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Keywords:

  • leptin;
  • colon cancer;
  • epithelial cell;
  • IL-6;
  • obesity;
  • adiponectin

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

We previously demonstrated that leptin, an adipose-derived hormone, induces cell proliferation in a model of preneoplastic (IMCE (ApcMin/+), but not normal (YAMC (Apc+/+), colon epithelial cells by inducing autocrine IL-6 production and trans-IL-6 signaling. Low serum adiponectin is associated with colon, prostate and breast cancer. Adiponectin is secreted by white adipose tissue; the levels of adiponectin in the blood decrease as body mass index (and leptin) increases. In our study, we tested whether murine recombinant globular adiponectin (gArcp30) could modulate leptin-induced cell proliferation, autocrine IL-6 production, trans-IL-6 signaling and other leptin-induced cell signaling events previously observed in IMCE cells but not YAMC cells. Under serum-free conditions, adiponectin (1 μg/ml) inhibited leptin-induced autocrine IL-6 production, soluble IL-6 receptor shedding, trans-IL-6 signaling and subsequent STAT3 phosphorylation in IMCE cells. Adiponectin inhibited leptin-induced cell proliferation in the IMCE cells and this inhibition was associated with IκB-α phosphorylation, IκB-α degradation and decreased NF-κB p65 DNA activation and binding. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin-induced NF-κB-dependent autocrine IL-6 production and trans-IL-6 signaling. We hypothesize that adiponectin may be an important regulator of colon epithelial cell homeostasis by linking the observed reduced risk for cancer in populations with high serum adiponectin concentrations to specific mechanisms of cell number homeostasis in a model of preneoplastic colon epithelial cells. These data may have broad implications for diet and lifestyle strategies for the prevention and treatment of obesity-associated cancers. © 2008 Wiley-Liss, Inc.

White adipose tissue secretes hormones and cytokines (also referred to as adipokines) that may play a role in the development of the systemic inflammatory state that is associated with obesity and subsequent cancer risk.1 Adipokines, such as leptin and adiponectin, and cytokines like interleukin-6 (IL-6) may play a direct role in a variety of inflammatory conditions both in vivo and in vitro.2 While these hormones/cytokines are critical to normal cell functioning at homeostatic concentrations, when levels are altered from normal (as in obesity) they may become pathologic. There is strong biological plausibility that an imbalance in these systemic mediators is causally related to obesity-associated cancers like colorectal cancer. However, the mechanisms underlying the obesity-cancer link are not understood.

Inflammation is associated with epithelial cell transformation and the process of carcinogenesis.3, 4 Truncating mutations in the adenomatous polyposis coli (Apc) gene are initiating events in colorectal carcinogenesis; a majority of adenomas in inherited and sporadic forms of colorectal cancers have mutations in Apc.5 In inflammation-associated colorectal cancer, such as cases associated with inflammatory bowel disease, nongenetic stimuli also encourage the survival and proliferation of initiated cells.3 We propose that an imbalance in adipokines, particularly those produced by visceral adipose tissue, may trigger inflammatory stimuli for preneoplastic cell proliferation and survival.

The adipocyte-derived hormone leptin, which plays a crucial role in regulating energy balance, is elevated in obese individuals.1 It is hypothesized that leptin may act locally within the gastrointestinal tract to influence intestinal functions, such as nutrient absorption.6 However, it is unclear whether elevated systemic leptin levels may have pathophysiologic implications for colon epithelial cells and the development of colon cancer, or if it may indirectly mediate the process via a wide variety of other cell types located in the gut. High serum leptin is associated with increased risk of several cancers including, colon,7 prostate8 and breast.7 In addition, leptin levels are positively correlated the likelihood of developing larger and riskier tumors.7, 9

In vitro data are much more consistent regarding the effects of leptin on cell fate. In tumor cell lines, leptin treatment induces cell proliferation in colon,10–12 breast,13, 14 gastric,14 prostate14, 15 and ovarian cancer.16 On the basis of these data, it is likely that leptin has cancer cell stage-specific and tissue-specific actions that ultimately result in a growth promoting effect on neoplastic cells. We previously demonstrated that leptin, a systemic mediator of inflammation associated with obesity, may act on the colon epithelial mucosal microenvironment to promote the survival and proliferation in a model of preneoplastic colon epithelial cells (IMCE (ApcMin/+)) but not normal colon epithelial cells (YAMC (Apc+/+)).17 The difference in leptin-induced cell proliferation we observed is likely due to a trans-IL-6 signaling mechanism activated in IMCE but not YAMC cells.18

Adiponectin is an adipocyte-derived hormone with diverse biological functions including, stimulation of glucose utilization, fatty-acid oxidation and inhibition of gluconeogenesis.19 Serum levels of adiponectin exceed 10 μg/ml, representing ∼0.01% of serum protein, in normal weight individuals.20 Adiponectin self-associates in serum into at least 3 oligomeric complexes, including trimer, hexamer and octadecamer (“high molecular weight;” HMW) forms.21 HMW forms of adiponectin predominate in serum of healthy human subjects; humans with diabetes and metabolic syndrome have a low percentage of HMW adiponectin relative to total adiponectin.22 However, as obesity increases, adiponectin levels decrease and this decrease is thought to be associated with an increased risk of diabetes and cardiovascular disease, implying an early role for adiponectin in disease development.23

To date, 2 specific adiponectin receptors have been identified. The receptors were identified primarily on muscle cells (AdipoR1) and liver cells (AdipoR2).24 AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity is correlated with reduced adiponectin sensitivity. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions which resulted in increased tissue triglyceride content, inflammation and oxidative stress leading to insulin resistance and marked glucose intolerance.25

A role for adiponectin receptors in colon cancer progression has not been identified. However, low serum adiponectin is associated with several cancers, including colon,26 breast7 and prostate cancers.27 The mechanism by which low adiponectin may be involved in cancer risk/progression is not understood. It is hypothesized that high serum adiponectin may impart some protective/preventative effect against chronic diseases like cancer.2 Findings from recent in vitro studies suggest that adiponectin may control cancer cell growth. Specifically, globular adiponectin induced cell growth arrest and even apoptosis in MDA-MB-231 breast cancer cells28 as well as antiproliferative and apoptotic responses in human MCF7 breast cancer cells.29

In our study, we tested whether murine recombinant adiponectin could block our previously observed cell proliferation and cell signaling activity induced by leptin. We utilized a model system of conditionally immortalized colon epithelial cell lines to dissect these early events. These cell lines, YAMC (Apc+/+) cells and IMCE (ApcMin/+) cells, respectively display phenotypes consistent with normal and preneoplastic colon epithelial cells observed in human colon epithelial carcinogenesis.30

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Chemicals

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise noted. Growth media, insulin/transferrin/selenium and murine interferon (IFN-γ) and were purchased from Life Technologies (Rockville, MD). Neonatal calf serum was purchased from Gemini Bio-Products (Woodland, CA). Recombinant murine leptin, IL-6 and g-adiponectin/gAcpr30 were purchased from R&D Systems (Minneapolis, MN).

Cells and cell culture conditions

The YAMC (Apc+/+) cells were developed from the transgenic SV40 large T antigen mouse.31 The IMCE (ApcMin/+) cells were derived from an F1 hybrid between the SV40 large T antigen transgenic mouse and the ApcMin/+ mouse.32 Both of these cell lines are nontumorigenic in nude mice, do not grow in soft agar and survive in culture only on extracellular matrix proteins such as collagen I.32 Both YAMC (Apc+/+) and IMCE (ApcMin/+) cells express the heat-labile SV40 large T antigen under the control of an IFN-γ-inducible promoter. At 33°C the temperature-sensitive SV40 large T antigen is active and drives cell proliferation. At 39°C the temperature-sensitive mutation yields an inactive protein, and cells behave as nonproliferating, differentiated colon epithelial cells.33 Cells were cultured as previously described.34 Prior to treatment cells were serum starved overnight and then all treatments were performed in serum free media.

Colon epithelial cell proliferation assays

IMCE (ApcMin/+) cells were grown in 96-well plates as described earlier. Briefly, ∼1,500 cells/well were seeded in 96-well plates coated with collagen I (BD Biosciences; San Jose, CA) as described earlier. Cells were left at 33°C overnight to adhere and reach 70% confluence. Plates were then moved to 39°C for 24 hr in serum-free and IFN-γ-free medium to allow for cessation of SV40 large T antigen-driven cell proliferation and to achieve stability. After the 24 hr stabilization period at 39°C the cells were treated (8 wells/treatment) with recombinant murine adiponectin at 0.01, 0.1, 0.5, 1, 10 or 25 μg/ml. Cells were also treated (8 wells/treatment) with leptin (50 ng/ml), IL-6 (50 ng/ml) or adiponectin cotreatment combinations. Concentrations of leptin and IL-6 were selected based on previously published data.17, 35 In addition, IMCE (ApcMin/+) cells were also treated a STAT3 inhibitor peptide, cell-permeable No. 573096 (Calbiochem, La Jolla, CA). This STAT3 inhibitor is a cell-permeable analog of the Stat3-SH2 domain binding phospho-peptide (Cat. No. 573095) and acts as a highly selective, potent blocker of Stat3 activation.

Cell proliferation was measured after 48 hr of treatment as previously described.17 Briefly, cell proliferation was measured using a commercially available compound, calcein AM (Molecular Probes, Eugene, OR), that is colorless, nonfluorescent and cell membrane permeable. The compound fluoresces when cleaved by nonspecific esterases in actively proliferating cells. After 48 hr of treatment, the cells were treated with 100 μl of 1 μM calcein AM in PBS for 30 min, and fluorescence was read at an excitation wavelength of 485 nm and emission wavelength of 530 nm in a Cytofluor® fluorescent plate reader (Millipore, Bedford, MA). We previously confirmed that this technique measures cell proliferation via flow cytometric analysis, as reported in a previous article.17

Caspase activity assay

The Caspase-Glo 3/7 and the Caspase-Glo 9 assays (Promega, Madison, WI) were utilized to detect caspase 3/7 and 9 activity. The assay utilizes a proluminescent caspase DEVD-aminoluciferin substrate and a proprietary thermostable luciferase in a reagent optimized for specific caspase, luciferase activity and cell lysis. A single reagent is added and results in cell lysis, followed by caspase cleavage of the substrate. Free aminoluciferin is released and is consumed by the luciferase, generating a luminescent signal. The stabilized signal is proportional to caspase activity of interest. Briefly, cells were cultured in 96-well plates and treated as described earlier (Promega). After 48 hr of treatment, 100 μl of Caspase-Glo reagent was added to each well according to the manufacturer's instructions. Plates were mixed on a plate shaker for 30 sec and incubated at room temperature for 3 hr. Luminescence was measured using the Synergy HT microplate reader (BIO-TEK, Winooski, VT).

IL-6 ELISA

The release of IL-6 into the culture medium was quantified by sandwich ELISA according to the manufacturer's instructions (R&D Systems; Minneapolis, MN). Briefly, 50 μl of undiluted culture medium was added to each well and incubated according to instructions. Upon completion of the assay procedure, the plate was read at 450 and 570 nm wavelength using a Synergy HT plate reader (Bio-Tek; Winooski, VT).

Nuclear activation assay

NF-kappaB (NF-κB) and STAT3 activation was measured in nuclear extracts using the TransAM™ Transcription Factor Assay Kit according to the manufacturer's instructions (Active Motif; Carlsbad, CA) as previously described.18 Briefly, 20 μg of nuclear extract was applied to the plates and incubated according to manufacturer instructions. Substrate-activated HRP-conjugated secondary antibody provided a sensitive colorimetric readout that was quantified by spectrophotometry at 450 nm wavelength using a Synergy HT plate reader (Bio-Tek; Winooski, VT).

Western blotting

Cells were grown in collagen I coated T-75 flasks and treated as described above prior to collection. Briefly, cells were washed twice with cold PBS and total cell lysate was harvested by scraping cells into 1 ml of cold lysis buffer (30 mM Tris pH 7.2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanidate, 1% NP-40 and 10% glycerol) per flask. The cell suspension was then sonicated to insure cell lysis and centrifuged at 4°C for 15 min at 14,000 rpm. Nuclear and cytoplasmic fractions were collected by scraping cells into 1 ml MENG-Mo buffer (25 mM MOPS, 2 mM EDTA, 0.02% NaN3, 10% glycerol and 20 mM Na2MoO4·2H2O, pH 7.4] plus Complete Mini protease inhibitor (Roche Diagnostics, Indianapolis, IN) and homogenized with 40 strokes in a Duall® 22 tissue homogenizer (Kimble/Kontes, Vineland, NJ) on ice.36 Cell disruption was confirmed by microscopic examination. Nuclear pellets were isolated by centrifugation of the cell lysates at 1,000g for 15 min. The supernatant was centrifuged at 2,000g for 15 m. Cytosolic fraction was removed and the nuclear pellet was resuspended and washed three times with MENG-Mo and once with MENG-Mo containing 50 mM NaCl. High salt nuclear extracts were prepared by suspension of the nuclear pellet in an equal volume of MENG-Mo containing 500 mM NaCl, incubation of this suspension at 4°C for 1 hr, and then centrifugation at 13,000g for 1 hr. Total protein in cytosolic and nuclear fractions was determined with the BCA protein assay (Pierce Chemical). For soluble receptor identification, conditioned medium was collected 48 hr posttreatment and concentrated 10-fold using Centricon concentrators (5,000 MW cut-off) (Millipore, Bedford, MA).

Protein content of the samples was determined by BCA assay (Bio-Rad Laboratories, Hercules, CA), and samples were loaded on an equal protein basis of ∼10 μg/lane. Samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with primary antibodies against AdipoR1 (#ADIPOR12-A) and AdipoR2 (#ADIPOR21-A; 1:750; Alpha Diagnostic International, San Antonio, TX) and sIL-6R (1:1,000; No. sc660), sgp130 (1:2,000; No. sc655), STAT3 (No. sc482) and pSTAT3 (Tyr-705; 1:1,000; No. sc8059), actin (1:1,000; No. sc1616) and pERK (1:1,000; No. sc7383) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) or phospho-specific antibody pairs for pIκβ-α (1:500; No. 9246), pp38 (1:500; No. 9215) from Cell Signaling Technology (Beverly, MA) with shaking overnight at 4°C. Incubation with the primary antibody was followed by the appropriate secondary antibody conjugated to horseradish peroxidase (included with the Pierce SuperSignal® West Femto Kit, Rockford, IL) and detected by chemiluminescence according to the manufacturer's instructions.

Samples, for both cell types, for either the receptor or signaling experiments were loaded on the same gel when possible. Pair matched proteins for phosho- and total proteins or nuclear and cyotplasmic proteins were run on separate gels at the same time and processed together. They were processed as a whole for all subsequent steps for optimal comparison. In addition, the same blots were used for each phospo-specific antibody pair. Each blot was stripped and rerun with other antibodies. Further, the experiments were repeated 3 times in the same manner. Densitometric analysis represents the actin corrected signal (mean ± SE) for the three repetitions. Blots shown are from one experiment representative of the three.

Statistical analysis

Cell proliferation and cell proliferation inhibition data were assessed statistically by comparing treated cell proliferation to control cell proliferation within each cell type. The experiments were repeated at least 3 times and data shown are from one of the experiments representative of all 3. The data shown is the mean ± error within one representative experiment. Differences in proliferation were compared using ANOVA in combination with Tukey's multiple comparisons test. IL-6 production data were assessed statistically by comparing supernants isolated from control cells to supernants from leptin-treated cells. Differences in densitometric analysis were compared using ANOVA in combination with Tukey's multiple comparisons test. Pair-wise differences were compared using ANOVA in combination with Tukey's multiple comparisons test. The Prism® software package (Graph Pad; San Diego, CA) was utilized for this analysis.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Adiponectin blocks cell proliferation with increasing concentration in the absence of leptin

YAMC (Apc+/+) and IMCE (ApcMin/+) cells were treated with concentrations of adiponectin ranging from 0.01 to 25 μg/ml in serum free media. These concentrations were chosen to represent a similar physiologic range of low to high circulating concentrations of adiponectin. Adiponectin (10 and 25 μg/ml) decreased cell proliferation at 48 hr relative to control in YAMC (Apc+/+) (p < 0.001; Fig. 1a). The IMCE (ApcMin/+) cells appear to be more sensitive to the antiproliferative effects of adiponectin. Adiponectin treatment decreased IMCE (ApcMin/+) cell proliferation at 0.01, and 1 μg/ml (p < 0.01) and a greater dose response was observed at 10 and 25 μg/ml (p < 0.001) (Fig. 1a).

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Figure 1. (a) The effect of adiponectin on the proliferation of IMCE (ApcMin/+) and YAMC (Apc+/+) colon epithelial cells as measured by fluorescence emission. Cells were placed at 37°C in serum free non-permissive media overnight and then treated with adiponectin from 0.01 to 25 μg/ml for 48 hr. Results are representative of 3 separate experiments. ω = p < 0.01 (compared to untreated IMCE control); * = p < 0.01 (compared to untreated YAMC control). (b) The effect of leptin treatment alone or leptin and adiponectin in combination cotreatment on the proliferation of IMCE (ApcMin/+) and YAMC (Apc+/+) colon epithelial cells. Cells were treated with recombinant murine leptin at 50 ng/ml and then cotreated with 0.1, 1 or 10 μg/ml recombinant murine adiponectin (adipo) for 48 hr. (c) The effect of adiponectin alone (1 μg/ml), leptin (50 ng/ml) treatment alone or leptin and adiponectin in combination cotreatment on caspase 3/7 or caspase 9 activity in IMCE (ApcMin/+) and YAMC (Apc+/+) colon epithelial cells. Activity was measured using Caspase-Glo® (Promega, Madison, WI). (CON, control; L50, leptin 50 ng/ml; A1, Adiponectin 1 μg/ml) * = p < 0.01 (compared to control); ** = p < 0.001 (compared to control).

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Adiponectin blocks leptin-induced cell proliferation

Cell proliferation was determined as previously described.17 YAMC (Apc+/+) and IMCE (ApcMin/+) cells were treated with leptin at 50 ng/ml and cotreated with adiponectin added at 0.1, 1 or 10 μg/ml in serum-free media. These concentrations were chosen to represent the physiologic range of low to high circulating concentrations of adiponectin. In YAMC (Apc+/+) cells, leptin did not induce proliferation (Fig. 1b).17 In contrast, leptin induced cell proliferation in IMCE (ApcMin/+) cells (Fig. 1b).17 Addition of adiponectin did not alter cell proliferation in YAMC (Apc+/+) cells. In contrast, adiponectin blocked leptin-induced IMCE cell proliferation at 0.1 μg/ml (p < 0.01) and at 1 and 25 μg/ml (p < 0.001) (Fig. 1b).

Caspase activation

Adiponectin (1 μg/ml in serum free media) decreased caspase 3/7 and 9 activity in IMCE (ApcMin/+) cells. However, cotreatment of leptin/adiponectin induced a significant increase in caspase 3/7 and 9 activity in IMCE (ApcMin/+) cells (Fig. 1c). Consistent with our previously published data, leptin treatment of YAMC cells increased caspase 3/7 and 9 activity. Adiponectin induced caspase 3/7 activation in YAMC cells. In addition, leptin/adiponectin cotreatment in serum-free media induced a significant increase in caspase 3/7 and 9 (Fig. 1c).

Adiponectin blocks IL-6-induced cell proliferation

YAMC (Apc+/+) and IMCE (ApcMin/+) cells were treated with IL-6 at 50 ng/ml and cotreated with adiponectin added at 0.1, 1 or 10 μg/ml. These concentrations were chosen to represent the physiologic range of low to high circulating concentrations adiponectin. In YAMC (Apc+/+) cells, IL-6 did not induce proliferation (data not shown). In contrast, IL-6-induced cell proliferation in IMCE (ApcMin/+) cells (Fig. 2a). Addition of adiponectin did not alter cell proliferation in YAMC (Apc+/+) cells. However, adiponectin blocked IL-6-induced IMCE (ApcMin/+) cell proliferation at 0.1 μg/ml (p < 0.01) and at 1 and 10 μg/ml (p < 0.001) (Fig. 2a).

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Figure 2. (a) The effect of interleukin-6 (IL-6) and IL-6/adiponectin cotreatment in serum-free media on the proliferation of IMCE (ApcMin/+). Cells were treated with recombinant murine IL-6 at 50 ng/ml and then cotreated with 0.1, 1 or 10 μg/ml recombinant murine adiponectin (adipo) for 48 hr. (b) Adiponectin receptor-1 (ADIPOR1) and -2 (ADIPOR2) protein in IMCE (ApcMin/+) and YAMC (Apc+/+) control and treated cells (C, control; L1, leptin 1 ng/ml; L50, leptin 50 ng/ml; IL6, interleukin-6 50 ng/ml) *, p < 0.001 (compared to IMCE control); **, p < 0.01 (compared to IL-6 treated IMCE). ***, p < 0.001 (compared to IL-6 treated IMCE). Results are representative of 3 separate experiments. Densitometric values represent the actin corrected means ± SE of two blots.

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Adiponectin receptors are present on both cell types

We employed western blot analysis to confirm the presence of adiponectin receptor-1 (AdipoR1) and/or -2 (AdipoR2) protein on YAMC (Apc+/+) and IMCE (ApcMin/+) cells. Total receptor protein confirms the presence of the receptors but is a crude estimate of functionality of the receptors. Both adiponectin receptors were found in relatively comparable quantities in these two cell lines (Fig. 2b). Treatment with either leptin (1 and 50 ng/ml) or IL-6 did increase protein content of adipoR1 levels in IMCE (ApcMin/+) cells. However, in YAMC (Apc+/+) cells, only 1 ng/ml leptin and 50 ng/ml IL-6 appear to increase AdipoR1 levels. No change was observed in AdipoR2 levels because of treatment in YAMC (Apc+/+) cells.

IL-6 production is mediated by adiponectin

IMCE (ApcMin/+) cells secrete moderate amounts of IL-6 under control, serum-free culture conditions (88.2 ± 2.5 pg/ml). Adiponectin treatment induced an increase in IL-6 production independent of concentration in IMCE (ApcMin/+) cells (Fig. 3a). Adiponectin increased IL-6 release by IMCE (ApcMin/+) cells to an average value of 172 ± 7.2 pg/ml. When IMCE (ApcMin/+) cells were treated with leptin, IL-6 increased to 372 ± 16.7 pg/ml (Fig. 3b). Adiponectin cotreatment with leptin reduced leptin-induced IL-6 to concentrations not different from control levels at 1 and 10 μg/ml adiponectin (Fig. 3b).

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Figure 3. (a) The effect of adiponectin (0.01–25 μg/ml) on IMCE (ApcMin/+) IL-6 release into conditioned media collected 48 hr after treatment; (b) The effect of leptin and leptin/adiponectin co-treatment on IMCE (ApcMin/+) IL-6 release into conditioned media. *, p < 0.01 (compared to untreated control); **, p < 0.001 (compared to leptin treatment). Results are representative of 3 separate experiments (Adipo = Adiponectin); (c) The effect of leptin (L50, 50 ng/ml), IL-6 (50 ng/ml), or adiponectin (A, 1 μg/ml) treatment or cotreatment or leptin + adiponectin (L+A) and IL-6 + adiponectin (IL-6 + A) on soluble IL-6R (sIL-6R) or soluble gp130 (sgp130) membrane receptors. Densitometric values represent means ± SE of two blots.

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Adiponectin mediates sIL-6R and sgp-130 release

Western blotting was used to detect changes in the presence of the soluble IL-6R (sIL-6R) and soluble gp130 (sgp130) membrane receptor protein in conditioned media. Previously we identified that in response to leptin treatment IMCE (ApcMin/+) cells shed sIL-6R and gp130 into conditioned media, while the YAMC (Apc+/+) cells did not.35 Adiponectin treatment alone significantly increased both sIL-6 and sgp130 release into conditioned media by IMCE cells (Fig. 3c). As previously observed, leptin treatment of IMCE (ApcMin/+) cells induced an increase in shedding of sIL-6R and sgp130 into conditioned media (Fig. 3c). IL-6 treatment of IMCE (ApcMin/+) cells did not induce shedding of sIL-6R but did induce sgp130 shedding into conditioned media (Fig. 3c). Cotreatment of leptin (50 ng/ml) with adiponectin (1 μg/ml) inhibited shedding of sIL-6R (Fig. 3c).

Adiponectin inhibits leptin-induced cell signaling activation

To establish whether adiponectin blocks leptin- and IL-6-dependent signaling pathways associated with the observed proliferative phenotype as previously described,17, 35 we surveyed cell signaling pathways previously demonstrated to be up-regulated by leptin or IL-6. Leptin induced the phosphorylation of p38 and p42/44 and subsequent NF-κB nuclear translocation (data previously published17). IL-6 induced the phosphorylation of STAT3 and subsequent nuclear translocation of pSTAT3 (data previously published35). Interestingly, adiponectin blocked leptin-induced p38 and p42/44 phosphorylation (Figs. 4a and 4b). In addition, adiponectin also blocked IL-6-induced STAT3 phosphorylation (Fig. 4c).

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Figure 4. The effect of leptin treatment (50 ng/ml) and leptin/adiponectin co-treatment on phosphorylation (activation) of p38 (a) and p42/44 (b), or IL-6 (50 ng/ml) or STAT3 (c) in IMCE (ApcMin/+) cells at 0, 15, 30 and 60 min posttreatment. Total protein controls for pair matched for p38, p42/44 and STAT3 are shown. (d) Actin, protein loading control. Results are representative of 2 separate experiments.

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NF-κB DNA binding

We previously reported that leptin-induced cell proliferation in IMCE (ApcMin/+) cells was dependent on NF-κB activity. Here we show that adiponectin treatment alone reduced NF-κB p65 DNA binding and activity below control IMCE (ApcMin/+) cells. In addition, when IMCE (ApcMin/+) cells are cotreated with leptin and adiponectin, leptin-induced NF-κB activity was restored to control levels (Fig. 5a).

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Figure 5. (a) The effect of leptin (50 ng/ml) treatment or cotreatment with adiponectin of IMCE (ApcMin/+) cells and nuclear NF-κB translocation in IMCE (ApcMin/+) cells. Cells were treated overnight in serum-free medium prior to exposure to treatment. Nuclear extracts were collected and 6 hr posttreatment is shown. *, p < 0.01 (compared to untreated control); †, p < 0.01 (reduced compared to untreated control). (b) Leptin treatment induced the degradation of cytoplasmic IκB-α while adiponectin inhibited IκB-α degradation and induced p-IκB-α while adiponectin inhibited IκB-α phosphorylation (Actin, protein loading control). Cytoplasmic extracts were isolated and collected and 1 hr posttreatment is shown. (c) Densitometric analysis of western blots in part B represent the means ± SE of two blots. *, p < 0.01 (compared to untreated control). (CON, control; L50, leptin 50 ng/ml; A1, Adiponectin 1 μg/ml).

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IκB-α degradation

Consistent with the DNA binding data shown above, leptin treatment induced IκB-α degradation, adiponectin blocked IκB-α degradation and leptin/adiponectin cotreatment IκB-α degradation did not differ from controls as measured by western blot analysis (Figs. 5b and 5c). In addition, leptin induced a significant increase in p-IκB-α. However, there was no effect of adiponectin or leptin/adiponectin cotreatment on p-IκB-α protein (Figs. 5b and 5c).

Leptin-induced STAT3 nuclear translocation and activation is attenuated by adiponectin

IL-6 treatment induced a time-dependent increase in nuclear accumulation of STAT3 in IMCE (ApcMin/+) cells, with maximal accumulation at 30 min (Fig. 6a). Cotreatment of IMCE (ApcMin/+) cells with IL-6 (50 ng/ml) and adiponectin (1 μg/ml) blocked the time-dependent nuclear accumulation of STAT3 (Fig. 6a). Adiponectin treatment alone reduced STAT3 DNA binding and activity in IMCE (ApcMin/+) cells. In addition, when IMCE (ApcMin/+) cells were cotreated with IL-6 and adiponectin, IL-6-induced STAT3 activity was restored to control levels (Fig. 6c).

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Figure 6. (a) IL-6 (50 ng/ml) treatment or cotreatment with adiponectin of IMCE (ApcMin/+) cells and nuclear STAT3 translocation in IMCE (ApcMin/+) cells 15, 30 or 60 min posttreatment. (b) The effect of IL-6 (50 ng/ml) and IL-6/STAT3 inhibitor cotreatment on the proliferation of IMCE (ApcMin/+) cells. Cells were treated with leptin or IL-6 at 50 ng/ml and then cotreated with STAT3 inhibitor at 0.01, 0.01, 0.1, 1, 10 or 100 mM for 48 hr. Results are representative of 3 separate experiments. ω = p < 0.01 (compared to untreated IMCE control); * = p < 0.05 (compared to leptin treated IMCE); ** = p < 0.001 (compared to leptin treated IMCE). (c) The effect of Il-6 (50 ng/ml) treatment or cotreatment with adiponectin (1 μg/ml) of IMCE (ApcMin/+) cells and nuclear STAT3 activity in IMCE (ApcMin/+) cells. Cells were treated overnight in serum-free medium prior to exposure to treatment. Nuclear extracts were collected and 6 hr posttreatment is shown. *, p < 0.01 (compared to untreated control); †, p < 0.01 (reduced compared to untreated control); (C, control; A1, Adiponectin 1 μg/ml; IL6, interleukin-6 50 ng/ml; INH, STAT3 inhibitor 1 mM Calbiochem No. 573096, 1 mM).

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STAT3 inhibitor inhibits interleukin-6-induced cell proliferation

To establish whether the IL-6-induced IMCE (ApcMin/+) cell proliferation was dependent on STAT3, we cotreated the IMCE (ApcMin/+) cells with a commercially available STAT3 inhibitor. The STAT3 inhibitor did not influence basal cell proliferation of IMCE (ApcMin/+) cells across a range of concentrations (data not shown). Concentrations were chosen based on recommendations given by the manufacturer (Calbiochem, San Diego, CA). The STAT3 inhibitor partially blocked IL-6-induced IMCE (ApcMin/+) cell proliferation at 0.01 mM (p < 0.05, Fig. 6b) and completely blocked IL-6-induced IMCE (ApcMin/+) cell proliferation at 0.1–10 mM (p < 0.01, Fig. 6b). In support, when IMCE (ApcMin/+) cells are cotreated with IL-6 and adiponectin or the STAT 3 inhibitor, IL-6-induced STAT3 activity was decreased approximately to control levels (Fig. 6c).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Our laboratory has established that leptin-induced signaling pathways, NF-κB and trans-IL-6 pathways, are up regulated in preneoplastic but not normal colon epithelial cells resulting in preneoplastic cell proliferation. In metastatic tumor cells and in mouse models of colitis adiponectin blocked cell proliferation and decreased tumor burden respectively.37–39 These phenotypic observations led us to explore a possible preventative role for adiponectin on our previously observed leptin-induced cell proliferation. In the present study our primary goal was to determine whether adiponectin could inhibit the preneoplastic (IMCE (ApcMin/+) cells) colon epithelial cell proliferation induced by leptin as we previously observed.

While not physiologic based on the fact that adiponectin or leptin do not exist in vivo alone but rather coexist in serum, we wanted to take advantage of our reductionist model system to identify adiponectin-specific phenotypic effects. Initially, we treated both the normal YAMC (Apc+/+) and preneoplastic IMCE (ApcMin/+) cells with adiponectin across a range of concentrations (0.01–25.0 μg/ml) to determine the concentration-dependent effects of adiponectin alone on basal cell proliferation. In serum-free conditions, adiponectin treatment alone slightly blocked YAMC (Apc+/+) and IMCE (ApcMin/+) cell proliferation at the highest concentrations of 10 and 25 μg/ml (Fig. 1a). Co-treatment of IMCE cells with leptin and adiponectin together inhibited leptin-induced cell proliferation (Fig. 1b) and increased caspase activation (Fig. 1c). These data are consistent with the hypothesis that adiponectin desensitizes the cells to proliferative signals evidenced by decreased cell numbers across adiponectin treatment alone or in combination with leptin. In addition, cotreatment of IMCE cells with leptin and adiponectin activated caspases consistent with the phenotypic observation in Figure 1b. It is interesting, while not physiologic, that treatment of IMCE cells with 1 μg/ml adiponectin alone decreased caspase activity (Fig. 1c) with no observed change in cell number (1A). Note that 1 μg/ml adiponectin treatment alone led to decreased NF-κB activation, increased total cytoplasmic IκB-α and decreased phospho- IκB-α (Fig. 5). These observations provide indirect but consistent evidence for IκK activation resulting in inhibition of NF-κB and may explain part of the decreased cell proliferation because of adiponectin treatment alone.

Adiponectin, independently and in the presence leptin, blocked cell proliferation of preneoplastic colon epithelial cells. We hypothesized this effect of adiponectin on leptin-induced cell proliferation could be explained by blocking NF-κB activation or trans-IL-6 signaling. Previously, we showed that leptin induced phosphorylation of p38 and p42/44 and increased NF-κB nuclear translocation and DNA binding.17 Evidence suggests that both p38 and p42/44 MAPKs are involved in NF-κB transactivation.40, 41 MAPKs can mediate the IκK complex and lead to IκB-α freeing bound p65 NF-κB from the cytoplasm and allowing nuclear translocation and DNA binding.42 In addition, MAPKs can directly phosphorylate p65 NF-κB resulting in transactivation and increased DNA binding potential.42 Indeed, in our model system adiponectin cotreatment blocked phosphorylation of p38 and p42/44. Consistent with this observation, cotreatment with adiponectin blocked leptin-induced IκB-α phosphorylation, IκB-α degradation and subsequent NF-κB DNA activation and binding. The inhibitory action of adiponectin on NF-κB activation is consistent with previously reported effects of HMW adiponectin. In vitro, adiponectin blocks proliferation in MCF-7 breast cancer cells,29 MDA-MB-231 breast cancer cells28 and prostate cancer cells.43 Adiponectin also decreases lipopolysaccharide-induced IL-8 production by intestinal epithelial cells.44 The growth inhibitory effects of adiponectin could be due to the ability of adiponectin to selectively bind to several mitogenic growth factors45 and the ability of adiponectin to activate caspases and induce apoptosis.46 Moreover, adiponectin may inhibit NF-kB activation,47 consistent with our findings (Fig. 5).

Previously we showed that leptin activated 2 independent pathways resulting in cell proliferation of preneoplastic colon epithelial cells. After characterizing the effect on NF-kB activation, we wanted to determine whether adiponectin also blocked leptin-induced IL-6 production and trans-IL-6 signaling as previously described. Given that the IL-6 promoter contains a putative NF-κB binding site, it was possible that this upstream action of adiponectin in blocking NF-κB nuclear activation and translocation might be responsible for inhibition of the trans-IL-6 signaling pathway activation in IMCE cells.

Here we show that adiponectin cotreatment blocks leptin-induced IL-6 production and shedding of the IL-6R by IMCE (ApcMin/+) cells (Fig. 3). Blocking IL-6 production resulted in decreased proliferative signal for the IMCE cells. Importantly, another novel observation was that adiponectin cotreatment with IL-6 and leptin blocked shedding of the sIL-6 receptor and increased the shedding of the sgp130 receptor. It is thought that the homeostatic signaling of IL-6 is through the membrane bound IL-6R whereas the proinflammatory process is likely regulated by IL-6 trans-signaling.48 We show that adiponectin can block the trans-IL-6 signaling mechanism induced by leptin. Given these observations, we hypothesized that NF-κB was likely not the only pathway being attenuated by adiponectin. Therefore, we cotreated the cells with IL-6 and adiponectin to identify whether adiponectin could block IL-6 signaling independent of NF-κB. The increase in IL-6 induced by adiponectin alone may constitute a homeostatic signal. Adiponectin treatment alone increased both soluble IL-6R and gp130 not altering the overall balance of the receptors. These data support the hypothesis that IL-6 production may be important for epithelial cell homeostasis.49

Adiponectin blocked key signaling molecules involved in IL-6 signaling (Figs. 4 and 6). Adiponectin cotreatment was able to inhibit IL-6-induced STAT3 phosphorylation, nuclear translocation and activity (Fig. 6). Further we show that the IMCE cell proliferation induced by IL-6 was dependent on STAT3. Chemical inhibition of STAT3 phosphorylation and nuclear translocation was associated with inhibition of IL-6-induced cell proliferation (Fig. 6). Therefore, we concluded that adiponectin could also block IL-6-induced cell proliferation via inhibition of STAT3 activation.

This mechanism is consistent with in vivo studies implicating a role for IL-6 and trans-IL-6 signaling in cancer. IL-6 is an important mediator of inflammatory bowel diseases which are a risk factor for colorectal cancer.50 Recent studies show that serum IL-6 levels were increased in patients with colon carcinoma and correlated with tumor size.51, 52 IL-6 also promotes cell proliferation in vitro in colon cancer cells.53 Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth.54 The use of STAT3 inhibitors to treat cancer is actively being pursued.55 Results from recent studies suggest that selective targeting of IL-6 trans-signaling may represent a viable strategy for treating cancers dependent on these signaling pathways.56–58 Future experiments will include the addition of recombinant sIL-6R and sgp130 to test the specific interactions between these mediators and leptin- and IL-6-induced IMCE cell growth.

We cannot neglect that the form of adiponectin is increasingly being shown to dictate specific signaling pathway activation. While a globular form (glycosylated homotrimer) of adiponectin was used in our studies, as well as many reported here, we cannot rule out the protein homotrimers associate in culture into oligomeric complexes. It was beyond the scope of this work to determine the distribution of adiponectin oligomers present and the receptors through which their effects are mediated. However, the inhibitory effect of adiponectin in NF-κB activation on these cells is consistent with previously reported effects of HMW adiponectin on NF-κB activation. Globular adiponectin induced NF-κB activation in several cell types while full length adiponectin inhibited activation.59, 60 Our data indicate that adiponectin treatment alone did not activate NF-κB consistent with the HMW adiponectin effect. Future research plans include characterizing the distributions of the forms of adiponectin found when used in culture.

The full length form of adiponectin predominates in serum and globular may be responsible for the proinflammatory effects sometimes observed with adiponectin treatment.61, 62 However, in breast cancer patients, modeling HMW instead of total adiponectin did not offer any additional predictive value of cancer risk.63In vivo, low total serum adiponectin is associated with several cancers including colon,26 prostate,9 breast,7 endometrial64 and gastric cancer.65 Data from the breast cancer study cited above suggests that low serum adiponectin levels and high serum leptin levels are associated with an increased risk for breast cancer.7 In addition, serum adiponectin levels were negatively associated with histologic grade and disease stage.7, 9 In a murine adiponectin KO model, adiponectin was protective against DSS- and TNBS-induced murine colitis.44 Paradoxically, Fayad et al.66 report, in an independently generated adiponectin KO mouse strain, that adiponectin deficiency protects mice from chemically induced colonic inflammation. There are several possible explanation for these differences. The authors attribute the observed disparate outcomes in these 2 adiponectin KO models to differences in the genetic background of mouse strains employed. Other important differences exist in the mouse model used by Fayed et al. These investigators reported the detection of adiponectin mRNA in normal and inflamed colonic tissue in addition to adipose tissue, a finding not observed by Nishihara. According to Nishihara, 7 days of 2.5% DSS treatment induces severe inflammation in both normal and adiponectin KO mice (Nishihara, personal communication). Overall, the preponderance of published data, in vivo and in vitro, supports an antiinflammatory role for adiponectin in inflammation-associated events in cancer.

Our in vitro data shows that adiponectin acts in a mechanistically distinct manner on models of normal and preneoplastic colon epithelial cells. These data indicate that adiponectin acts on preneoplastic colon epithelial cells to regulate cell growth via 2 distinct pathways inhibiting leptin-induced NF-κB-dependent autocrine IL-6 production and trans-IL-6 signaling. As such, adiponectin may be an important stage-dependent regulator of colon epithelial cell homeostasis. In summary, our data provide the first mechanistic evidence that adiponectin may abrogate the proliferative effect of leptin in a model of preneoplastic colon epithelial cells. These findings, if confirmed in relevant human model systems, enhances the biologic plausibility that adiponectin may act at an early stage of carcinogenesis to block leptin-induced colon epithelial cell proliferation.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
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