Journal: Cancer & Metabolism

Article Title: Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia

doi: 10.1186/2049-3002-2-18

Figure Lengend Snippet: Ketone bodies inhibit tumor cell-conditioned medium-induced degradation of myofibers and adipolysis. Differentiated C2C12 cells were treated with S2-013 (A) and Capan1 (B) cell-conditioned medium with or without solvent control and 10 and 20 mM NaHB and LiAcAc for 72 h, and bright-field images were represented for individual treatments. (C) Differentiated C2C12 cells were cultured in Capan1 and S2-013 cell-conditioned medium with or without ketone body treatment for 24 h. Total RNA was isolated and relative mRNA levels of MuRF1 and Atrogin were determined by performing qRT-PCR. β-Actin was utilized as an internal control. Differentiated 3T3L1 cells were cultured in S2-013 (D) and Capan1 (E) cell-conditioned medium with or without ketone body treatment for 72 h and stained with nile red. Fluorescent and bright-field images for individual treatments are presented. (F) Differentiated 3T3L1 cells were cultured in Capan1 and S2-013 cell-conditioned medium with or without ketone body treatment for 24 h. Total RNA was isolated and relative mRNA levels of Zag and HSL were determined by qRT-PCR. β-Actin was utilized as an internal control. Values represented are mean ± SEM. All statistical analyses were conducted with one-way ANOVA with Dunnett’s post hoc test and CM as the reference group. * P < 0.05; ** P < 0.01.

Article Snippet: The human pancreatic cancer cell line Capan1, mouse myoblast C2C12, and mouse embryo fibroblast (preadipocyte) 3T3L1 were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Solvent, Control, Cell Culture, Isolation, Quantitative RT-PCR, Staining

Journal: Cancer & Metabolism

Article Title: Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia

doi: 10.1186/2049-3002-2-18

Figure Lengend Snippet: Pretreatment of tumor cells with ketone bodies or glycolytic inhibition diminishes their cachectic potential. S2-013 cells were treated with solvent control, 20 mM NaHB (NaHB-S2-013), 20 mM LiAcAc (LiAcAc-S2-013), and 10 μM 3-bromopyruvic acid (BPA-S2-013) for 24 h. The cells were then washed twice with phosphate-buffered saline and cultured in serum-free DMEM. After 24 h, the conditioned medium was collected. The conditioned medium was also prepared from GLUT1 knockdown S2-013 (S2-013-sh GLUT1 ) and control cells (S2-013-shScr). Differentiated myotubes from C2C12 cells were cultured in (A) control, S2-013-CM, NaHB-S2-013-CM, LiAcAc-S2-013-CM, and BPA-S2-013-CM or (B) control, S2-013-shScr-CM, and S2-013-sh GLUT1 -CM for 72 h, and bright-field images were represented for individual treatments. Differentiated 3T3L1 cells were cultured in (C) control, S2-013-CM, NaHB-S2-013-CM, LiAcAc-S2-013-CM, and BPA-S2-013-CM or (D) control, S2-013-shScr-CM, and S2-013-sh GLUT1 -CM for 72 h and stained with nile red, and images for individual treatments are represented. (E) Differentiated myotube form C2C12 cells were cultured in similar conditions for 24 h. Total RNA was isolated and relative mRNA levels of MuRF1 and Atrogin were determined by qRT-PCR. β-Actin was utilized as an internal control. (F) Differentiated 3T3L1 cells were cultured in the above-mentioned conditions for 24 h. Total RNA was isolated and relative mRNA levels of Zag and HSL were determined by qRT-PCR. β-Actin was utilized as an internal control. Values represented are mean ± SEM. All statistical analyses were conducted with one-way ANOVA with Dunnett’s post hoc test and S2-013-CM as the reference group.* P < 0.05; ** P < 0.01.

Article Snippet: The human pancreatic cancer cell line Capan1, mouse myoblast C2C12, and mouse embryo fibroblast (preadipocyte) 3T3L1 were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Solvent, Control, Saline, Cell Culture, Knockdown, Staining, Isolation, Quantitative RT-PCR

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A, THP-1 cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of resveratrol for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. B-E, THP-1 cells were treated with vehicle or the indicated concentration of resveratrol for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Enzyme immunoassay was used to measure TNF-α (B), IL-1β (C) and PGE2 (E) in the conditioned medium. Western blot analysis was used to determine levels of COX-2 protein (D). F and G, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of resveratrol. F, real-time PCR was used to quantify aromatase mRNA; G, aromatase activity was measured in a microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Transfection, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A, THP-1 cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of curcumin for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. B-E, THP-1 cells were treated with vehicle or the indicated concentration of curcumin for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Enzyme immunoassay was used to measure TNF-α (B), IL-1β (C) and PGE2 (E) in the conditioned medium. Western blot analysis was used to determine levels of COX-2 protein (D). F and G, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of curcumin. F, real-time PCR was used to quantify aromatase mRNA; G, aromatase activity was measured in a microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Transfection, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A, THP-1 cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of Zyflamend® for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. B-E, THP-1 cells were treated with vehicle or the indicated concentration of Zyflamend® for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Enzyme immunoassay was used to measure TNF-α (B), IL-1β (C) and PGE2 (E) in the conditioned medium. Western blot analysis was used to determine levels of COX-2 protein (D). F and G, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of Zyflamend®. F, real-time PCR was used to quantify aromatase mRNA; G, aromatase activity was measured in microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Transfection, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Real-time Polymerase Chain Reaction

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: Cells were treated with vehicle or the indicated concentration of LY294002 for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 2 hours. Western blot analysis was used to determine levels of Phospho-Akt and Akt (A) or Phospho-p65 and p65 (C). B, Cells were transfected with 1.8 μg NF-κB-luciferase and 0.2 μg pSVβgal. Cells were then treated with vehicle or the indicated concentration of LY294002 for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 24 hours. Luciferase activity represents data that have been normalized to β-galactosidase. D, Preadipocytes were incubated for 24 hours with conditioned medium from THP-1 cells that had been treated with vehicle, stearic acid (10 μmol/L) or stearic acid plus the indicated concentration of LY294002. Aromatase activity was measured in a microsomal preparation and is expressed as femtomoles/μg protein/minute. Columns, means (n=6); bars, SD. *P < 0.01. E-H, Cells were treated with vehicle or the indicated concentration of resveratrol (E), curcumin (F), EGCG (G) or Zyflamend® (H) for 2 hours. Subsequently, the cells received vehicle or 10 μmol/L stearic acid for 2 hours. Western blot analysis was used to determine levels of Phospho-Akt and Akt.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Concentration Assay, Western Blot, Transfection, Luciferase, Activity Assay, Incubation

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Dietary Polyphenols Suppress Elevated Levels of Proinflammatory Mediators and Aromatase in the Mammary Gland of Obese Mice

doi: 10.1158/1940-6207.CAPR-13-0140

Figure Lengend Snippet: A and B, Western blot analysis was used to determine levels of Phospho-Akt and Akt (A) and Phospho-p65 and p65 (B) in the mammary glands of four mice in each of the indicated treatment groups; C, 5 μg of nuclear protein was incubated with a 32P-labeled oligonucleotide containing NF-κB binding sites. Binding of nuclear protein from mammary glands is shown for each of the indicated treatment groups. D, Paracrine interactions between macrophages and other cell types, e.g., preadipocytes can explain the elevated levels of aromatase in the mammary glands of obese mice. In obesity, lipolysis is increased resulting in increased concentrations of free fatty acids. Saturated fatty acids stimulate the PI3K/Akt pathway leading to activation of NF-κB in macrophages and increased production of proinflammatory mediators (TNF-α, IL-1β and PGE2). Each of these proinflammatory mediators can act in a paracrine manner to induce aromatase. Polyphenols block the activation of the PI3K/Akt/NF-κB pathway in macrophages and thereby suppress the induction of proinflammatory mediators and aromatase.

Article Snippet: Tissue culture Human visceral preadipocytes (ScienCell) were grown in preadipocyte medium containing 10% FBS.

Techniques: Western Blot, Incubation, Labeling, Binding Assay, Activation Assay, Blocking Assay