ω3-PUFAs exert anti-inflammatory activity in visceral adipocytes from colorectal cancer patients

Massimo D'Archivio, Beatrice Scazzocchio, Stefania Giammarioli, Maria L Fiani, Rosaria Varì, Carmela Santangelo, Augusto Veneziani, Annunziata Iacovelli, Claudio Giovannini, Sandra Gessani, Roberta Masella, Massimo D'Archivio, Beatrice Scazzocchio, Stefania Giammarioli, Maria L Fiani, Rosaria Varì, Carmela Santangelo, Augusto Veneziani, Annunziata Iacovelli, Claudio Giovannini, Sandra Gessani, Roberta Masella

Abstract

Objective: The aim of this study was to correlate specific fatty acid profiles of visceral white adipose tissue (WAT) with inflammatory signatures potentially associated with colorectal cancer (CRC).

Methods: Human adipocytes were isolated from biopsies of visceral WAT from 24 subjects subdivided in four groups: normal-weight (BMI 22.0-24.9 Kg/m2) and over-weight/obese (BMI 26.0-40.0 Kg/m2), affected or not by CRC. To define whether obesity and/or CRC affect the inflammatory status of WAT, the activation of the pro-inflammatory STAT3 and the anti-inflammatory PPARγ transcription factors as well as the expression of adiponectin were analyzed by immunoblotting in adipocytes isolated from each group of subjects. Furthermore, to evaluate whether differences in inflammatory WAT environment correlate with specific fatty acid profiles, gas-chromatographic analysis was carried out on WAT collected from all subject categories. Finally, the effect of the ω3 docosahexaenoic acid treatment on the balance between pro- and anti-inflammatory factors in adipocytes was also evaluated.

Results: We provide the first evidence for the existence of a pro-inflammatory environment in WAT of CRC patients, as assessed by the up-regulation of STAT3, and the concomitant decrease of PPARγ and adiponectin with respect to healthy subjects. WAT inflammatory status was independent of obesity degree but correlated with a decreased ω3-/ω6-polyunsaturated fatty acid ratio. These observations suggested that qualitative changes, other than quantitative ones, in WAT fatty acid may influence tissue dysfunctions potentially linked to inflammatory conditions. This hypothesis was further supported by the finding that adipocyte treatment with docosahexaenoic acid restored the equilibrium between STAT3 and PPARγ.

Conclusion: Our results suggest that adipocyte dysfunctions occur in CRC patients creating a pro-inflammatory environment that might influence cancer development. Furthermore, the protective potential of docosahexaenoic acid in re-establishing the equilibrium between pro- and anti-inflammatory factors might represent a useful tool for preventive and therapeutic strategies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Immunoblotting analysis of pSTAT3, PPARγ,…
Figure 1. Immunoblotting analysis of pSTAT3, PPARγ, and adiponectin.
Human visceral adipocytes, collected from the four groups of subjects, were serum-starved for 18 h. Whole cell extracts and nuclear protein extracts were separated by SDS-PAGE and analyzed using anti-pSTAT3 (A), anti-PPARγ (B) and anti-adiponectin (C) antibodies. Results were normalized to STAT3, Lamin B and GAPDH protein content, respectively. NW: normal weight subjects (n=5); Ob: overweight/obese subjects (n=5); NWCC: normal weight with colorectal cancer (n=7); ObCC: overweight/obese with colorectal cancer (n=7). The data are expressed as means ± SEM. *, P<0.05 compared with NW; **, P<0.05 compared with NW and Ob; ***, P<0.05 compared with NW, Ob, and NWCC. Representative blots are shown.
Figure 2. Linear regression analysis between ω3-/ω6-PUFA…
Figure 2. Linear regression analysis between ω3-/ω6-PUFA ratio and Body Mass Index (BMI).
A: all subjects; B: subjects without colorectal cancer; C: subjects affected by colorectal cancer.
Figure 3. Immunoblotting analysis of pSTAT3, and…
Figure 3. Immunoblotting analysis of pSTAT3, and evaluation of IL-6 secretion after DHA treatment.
A: Human visceral adipocytes, collected from the four groups of subjects, were serum-starved for 18 h. Whole cell extracts were separated by SDS-PAGE and analyzed using anti-pSTAT3 antibody. Results were normalized to STAT3 protein content. The data are expressed as means ± SEM. Representative blots are shown. B: IL-6 release was evaluated in the culture media by Elisa as described in Materials and Methods. Data are expressed as means ± SEM. *, P<0.05 with respect to the untreated paired cells. NW: normal weight subjects (n=5); Ob: overweight/obese subjects (n=5); NWCC: normal weight with colorectal cancer (n=7); ObCC: overweight/obese with colorectal cancer (n=7).
Figure 4. Linear regression analysis between ω3-/ω6-PUFA…
Figure 4. Linear regression analysis between ω3-/ω6-PUFA ratio, Body Mass Index (BMI) and pSTAT3 decrease.
Correlation between the ω3-/ω6-PUFA ratio and the percentage of pSTAT3 decrease after DHA treatment in control subjects (n=10) (A) and cancer subjects (n=14) (B). Correlation between the BMI and the percentage of pSTAT3 decrease after DHA treatment in control subjects (C) and cancer subjects (D).
Figure 5. Immunoblotting analysis of PPARγ, and…
Figure 5. Immunoblotting analysis of PPARγ, and adiponectin after DHA treatment.
Human visceral adipocytes, collected from the four groups of subjects, were serum-starved for 18 h and incubated with 10µM DHA, as described in Material and Methods. Nuclear protein extracts and whole cell extracts were separated by SDS-PAGE and analyzed using anti-PPARγ (A) and anti-adiponectin (B) antibodies. Results were normalized to Lamin B and GAPDH protein content, respectively. NW: normal weight subjects (n=5); Ob: overweight/obese subjects (n=5); NWCC: normal weight with colorectal cancer (n=7); ObCC: overweight/obese with colorectal cancer (n=7). The data are expressed as means ± SEM. *, P<0.05; **, P<0.01; ***, P<0.01 compared with untreated paired cells. Representative blots are shown.

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