Tumor invasion and metastasis, which are fundamental properties of malignant cancer cells, are a multistep, complex process, which includes cell division and proliferation, as well as proteolytic digestion of the extracellular matrix (ECM) and basement membrane.1 Proteolytic degradation of ECM components, especially by matrix metalloproteinases (MMPs), which are a family of zinc-dependent endopeptidases, is involved in both physiological and pathological processes, such as development, tissue remodeling, inflammation, tumor cell invasion and tumor metastasis.2 MMP-2 and MMP-9 of the MMP family are known to degrade type IV collagen, a major constituent of the basement membrane during cancer invasion and metastasis.3
Platelet-activating factor (PAF), which is produced by a variety of inflammatory cells, is a potent lipid messenger involved in cellular activation, fertilization, intracellular signaling, apoptosis and a diverse range of inflammatory reactions.4, 5, 6, 7, 8 During the past decade, in vitro studies and animal models have highlighted a potential role of PAF in cancer and tumor metastasis. Thus, PAF acts on the growth of various human tumor cell lines,9, 10 increases adhesiveness of tumor cells to vascular endothelia,11 enhances oncogene expression12 and can contribute to tumor development by enhancing cell motility and stimulating the angiogenic response.10, 13 Furthermore, PAF is produced by certain tumor cells,14, 15 elevated levels of which have been reported in human tumor metastasis.16, 17 In addition, direct evidence that PAF promotes experimental pulmonary metastasis by B16F10 murine melanomas has also been reported in our previous study.18 However, the mechanism for PAF-induced tumor metastasis remains to be elucidated.
In this study, to improve our knowledge concerning the role of PAF in tumor metastasis, our attention was focused on the potential involvement of MMP-2 and MMP-9, based on reports that PAF induces the expression of MMP-9 in a number of cell types, such as transformed human vascular endothelial cell line,19 bronchial epithelial cells,20 neutrophils21 and corneal epithelial cells,22 and the expression of MMP-2 in endothelial cells.23, 24
Material and methods
Specific pathogen-free female C57BL/6 mice were obtained from the Korean Institute of Chemistry Technology (Daejeon, Republic of Korea) and kept in our animal facility for at least 2 weeks before use. All mice were used at 8–10 weeks of age.
PAF (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine), curcumin and 1,10-phenanthroline were obtained from Sigma Chemical Co. (St. Louis, MO). The PAF receptor antagonist, CV 6209, and the NF-κB inhibitor, parthenolide, were from BIOMOL Research Laboratories (Plymouth Meeting, PA).
Small interfering RNA (siRNA) strands for mouse p65, c-jun, MMP-2 and MMP-9 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In vivo delivery of siRNA was performed via intratracheal injection of 0.01 μM siRNA per gram of body weight using the TransIT In Vivo Gene Delivery System (PANVERA, Madison), according to the instructions of manufacturer.
The B16F10 mouse melanoma, which is metastatic in the lungs of C57BL/6 mice, was originally supplied by the Tumor Repository of the National Cancer Institute (Bethesda, MD), and maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Cambrex Co., Walkersville, MD) at 37°C in a 5% CO2 atmosphere.
Electrophoretic mobility shift assay
Nuclear extracts were prepared from the lungs, as previously described.19 To inhibit the endogenous protease activity, 1 mM PMSF was added. An oligonucleotide containing the Igκ-chain-binding site (κB, 5′-CCGGTTAACAGAGGGGGCTTTCCGAG-3′) or AP-1-binding site (AP-1, 5′-AAGGCGCTTGATGACTCAGCCGGAA-3′) was synthesized as a probe for the gel retardation assay. The 2 complementary strands were annealed and labeled with [α-32P]dCTP. Labeled oligonucleotides (10,000 cpm), 15 μg nuclear extracts and binding buffer [10 mM Tris-HCl (pH 7.6), 500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng poly (deoxyinosinic-deoxycytidylic acid) and 1 mM DTT] were incubated for 30 min at room temperature in a final volume of 20 μL. For the supershift/inhibition assay, 1 μg specific supershifting antibody against mouse p50, p65, c-fos or c-jun (Santa Cruz Biotechnology) was incubated with the nuclear extract on ice for 1 hr, followed by the addition of the labeled oligonucleotide to the binding reaction. Normal rabbit IgG antibody (Santa Cruz Biotechnology) was used as a control. The reaction mixture was analyzed by electrophoresis on a 5% polyacrylamide gel in 0.5× Tris-borate buffer. Specific binding was controlled by competition with a 50-fold excess of cold κB, AP-1 or cyclic AMP response element (CRE) oligonucleotide.
Real-time reverse transcription-PCR
RNA was prepared as previously described.19 Reverse transcription was performed using 0.5 μg of total RNA in 10 μL of reaction mixture (Promega, Madison, WI) containing oligo(dT) and avian myeloblastosis virus reverse transcriptase. PCR was performed on the Rotor-Gene 3000 System (Corbett Research, Morklake, Australia) using the SYBR Green PCR Master Mix Reagent Kit (Qiagen, Valencia, CA). Mouse specific primers used were as follows: MMP-2 for real-time PCR, 5′-CTGGAATGCCATCCCTGATAA-3′ and 5′-CAAACTTCACGCTCTTGAGACTTT-3′; MMP-2 for the visualization of the results, 5′-CTCAGATCCGTGGTGAGATCT-3′ and 5′-CTTTGGTTCTCCAGCTTCAGG-3′; MMP-9 for real-time PCR, 5′-TCGTGGCTCTAAGCCTGACC-3′ and 5′-GACACATAGTGGGAGGTGCT-3′; MMP-9 for the visualization of the results, 5′-ATCCAGTTTGGT-GTCGCGGAGC-3′ and 5′-GAAGGGGAAGACGCACAGCT-3′ and β-actin, 5′-CTGAAGTCACCCATTGAACATGGC-3′ and 5′-CAGAGCAGTAATCTCCTTCTGCA-3′. The relative levels of mRNA were calculated using the standard curve generated from sequential cDNA dilutions. The mean cycle threshold (Ct) values from quadruplicate measurements were used to calculate the gene expression, with normalization to β-actin as an internal control. Calculations of the relative levels of gene expression were conducted using the complementary computer software (Corbett Research) employing a standard curve. The results were expressed as fold increase over untreated mice. cDNA, amplified by PCR (Perkin Elmer System 2400, Norwalk, CT), was visualized after staining with ethidium bromide.
Small lung specimens were homogenized in cell lysis buffer [250 mM sucrose and 40 mM Tris-Cl, (pH 7.4)], which contained protease inhibitors, with equal amounts of the lysates separated on 8.5% SDS polyacrylamide gel under reducing conditions. The lysates were then transferred onto Protran nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Equal protein loading was verified by staining both the gel and the membrane with Coomassie brilliant blue R-250 and Ponceau S (Sigma Chemical), respectively. Membranes were blocked by incubation for 1 hr at room temperature in 5% skim milk in TBS, followed by a further 2 hr of incubation with primary antibodies against mouse MMP-2 and MMP-9 (Oncogene Research Products, San Diego, CA), which recognize only the latent form under reducing conditions, p65, c-jun as well as β-actin (Sigma Chemical). Blots were washed for 15 min with three changes of TBS-0.05% Tween 20 solution, followed by incubation for 1 hr at room temperature with the alkaline phosphatase-conjugated antirabbit IgG antibody (Santa Cruz Biotechnology). The alkaline phosphatase activity was detected using the nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color development system (Promega), with β-actin used as a loading control.
Equal amounts of the lung extracts were separated by electrophoresis on 8.5% SDS-polyacrylamide gel containing 1 mg/mL gelatin (Sigma Chemical). After the electrophoresis, the gel was washed at room temperature for 1 hr in washing buffer [50 mM Tris-Cl (pH 7.4) and 2.5% Triton X-100] and incubated at 37°C overnight in the incubation buffer [50 mM Tris-Cl (pH 7.4), 75 mM NaCl and 2.5 mM CaCl2]. The gel was stained with 0.2% Coomassie brilliant blue R-250 (Sigma Chemical) in a mixture of methanol:acetic acid:water (2:1:7) for 2 hr and destained in the same solution without dye. Clear zones against the blue background indicated the presence of gelatinolytic activity.
The lungs were removed 12 hr after treatment with 1 μg PAF and then fixed in 4% neutral buffered formalin (Sigma Chemical) overnight at 4°C, paraffin embedded and sectioned at 4 μm. Immunoperoxidase staining was performed at room temperature using Histostain-Plus kit (ZYMED Lab., San Francisco, CA), according to the instructions of the manufacturer. In brief, deparaffinized sections were treated with 3% H2O2 in methanol for 3 min to reduce the endogenous peroxidase activity. Sections were incubated with serum block solution for 30 min, followed by incubation with one of the primary antibodies against mouse MMP-2 and MMP-9 (Santa Cruz Biotechnology) for 1 hr and then washed in PBS. The specimens were incubated with the corresponding biotinylated secondary antibodies for 10 min and then HRP-streptavidin complex for a further 10 min. The color was allowed to develop with HRP substrate for 3 min. Sections were counterstained with hematoxylin. All magnifications shown are 200×.
Lung colonization assay
A single-cell suspension (>95% viability by trypan blue exclusion assay), at a concentration of 1.5 × 105 in 100 μL, in PBS was injected into the lateral tail veins of C57BL/6 mice, which were autopsied 14 days later. Lungs were removed and fixed in Bouin's solution (Sigma Chemical), and the number of surface lung colonies counted under a dissecting microscope.
Data are represented as the mean ± SE. Statistical significance was determined by a one-way ANOVA test (StatView; Abacus Concepts, Berkeley, CA). All experiments were conducted at least twice. Reproducible results were obtained and representative data, therefore, are shown in the figures.
In vivo effect of PAF on the activation of NF-κB and AP-1
Initially, the effects of PAF on the activation of NF-κB and AP-1 were examined. Injection of PAF resulted in the activation of NF-κB in the lungs, which was blocked by pretreatment with the PAF antagonist, CV6209, the NF-κB inhibitor, parthenolide or p65 siRNA, but not by the nonsense mismatched control (Fig. 1a). Complete blocking of NF-κB mobilization, by the addition of the cold competitor, but not the irrelevant motif, CRE, indicated the specificity of NF-κB binding. In the supershift assay, anti-p50 and anti-p65 antibodies reduced the intensity of the DNA-binding activity, suggesting that the p50/p65 heterodimer is the activated form of the NF-κB induced by PAF (Fig. 1a). To confirm that the p65 siRNA used really block the synthesis of its target, an immunoblotting analysis was performed. The synthesis of p65 was strongly blocked by p65 siRNA, but not by control siRNA (Fig. 1b). In a manner parallel to the NF-κB activation, PAF also induced AP-1 activation, which was blocked by pretreatment with CV6209 and the AP-1 inhibitor, curcumin or c-jun siRNA (Fig. 1c). A competitive binding assay with cold AP-1 revealed the specificity of AP-1 binding. In the supershift assay, anti-c-jun, but not anti-c-fos antibody, reduced the intensity of the DNA-binding activity, suggesting that c-jun is the activated form of the AP-1 induced by PAF (Fig. 1c). The synthesis of c-jun was also blocked by c-jun siRNA, but not by control siRNA (Fig. 1d).
In vivo effect of PAF on the expression of MMP-2 and MMP-9
PAF significantly increased the mRNA expressions of both MMP-2 and MMP-9 in the lungs in a time-dependent manner (Fig. 2a). The increased MMP-9 expression was inhibited by both NF-κB inhibitors (parthenolide and p65 siRNA) and AP-1 inhibitors (curcumin and c-jun siRNA), whereas the increased MMP-2 expression was inhibited by only AP-1 inhibitors, curcumin and c-jun siRNA (Fig. 2a). The protein levels (Fig. 2b) and activities (Fig. 2c) of MMP-2 and MMP-9 in the lungs showed similar kinetics to that of the mRNA expression. The protein levels and activities of MMP-9 were also inhibited by both NF-κB inhibitors and AP-1 inhibitors, whereas those of MMP-2 were inhibited by only AP-1 inhibitors (Figs. 2b and 2c). The increased mRNA expression, protein synthesis and enzymatic activity in response to PAF were stronger for MMP-9 than those for MMP-2.
To further verify the effects of PAF on the expressions of MMP-2 and MMP-9, an immunohistochemical analysis was performed. Sections of the lungs of PAF-treated mice were immunostained with anti-MMP-2 and MMP-9 antibodies. MMP-2 expression was observed only in bronchial epithelial cells, and was blocked by both CV6209 and c-jun siRNA, but not by p65 siRNA, whereas MMP-9 was expressed in bronchial epithelial cells as well as the walls of blood vessels, which was blocked by CV6209, p65 siRNA and c-jun siRNA (Fig. 3). Taken together, these data clearly indicate that PAF enhances the expression of MMP-2 through AP-1 activation and that of MMP-9 through both NF-κB and AP-1 activation.
Effects of MMP inhibitors on the PAF-induced enhancement of pulmonary metastasis of B16F10
As previously described,18 PAF significantly enhanced the pulmonary metastasis of B16F10 about 3.5-fold, which was inhibited by both NF-κB inhibitors (parthenolide and p65 siRNA) and c-jun siRNA (Fig. 4). The upper panel of Figure 4 showed the gross pathological pictures of lungs. Finally, the effect of MMP-2 and MMP-9 inhibitors on PAF-induced enhancement of the pulmonary metastasis of B16F10 was examined. First, each inhibitor, at the concentration used in this metastasis experiment, was verified as being effective at preventing the PAF-induced enhanced production and activity of its target MMP (Figs. 5a and 5b). The MMP inhibitor, phenanthroline or MMP-9 siRNA, but not MMP-2 siRNA, completely inhibited the PAF-induced B16F10 metastasis (Fig. 5c). The upper panel of Figure 5c showed the pathological pictures of lungs. These data indicate that MMP-9 plays an important role in the PAF-induced enhancement of pulmonary metastasis of B16F10.
Although PAF has been suggested to be involved in metastasis of human breast cancer,16, 17 little is known about the role of PAF in tumor metastasis, with the exception of our direct evidence that PAF enhances the pulmonary metastasis of B16F10.18 Herein, it has been demonstrated that such an effect of PAF was mediated by MMP-9, the expression of which was regulated by NF-κB and AP-1.
Injection of PAF resulted in enhanced mRNA expression, protein synthesis and enzymatic activities of both MMP-2 and MMP-9 in the lungs, but those of MMP-2 were lower than those of MMP-9 (Fig. 2). In addition, our immunohistochemical analysis revealed that MMP-9 was expressed in bronchial epithelial cells as well as in the walls of blood vessels, whereas MMP-2 expression was observed only in bronchial epithelial cells. These data agree with the previously reported findings that MMP-9 expression is seen in various types of epithelial cells.19, 20, 21, 22 Although the in vitro expression of MMP-2 has been reported in endothelial cells,23, 24 the expression of MMP-2 in pulmonary blood vessel endothelial cells in response to PAF injection was not demonstrated in this study. This may account for the weaker mRNA expression, protein synthesis and increased enzymatic activity of MMP-2 compared to those of MMP-9 in the lungs. In fact, the specific activity of MMP-2 against gelatin has been reported to be much lower than that of MMP-9 in bronchial epithelial cells.20
The promoter of the MMP-9 gene contains putative binding sites for AP-1, NF-κB, Sp1 and PEA3,25 and the promoter region of MMP-2 has AP-1, AP-2, SP-1 and SP-3 binding sites.26 In accordance with these findings, the PAF-induced MMP-2 expression/activity was demonstrated to be prevented by AP-1 inhibitors, whereas the MMP-9 expression/activity was prevented by both NF-κB and AP-1 inhibitors. Furthermore, our findings were in good agreement with those of previous studies, in that the NF-κB and AP-1 activities were associated with the regulation of MMP-9 expression,19, 27, 28, 29 and AP-1 activity with the regulation of MMP-2 expression.30, 31
Importantly, herein, the PAF-induced pulmonary metastasis of B16F10 has been shown to be efficiently blocked by MMP-9 inhibitor, but not by MMP-2 inhibitor, suggesting that MMP-9 plays an important role in PAF-induced tumor metastasis. The reason MMP-2 inhibitor displayed a no inhibitory effect was probably due to the fact that MMP-2 expression does not occur in the pulmonary blood vessel in response to PAF (Fig. 3). MMP-9 has been shown to have a direct role in tumor metastasis in certain systems.32 Furthermore, several recent studies have revealed that more invasive cells had a higher MMP-9 expression,33, 34 which was overexpressed in advanced stage melanoma cells35 and highly expressed by tumors characterized by frequent vascular invasion.36, 37 MMP-2 has also been found to not only correlate with enhanced metastasis and poor prognosis,38, 39 but to also be associated with the risk of a relapse in breast cancer patients.40 Nevertheless, through all these data, MMP-9 is certainly one of the key enzymes involved in tumor invasion and metastasis.
In addition to metastasis, MMP-9 has been reported to be required for angiogenesis.41 During angiogenesis, proteolytic activity is required during the formation of the capillary bud to enable the endothelial cell to migrate out through the pericapillary membrane and the ECM. In addition, capillary elongation, lumen formation and ECM remodeling all require proteolytic activity.42 Recent studies have implicated MMP-9 as an important protease component in angiogenesis, perhaps as a proteolytic activator43 as well as a downstream modulator of known angiogenesis-related molecules.43, 44, 45 Therefore, together with the information that PAF promotes the production of the potent angiogenic factor, vascular endothelial growth factor,46 it is possible that MMP-9 inhibitor inhibits PAF-induced tumor metastasis by blocking MMP-9-mediated ECM remodeling as well as angiogenesis.
Our data obtained in mouse models regarding the influence of PAF on angiogenesis and tumor metastasis may not be directly translatable to the human because humans have developed powerful systems to limit PAF effects. For example, PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A2, is known to inactivate PAF by formation of lyso-PAF and acetate. Recently, another PAF-hydrolyzing enzyme, human group X secreted phospholipase A2, has been described, and the existence of this enzyme may account for that PAF-AH deficient patients are not susceptible to the biological effects of PAF.47
In summary, our present data demonstrate that PAF enhances the pulmonary metastasis of melanoma cells mainly through the induction of MMP-9 in the lungs. Thus, it is possible that PAF, produced locally from tumor cells,14, 15 as well as tumor-infiltrating inflammatory cells,48 can induce ECM solubilization, resulting in the creation of a microenvironment suitable for facilitating tumor cell migration. Thus, this study has provided evidence for better understanding of the mechanism of tumor metastasis and for targeted therapeutic intervention in cancer.