA) and then fixed in 4% paraformaldehyde in PBS solution overnight at 4 °C. Cells were made permeable the next day via incubation in 0.1% sodium citrate-0.1% Triton X-100 solution at 4 °C for 2 min and then labeled for apoptotic DNA strand breaks using the in situ cell death detection kit, AP (Roche Applied Science, Indianapolis, IN) in accordance with manufacturer’s instructions. Cells were stained in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing DAPI (4,6-diamidino-2-phenylindole) to counterstain DNA. Fluorescein-labeled DNA strand breaks (TUNEL-positive cells) were then visualized using a fluorescence microscope (Leica Microsystems Inc., Bannockburn, IL), and 5 representative pictures of the slide field were taken with a digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). TUNEL-positive nuclei (green) were scored and compared with DAPI-stained nuclei (blue) to determine the induction percentage of apoptosis for each treatment group. Annexin V and propidium iodide staining were performed with the Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) as follows: pelleted cells were washed twice with cold PBS and then briefly resuspended in a 1× binding buffer before incubation with annexin V-FITC and propidium iodide according to the manufacturer’s instructions. Flow cytometry was performed using a FACScan flow cytometer (Becton–Dickinson), with analysis using FLOW-JO software (Tree Star, Ashland, OR) to assess the Annexin-positive cell population.
Caspase enzymatic activity assay and use of irreversible caspase inhibitors
Enzymatic activities of caspases-3, -8, and -9 were determined using each respective fluorogenic substrate (Calbiochem, EMD Biosciences, San Diego, CA). Protein extract (20 µg) was incubated in a reaction buffer containing 50 mM Tris (pH 7.5) and caspase substrate at a final concentration of 20 µM in 96-well plates. After 3-h incubation at 37 °C, liberated fluorescent 7-amido-4-methyl-coumarin groups were quantified using a multi-well plate VersaFluorTM Fluorometer with an excitation filter of 380 nm and an emission filter of 460 nm (Bio-Rad Laboratories, Hercules, CA). When irreversible inhibitors of caspases-3, -8, and -9 were used (Calbiochem, EMD Biosciences), each inhibitor was used at a final concentration of 10 µM and incubated with the treatment group 4 h before treatment with tocotrienol. Tocotrienol was then added to the media containing the inhibitor at the indicated final concentration and treated for the desired length of time.
Western blot analysis
To prepare whole cell protein lysates, cells were seeded at 1 × 106 cells per 100-mm tissue culture dish and treated the following day with δ-tocotrienol. After desired treatment times, cells were collected, washed with PBS, pelleted, lysed on ice for 5 min, and then stored at − 20 °C. Tumor tissues were cut into small pieces and homogenized with tissue protein extraction reagent (T-PER, Pierce) and centrifuged at 10 000 RPM for 10 min. The extracted proteins were stored at − 20 °C. Protein concentration was determined to ensure equal protein loading. Protein extracts were resolved using a 12.5% SDS-PAGE gel and then transferred to nitrocellulose membrane. Protein separation was briefly assessed using Ponceau S solution (Pierce). Membranes were then washed with fresh Tris-buffered saline containing .1% Tween 20 (1× TBS-T) and blocked with 5% nonfat dry milk solution. Primary antibodies were incubated in 3% BSA solution either overnight at 4 °C or for 1 h at 25 °C. When reblotting was necessary, antibodies were stripped from membrane by incubating with Western blot stripping buffer (Pierce) for 0.5 h at 25 °C. High-affinity antibodies were stripped from membrane by washing for 5 min with a 0.2 M NaOH solution 3 times at 25 °C, followed by 3 × 5-min washes with deionized water. Membranes were thoroughly washed in 1× TBS-T at 25 °C and incubated with secondary antibody (horseradish peroxidase-conjugated) at 1:2000 dilutions for 1 h at room temperature. The washed blot was then treated with SuperSignal West Pico chemiluminescent substrate (Pierce Biotech) for positive antibody reaction. Membranes were exposed to x-ray film for visualization and densitometric quantization of protein bands using AlphaEaseFC software (Alpha Innotech, Santa Clara, CA).
Generation of MiaPaCa-2 cells stably expressing GFP-labeled c-FLIPs
MiaPaCa-2 cells were transfected with a plasmid-containing full-length open-reading frame cDNA of c-FLIPs fused with GFP at the C-terminus in a pCMV6-AC-GFP vector. MiaPaCa-2 cells transfected with the pCMV6-AC-GFP vector containing GFP alone served as control. All transfections were performed using Lipofectamine™ 2000 reagent (Invitrogen) at a 1 µg:1 µL DNA-to-Lipofectamine 2000 ratio, per manufacturer’s instructions. Cells were selected for 2 weeks with G418 sulfate at a final concentration of 500 µg/mL.
Immunoprecipitation for detection of c-FLIP ubiquitination
Mia-FLIPs cells, stably expressing GFP-FLIPs, were transfected with HA-ubiquitin plasmid (Addgene) using the Lipofectamine 2000 transfection reagent, in accordance with the manufacturer’s instructions. After 24 h, cells were treated with δ-tocotrienol alone (50 µM) or in combination with MG-132 (25 µM) for 6 h and then lysed for immunoprecipitation of ubiquitin-bound c-FLIPs, using GFP polyclonal antibody (Abcam). Harvested cells were washed 3 times with ice-cold PBS and resuspended in 1 mL of ice-cold CelLytic™ M cell lysis reagent (Sigma) containing protease inhibitors. Cleared lysates were then incubated with 10 µg of GFP polyclonal antibody and 100 μL of 50% slurry of Protein A/G Agarose (Santa Cruz, sc-2003) into the lysate, and the mixture was rotated overnight at 4 °C. Beads were washed three times with lysate buffer followed by spinning for 5 s at 10,000 g. After the washes, 50 μL of 1 × sample buffer was added to the bead pellet and boiled at 100 °C for 5 min followed by the detection of ubiquitin-bound c-FLIPs with Western blotting using anti-HA antibody (Santa Cruz, sc-57592).
Quantitative PCR analysis
RNA was isolated from cells using the AllPrep RNA/Protein kit from Qiagen (Germantown, MD), in accordance with the manufacturer’s instructions. RNA extracts were analyzed by quantitative PCR by the Molecular Biology Core at the Moffitt Cancer Center using the TaqMan gene expression assay kit to assess c-FLIP mRNA levels. 18S rRNA was used as an internal control.
In vivo tumor xenograft model
MiaPaCa-2 cells were harvested and resuspended in fresh media, and the number of viable cells was determined. Cells were then injected subcutaneously into flanks of NIH-III nude SCID mice (Charles River Laboratories, Boston, MA) at a density of 1 × 106 cells/per flank in a 1:1 solution of PBS to Matrigel Matrix (BD BioSciences). Mice were randomized into control and treated groups, with controls receiving ethanol-extracted olive oil (vehicle control) alone and treated mice receiving 200 mg/kg of δ-tocotrienol in vehicle. All treatments were administered orally by gavage 12 times/week (weekdays, 2×/day; weekends, 1×/day), and mouse weights were recorded twice per week.
For the drug combination (TRAIL and VEDT) experiment, female Athymic nude mice (n = 20) were injected subcutaneously with MiaPaCa-2 cells into flanks at a density of 1 × 106 cells/per flank in a 1:1 solution of PBS to Matrigel Matrix. Mice were randomized into 4 groups of 5 animals each and treated as follows: (Group 1) Vehicle control mice were given orally ethanol-extracted olive oil twice a day and intraperitoneal (IP) injection of PBS on alternate days for 4 weeks, (Group 2) TRAIL mice were injected with TRAIL 20 µg/kg IP on alternate days for 4 weeks, (Group 3) VEDT mice were given orally VEDT (200 mg/kg) twice a day for 4 weeks, and (Group 4) TRAIL + VEDT mice were injected with TRAIL 20 µg/kg IP on alternate days and VEDT orally (200 mg/kg) twice a day for 4 weeks. Tumors were measured daily with calipers using the formula T = L × W × [(L + W)/2] × 0.5236, where T is tumor volume, L is smallest tumor diameter, and W is largest tumor diameter. Animal data are representative of 2 independent experiments. Animals were sacrificed after 4 weeks and tumor weights were recorded and half tumor tissues were fixed in buffered formalin for histology staining and the other half were immersed in liquid nitrogen then frozen at − 80 °C for further biochemical analyses.
Histological and immunohistochemical analyses
At the end of the study, mice were killed, and tumors were extracted and embedded in paraffin sections for further analyses. Immunohistochemistry was performed using the Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ), per manufacturer’s protocol, with proprietary reagents. Slides were deparaffinized on the automated system with EZ Prep solution. Sections were heated for antigen retrieval. For immunohistochemistry, tissue sections were incubated with antibodies. Detection was performed using the Ventana OmniMap kit. Apoptosis by TUNEL staining, cleaved caspase-8, cleaved caspase-3, and Ki-67 staining and hematoxylin and eosin staining were quantified by the Moffitt Anatomic Pathology Core.
Statistical analyses
Data, expressed as mean ± standard error of the means, were analyzed statistically using unpaired t-tests or 1-way analysis of variance (ANOVA), as appropriate. ANOVA was followed by Duncan’s multiple range tests using SAS statistical software for comparisons between different treatment groups. Analyses of in vivo data were performed using the GraphPad Prism program (GraphPad Software, San Diego, CA) on the entire tumor growth curve using a 2-way ANOVA, mixed-effect model for repeated measures, with respect to the column factor corresponding to δ-tocotrienol treatment. Statistical significance was set at P < .05.
Discussion
Pancreatic cancer is highly aggressive, with only 6% of patients surviving 5 years after diagnosis. Suppression of programed cell death is a hallmark of this cancer, which it displays as increased viability and resistance to cytotoxic therapies [
26]. One example of this resistance to therapy is pancreatic cancer’s varied sensitivity to TRAIL, a tumor-selective cytokine, which activates the extrinsic apoptotic pathway [
27‐
31]. Therefore, there is an important need for proapoptotic agents that can sensitize pancreatic ductal adenocarcinoma cells to TRAIL and thereby overcome pancreatic cancer resistance to apoptosis. One intriguing finding that has been shown consistently by our group and others is the selective killing of pancreatic cancer cells by VEDT [
5,
16,
19‐
21]. We have also shown selective induction of apoptosis by VEDT of pancreatic-transformed and malignant epithelial cells but not normal immortalized human pancreatic ductal epithelial cells [
16,
19]. Additionally, our animal studies showed that VEDT not only caused a statistically significant inhibition of tumor growth but also resulted in apoptosis of tumor tissues [
16,
20,
21]. These results clearly demonstrate that induction of apoptosis is implicated in the antitumor activity of VEDT.
In the current study, we found that VEDT alone triggered a caspase-8-dependent apoptosis in pancreatic cancer cells; however, when combined with TRAIL, VEDT significantly augmented and potentiated the TRAIL-induced apoptosis of pancreatic cancer cells. Specifically, we were able to demonstrate in vitro and in vivo increased cleaved caspase-8 and caspase-3 and induction of apoptotic cell death with VEDT treatment versus controls. Conversely, pharmacological inhibition of caspase-8 and caspase-3 demonstrated significant attenuation of VEDT-induced apoptosis. Together, these results strongly suggest that VEDT induces apoptosis through activation of the caspase-8 cascade. The role of caspase-8 in death receptor-mediated apoptosis has been extensively studied [
24,
32,
33]. Interestingly, whereas pancreatic cancer cells have been shown to express all of the proteins of the extrinsic apoptotic pathway, most cell lines remain relatively insensitive to TRAIL-induced apoptosis. Both recombinant and antibody-based TRAIL therapies have been developed to date for the treatment of many cancers, but resistance to death receptor-mediated apoptosis remains a significant barrier in the field of pancreatic cancer [
34].
A possible molecular target for VEDT is c-FLIP
s, an inhibitor of caspase-8 in pancreatic cancer cells. We had shown that VEDT but not tocopherols augment TRAIL activity (unpublished data). Our data show that VEDT decreased c-FLIP
s levels without consistently modulating the expression of decoy death receptors 1, 2, and 3 or death receptors 4 and 5. Furthermore, VEDT was found to inhibit c-FLIP
s expression and induce caspase-8-dependent apoptosis in pancreatic cancer cells, followed by caspase-3 and PARP1 cleavage in a time-dependent manner. The activation of caspases-8 and -3 were required for VEDT induction of apoptosis, as demonstrated by rescue with specific caspase inhibitors. Moreover, the growth of pancreatic xenograft tumors in mice was significantly inhibited when treated with VEDT. This inhibition was also accompanied by an induction of apoptosis in tumors and depletion of c-FLIP
s protein expression. Although VEDT inhibited c-FLIP
s protein levels by 6 h, mRNA levels were actually increased at this time point, suggesting that VEDT may be inhibiting c-FLIP
s through protein degradation. From these analyses, we concluded that the mechanism of c-FLIP
s down-regulation is unlikely caused by transcriptional repression but rather an ubiquitin-mediated, proteasome-dependent process. Moreover, ferroptosis is a newly discovered type of cell death that differs from traditional apoptosis and necrosis and results from iron-dependent lipid peroxide accumulation and autophagy [
35,
36]. Pancreatic ductal adenocarcinoma with a mutant
KRas gene is more susceptible to ferroptosis, and it might be related to the tumorigenesis of pancreatic carcinoma [
37]. Therefore, VEDT may kill pancreatic cancer cells through ferroptosis. This possibility will be investigated in our future studies.
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