Background
Pancreatic cancer is an exceptionally devastating and incurable disease, which is becoming the fourth killer of patients with cancer [
1] It was estimated that 34,300 people died of pancreatic cancer in the United States and 40,000 in Europe in 2008 alone [
1,
2]. The treatment of pancreatic cancer has largely been unsuccessful due to its uncontrolled growth, high rate metastasis, and common resistance to conventional approaches including surgery, radiation and/or chemotherapy [
3‐
5]. Therefore, there is a need for development of new and effective chemotherapeutic agents, which can target multiple signaling pathways to induce responsiveness of pancreatic cancer cells to death signals [
6,
7].
TNFα, a key proinflammatory factor, has been shown to have anti-tumor activity in several preclinical models and in non-comparative clinical trials [
8‐
12]. However, the curative effect seems not perfect as anticipated due to its systemic cytotoxicities and resistance to tumor cells, which prevents TNFα from becoming an effective anticancer agent [
10,
13]. Recent studies have shown that the abnormal activation of NF-κB is the main reason for TNFα tolerance in many types of tumor [
8,
10,
13,
14]. When binding to its receptor (tumor necrosis factor receptor, TNFR) on the cells, TNFα can induce apoptosis via recruiting TNFR-associated death domain protein (TRADD), FADD, and Caspase-8, and then modulate Caspase-9 and Caspase-3. The activated Caspase-3 can induce poly (ADP-ribose) polymerase (PARP) cleavage and switch on cell apoptosis programs. On the other hand, bound receptor TNFR has been shown to activate IκBα kinase (IKK). IKK, in turn, phosphorylates the IκBα protein, resulting in the ubiquitination and dissociation of IκBα from NF-κB complex, and eventually leading to the degradation of IκBα by the proteosome. The activated NF-κB is then translocated into the nucleus where it binds to specific sequences of DNA and activates the expression of some anti-apoptosis genes, such as Bcl-2, Survivin, XIAP, and IAPs [
15,
16]. Therefore, agents that can suppress TNFα-induced NF-κB activation, and at the same time enhance TNFα-induced cell apoptotic activation will significantly improve the anti-tumor activities of TNFα [
8,
10,
13].
Natural products have been a successful source of therapeutic agents and drug leads. MA, a pentacyclic triterpene acid, is widely present in dietary plants, especially abundant in olive fruit skins. The compound has attracted much interest due to its proven pharmacological safety and its many biological activities such as anti-inflammation[
17], anti-virus [
18], anti-oxidation [
17,
19], and anti-diabetogenic activities [
20,
21]. Recently, some studies have shown that MA has moderate anti-cancer activities in colon cancer [
22] and astrocytoma cell [
23]. However, the mechanisms of MA action in inflammation and cancers are still not clear, and the synergistic effects of MA and TNFα in inhibiting tumor growth and proliferation have not been investigated.
In this study, we demonstrated that MA greatly potentiated the inhibitory effect of TNFα on the growth of pancreatic cancer through activation of apoptosis. MA activates TNFα-induced caspase and PARP apoptotic signaling pathway while suppresses TNFα-induced NF-κB activation in a dose-dependent manner. Moreover, MA inhibited TNFα-induced IκBα degradation, p65 phosphorylation and nuclear translocation, as well as NF-κB mediated gene expression. Finally, in vivo athymic nu/nu mouse model, MA suppressed pancreatic tumor growth and induced tumor cell apoptosis by inhibiting NF-κB-regulated anti-apoptosis genes, such as Survivin and Bcl-xl. Therefore, our results demonstrated that MA potentiated the efficacy of TNFα susceptibility to pancreatic tumor by enhancing caspase apoptotic signaling pathway and by inhibiting NF-κB activation and its down-stream gene expression.
Discussion
The aim of this study was to determine whether MA, a nontoxic microchemical ingredient and widely present in the diet, could sensitize pancreatic cancer to the treatment of TNFα. In various pancreatic cancer cell lines, MA or TNFα alone moderately inhibited cancer cell growth and induced apoptosis. When combined together, these anti-tumor cell effects were dramatically increased. To understand the underlying mechanisms, we demonstrate that MA could enhance TNFα induced caspase apoptotic pathway and suppress TNFα induced NF-κB activation (Fig.
6D). MA can inhibit TNFα induced IκBα degradation, block p65 nuclear translocation and phosphorylation, and finally, down-regulate the expression levels of NF-κB-mediated genes/proteins involved in proliferation (Cyclin D1, COX-2 and c-Myc), apoptosis (Survivin, Bcl-2, Bcl-xl, XIAP, IAP1), invasion (MMP-9 and ICAM-1), and angiogenesis (VEGF). In athymic nu/nu mice model, MA suppressed pancreatic tumor growth, induced apoptosis, and inhibited NF-κB-regulated antiapoptosis genes (Survivin and Bcl-xl).
Previous studies indicate that TNFα have multiple roles in pancreatic cancer [
13,
26,
27]. Several evidences found that TNFα could induce pancreatic cancer cell apoptosis
in vitro and
in vivo[
11,
27]. However, it is also reported that pancreatic tumor secreting TNFα may be the crucial trigger of tumor recurrence and metastasis after surgical resection [
8,
28]. Many studies also showed that tumor secreted-TNFα could prevent pancreatic cancer cell from apoptosis mediated by exogenous TNFα by activating NF-κB signaling pathway [
8,
14]. Furthermore, the apoptosis potency of TNFα can be increased in certain tumors when NF-κB activity is inhibited [
2,
13,
16,
27,
29‐
32]. Therefore, inhibition of NF-κB activation by pharmacologic approaches has become an attractive strategy for improving the anti-tumor activity of TNFα [
2,
10,
13,
27]. In the current study, we found that MA or TNFα alone showed moderate apoptosis activities in pancreatic cancer cell lines. However, MA dramatically increased cell apoptosis and inhibited NF-κB activity induced by TNFα in various pancreatic cancer cell lines, suggesting a synergistic effect of MA together with TNFα. And finally, we demonstrated that the expression levels of NF-κB-mediated downstream genes, including anti-apoptosis genes, proliferation genes, and metastasis related genes induced by TNFα, were all inhibited by MA in the pancreatic cancer cells.
In this study, we found that MA alone can effectively suppress pancreatic tumor growth, induce tumor apoptosis, and inhibit the expression levels of NF-κB-regulated anti-apoptosis genes in xenograft nude mouse models. Previous studies have shown that the secretion of TNFα was highly increased in pancreatic tumor tissues and even in serum
in vivo[
8,
27,
29]. Human plasma levels of TNFα were significantly higher in pancreatic adenocarcinoma patients (32.7 pg/ml) compared with chronic pancreatitis patients (3.2 pg/ml) and control group (<1.6 pg/ml; p < 0.01) [
33], suggesting that MA could be an effective agent in the treatment of pancreatic cancer due to the high level of TNFα secretion in the patients.
Methods
Materials
MA was synthesized in our laboratory. The antibodies used in this study were all obtained from Cell Signaling Technology (Danvers, MA). The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was obtained from Millipore Biotechnology. Penicillin, streptomycin, MEM/F12, DMEM (high glucose) and fetal bovine serum (FBS) were obtained from Invitrogen. Bacteria-derived recombinant human TNFα, Tris, glycine, NaCl, SDS, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO).
Cell lines and cell culture
The Panc-28 cell line was a generous gift from Dr. Bharat B. Aggarwal (The University of Texas M.D. Anderson Cancer Center Houston, TX). Panc-1, BxPC-3, AsPC-1 and A293 were obtained from American Type Culture Collection. Panc-28 cells were cultured in DME/F12 supplemented with 10% FBS. Other cells were cultured in DMEM supplemented with 10% FBS.
Proliferation assay
The proliferation effect of MA and TNFα was determined by the MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m)) method following the manual of CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega) with VERSAmax microplate reader (Molecular Devices). Briefly, 5000 cells were incubated with MA for 6 h in triplicate on 96-well plates and then treated with 0.1 nM TNFα for 36 h at 37°C. Thereafter, 20 microL MTS solution-I was added to each well. After 2~4 h of incubation at 37°C, the OD was measured at 490 nm using a 96-well multiscanner.
Transwell invasion assay
To test the effect of MA on cell invasion activity, we performed transwell invasion assay as previously described {Yi, 2008 #37}. Briefly, the starved cells (1 × 105/well) were seeded onto the top chambers of the transwell (Corning, with an 8 micro meter pore polycarbonate filter insert) coated with 0.1% gelatin (Corning). The bottom chambers were filled with DMEM/F12 with 10% FBS supplemented with 0.1 nM TNFα. The top and bottom chamber medium contain the same concentration of MA. PANC28 cells were allowed to migrate for 12 h. Then scraped the cells on the top surface of membrane and stained, counted the cells on the bottom side of the membranes (migrated cells) using OLYMPUS inverted microscope.
Live and Dead assay
To assess the cytotoxicity of MA and TNFα, we performed Live/Dead assay as previously described [
16]. Briefly, 1 × 10
5 cells were incubated with 25 microM MA for 12 h, and then incubated with 0.1 nM TNFα for 24 h at 37°C. Cells were stained with Live/Dead reagent (5 microM ethidium homodimer and 5 microM calcein-AM) and then incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (DM 4000B, Leica).
Annexin V and Tunnel assays
To detect whether MA induces pancreatic cancer cell apoptosis, we stained the treated cells with annexin V kit (sigma) as previously described [
16]. Briefly, 1 × 10
6 cells were pretreated with 25 microM MA for 3 h, treated with 0.1 nM TNFα for 12 h, and then subjected to annexin V staining. The results were analyzed with a flow cytometer (FACSAria; BD Biosciences) Cell apoptosis was also measured by the TUNEL assay using an
in situ cell death detection reagent (Millipore) as previously described. Stained cells were analyzed with a flow cytometer.
Electrophoretic mobility shift assay
To assess NF-κB activation by TNFα, we performed electrophoretic mobility shift assay (EMSA) using the Odyssey Infrared EMSA Kit following the manufactiure protocol. Briefly, nuclear extracts were prepared as previously described [
16]. Nuclear extracts (5 micro g/sample) were incubated with NF-κB IRDye™ 700 Infrared Dye Labeled Oligonucleotides, 5' AGTTGA
GGGGACTTTCCC AGG C 3' and 3' TCAACT
CCCCTGAAAGGG TCCG 5' (Boldface indicates NF-κB binding sites) in reaction buffers, for 30 min at 37°C. DNA-protein complex was separated from free oligonucleotide on 6.6% native polyacrylamide gels. The gels were visualized with Odyssey Infrared system and were quantitated using Imagequant software (LICOR Biosciences).
NF-κB-dependent luciferase reporter gene assays
The effect of MA on TNFα-induced NF-κB-dependent luciferase reporter assays were performed as previously described [
16].
Chromatin immunoprecipitation (CHIP) assay
CHIP assay was performed as described previously with some modification. Panc-28 (2 × 107 cells) were incubated with 25 microM MA for 6 h and then treated with 0.1 nM TNFα for the indicated time. Cells were then cross-linked with formaldehyde, quenched with glycine, and sonicated on ice and centrifuged at 4°C. Mixtures were incubated with anti-p65 antibody with rotation at 4°C overnight and then incubated with 100 micro L of protein A beads at 4°C for 6 h. After gentle centrifugation (2000 rpm), beads were resuspended in 1 mL of wash buffer, washed 3 times. Finally, immunocomplexes were washed and eluted with elution buffer (500 μg/ml each) at 37°C for 30 minutes. Extracted and dissolved Immunoprecipitated DNA was prepared for PCR assays. PCR products were run on 1.5% polyacrylamide gel and stained with ethidium bromide. Stained bands were visualized under UV light and photographed. The primers corresponding to -434/-414 and -319/-337 of human COX-2 promoter sequence: 5' AAAGACATCTGGCGGAAACCT 3' and 5' AGGAAGCTGCCCCAATTTG 3', were used in the PCR reactions.
Immunocytochemical analysis of NF-κB/p65 localization
The effect of MA on the nuclear translocation of p65 was examined by immunocytochemistry as previously described. Briefly, cells were pretreated with 25 microM MA following seeded on a gelatin-coated glass, after stimulation with or without 0.1 nM TNFα for 20 minutes, fixed with 4% paraformaldehyde after permeabilization with 0.2% Triton X-100. After being washed in PBS, blocked and then incubated with rabbit polyclonal anti-human p65 antibody at a 1/25 dilution overnight incubation at 4°C, washed, incubated with goat anti-rabbit IgG-Alexa Fluor 594 (Molecular Probes) at a 1/500 dilution for 1 h, and counterstained for nuclei with DAPI (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium purchased from Sigma-Aldrich and analyzed under a fluorescence microscope.
Xenograft mouse model of pancreatic cancer
Xenograft mouse models were performed as described. 4-5 week-old athymic nu/nu male mice purchased from SIBS, Shanghai. Maintain, use, and treatment of all animals in this study were in accordance with accepted standards of the Ethics Committee at ECNU. Mice weighing about 20 g were divided with six mice per group. Panc-28 tumor cells were s.c. injected (3.3 × 106 cells per mouse) into the mice. After the tumors had established (about 100 mm3), the mice were subcutaneous injected with 10 mg/kg or 50 mg/kg MA every two day. The control mice were injected with DMSO. The mice body weight and tumor sizes were recorded every two-day and the tumor size were determined by Vernier caliper measurements and calculated as length × width × height. After 36 days, mice with tumors were sacrificed.
Histology and immnohistochemistry
Tumor were removed, weighed, fixed with 10% formalin, and embedded with paraffin. TUNEL assay was performed to detect apoptotic cells using the in situ Cell Death Detection kit from Chemicon according to the manufacture's instructions. The expression of Survin and Bcl-xl were examined using an immunohistochemical method described previously.
Statistical analysis
Data are represented as mean ± SD for the absolute values as indicated in the vertical axis legend of figures. The statistical significance of differential findings between experiments and controls was determined by t-test or Dunnett test as implemented by SPSS10.0 software. P values smaller than 0.05 were considered statistically significant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CL, ZY, CZ, WQ and LW performed the experiments, WQ and JT synthesized the compound, CL, DL, ZY, LW, JT, MQ, JL, and ML analyzed the data, CL, JL and ML designed the experiments and prepared the manuscript. All authors read and approved the final manuscript.