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, 6 (7), e1328338
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IL-32α-induced Inflammation Constitutes a Link Between Obesity and Colon Cancer

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IL-32α-induced Inflammation Constitutes a Link Between Obesity and Colon Cancer

Victoria Catalán et al. Oncoimmunology.

Abstract

Growing evidence indicates that adipose tissue inflammation is an important mechanism whereby obesity promotes cancer risk and progression. Since IL-32 is an important inflammatory and remodeling factor in obesity and is also related to colon cancer (CC) development, the aim of this study was to explore whether IL-32 could function as an inflammatory factor in human obesity-associated CC promoting a microenvironment favorable for tumor growth. Samples obtained from 84 subjects [27 lean (LN) and 57 obese (OB)] were used in the study. Enrolled subjects were further subclassified according to the established diagnostic protocol for CC (49 without CC and 35 with CC). We show, for the first time, that obesity (p = 0.009) and CC (p = 0.026) increase circulating concentrations of IL-32α. Consistently, we further showed that gene (p < 0.05) and protein (p < 0.01) expression levels of IL-32α were upregulated in VAT from obese patients with CC. Additionally, we revealed that IL32 expression levels are enhanced by hypoxia and inflammation-related factors in HT-29 CC cells as well as that IL-32α is involved in the upregulation of inflammation (IL8, TNF, and CCL2) and extracellular matrix (ECM) remodeling (SPP1 and MMP9) genes in HT-29 cancer cells. Additionally, we also demonstrate that the adipocyte-conditioned medium obtained from obese patients stimulates (p < 0.05) the expression of IL32 in human CC cells. These findings provide evidence of the potential involvement of IL-32 in the development of obesity-associated CC as a pro-inflammatory and ECM remodeling cytokine.

Keywords: Adipose tissue; IL-32; colon cancer; inflammation; obesity.

Figures

Figure 1.
Figure 1.
IL-32 levels in obesity and colon cancer (CC). (A) Circulating concentrations of IL-32α of lean (LN) and obese (OB) volunteers classified according to the presence or not of CC. Gene (B) and protein levels (C) of IL32 in visceral adipose tissue (VAT). The gene and protein expression in patients without CC was assumed to be 1. Representative blots are shown at the bottom of the histograms. The intensity of the bands was normalized with total protein values. (D) Immunohistochemical detection of IL-32α in histological sections of human VAT. Representative images of at least three separate experiments are shown. (E) Gene expression levels of IL32 in small intestine from OB volunteers classified into three groups [normoglycemia (NG), impaired glucose tolerance (IGT) and type 2 diabetes (T2D)]. Differences between groups were analyzed by two-way ANCOVA, one way ANCOVA as well as by two-tailed unpaired Student's t tests, where appropriate. Bars represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs patients without CC. ††p < 0.01 vs OB-NG and ‡‡p < 0.01 vs OB-IGT.
Figure 2.
Figure 2.
Impact of obesity and colon cancer (CC) on inflammation. Circulating concentrations of (A) interleukin (IL)-6, (B) IL-8, (C) IL-4 and (D) IL-13 of lean (LN) and obese (OB) volunteers classified according to the presence or not of CC. Bars represent the mean ± SEM. Differences between groups were analyzed by two-way ANCOVA or one way ANCOVA followed by Tukey's tests in the case of IL-8 due to interaction (*p < 0.05 vs LN non-CC).
Figure 3.
Figure 3.
Effect of obesity and colon cancer (CC) on extracellular matrix remodeling factors. Circulating concentrations of (A) vascular endothelial growth factor A (VEGFA), (B) WNT1 inducible signaling pathway protein 1 (WISP1), (C) secreted protein acidic and cysteine rich (SPARC) and (D) secreted phosphoprotein 1 [osteopontin (OPN)] of lean (LN) and obese (OB) volunteers classified according to the presence or not of CC. Bars represent the mean ± SEM. Differences between groups were analyzed by two-way ANCOVA.
Figure 4.
Figure 4.
Impact of inflammation-related factors and hypoxia on IL32 gene expression levels in HT-29 colon cancer cells. Bar graphs show the effect of TNF-α (A), LPS (B), IL-13, (C), IL-4 (D), and CoCl2 (E) incubated for 24 h on the transcript levels of IL32 in HT-29 cells. Gene expression levels in unstimulated cells were assumed to be 1. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by one-way ANOVA followed by Dunnet's tests. *p < 0.05, **p < 0.01 and ***p < 0.001 vs unstimulated cells.
Figure 5.
Figure 5.
Effect of IL-32α treatment on the expression of inflammatory and ECM remodeling markers in HT-29 cells. Gene expression levels of pro-inflammatory markers as well as extracellular matrix remodeling-related molecules in HT-29 cells stimulated with recombinant IL-32α (1, 10, and 100 nmol/L) for 24 h. Gene expression levels in unstimulated cells were assumed to be 1. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by one-way ANOVA followed by Dunnet's tests. *p < 0.05, **p < 0.01, and ***p < 0.001 vs unstimulated cells.
Figure 6.
Figure 6.
Adipocyte-conditioned media (ACM) induces gene expression levels of IL32 in HT-29 cells. Bar graphs show the effect of adipocyte CM (20% and 40%) from obese subjects incubated for 24 h on the transcript levels of IL32 in HT-29 cells. Values are the mean ± SEM (n = 6 per group). Differences between groups were analyzed by one-way ANOVA followed by Dunnet's tests. *p < 0.05 vs unstimulated cells.

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