Adipokine regulation of colon cancer: adiponectin attenuates interleukin-6-induced colon carcinoma cell proliferation via STAT-3

Abstract

Obesity results in increased circulating levels of specific adipokines, which are associated with colon cancer risk. The disease state is associated with increased leptin, insulin, IGF-1, and IL-6. Conversely, adiponectin levels are decreased in obese individuals. Previously, we demonstrated adipokine-enhanced cell proliferation in preneoplastic, but not normal, colon epithelial cells, demonstrating a differential effect of adipokines on colon cancer progression in vitro. Using a model of late stage carcinoma cancer cell, namely murine MC-38 colon carcinoma cells, we compared the effect of obesity-associated adipokines (leptin, insulin, IGF-1, and IL-6) on MC-38 cell proliferation and determined whether adiponectin (full length or globular) could modulate adipokine-induced cell proliferation. We show that insulin and IL-6, but not leptin and IGF-1, induce proliferation in MC-38 cells. Adiponectin treatment of MC-38 cells did not inhibit insulin-induced cell proliferation but did inhibit IL-6-induced cell proliferation by decreasing STAT-3 phosphorylation and activation. Nitric oxide (NO) production was increased in MC-38 cells treated with IL-6; co-treatment with adiponectin blocked IL-6-induced iNOS and subsequent NO production. These data are compared to previously reported findings from our laboratory using the YAMC (model normal colon epithelial cells) and IMCE (model preneoplastic) cells. The cell lines are utilized to construct a model summarizing the hormonal consequences of obesity and the impact on the differential regulation of colon epithelial cells along the continuum to carcinoma. These data, taken together, highlight mechanisms involved in obesity-associated cancers and may lead to potential-targeted therapies.

Figure 1

The effect of leptin, IL-6, IGF-1 and insulin on the proliferation of MC-38 cells and specific adipokine receptor expression. Cells were treated with (A) leptin (0.01-50 ng/ml), (B) IL-6 (0.01-50 ng/ml), (C) IGF-1 (0.1-200 ng/ml), or (D) insulin (0.001-100 μg/ml) for 24 hrs and proliferation measured. (E) Characterization of specific adipokine receptors in MC-38 cells. Western blot analysis was performed and protein levels for IL-6 (IL-6R and gp130), adiponectin (AdipoR1 and AdipoR2), leptin (Ob-R), insulin (INSR-a and INSR-b), IGF-1 (IGF-R1) and actin loading control were determined. CON; control. Results are representative of 3 separate experiments. * = p<0.05 (compared to control); ** = p<0.01(compared to control).

Figure 2

The effect of adiponectin treatment on the proliferation of MC-38 cells. Cells were treated with (A) globular adiponectin (gAdiponectin, 0.001-1 μg/ml) or (B) full-length length adiponectin (fAdiponectin, 0.001-1μg/ml) for 24 hrs. (C) Cells were treated with insulin (1 and 10 μg/ml) alone, fAdiponectin (1 and 10 μg/ml) alone, or in combination for 24 hrs. (D) Cells were treated with insulin (1 and 10 μg/ml) alone, gAdiponectin (1 and 10 μg/ml) alone, or in combination for 24 hrs. Con; Control, *=p<0.05 (compared to control). Results are representative of 3 separate experiments.

Figure 3

IL-6 treatment of MC-38 cells specifically activates STAT-3. (A) The effect of IL-6 (50 ng/ml) on STAT-3 activation measured by western blot. Cells were treated overnight in serum-free medium prior to exposure to IL-6, and then treated with IL-6 for 0-60 min. At the indicated times, cellular extracts were prepared and western blot analysis was performed. Actin and STAT-3 shown as total protein loading controls. (B) IL-6 (50 ng/ml) treatment or co-treatment with anti-IL-6R of MC-38 cells and activation of nuclear STAT-3 in MC-38 cells using the TransAM™ STAT-3 Transcription Factor Assay. Cells were treated with IL-6 alone (50ng/ml) or in combination with anti-IL-6R antibody (1 μg/ml) and STAT-3 activation measured at indicated times. *=p<0.05 (compared to IL-6/anti-IL-6R treated cells at same time point), **=p<0.01 (compared to IL-6/anti-IL-6R treated cells at same time point). (C) Toxicity of a STAT-3 inhibitor on MC-38 cells. Cells were treated with a STAT-3 inhibitor (0.01-100 mM) for 24 hrs and cell proliferation was measured as described in the methods section. (D) Effect of STAT-3 inhibitor on IL-6-induced cell proliferation in MC-38s. Cells were treated with either IL-6 (50 ng/ml) alone, STAT-3 inhibitor (0.1-100 mM) alone or in combination for 24 hrs. *= p<0.05 (compared to IL-6 alone treatment); **, p<0.01 (compared to IL-6 alone treatment); ◆=p<0.05 compared to untreated control. Results are representative of 3 separate experiments.

Figure 4

Adiponectin (globular and full-length) treatment attenuates IL-6-induced signaling in MC-38 cell. (A) The effect of 24 hr treatment of IL-6 alone (50 ng/ml), gAdipo alone (1 μg/ml), fAdipo alone (1 μg/ml), or in combination, on MC-38 cell proliferation. *=p<0.05 (compared to untreated control), **=p<0.01 (compared to IL-6 alone treatment). (B) Co-treamtent with neutralizing anti-adiponectin receptor 1(AdipoR1) or receptor 2 (AdipoR2) on the effect of 24 hr cell proliferation induced by IL-6 *=p<0.05 (compared to untreated control), **=p<0.01 (Different from both IL-6 treatment and CON). (C) The effect of IL-6 (50 ng/ml) alone or IL-6 co-treatment with gAdiponectin, anti-AdipoR1 or R2 (1μg/ml) on STAT-3 activation measured by western blot. MC-38 cells were treated for 0-60 min, nuclear extracts collected and membranes probed for pSTAT-3. STAT-3 and actin loaded for control. gAdipo; globular adiponectin. fAdipo; full-length length adiponectin. Results are representative of 3 separate experiments.

Figure 5

IL-6-induced nitric oxide production is inhibited by gAdiponectin treatment in MC-38 cells. (A) The effect of IL-6 treatment on nitrous oxide (NO) production in MC-38 cells. Cells were treated with 50 ng/ml IL-6 and NO measured after indicated times between 3 and 24 hrs. * = p<0.05 (compared to 3 hr time) (B) The effect of IL-6 (1 and 50 ng/ml) and gAdiponectin (1 μg/ml) on NO production. Cells were treated with above described concentrations and NO measured after 24 hrs. CON; control. * = p<0.05 (compared to control); ** = p<0.01(compared to control); ***=P<0.01 (compared to IL-6 50 ng treatment). (C) IL-6 treatment or co-treatment with adiponectin of MC-38 cells and phosphorylation (activation) of iNOS. Cells were incubated overnight in serum-free medium and then treated with either IL-6 alone (1 ng/ml or 50 ng/ml) or in combination with adiponectin (1 μg/ml, IL-6 at 50 ng/ml). Cellular extracts were then prepared and western-blot analysis was performed. Actin shown as total protein control. Results are representative of 3 separate experiments.

Figure 6

Hypothesized role of Adipokines on Colon Epithelial Cell Homeostasis