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1 Department of Surgery and Surgical Basic Science and 2 Department of Bioorganic Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
Requests for Reprints:Ryuichiro Doi, Department of Surgery and Surgical Basic Science, Kyoto University, 54-Shogoin Kawara-cho, Sakyo, Kyoto 606-8507, Japan. Phone: 81-75-751-3671; Fax: 81-75-751-3219. E-mail: doir{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Key Words: CXCR4 • SDF • migration • invasion • receptor antagonist
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Introduction |
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Recently, several studies have been conducted to detect the expression of CXCR4 and SDF-1 in solid tumors. The results are not uniform, and the relevance to cancer progression and tumor angiogenesis is unclear (15–17). In breast cancer, the SDF-1/CXCR4 system has been implicated in the formation of metastasis (18). Before this, we found that SDF-1 mRNA expression is detected in pancreatic cancer tissues, but is not detected in pancreatic cancer cell lines, whereas CXCR4 mRNA expression is detected in both pancreatic cancer tissues and cancer cell lines (19). To date, however, the role of interaction between SDF-1 and CXCR4 in pancreatic cancer progression has not been defined.
The specific ligands for these receptors are able to inhibit HIV infection (20–23). As for the malignant solid tumors, neutralizing the interactions of SDF-1/CXCR4 by administration of an antibody to CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph node and lung in the animal model. For this reason, efforts have focused on developing a specific antagonist for chemokine receptors.
A CXCR4 antagonist, T22, was previously discovered as an anti-HIV peptide based on chemical conversions of horseshoe crab self-defense peptides, tachyplesins and polyphemusins (24). On the basis of T22 structure, we reported the synthesis of a novel CXCR4 inhibitor, T140 (25). T140 strongly antagonizes CXCR4 function, though it is not stable in serum due to the cleavage of COOH-terminal Arg14. Consequently, we reported that the COOH-terminal amidation and the double-L-citrulline (Cit)-scanning of T140 led to development of a novel effective CXCR4 inhibitor, TN14003, which possesses high selectivity index and complete stability in serum, without significant change in the secondary structure (25). In this study, we investigated the role of SDF-1/CXCR4 axis in human pancreatic cancer cells, and tested the inhibitory effect of the novel CXCR4 inhibitor, TN14003, on in vitro cell functions that are relevant to metastasis.
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Materials and Methods |
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Reagents and Antibodies
The following antibodies and reagents were purchased: An affinity-purified goat anti-CXCR4 polyclonal antibody (sc-6190, Santa Cruz Biotechnology, Santa Cruz, CA); a mouse anti-human CXCR4 monoclonal antibody (MAB172, R&D Systems, Minneapolis, MN); anti-phospho-p44/42 mitogen-activated protein (MAP) kinase antibody (#9106, Cell Signaling Technology, Beverly, MA); anti-p44/42 MAP kinase antibody (#9102, Cell Signaling Technology); PD98059 (Calbiochem, San Diego, CA); Cy3-conjugated Affinipure goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA); and rhodamine-phalloidin (R-415, Molecular Probes, Eugene, OR).
Cell Line and Culture Conditions
Human pancreatic cancer cell lines (CFPAC-1, Capan-2, AsPC-1, PANC-1, BxPC-3, and SUIT-2) were maintained in the following media at 37°C in a humid atmosphere of 5% CO2/95% air. CFPAC-1 cells were cultured in Iscove's modified Dulbeco's medium with 10% fetal bovine serum (FBS). PANC-1 cells were cultured in DMEM with 10% FBS. BxPC-1 cells, Capan-2 cells, AsPC-1 cells, and SUIT-2 cells were cultured in RPMI 1640 with 10% FBS. Each medium contained 100 units/ml penicillin and 0.1 mg/ml streptomycin.
Expression of CXCR4 in Pancreatic Cancer Cells
Serum-starved subconfluent cells were harvested, washed in cold PBS, and lysed in ice-cold lysis buffer [10 mM PBS (pH 7.4), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA] supplemented with 1% phenylmethylsulfonyl fluoride and 0.02 mg/ml gabexate mesilate, a synthetic protease inhibitor (FOY, Ono Pharmaceutical, Osaka, Japan) for 30 min at 4°C. The lysate was homogenized and centrifuged at 15,000 rpm for 30 min at 4°C to remove debris, and the protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL). The extracted protein was subjected to Western blotting, as previously described (26). Equal amounts of protein were loaded onto 10% SDS-polyacrylamide gels and the proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA). Blots were blocked at 4°C overnight with 5% (w/v) nonfat milk in TTBS buffer (10 mM Tris-HCl, 150 mM NaCl, 0.5% Tween 20). The blots were incubated for 1 h at room temperature with the primary antibody against CXCR4 (sc-6190, Santa Cruz Biotechnology) diluted at 1:100 with TTBS. The blots were incubated for 1 h with a second antibody (200 ng/ml alkaline phosphatase-conjugated anti-goat IgG, Pierce). Immunoreactive proteins were visualized using alkaline phosphatase solution supplemented with 100 mM Tris-HCl, 100 mM HCl, 5 mM MgCl2, 0.03% nitroblue tetrazolium, and 0.017% 5-bromo-indolylphosphate P-toluidine salt.
Immunocytochemistry
Pancreatic cancer cells were seeded on coverslips and incubated for 24 h at 37°C in a humid atmosphere of 5% CO2/95% air. The coverslips with cells were then fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS, permeabilized in 1% Triton X-100 in PBS for 15 min, washed, and blocked with TTBS with 1% BSA. For CXCR4 staining, fixed and permeabilized cells were incubated with mouse anti-human CXCR4 monoclonal antibody (MAB172, R&D Systems) diluted in 1% BSA-TTBS (0.01 mg/ml) for 2 h at 37°C, and rinsed 3 times with TTBS, and then incubated for 30 min with secondary antibody (Cy3-conjugated Affinipure goat anti-mouse IgG, Jackson Immunoresearch) diluted in 1% BSA-TTBS (1:100). After the final wash, coverslips were mounted on the slide-glass using 50% solution of glycerol in PBS. The cells were examined under a fluorescence microscope (Olympus, Tokyo, Japan).
In Vitro Proliferation Assay
Pancreatic cancer cells (AsPC-1, PANC-1, and SUIT-2) were seeded at a density of 5000 cells per well into 96-well plates in culture medium containing 10% FBS. After 24 h, the cultures were washed and refed with medium alone (control) or with medium containing SDF-1 or TN14003 at various concentrations. After 3 days, the number of viable cells was counted using Cell Counting Kit 8 (Dojindo Co., Kumamoto, Japan) according to the manufacturer's instructions. The assay reagent is a tetrazolium compound (WST-8) that is reduced by live cells into a colored formazan product measured at 450 nm. The quantity of formazan product measured at 450 nm is directly proportional to the number of live cells in the culture. The experiments were repeated in triplicate wells.
In Vitro Migration Assays
Migration of cancer cells was assayed using 6.5-mm-diameter chambers with 8-µm pore filters (Transwell, 24-well cell culture, Coster, Boston, MA). Pancreatic cancer cells were suspended at 2 x 105 cells/ml in serum-free media, and then 0.2 ml cell suspension was added to the upper chamber. Then, 0.5 ml serum-free media with various concentrations of SDF-1 was added to the lower chamber. In another set of experiments, 0.5 ml serum-free media with 100 ng/ml of SDF-1 (fixed concentration) plus various concentrations of TN14003 was added to the lower chamber. The chambers were incubated for 12 h at 37°C in a humid atmosphere of 5% CO2/95% air. After incubation, the filters were fixed and stained with Diff-Quick reagent (Dade Behring, Dugen, Switzerland). The upper surface of the filters was scraped twice with cotton swabs to remove non-migrating cells. The experiments were repeated in triplicate wells, and the number of migrating cells in five high-power fields per filter was counted microscopically at x400 magnification. Because the background migration without SDF-1 varied among experiments, data were normalized as the migration index: the number of migrating cells in an experimental chamber/the number of migrating cells in control chamber without SDF-1. In another experiment, cells were pretreated with 30 µM PD98059 (Calbiochem), a MEK1 inhibitor, before SDF-1 treatment.
In Vitro Invasion Assays
Invasion of cancer cells was assayed using a Biocoat Matrigel invasion chamber (Becton Dickinson, Bedford, MA), which consists of an 8-µm pore size polyethylene terephthalate (PET) membrane that has been overlaid with Matrigel (basement membrane matrix). PANC-1, SUIT-2, or AsPC-1 cells were suspended at 2 x 105 cells/ml in serum-free media, and then 0.2 ml cell suspension was added to the upper chamber. Next, 0.5 ml serum-free media with 100 ng/ml of SDF-1 (fixed concentration) plus various concentrations of TN14003 was added to the lower chamber. The chambers were incubated for 12 h at 37°C in a humid atmosphere of 5% CO2/95% air. After incubation, the filters were fixed and stained with Diff-Quick reagent (Dade Behring). The upper surface of the filters was scraped twice with cotton swabs to remove non-invading cells. The experiments were repeated in triplicate wells, and the number of invading cells in five high-power fields per filter was counted microscopically at x400 magnification. The data were processed by the method described in migration assay.
Detection of p44/42 MAP Kinases in Pancreatic Cancer Cells
Cells were put in DMEM containing low levels of BSA (0.5%) to render the cells quiescent. Then cells were incubated with 100 ng/ml SDF-1 or 100 ng/ml SDF-1 plus 100 nM TN14003 for 5 min to 1 h at 37°C. In another setting, PANC-1 cells were preincubated for 1 h at 37°C with 10 µM PD98059 (Calbiochem), a MEK1 inhibitor, before SDF-1 treatment. After treatment with SDF-1, cells were lysed for 60 min in phosphorylation-inhibitory radioimmunoprecipitation assay (RIPA) buffer containing 50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% NP40, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml gabexate mesilate, then the lysate was sonicated for 10 s. Total extracts were cleaned by centrifugation at 12,000 rpm for 10 min at 4°C, and the supernatants were collected. Protein concentrations were measured using a protein assay kit (Tonein-TP, Otsuka Pharmaceutical, Tokyo, Japan). The lysates were resuspended in 1 volume of the gel loading buffer that contained 50 mM Tris-HCl (pH 6.7), 4% SDS, 0.02% bromophenol blue, 20% glycerol, and 4% 2-mercaptoethanol, and then boiled at 95°C for 90 s. The extracted protein was subjected to Western blotting, as previously described (26). In brief, 30-µg aliquots of protein were size-fractionated to a single dimension by SDS-PAGE (12% gels) and transblotted to a 0.45-µm polyvinylidene difluoride membrane (Bio-Rad) in a semidry electroblot apparatus (Bio-Rad). The blots were then washed 3 times with TTBS buffer and incubated for 2 h at room temperature in the first antibody solution containing anti-phospho-p44/42 MAP kinase antibody (#9106, Cell Signaling Technology). After three washings in TTBS buffer, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody. After three washings in TTBS buffer, membranes were treated with enhanced chemiluminescence reagents (Amersham Life Sciences, Amersham, United Kingdom) according to the manufacturer's protocol. Membranes were exposed to X-ray film for 50 s. Protein expression was measured by a densito-analyzer system (AE-6920M, ATTO Corporation, Tokyo Japan). After stripping and blocking, the same blots were re-probed with anti-p44/42 MAP kinase antibody (#9102, Cell Signaling Technology) to measure total MAP kinase.
Actin Cytoskeleton
Pancreatic cancer cells were seeded on coverslips and incubated for 24 h at 37°C in a humid atmosphere of 5% CO2/95% air. Then the cells were incubated in serum-free medium containing 0.1% BSA with 100 ng/ml SDF-1 or with 100 ng/ml SDF-1 plus 100 nM TN14003 for 1 h at 37°C. The coverslips with cells were then fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS, permeabilized in 1% Triton X-100 in PBS for 15 min, washed, and blocked with TTBS with 1% BSA. For visualization of filamentous actin, the cells were exposed to rhodamine-phalloidin for 30 min at 37°C and washed with TTBS. After final wash, coverslips were mounted on the slide-glass using 50% solution of glycerol in PBS. The cells were examined under a fluorescence microscope (Olympus).
Adhesion Assay
The adhesion of pancreatic cancer cells to the elements of extracellular matrix (ECM) was evaluated. Pancreatic cancer cells were grown to subconfluent state and then harvested by 0.25% trypsin/EDTA (Invitrogen Corp., Carlsbad, CA) in 1 min. Cells were preincubated with SDF-1 or with SDF-1 plus TN14003. The same volume of vehicle was added to cells as a control. Preincubated cells were plated onto a 96-well microplate which was precoated with various kinds of ECM elements, that is, 0.01 mg/ml plasma fibronectin, 0.01 mg/ml vitronectin or Matrigel, or 96-well Microtest Plate (BioCoat, Becton-Dickinson Japan, Tokyo, Japan) precoated with collagen I, collagen IV, or laminin. Then the cells were incubated for 3 h at 37°C in 5% CO2/95% air to allow cell attachment. Cells were washed gently with PBS 3 times to remove detached cells. The number of adherent cells was measured by WST method using Cell Counting Kit 8 (Dojindo). Experiments were repeated 3 times in triplicate wells.
Statistical Analysis
Statistical comparisons were performed by a two-way ANOVA for repeated measures, followed by a post hoc Turkey test, or Student's two-tailed t test. P < 0.05 was considered to be significant.
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Effect of TN14003 on SDF-1-Induced Migration and Invasion of Pancreatic Cancer Cells
SDF-1 stimulated the migration of pancreatic cancer cells (Fig. 3). Maximal effect was observed at 100 ng/ml of SDF-1 in all pancreatic cancer cell lines, and maximal migration indices were 214 ± 27% in PANC-1 cells, 191 ± 35% in AsPC-1 cells, and 159 ± 17% in SUIT-2 cells, respectively. Supramaximal suppression was observed at concentrations over 100 ng/ml in this system. Next, the inhibitory effect of TN14003 on SDF-1-induced migration was tested. The migration induced by SDF-1 at 100 ng/ml was inhibited by TN14003 in PANC-1, AsPC-1, and SUIT-2 cells, and was completely blocked by TN14003 at 100 nM (Fig. 4).
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Cell Adhesion Assay
The effects of SDF-1 on pancreatic cancer cell adhesion to the elements of ECM were tested. The number of adherent cells to fibronectin, vitronectin, and collagen I was not changed by SDF-1 (Fig. 8, A–C). In contrast, the number of adherent cells to Matrigel was increased by SDF-1 (Fig. 8D). Matrigel is a solubilized basement membrane matrix extracted from the Engelbreth-Holm-Swarm mouse tumor, and is composed of collagen IV, laminin, and other elements. Therefore, we tested the effects of SDF-1 on cell adhesion to collagen IV and laminin-coated plate. SDF-1 induced increase of pancreatic cancer cell adhesion to laminin but not to collagen IV (Fig. 8, E and F). The increasing effect of SDF-1 on pancreatic cancer cell adhesion to laminin was not reversed by an addition of TN14003 (data not shown).
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Discussion |
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B regulates the motility of cancer cells by directly up-regulating the expression of CXCR4 (31). In pancreatic cancer cell lines, we showed that SDF-1 had no direct effect on cell proliferation. Our result is in accordance with the recent study which showed that SDF-1 had no proliferative effects on rhabdomyosarcoma (32). It also agrees with a report demonstrating lack of a proliferative effect by SDF-1 on colony-forming units-megakaryocyte (CFU-MK) (33) or lymphohematopoietic cells (34). In contrast, recent reports demonstrated that SDF-1 stimulates proliferation of small cell lung cancer cells (NCI-H69) in the presence of serum (16), that SDF-1 acts together with thrombopoietin to enhance the development of CFU-MK in a murine model (35) and that SDF-1 at low doses enhances the proliferation of peripheral blood CD34+ cells (36). In addition, antisense CXCR4 overexpression in glioblastoma cells caused inhibition of cell proliferation, suggesting that SDF-1/CXCR4 system is involved in cell proliferation in glioblastoma cell lines as well (15, 37). We suggest that these differences may be due to the different culture system or to the different target cells.
Although we did not find any effect of SDF-1 on proliferation of pancreatic cancer cells, we found that SDF-1 stimulates cell metastasis and invasive behavior. In migration assay by Transwell chamber, SDF-1 significantly increased migration of pancreatic cancer cells. The cancer cells responded to SDF-1 in a similar manner, and maximal effect by SDF-1 was observed at 100 ng/ml. Invasion assay by Matrigel-coated invasion chamber also showed that SDF-1 significantly stimulated invasion of pancreatic cancer cell. Previous reports similarly showed that invasion through Matrigel was stimulated by SDF-1 in CXCR4-expressing ovarian cancer cells (38), prostate cancer cells (39), and myeloma cells (40). Video microscopic examination revealed that SDF-1 stimulated the motility of small cell lung cancer cells (16).
It has recently been reported that CXCR4 was highly expressed in malignant but not normal breast tissue, and that its ligand, SDF-1, is expressed in those organs where breast cancer metastasis is frequently found (bone marrow, lymph node, lung, and liver) (18). Furthermore, neutralizing the interactions of SDF-1/CXCR4 by administration of an antibody to CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph node and lung in their breast cancer metastasis model. These reports, taken together with the current results, indicate that SDF-1/CXCR4 interaction may be generally important for the metastasis of solid tumors that express CXCR4.
In tumor cells, high levels of actin polymerization are required for the formation of stress fiber and pseudopodia, which in turn are implicated in the enhancement of cell migration and invasion. We showed that the treatment of pancreatic cancer cells with SDF-1 resulted in a dramatic increase in actin polymerization, which is needed for the invasion of malignant cells into tissues and for efficient metastasis. These findings suggest that SDF-1/CXCR4 ligand receptor system plays an important role in invasion as well as metastasis in pancreatic cancer.
Since CXCR4 was identified as a co-receptor for the entry of T-cell line-tropic (T-tropic) HIV-1 (13, 14), development for CXCR4 antagonists seemed an ideal approach to discover an effective anti-HIV agent. Several specific antagonists for CXCR4 have been developed to date; however, there was no effective agent that satisfied both pharmacological and clinical requirements. The CXCR4 antagonist T22, which was derived from chemical conversions of horseshoe crab self-defense peptides, tachyplesins, and polyphemusins, had previously been discovered as an anti-HIV peptide (24).
On the basis of the structure-activity relationship study (SAR) of T22, we previously synthesized a more effective analogue, T140 (27). This agent showed the highest level of anti-HIV activity and antagonism of target cell entry by X4-HIV-1 among all the CXCR4 antagonists that have been reported to date. However, T140 is not stable in serum due to the cleavage of the COOH-terminal Arg, indispensable for anti-HIV activity. The COOH-terminal amidation and the double-Cit-scanning of T140 have led to development of the novel effective CXCR4 inhibitor, TN14003 (25). TN14003 has a very high selectivity index (SI [CC50/EC50] = 680,000) (25), that is, it showed high anti-HIV activity (EC50 = 0.6 nM) which was based on the protection against HIV-induced cytopathogenicity in MT-4 cells, whereas TN14003 showed low cytotoxicity (CC50 = 410 µM) which was determined by the trypan blue exclusion staining method in human peripheral blood mononuclear cells. In addition, unlike T140, TN14003 possesses complete stability in serum.
Because of the high selectivity index of TN14003 for this system, we applied this agent on CXCR4-expressing human pancreatic cancer cells. We demonstrated that SDF-1-induced migration and invasion of pancreatic cancer cells were completely blocked by 100 nM TN14003. The IC50 of TN14003 on cancer cell migration and invasion was almost one-tenth that of T22. We demonstrated that TN14003 inhibited SDF-1-induced actin polymerization of pancreatic cancer cells. In contrast, SDF-1 stimulated adhesion of pancreatic cancer cells to laminin, one of the ECM components, but the addition of TN14003 did not reverse the effect of SDF-1. Therefore, TN14003 seems to mainly antagonize the stimulatory effect of SDF-1 on the mobility of pancreatic cancer cells. The Cit-substitution for a reduction of total cationic charges in the molecule is thought to be useful for developing effective anti-metastatic peptides, which could be a new type of anti-cancer agents against CXCR4.
In this study, we found that human pancreatic cancer cells express the chemokine receptor CXCR4, which mediate ligand-dependent cell migration and invasion in vitro, and that migration and invasion of pancreatic cancer cells induced by SDF-1 were completely blocked by the novel CXCR4 inhibitor TN14003. These results suggest that novel therapeutic strategies in metastasis of pancreatic cancer can be expected based on manipulation of the SDF-1 and CXCR4 ligand receptor system. This stimulative effect by SDF-1 was mediated via the MAP kinase pathway as shown by detecting increased level of phosphorylated MAP kinases, and by the fact that MAP kinase kinase inhibitor suppressed phosphorylated MAP kinases and cell mobility. These results are consistent with the previous report that showed that SDF-1 selectively activated p44/42 MAP kinase (41). The novel CXCR4 inhibitor TN14003 also suppressed SDF-1-induced phosphorylation of MAP kinases.
In conclusion, the results in this study indicate that the SDF-1/CXCR4 ligand receptor axis may play an important role in metastasis of pancreatic cancer, and that CXCR4 receptor antagonists could inhibit metastatic behavior of pancreatic cancer cells. Therefore, it is strongly suggested that TN14003 could be an effective anti-metastatic agent. Small-molecule antagonists of chemokine receptors like TN14003 may be useful in treating cancer patients.
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Footnotes |
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Grant support: Grant-in-Aid for Scientific Research (#15390395) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Note: T. Mori and R. Doi contributed equally.
Received 8/11/03; revised 10/13/03; accepted 10/16/03.
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 J. Kim, H. Takeuchi, S. T. Lam, R. R. Turner, H.-J. Wang, C. Kuo, L. Foshag, A. J. Bilchik, and D. S.B. Hoon Chemokine Receptor CXCR4 Expression in Colorectal Cancer Patients Increases the Risk for Recurrence and for Poor Survival J. Clin. Oncol., April 20, 2005; 23(12): 2744 - 2753. [Abstract] [Full Text] [PDF]
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 C. Laverdiere, B. H. Hoang, R. Yang, R. Sowers, J. Qin, P. A. Meyers, A. G. Huvos, J. H. Healey, and R. Gorlick Messenger RNA Expression Levels of CXCR4 Correlate with Metastatic Behavior and Outcome in Patients with Osteosarcoma Clin. Cancer Res., April 1, 2005; 11(7): 2561 - 2567. [Abstract] [Full Text] [PDF]
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C. C. Schimanski, S. Schwald, N. Simiantonaki, C. Jayasinghe, U. Gonner, V. Wilsberg, T. Junginger, M. R. Berger, P. R. Galle, and M. Moehler Effect of Chemokine Receptors CXCR4 and CCR7 on the Metastatic Behavior of Human Colorectal Cancer Clin. Cancer Res., March 1, 2005; 11(5): 1743 - 1750. [Abstract] [Full Text] [PDF]
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 F. Marchesi, P. Monti, B. E. Leone, A. Zerbi, A. Vecchi, L. Piemonti, A. Mantovani, and P. Allavena Increased Survival, Proliferation, and Migration in Metastatic Human Pancreatic Tumor Cells Expressing Functional CXCR4 Cancer Res., November 15, 2004; 64(22): 8420 - 8427. [Abstract] [Full Text] [PDF]
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, AsPC-1; 
, PANC-1; 
, SUIT-2. Columns, mean of three separate experiments; bars, SE. *, P < 0.05 compared with control.