Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

<div><p>Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, <i>p<0.05</i>) and maximal (50%, <i>p<0.05</i>) OCR and gene expression of mitochondrial proteins and Bax without affecting cell viability or expression of glycolytic enzymes. Similar changes could be recapitulated by incubating cells with leptin, whereas, leptin-receptor specific antagonist inhibited the reduced OCR induced by conditioned media from obese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues.</p> </div

Effects of the obese CM on HCT116 glycolysis.

<p>HCT 116 cells were treated for 24 hours with CM collected from visceral AT of non-obese subjects (<i>n</i>=4) or obese subjects (<i>n</i>=9). (<i>A</i>) Bax gene expression levels were detected using quantitative real-time PCR. *, <i>P</i>< 0.05 vs. respective non-obese sample of each gene (Mann Whitney test). (<i>B</i>) Cell lysates were analyzed by Western blot. HM-7 and Caco₂ were used as controls (see Results) (<i>C</i>), Densitometric analysis of the Western blot data. *, <i>P</i>> 0.01, **, <i>P</i><0.05 (Two Way ANOVA, Bonferroni test).</p

Effect of leptin on HCT116 cells mitochondria.

<p>HCT116 cells were treated with DMEM (control) vs. leptin (100 ng/ml), for 24 hours. (<i>A</i>, <i>B</i>) Gene expression levels were detected using quantitative real-time PCR (<i>n</i>=4). *, <i>P</i>< 0.05, **, <i>P</i>< 0.01 vs. respective control of each gene (Student’s <i>t</i>-test). (<i>C</i>) Cell lysates were analyzed for cytochrome C (CytC, top panel) and β-actin (bottom panel) by Western blot, and densitometric analysis of the data was made. * <i>P</i>> 0.01, vs. control (Student’s <i>t</i>-test).</p

Effects of the obese CM on HCT116 cells mitochondria.

<p>HCT116 cells were treated for 24 hours with DMEM (control), CM collected from visceral AT of non-obese subjects. (<i>A</i>, <i>B</i>) Gene expression levels were detected using quantitative real-time PCR. Obese (<i>n</i>=8), non-obese (<i>n</i>=4). *, <i>P</i>< 0.05, **, <i>P</i>< 0.01 vs. respective non-obese sample of each gene (Mann Whitney test). (<i>C</i>) Gene expression levels were detected using quantitative real-time PCR. Control (<i>n</i>=3), non-obese (<i>n</i>=4), obese (<i>n</i>=9). *, <i>P</i>< 0.05, vs. control (Mann Whitney), ** <i>P</i>< 0.05, vs. non-obese (Mann Whitney). (<i>D</i>) Cell lysates were analyzed for Bax (top panel) or β-actin (bottom panel) antibodies, by Western blot and densitometric analysis was made. Vertical white lines denote image splicing to present only relevant bands, for clarity (shown is a single blot). Control (<i>n</i>=3), non-obese (<i>n</i>=3), obese (<i>n</i>=6). *, <i>P</i>> 0.01 vs. Control (One Way ANOVA, Tukey test). **, <i>P</i>< 0.001, vs. the non-obese samples (One Way ANOVA, Tukey test).</p

Leptin involvement in mediating obesity-reduced OCR.

<p>HCT116 cells were treated for 24 hours with CM collected from visceral AT of non-obese or obese subjects, with or without leptin antagonist (1 ng/ml). OCR levels were measured using the XF24 Analyzer. Non-obese (<i>n</i>=4), obese (<i>n</i>=7), non-obese antagonist (LepA non-obese, <i>n</i>=3), obese antagonist (LepA Obese, <i>n</i>=6). *, <i>P</i>< 0.05 vs. samples from obese (Student’s t-test). Results were normalized to protein concentration and expressed as percentage of control.</p