Limits...
The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil.

Sánchez-Aragó M, Cuezva JM - J Transl Med (2011)

Bottom Line: We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment.Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells.Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras CIBERER-ISCIII, Instituto de Investigación Hospital 12 de Octubre, Universidad Autónoma de Madrid, 28049 Madrid, Spain.

ABSTRACT

Background: Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU).

Methods: The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells.

Results: We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable.

Conclusions: Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

Show MeSH

Related in: MedlinePlus

The Bioenergetic Signature correlates with the cell-death response to chemotherapy . HCT116 cells were treated as indicated [11] to produce cells with low (G-cells) (A), medium (M-cells) (B) and high (SM-cells) (C) bioenergetic signatures (β-F1/GAPDH ratio). The rates of aerobic glycolysis in G-, M- and SM-cells are also indicated. Cells were exposed to the following agents: 8 μM 3BrP, 100 μM IA, 10 μM 5-FU or were left untreated (Control). Cells were double-stained with Hoechst 33342 and propidium iodide and visualized using fluorescence microscopy at 20x magnification. The percentage of dead cells (red cells, PI positive) was determined by examination of different randomly selected fields. Histograms shown (A-C) represent the means ± SEM of 10-25, 10-24 and 10-23 independent determinations in G-, M-, and SM-cells respectively. *, P < 0.05 for multiple comparisons by ANOVA and post hoc Dunnett's test. Plots in (D) illustrate the inverse (3BrP and IA) and direct (5-FU) correlation that exists between the bioenergetic signature of the cells and the death-response to the chemotherapeutic agents.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045315&req=5

Figure 1: The Bioenergetic Signature correlates with the cell-death response to chemotherapy . HCT116 cells were treated as indicated [11] to produce cells with low (G-cells) (A), medium (M-cells) (B) and high (SM-cells) (C) bioenergetic signatures (β-F1/GAPDH ratio). The rates of aerobic glycolysis in G-, M- and SM-cells are also indicated. Cells were exposed to the following agents: 8 μM 3BrP, 100 μM IA, 10 μM 5-FU or were left untreated (Control). Cells were double-stained with Hoechst 33342 and propidium iodide and visualized using fluorescence microscopy at 20x magnification. The percentage of dead cells (red cells, PI positive) was determined by examination of different randomly selected fields. Histograms shown (A-C) represent the means ± SEM of 10-25, 10-24 and 10-23 independent determinations in G-, M-, and SM-cells respectively. *, P < 0.05 for multiple comparisons by ANOVA and post hoc Dunnett's test. Plots in (D) illustrate the inverse (3BrP and IA) and direct (5-FU) correlation that exists between the bioenergetic signature of the cells and the death-response to the chemotherapeutic agents.

Mentions: Because of the regulated expression of β-F1-ATPase, development of HCT116 colon cancer cell lines, displaying low (G-cells), medium (M-cells) or high (SM-cells) bioenergetic signatures (see Figure 1, and additional file 1) was accomplished by modification of cell culture conditions [11]. As recently detailed [11], the bioenergetic signature of each cell line was found to inversely correlate with the rate of aerobic glycolysis, where G-cells > M-cells > SM-cells (Figure 1A-C). Evaluation of cell death responses were assessed using fluorescence microscopy after double labeling with Hoechst 33342 and propidium iodide (PI) (Figure 1A-C). Our results show that death responses (% PI positive cells) to both metabolic inhibitors (3BrP and IA) decreased as the bioenergetic signature of the cells increased. Thus, the lower the bioenergetic signature of a cell the greater the death response to the glycolytic inhibitor treatment (G > M > SM) (Figure 1D). In fact, significant inverse correlations were uncovered between the bioenergetic signature of a cell and the extent of cell death following 3BrP (R = -0.633; n = 36, P < 0.01) and IA (R = -0.616; n = 36, P < 0.01) treatment, supporting the relevance of these glycolytic inhibitors in cancer treatment [7,23]. Specifically, in M-cells, 3BrP treatment was more effective than IA treatment at triggering cell death (Figure 1). In contrast, cell death in response to 5-FU treatment was found to directly correlate with bioenergetic signature (R = 0.519; n = 27, P < 0.01) (Figure 1D): as the activity of aerobic glycolysis is diminished cell death in response to 5-FU treatment is augmented (SM-cells > M-cells > G-cells), suggesting the participation of mitochondrial oxidative phosphorylation in the mechanism of 5-FU mediated cell death.


The bioenergetic signature of isogenic colon cancer cells predicts the cell death response to treatment with 3-bromopyruvate, iodoacetate or 5-fluorouracil.

Sánchez-Aragó M, Cuezva JM - J Transl Med (2011)

The Bioenergetic Signature correlates with the cell-death response to chemotherapy . HCT116 cells were treated as indicated [11] to produce cells with low (G-cells) (A), medium (M-cells) (B) and high (SM-cells) (C) bioenergetic signatures (β-F1/GAPDH ratio). The rates of aerobic glycolysis in G-, M- and SM-cells are also indicated. Cells were exposed to the following agents: 8 μM 3BrP, 100 μM IA, 10 μM 5-FU or were left untreated (Control). Cells were double-stained with Hoechst 33342 and propidium iodide and visualized using fluorescence microscopy at 20x magnification. The percentage of dead cells (red cells, PI positive) was determined by examination of different randomly selected fields. Histograms shown (A-C) represent the means ± SEM of 10-25, 10-24 and 10-23 independent determinations in G-, M-, and SM-cells respectively. *, P < 0.05 for multiple comparisons by ANOVA and post hoc Dunnett's test. Plots in (D) illustrate the inverse (3BrP and IA) and direct (5-FU) correlation that exists between the bioenergetic signature of the cells and the death-response to the chemotherapeutic agents.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045315&req=5

Figure 1: The Bioenergetic Signature correlates with the cell-death response to chemotherapy . HCT116 cells were treated as indicated [11] to produce cells with low (G-cells) (A), medium (M-cells) (B) and high (SM-cells) (C) bioenergetic signatures (β-F1/GAPDH ratio). The rates of aerobic glycolysis in G-, M- and SM-cells are also indicated. Cells were exposed to the following agents: 8 μM 3BrP, 100 μM IA, 10 μM 5-FU or were left untreated (Control). Cells were double-stained with Hoechst 33342 and propidium iodide and visualized using fluorescence microscopy at 20x magnification. The percentage of dead cells (red cells, PI positive) was determined by examination of different randomly selected fields. Histograms shown (A-C) represent the means ± SEM of 10-25, 10-24 and 10-23 independent determinations in G-, M-, and SM-cells respectively. *, P < 0.05 for multiple comparisons by ANOVA and post hoc Dunnett's test. Plots in (D) illustrate the inverse (3BrP and IA) and direct (5-FU) correlation that exists between the bioenergetic signature of the cells and the death-response to the chemotherapeutic agents.
Mentions: Because of the regulated expression of β-F1-ATPase, development of HCT116 colon cancer cell lines, displaying low (G-cells), medium (M-cells) or high (SM-cells) bioenergetic signatures (see Figure 1, and additional file 1) was accomplished by modification of cell culture conditions [11]. As recently detailed [11], the bioenergetic signature of each cell line was found to inversely correlate with the rate of aerobic glycolysis, where G-cells > M-cells > SM-cells (Figure 1A-C). Evaluation of cell death responses were assessed using fluorescence microscopy after double labeling with Hoechst 33342 and propidium iodide (PI) (Figure 1A-C). Our results show that death responses (% PI positive cells) to both metabolic inhibitors (3BrP and IA) decreased as the bioenergetic signature of the cells increased. Thus, the lower the bioenergetic signature of a cell the greater the death response to the glycolytic inhibitor treatment (G > M > SM) (Figure 1D). In fact, significant inverse correlations were uncovered between the bioenergetic signature of a cell and the extent of cell death following 3BrP (R = -0.633; n = 36, P < 0.01) and IA (R = -0.616; n = 36, P < 0.01) treatment, supporting the relevance of these glycolytic inhibitors in cancer treatment [7,23]. Specifically, in M-cells, 3BrP treatment was more effective than IA treatment at triggering cell death (Figure 1). In contrast, cell death in response to 5-FU treatment was found to directly correlate with bioenergetic signature (R = 0.519; n = 27, P < 0.01) (Figure 1D): as the activity of aerobic glycolysis is diminished cell death in response to 5-FU treatment is augmented (SM-cells > M-cells > G-cells), suggesting the participation of mitochondrial oxidative phosphorylation in the mechanism of 5-FU mediated cell death.

Bottom Line: We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment.Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells.Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Centro de Investigación Biomédica en Red de Enfermedades Raras CIBERER-ISCIII, Instituto de Investigación Hospital 12 de Octubre, Universidad Autónoma de Madrid, 28049 Madrid, Spain.

ABSTRACT

Background: Metabolic reprogramming resulting in enhanced glycolysis is a phenotypic trait of cancer cells, which is imposed by the tumor microenvironment and is linked to the down-regulation of the catalytic subunit of the mitochondrial H+-ATPase (β-F1-ATPase). The bioenergetic signature is a protein ratio (β-F1-ATPase/GAPDH), which provides an estimate of glucose metabolism in tumors and serves as a prognostic indicator for cancer patients. Targeting energetic metabolism could be a viable alternative to conventional anticancer chemotherapies. Herein, we document that the bioenergetic signature of isogenic colon cancer cells provides a gauge to predict the cell-death response to the metabolic inhibitors, 3-bromopyruvate (3BrP) and iodoacetate (IA), and the anti-metabolite, 5-fluorouracil (5-FU).

Methods: The bioenergetic signature of the cells was determined by western blotting. Aerobic glycolysis was determined from lactate production rates. The cell death was analyzed by fluorescence microscopy and flow cytometry. Cellular ATP concentrations were determined using bioluminiscence. Pearson's correlation coefficient was applied to assess the relationship between the bioenergetic signature and the cell death response. In vivo tumor regression activities of the compounds were assessed using a xenograft mouse model injected with the highly glycolytic HCT116 colocarcinoma cells.

Results: We demonstrate that the bioenergetic signature of isogenic HCT116 cancer cells inversely correlates with the potential to execute necrosis in response to 3BrP or IA treatment. Conversely, the bioenergetic signature directly correlates with the potential to execute apoptosis in response to 5-FU treatment in the same cells. However, despite the large differences observed in the in vitro cell-death responses associated with 3BrP, IA and 5-FU, the in vivo tumor regression activities of these agents were comparable.

Conclusions: Overall, we suggest that the determination of the bioenergetic signature of colon carcinomas could provide a tool for predicting the therapeutic response to various chemotherapeutic strategies aimed at combating tumor progression.

Show MeSH
Related in: MedlinePlus