Metabolic reprogramming induced by ketone bodies diminishes pancreatic cancer cachexia

Abstract

Background: Aberrant energy metabolism is a hallmark of cancer. To fulfill the increased energy requirements, tumor cells secrete cytokines/factors inducing muscle and fat degradation in cancer patients, a condition known as cancer cachexia. It accounts for nearly 20% of all cancer-related deaths. However, the mechanistic basis of cancer cachexia and therapies targeting cancer cachexia thus far remain elusive. A ketogenic diet, a high-fat and low-carbohydrate diet that elevates circulating levels of ketone bodies (i.e., acetoacetate, β-hydroxybutyrate, and acetone), serves as an alternative energy source. It has also been proposed that a ketogenic diet leads to systemic metabolic changes. Keeping in view the significant role of metabolic alterations in cancer, we hypothesized that a ketogenic diet may diminish glycolytic flux in tumor cells to alleviate cachexia syndrome and, hence, may provide an efficient therapeutic strategy.

Results: We observed reduced glycolytic flux in tumor cells upon treatment with ketone bodies. Ketone bodies also diminished glutamine uptake, overall ATP content, and survival in multiple pancreatic cancer cell lines, while inducing apoptosis. A decrease in levels of c-Myc, a metabolic master regulator, and its recruitment on glycolytic gene promoters, was in part responsible for the metabolic phenotype in tumor cells. Ketone body-induced intracellular metabolomic reprogramming in pancreatic cancer cells also leads to a significantly diminished cachexia in cell line models. Our mouse orthotopic xenograft models further confirmed the effect of a ketogenic diet in diminishing tumor growth and cachexia.

Conclusions: Thus, our studies demonstrate that the cachectic phenotype is in part due to metabolic alterations in tumor cells, which can be reverted by a ketogenic diet, causing reduced tumor growth and inhibition of muscle and body weight loss.

Keywords: Cancer cachexia; Cancer metabolism; Ketone bodies; Pancreatic cancer.

Figure 1

Ketone bodies inhibit growth and induce apoptosis in pancreatic cancer cell lines. Capan1 (A) and S2-013 (B) cells were treated with different concentrations of sodium-3-hydroxybutyrate (NaHB) and lithium acetoacetate (LiAcAc) for 72 h, and cell viability was determined by MTT assay. Bar represents percent viability under indicated treatments relative to treatment with solvent control. Representative bright-field images of Capan1 (C) and S2-013 (D) cells under treatment with 10- and 20-mM concentrations of NaHB and LiAcAc for 72 h. (E) Multiple pancreatic cancer cell lines were treated with 10- and 20-mM concentrations of NaHB and LiAcAc for 72 h, and relative cell viability determined by MTT assay is plotted in the bar charts. (F) Capan1 and S2-013 cells treated with 10- and 20-mM concentrations of sodium-3-hydroxybutyrate and lithium acetoacetate for 48 h and the relative caspase 3/7 activity are plotted. Values represented are mean ± SEM. *P < 0.05; **P < 0.01.

Figure 2

Ketone bodies induce metabolic alterations in pancreatic cancer cell lines. S2-013 (A) and Capan1 (B) cells were treated with different doses of ketone bodies for 24 h, and glucose uptake was determined by performing ³H-2DG uptake assay. Bars represent counts normalized with cell number and plotted relative to control. Lactate release was determined by colorimetric assay using culture medium of S2-013 (C) and Capan1 (D) cells treated with different concentrations of NaHB and LiAcAc for 24 h. Values were normalized with total cell number and represented relative to controls. S2-013 (E) and Capan1 (F) cells were treated with indicated concentrations of ketone bodies for 24 h, and glutamine uptake was determined by performing tritiated Glutamine, l-[3,4-³H(N)] uptake assays. Counts were normalized with cell number and plotted relative to control. ATP levels in S2-013 (G) and Capan1 (H) cells post 24-h treatment with ketone bodies were determined by performing ATP bioluminescence assays. Values were normalized to total protein concentration and represented relative to control. Reactive oxygen species level of S2-013 (I) and Capan1 (J) cells under treatment with ketone bodies was determined by utilizing a fluorescence probe, dihydroethidium (DHE), and fluorescence intensity normalized to cell count was plotted. Values represented are mean ± SEM. *P < 0.05; **P < 0.01.

Figure 3

Ketone bodies repress the expression of key glycolytic enzymes. Relative mRNA expression levels of GLUT1, HKII, and LDHA in Capan1 (A) and S2-013 (B) cells treated with 10- and 20-mM concentrations of NaHB and LiAcAc for 24 h. Total RNA was isolated from NaHB- and LiAcAc-treated as well as control cells, and relative mRNA levels of different genes were determined by performing qRT-PCR. β-Actin was utilized as an internal control. Protein expression of GLUT1 and HKII was determined by immunoblotting the total cell lysates from S2-013 (C) and Capan1 (D) cells treated with 10 and 20 mM NaHB and LiAcAc for 48 h. β-Tubulin was utilized as an internal control. Values shown are mean ± SEM. *P < 0.05; **P < 0.01.

Figure 4

Ketone bodies reduce c-Myc expression and its recruitment to glycolytic gene promoters. Recruitment of c-Myc onto GLUT1(A) and LDHA(B) promoters in S2-013 cells under treatment with 20 mM NaHB, LiAcAc, or control was confirmed by performing ChIP using anti-c-Myc Ab and IgG control, followed by qRT-PCR analysis. Relative c-Myc mRNA levels in Capan1 (C) and S2-013 (D) cells treated with 10 and 20 mM NaHB, LiAcAc, or control for 24 h. Total RNA was isolated and relative mRNA level of c-Myc was determined by qRT-PCR. β-Actin was utilized as an internal control. Capan1 (E) and S2-013 (F) cells were treated with indicated doses of ketone bodies for 48 h, and c-Myc protein level was determined by immunoblotting the whole cell lysates. HSP90 was used as an internal control. (G) c-Myc-promoter-firefly luciferase reporter and Renilla luciferase reporter plasmids were transiently transfected into S2-013 cells. After 16 h of transfection, cells were treated with solvent control or ketone bodies for 24 h. Normalized firefly to Renilla luciferase activity ratio is plotted in the bar chart. Values represented are mean ± SEM. *P < 0.05; **P < 0.01.

Figure 5

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.

Figure 6

Ketone bodies modulate metabolite levels in pancreatic cancer cells. (A) OPLS-DA score plot generated from 1D ¹H NMR spectra collected from cell lysates of S2-013 cells (red square) and S2-013 cells treated with 20 mM NaHB (green diamond); each point in the OPLS-DA score plot represents a single 1D ¹H NMR spectrum. Ellipses enclose the 95% confidence intervals estimated by the sample means and covariances of each class. The leave-n-out cross validation yielded a quality assessment (Q²) value of 0.959 and R² value of 0.997. The OPLS-DA model was validated using CV-ANOVA yielding a p value of 8.74 × 10⁻⁶. (B) Heat map generated from 2D ¹H-¹³C HSQC NMR spectral data for S2-013 cells. The heat map represents triplicate measurements of metabolite intensities recorded (*P < 0.1; **P < 0.05; ***P < 0.001). (C) Metabolic pathway depicts ¹³C carbon flow from glucose to the intermediates of glycolytic pathway, citric acid cycle, amino acid metabolism, and nucleotide analogues. The arrows represent relative increase (green arrow up) or decrease (red arrow down) in metabolite concentrations due to ketone body treatment.

Figure 7

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-shGLUT1) 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-shGLUT1-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-shGLUT1-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.

Figure 8

A ketogenic diet reduces tumor growth and proliferation and reverts the cachectic phenotype. Into the pancreas of athymic nude mice, 0.5 × 10⁶ S2-013 cells were orthotopically implanted. After 1 week of implantation, mice were divided in two groups and fed with either a control diet or a ketogenic diet. Three weeks post treatment, mice were sacrificed and tumor weight (A) and tumor volume (B) were measured. (C) Masson’s trichrome staining (blue stain indicates desmoplastic region) and immunohistochemistry images from the control diet and the ketogenic diet-fed mice tumor sections. (D) Blood glucose and ketone levels in the control and the ketogenic diet-fed mice before necropsy. (E) Muscle weight and carcass weight of the control and the ketogenic diet-fed mice. (F) Immunohistochemistry of c-Myc in the control and the ketogenic diet-fed mice tumor sections. Values shown are mean ± SEM. *P < 0.05; **P < 0.01.