Figure 1.
Body composition, blood glucose, and serum factors in C57BL/6 mice following 21 weeks of diet treatment.
Calorie restriction (CR) decreased A, body weight (reported up to 20 weeks on diets, before tumor cell injection); B, percent body fat (at 21 weeks on diet); and C, fasting blood glucose (at 21 weeks on diet) relative to CON diet (n = 15/group). Data shown represent mean ± SD. CR altered mean fasting serum levels of D, insulin; E, IGF-1; F, leptin; and G, adiponectin (n = 8/group). Data shown represent mean ± SEM. * denotes significant differences between CR and CON. CR, calorie restriction; CON, control diet; IGF-1, insulin-like growth factor-1.
Figure 2.
CR reduced Panc02 tumor volume and inflammatory profile.
CR decreased A, tumor growth and B, median tumor weight compared to CON mice (n = 15/group). Data shown are mean ± SD with the exception of the line in scatter plot representing the median tumor weight. C, CR reduced mRNA expression within the tumor microenvironment compared to CON mice (CON, n = 6; CR, n = 5). Data represent CR gene expression relative to CON. D, Mean fasting serum levels of MCP-1 (n = 8/group, randomly selected). E, Representative micrographs of p-p65-staining tumor sections (40X). F, Quantification of staining. Bar graph represents total percentage of p-p65-positive cells, with further illustration of intracellular localization (nuclear, black; cytoplasmic, white) (CON, n = 10 and CR, n = 7). Data shown represent mean ± SE. * denotes significant differences between CR and CON. CR, calorie restriction; CON, control diet; MCP-1, monocyte chemoattractant factor-1; p-p65, phosphorylated p65.
Figure 3.
Effect of IGF-1 on p65 nuclear localization and NF-κB pathway activation in Panc02 cells.
A, Representative fluorescent images of p65 localization at various time points following IGF-1 treatment (400 ng/mL) using 300 nM DAPI alone, a p65 antibody alone (p65), or p65 antibody counterstained with DAPI (merged). Data representative of 4 separate assays. B, ELISA of p65 DNA binding in response to 4-hour IGF-1 treatment (400 ng/mL). C, NF-κB luciferase reporter assay after 6-hour IGF-1 treatment (400 ng/mL). *denotes significant differences between SF and IGF-1. D, PCR analysis of mRNA expression of NF-κB downstream gene targets after 18-hour IGF-1 treatment. E, ELISA measurement of RANTES, LIF, and VEGF (n = 3) expression in Panc02 supernatant after 24 hours of IGF-1 treatment. *denotes significant differences between SF and IGF-1 with respect to each respective gene or protein of interest. All experiments used 400 ng/mL of IGF-1. Bar graphs represent mean ± SEM (n = 3 separate experiments performed in triplicate for B, C, and D). SF, serum-free; IGF-1, insulin-like growth factor-1; DAPI, 4′-6-diamidino-2-phenylindole; RANTES, Regulated on Activation, Normal T cell Expressed and Secreted; LIF, leukemia inhibitory factor; VEGF, vascular endothelial growth factor.
Figure 4.
Effect of silenced p65 on IGF-1-induced expression of downstream NF-κB targets in Panc02 cells.
A, Expression of p65 mRNA in Panc02 cells after exposure to either scrambled or p65-specific small interfering RNA. B, Ccdn1; Vegfa; Birc5; and COX-2 mRNA expression after 18-hour IGF-1 treatment (400 ng/mL). Bar graphs represent mean ± SEM (n = 3 separate experiments performed in triplicate for each gene). Different letters or asterisk denotes significance between groups. IGF-1, Insulin-like growth factor-1.
Figure 5.
Diet effects on MiaPaCa-2 tumor burden and gene expression.
A, Mean body weight of athymic nude mice per group after 16 weeks of diet treatment (prior to tumor injection). B, MiaPaCa-2 tumor volume measured over 6 weeks of tumor growth. Line graphs represent mean ± SD. C, MiaPaCa-2 tumor weight measured at study termination; line in scatter plot represents median tumor weight. A, B, & C, n = 14/group. D, Diet effects on mRNA expression of NF-κB downstream targets. Data represent CR, relative to CON, mRNA expression, mean ± SEM (n = 6/group). Significance (p<0.05) is denoted by *. CON, control; CR, calorie restriction.