Department of Food Science and Human Nutrition, Michigan State University, 2110 Anthony Hall East Lansing, MI 48824, USA and
1 Department of Animal Science, Michigan State University, E. Lansing, MI 48824, USA
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Abstract |
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Abbreviations: APC, adenomatous polyposis coli; EGF, epidermal growth factor; HGF, hepatocyte growth factor; IMCE, immortomouse/Min colon epithelial; INF-, interferon-
; MMP, matrix metalloproteinase; MT, membrane type; YAMC, young adult mouse colon.
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Introduction |
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Specific dietary factors, such as curcumin, present in the spice turmeric, and citrus flavonoids have been isolated from plant sources and inhibit tumorigenesis in experimental models (1012); however, their mechanism of action is poorly understood. The high level of curcumin consumption in India has been hypothesized to contribute to the low rate of colon cancer seen there (13). Curcumin has many chemopreventive properties including antiproliferative, antioxidant, anti-inflammatory and anti-angiogenic effects (1417). In addition, the chemopreventive efficacy of curcumin was demonstrated in animal models (18,19). These effects of curcumin may be mediated by its inhibitory effect on a host of cell-signaling components including protein kinase C, epidermal growth factor (EGF) receptor tyrosine kinase and IkappaB kinase (20,21). Mutations in the tumor suppressor gene adenomatous polyposis coli or Apc have been demonstrated to play a role in the human inherited colon cancer susceptibility syndrome, familial adenomatous polyposis, and are found in over 85% of sporadic colorectal tumors in humans (22,23). Apc heterozygote mice, including transgenic Apc mice and Min (Multiple intestinal neoplasia) mice, which carry germline Apc mutations, are highly susceptible to intestinal neoplasia early in life (24).
Notably, the Apc genotype may affect colon epithelial cell migration; expression of the migratory phenotype is required for normal cell differentiation and apoptosis (25). Colon epithelial cells normally migrate up the surface of the colon and experience cell death. These events take ~25 days and are normal in the life cycle of colon epithelial cells (23). As such, truncated APC protein may stall cell migration, delaying cell differentiation and death, and allow for accumulation of additional mutations leading to polyp formation, a precursor lesion in colorectal cancer (26). However, this hypothesis has not been proven. The role of the Apc as the `gate-keeper' gene in colonic carcinogenesis and its link with the downstream transcriptional regulator ß-catenin/Tcf-LEF complexes has been recently established (25). As such, full-length APC effectively binds ß-catenin, targeting it to the ubiquitinproteosome pathway for degradation, thus tightly regulating free intracellular ß-catenin levels. C-terminus truncated APC, on the other hand, cannot bind ß-catenin and regulate its degradation, thus nuclear levels of ß-catenin/Tcf-LEF accumulate and induce the expression of growth regulatory genes such as c-myc (25,27).
Circumstantial evidence links APC protein function and the regulation of specific enzymes required for cell movement called matrix metalloproteinases (MMPs). MMPs are a family of zinc-dependent proteinases that degrade extracellular matrix components. One specific type of MMP, the membrane-type MMPs (MT-MMPs), plays a critical role in cell migration (28). MT-MMPs are unique from other MMPs because they possess a transmembrane domain and a cytoplasmic tail at the C-terminus (29). Interestingly, both MT-MMPs and APC localize to the leading edge of the plasma membrane under conditions associated with migration (30). In addition, the connection between APC and MMPs has also been strengthened by evidence that one MMP (MMP-7) is transcriptionally regulated by the ß-catenin/Tcf-LEF transcription complex (31).
The objectives of this study were: (i) to characterize the Apc genotype-dependent migratory response induced by classical motorgen growth factors, EGF and hepatocyte growth factor (HGF), (ii) to determine whether a dietary factor known to prevent colon cancer experimentally can alter cell migration in an Apc-dependent fashion and (iii) to determine whether migration caused by curcumin is dependent on the Apc genotype. The mechanisms of growth factor- and curcumin-dependent cell migration were further studied to determine if this phenotype was MMP-dependent.
In this study, the conditionally immortal murine colonic epithelial cell lines immortomouse/Min colon epithelial (IMCE, ApcMin/+) and young adult mouse colon (YAMC, Apc+/+) were used to quantify the ability of EGF, HGF and curcumin to elicit the migratory phenotype. These non-transformed and non-tumorigenic cells have an average life span of ~10 days under non-transforming conditions (22,23). These unique characteristics support their use as an excellent model system in which to study the early events in colorectal carcinogenesis.
Materials and methods
Chemicals
All chemicals were purchased from Sigma (St Louis, MO) unless noted. Growth media, insulin, transferrin, selenium, murine interferon- (IFN) and type IV collagen were purchased from Life Technologies (Rockville, MD). Neonatal calf serum and antibiotics were purchased from Gemini Bio-Products (Woodland, CA).
Cells and cell culture conditions
Experiments were carried out using two conditionally immortalized murine colonic epithelial cell lines contrasting in Apc genotype developed by Dr Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne, Australia). The normal (YAMC) cells (Apc+/+) were developed from the transgenic SV40 large T antigen mouse (18). The ApcMin/+ (IMCE) are derived from the F1 hybrid mouse from the mating of SV40 large T antigen transgenic mouse and the ApcMin/+ mouse. The Apc genotype was confirmed in our laboratory by allele-specific polymerase chain reaction (PCR)/DNA electrophoresis. These cells are non-tumorigenic in nude mice, do not grow in soft agar and survive in culture only on extracellular matrix proteins such as collagen I. These cells serve as an excellent model system to study early events in colorectal carcinogenesis (19). Both YAMC and IMCE cells express the heat-liable SV40 large T antigen with an IFN--inducible promoter. The temperature-sensitive SV40 large T antigen with an IFN-
-inducible promoter is only active at 33°C. The temperature-sensitive mutation in SV40 large T yields an inactive protein at 39°C. All cells were grown on 75 cm2 culture flasks coated with 5 µg/cm2 type I collagen (BD Biosciences, Bedford, MA) in RPMI 1640 media supplemented with 5% neonatal calf serum, ITS® (BD Biosciences; insulin 6.25 µg/ml, transferrin 6.25 µg/ml and selenous acid 6.25 ng/ml), 5 IU/ml of murine IFN-
, and 100 000 IU/l penicillin and 100 mg/l streptomycin. Cells were cultured at 33°C with 5% CO2 in media plus all the aforementioned supplements until they reached ~70% confluence. Upon 70% confluence, the cells were transferred to 39°C under non-transforming conditions in serum-free and IFN-
-free media for 72 h before each experiment.
Cell migration
Cell migration over slides coated with collagen IV was assessed using the Cell the company protocol and Sedimentation Manifold (CSM) System developed by Creative Scientific Methods (Phoenix, AZ) (32,33). We have optimized this system for our cell culture model system. Briefly, 10 well slides are pre-coated with collagen IV and 2500 cells were dispensed into each channel of the manifold. Cells were cultured under permissive conditions at 33°C to facilitate cell adhesion overnight. Slides were then transferred to 39°C under non-transforming conditions for 4 h pre-incubation prior to treatment for 24 h. Cells were treated with specific growth factors (HGF or EGF) in a dose-dependent fashion (0.150 ng/ml) or curcumin (0.1100 µM) in a dose-dependent fashion. Cells induced to migrate were then co-treated with the global MMP inhibitor Ilomastat® (Chemicon, Temecula, CA) in a dose-dependent fashion (10, 25 or 50 µM) to inhibit migration. Cells induced to migrate were also co-treated with 1 µg/ml anti-MT1-MMP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or 1 µg/ml anti-MT-1, -2 and -3-MMP antibodies (Santa Cruz Biotechnology).
Cell treatments
Different concentrations of EGF, HGF or curcumin and Ilomastat® were made in the RPMI non-permissive media and applied to either the flasks or slides for 24 h. Control cells received non-permissive media only. Each experiment was repeated at least three times with 10 well slides (n = 10) for each experiment.
Gelatin-substrate zymography
Gelatinolytic proteinases were assayed by gelatin-substrate zymography. Pig gelatin type A (3.8 mg) was embedded in a 12% acrylamide gel. Seventy percent confluent T-75 flasks of both cell types were treated as described above and conditioned media was recovered from each flask, aliquoted and stored at 80°C until analysis. Conditioned media samples (40 µl) were treated 1:2 with non-reducing Laemelli sample buffer and 40 µl was electrophoresed. Gels were washed two times for 30 min in buffer containing 2.5% Triton X-100 and incubated at 37°C for 24 h in buffer containing 50 mM TrisHCl pH 7.5, 200 mM NaCl, 10 mM CaCl2, 10 µM ZnCl2, 0.02% Brij 35. Gels were then stained with Coomassie Blue for 1 h and destained until clear bands appeared on the blue background. To demonstrate that this activity was due to MMPs and not another protease, bands were inhibited with 1,10- phenanthroline, a specific inhibitor of MMPs. In addition, MMP-2 standard was included on all zymograms.
Immunocytochemical staining
Cells were grown on 10 well slides and treated following the CSM methodology from above. Cells were fixed in Carnoy's solution for 10 min, washed 3x 5 min in PBS/0.1% BSA and incubated for 90 min with 1:200 dilution of anti-MT1-MMP (Chemicon). Incubation was followed by 3x 5 min washes with PBS and 1x 5 min wash with PBS/0.1%BSA and by a 30 min incubation with R-Phycoerythrin-conjugated donkey anti-rabbit secondary antibody (Jackson Immunoresearch, West Grove, PA) at a 1:100 dilution. Slides were washed 3x 5 min with PBS and treated with Slow Fade (Molecular Probes, Eugene, OR), coverslipped and sealed with nail polish. The negative control used for the primary antibody was a non-specific murine IgG2b monoclonal antibody. As such, this antibody did not inhibit HGF- or curcumin-induced migration in either cell type (data not shown). Digital photographs of stained cells were obtained using a Leica fluorescence microscope (100x) associated with a Dell PC (Pentium® 300 MHz) using Spot® software (version 3.2.4; Diagnostic Instruments, Sterling Heights, MI).
Western immunoblotting
Briefly, cells were washed twice with cold PBS and harvested by solubilizing cells in 1 ml of Triton X-114 buffer (1.5% Triton X-114 in TBS consisting of 50 mM TrisHCl pH 7.5 and 150 mM NaCl containing 1 mM CaCl2, 1 mM MgCl2, 1 mM PMSF and 5 mM EDTA) per flask. Flasks were on ice rocking for 2030 min until cells detached from the flask. Solution was passed through a Dounce homogenizer and centrifuged 14 000 g for 15 min at 4°C. The supernatant was collected, warmed for 2 min at 37°C and centrifuged 14 000 g for 10 min at 22°C to separate lower, detergent and upper, aqueous phase. The aqueous phase was collected and stored at 80°C until use. Samples were electrophoresed and transferred to PVDF membrane. Blots were probed with the MT1-MMP (1:250) or actin (1:1000) primary antibody (Santa Cruz Biotechnology) followed by a secondary anti-goat IgG antibody conjugated to horseradish peroxidase (1:2500) and detected by chemiluminescence using the femtoLucent kit (Geno Technology, St Louis, MO).
Statistical analysis
Cell motility was assessed by measuring the total area encompassed by cells migrating across a two-dimensional surface coated with type IV collagen. Two-dimensional migration was quantified by computing the areas occupied by epithelial cells using NIH Image® software (National Institutes of Health, Bethesda, MD). Even though the cells were seeded 2500 cells/well, the IMCE control cells covered more area than the YAMC cells. Therefore, data were transformed to percent of control because the two cell types are different sizes and covered a different starting area. Differences in the size of the area covered by the cells were compared using ANOVA in combination with Tukey's multiple comparison procedure. Statistical significance was considered at P < 0.05.
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Results |
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Discussion |
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Data exist indicating a role for MT-MMP activity in the migration of endothelial cells (35). These studies employed monoclonal antibodies against MT1-MMP to effect the partial inhibition of migration of endothelial cells and led us to investigate a role for this family of extracellular matrix remodeling enzymes in IMCE and YAMC cell migration. Gelatin zymography indicates that YAMC cells secrete higher amounts of both MMP-2 and -9 in cell culture supernatant. The fact that neither EGF nor HGF treatment altered the activity of MMP-2 or -9 suggests that the migration-enhancing ability of these growth factors may be independent of the activity of these enzymes. However, the addition of a global MMP inhibitor (Ilomastat®) successfully inhibited migration indicating that other MMPs may be involved in migration. Based on the recent evidence that MT1-MMP is involved in migration in other cell types, we deduced that Ilomastat® may be inhibiting the activity, localization and/or translation of MT1-MMP. The authors recognize that MT-MMPs are known to bind secreted MMPs such as MMP-2 and enhance their activity and cannot rule out the contribution of this mechanism to migration.
Nascent MT-MMPs localize to the ER where they are synthesized and stored until signaled to localize in the plasma membrane (36). We observed a shift in localization in our cells that is consistent with observations in MDCK cells where HGF was shown to elicit a similar pattern of MT1-MMP redistribution as well as activation of pericellular collagenase activity, the putative mechanism enabling cell migration (28). Thus, these compounds that cause cell migration, ostensibly via different signaling mechanisms, all result in the redistribution of MT1-MMP, consistent with observations in MDCK cells (28).
Evidence from mouse models suggests that cells heterozygous for Apc may have less migratory potential than cells expressing only wild-type Apc (30). Consistent with these observations, these cells were less migratory than YAMC cells in response to EGF and HGF. Comparable migratory responses in both cell types elicited by curcumin suggest that, like EGF- and HGF-induced migration, this phenotype is associated with MMP activity and associated with a redistribution of MT1-MMP from the ER to the plasma membrane. Unlike the EGF- and HGF-induced migration, curcumin may initiate signaling pathways in these cells not related to the Apc genotype and, therefore, not linked to activation of the Wnt/ß-catenin/Tcf-LEF axis. The authors are currently working to identify possible signaling pathways differentially activated by these motorgenic compounds. The fact that co-treatment of HGF or EGF with curcumin did not have an additive effect on the migration of either cell type suggests that signals regulated by these compounds overlap in a manner that does not increase the overall migratory response.
In addition to causing the redistribution of MT1-MMP, curcumin may have caused increased activation of MT1-MMP enzymatic activity leading to increased migration. We could not assess the role of curcumin in the activation of MT1-MMP. These results suggest that curcumin may initiate cell-signaling pathways that cause MT-MMP redistribution and cell migration.
The differential effect of Ilomastat® on HGF- and curcumin-induced migration may be explainable in light of zymographic and western blot data. The fact that HGF-induced migration was inhibited at the lowest concentration of Ilomastat® in IMCE cells may reflect a decreased concentration required given the lower amount of MMP-2 and -9 secreted by these cells as well as the lower level of MT1-MMP protein in HGF-induced IMCE cells. Higher concentrations of Ilomastat® were required to inhibit migration in YAMC cells, consistent with the higher amounts of MMP-2 and -9 secreted activity and increased HGF-induced MT1-MMP protein in these cells.
Using neutralizing polyclonal antibodies against MT1-, 2- or 3-MMPs, we were able to establish the role of MT-MMP activity in colon epithelial cell migration. As in endothelial cells (35), antibodies against MT1-MMP resulted in the partial inhibition of cell migration. The inhibition of migration resulting from treatment with antibodies against MT1-, 2- and 3-MMPs demonstrated a role for multiple MT-MMPs in cell migration. The data provide the first evidence for a role for MT-MMP localization and activity in the migration of non-tumorigenic colon epithelial cells.
Data presented here clearly show that curcumin induces dose-dependent effects on cell migration. The unique model system employed here utilizes non-tumorigenic, differentiated, non-proliferating colon epithelial cells in serum-free media. Reductionist approaches such as these have certain limitations. Colon epithelial cells in vivo are exposed to a tremendous variety of autocrine, paracrine, endocrine, luminal and dietary substances that exert heterogeneous cellular and physiologic responses. Even so, the addition of single agents to epithelial cells in serum-free media provides the opportunity to identify agent-specific effects on specific phenotypes that are not possible to identify in vivo. The data provide evidence that curcumin, a common dietary constituent in certain cultures, stimulates the migratory phenotype at low concentrations. These concentrations are ostensibly achievable in the lumen by dietary means. As such, this suggests that dietary compounds may exert their anticarcinogenic effects, in part, through the enhancement of epithelial cell migration and concomitant increase in villus-associated apoptosis.
The data present here are consistent with the hypothesis that cell migration elicited by curcumin is a potent stimulator of MT-MMP-mediated cell migration relative to that elicited by EGF and HGF. This migration requires MT-MMP activity but is independent of the Apc genotype of IMCE and YAMC cells. Whether the migratory response elicited by curcumin is regulated via different signaling mechanisms than EGF or HGF is a matter of current investigation. The data suggest a potential mechanism by which curcumin may allow cells heterozygous for Apc to overcome defective cell migration, a phenotype associated with cell differentiation and apoptosis.
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Notes |
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Acknowledgments |
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References |
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