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Dichloroacetate, a selective mitochondria-targeting drug for oral squamous cell carcinoma: a metabolic perspective of treatment.

Ruggieri V, Agriesti F, Scrima R, Laurenzana I, Perrone D, Tataranni T, Mazzoccoli C, Lo Muzio L, Capitanio N, Piccoli C - Oncotarget (2015)

Bottom Line: In this study we tested comparatively the effects of DCA on three different OSCC-derived cell lines, HSC-2, HSC-3, PE15.DCA treatment of the three OSCC cell lines, at pharmacological concentrations, resulted in stimulation of the respiratory activity and caused a remarkably distinctive pro-apoptotic/cytostatic effect on HSC-2 and HSC-3.This was accompanied with a large remodeling of the mitochondrial network, never documented before, leading to organelle fragmentation and with enhanced production of reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pre-Clinical and Translational Research, IRCCS, CROB, Rionero in Vulture, Potenza, Italy.

ABSTRACT
Reprogramming of metabolism is a well-established property of cancer cells that is receiving growing attention as potential therapeutic target. Oral squamous cell carcinomas (OSCC) are aggressive and drugs-resistant human tumours displaying wide metabolic heterogeneity depending on their malignant genotype and stage of development. Dichloroacetate (DCA) is a specific inhibitor of the PDH-regulator PDK proved to foster mitochondrial oxidation of pyruvate. In this study we tested comparatively the effects of DCA on three different OSCC-derived cell lines, HSC-2, HSC-3, PE15. Characterization of the three cell lines unveiled for HSC-2 and HSC-3 a glycolysis-reliant metabolism whereas PE15 accomplished an efficient mitochondrial oxidative phosphorylation. DCA treatment of the three OSCC cell lines, at pharmacological concentrations, resulted in stimulation of the respiratory activity and caused a remarkably distinctive pro-apoptotic/cytostatic effect on HSC-2 and HSC-3. This was accompanied with a large remodeling of the mitochondrial network, never documented before, leading to organelle fragmentation and with enhanced production of reactive oxygen species. The data here presented indicate that the therapeutic efficacy of DCA may depend on the specific metabolic profile adopted by the cancer cells with those exhibiting a deficient mitochondrial oxidative phosphorylation resulting more sensitive to the drug treatment.

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Related in: MedlinePlus

Comparative analysis of the metabolic profiles of different OSSC-derived cell lines(A) Graphical representation of OCR values in intact cells measured by respirometric assay: light green columns represent the OCRLEAK values, obtained after addition of the FoF1-ATP synthase inhibitor oligomycin to intact cells; dark green columns indicate the OCRATP values calculated as the difference between the overall endogenous OCR and the oligomycin-insensitive OCR. The values reported are mean (± SEM) of six independent experiments with the OCRs normalized to the protein content. Statistical significance: (*) P < 0.05, (**) P < 0.01 for OCRLEAK; (#), P < 0.05, (##) P < 0.01 for OCRATP. (B) Cellular content of NAD+ (empty columns), NADH (black columns) and of their ratio (grey columns) normalized to the protein content of each sample, calculated from three independent experiments. (*) P < 0.05 as total NAD content. (C) Flow cytometric analysis of ΔΨm in OSCC stained with the specific probe TMRE; 10,000 events for each sample were acquired and analyzed with the CellQuest software. (D) Measurement of lactate in culture medium; 2 × 106 cells were plated and, after 24 h of incubation, the lactate released were determined as indicated in Material and Methods and normalized to the cellular proteins. The data reported means (±SEM) of three independent experiments. (*) P < 0.05, (**) P < 0.01. (E) Analysis of the OxPhos/Glycolysis metabolic flux ratio calculated as the ratio between the OCRATP (see panel A) and the lactate amounts (see panel D). Statistical significance, (*) P < 0.05, (**) P < 0.005. (F) NADH dehydrogenase (CI) and cytochrome c oxidase (CIV) enzymatic activities measured spectrophotometrically as detailed in Materials and Methods; the results are means (± SEM) of three independent experiments, (*), P < 0.05. The inset shows the citrate synthase (CS) activity measured on the same samples. (G) Protein expression levels of the five OxPhos complexes (CI to CV), determined by immunoblot assay on total cell lysates using a cocktail of specific antibodies; β-actin was used as loading control. The blotting is representative of three independent experiments.
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Figure 1: Comparative analysis of the metabolic profiles of different OSSC-derived cell lines(A) Graphical representation of OCR values in intact cells measured by respirometric assay: light green columns represent the OCRLEAK values, obtained after addition of the FoF1-ATP synthase inhibitor oligomycin to intact cells; dark green columns indicate the OCRATP values calculated as the difference between the overall endogenous OCR and the oligomycin-insensitive OCR. The values reported are mean (± SEM) of six independent experiments with the OCRs normalized to the protein content. Statistical significance: (*) P < 0.05, (**) P < 0.01 for OCRLEAK; (#), P < 0.05, (##) P < 0.01 for OCRATP. (B) Cellular content of NAD+ (empty columns), NADH (black columns) and of their ratio (grey columns) normalized to the protein content of each sample, calculated from three independent experiments. (*) P < 0.05 as total NAD content. (C) Flow cytometric analysis of ΔΨm in OSCC stained with the specific probe TMRE; 10,000 events for each sample were acquired and analyzed with the CellQuest software. (D) Measurement of lactate in culture medium; 2 × 106 cells were plated and, after 24 h of incubation, the lactate released were determined as indicated in Material and Methods and normalized to the cellular proteins. The data reported means (±SEM) of three independent experiments. (*) P < 0.05, (**) P < 0.01. (E) Analysis of the OxPhos/Glycolysis metabolic flux ratio calculated as the ratio between the OCRATP (see panel A) and the lactate amounts (see panel D). Statistical significance, (*) P < 0.05, (**) P < 0.005. (F) NADH dehydrogenase (CI) and cytochrome c oxidase (CIV) enzymatic activities measured spectrophotometrically as detailed in Materials and Methods; the results are means (± SEM) of three independent experiments, (*), P < 0.05. The inset shows the citrate synthase (CS) activity measured on the same samples. (G) Protein expression levels of the five OxPhos complexes (CI to CV), determined by immunoblot assay on total cell lysates using a cocktail of specific antibodies; β-actin was used as loading control. The blotting is representative of three independent experiments.

Mentions: Figure 1A shows the results of a comparative analysis of the rates of endogenous respiration in intact OSCC cells performed by high-performance oxymetry. The oxygen consumption rates (OCRs) were normalized to the cellular protein content and corrected for the residual oxygen consumption after the addition of the Complex I inhibitor rotenone, and therefore attributable to the mitochondrial respiratory chain activity. The HSC-2 cell line displayed the lowest OCR when compared to the HSC-3 and PE15 cell lines. In the presence of the FoF1-ATP synthase inhibitor oligomycin, the endogenous OCR was depressed but at different extent in the three OSCC samples (lighter colored columns in Fig. 1A). The difference between the overall endogenous OCR and that in the presence of oligomycin (darker colored columns in Fig. 1A) provides a measure of the oxygen consumption linked to the oxidative phosphorylation (OxPhos) (OCRATP) and indicate a different OxPhos efficiency among the three OSCC cell lines in the sequence HSC-2<HSC-3<PE15. The oligomycin-insensitive OCR is a measure of the respiratory chain activity not linked to the synthesis of ATP and controlled by dissipative proton fluxes across the membrane also known as proton leaks (OCRLEAK). To notice, the residual OCRLEAK displayed a 2–3 fold larger value in HSC-2 as compared with HSC-3 and P15 and the lowest OCRATP.


Dichloroacetate, a selective mitochondria-targeting drug for oral squamous cell carcinoma: a metabolic perspective of treatment.

Ruggieri V, Agriesti F, Scrima R, Laurenzana I, Perrone D, Tataranni T, Mazzoccoli C, Lo Muzio L, Capitanio N, Piccoli C - Oncotarget (2015)

Comparative analysis of the metabolic profiles of different OSSC-derived cell lines(A) Graphical representation of OCR values in intact cells measured by respirometric assay: light green columns represent the OCRLEAK values, obtained after addition of the FoF1-ATP synthase inhibitor oligomycin to intact cells; dark green columns indicate the OCRATP values calculated as the difference between the overall endogenous OCR and the oligomycin-insensitive OCR. The values reported are mean (± SEM) of six independent experiments with the OCRs normalized to the protein content. Statistical significance: (*) P < 0.05, (**) P < 0.01 for OCRLEAK; (#), P < 0.05, (##) P < 0.01 for OCRATP. (B) Cellular content of NAD+ (empty columns), NADH (black columns) and of their ratio (grey columns) normalized to the protein content of each sample, calculated from three independent experiments. (*) P < 0.05 as total NAD content. (C) Flow cytometric analysis of ΔΨm in OSCC stained with the specific probe TMRE; 10,000 events for each sample were acquired and analyzed with the CellQuest software. (D) Measurement of lactate in culture medium; 2 × 106 cells were plated and, after 24 h of incubation, the lactate released were determined as indicated in Material and Methods and normalized to the cellular proteins. The data reported means (±SEM) of three independent experiments. (*) P < 0.05, (**) P < 0.01. (E) Analysis of the OxPhos/Glycolysis metabolic flux ratio calculated as the ratio between the OCRATP (see panel A) and the lactate amounts (see panel D). Statistical significance, (*) P < 0.05, (**) P < 0.005. (F) NADH dehydrogenase (CI) and cytochrome c oxidase (CIV) enzymatic activities measured spectrophotometrically as detailed in Materials and Methods; the results are means (± SEM) of three independent experiments, (*), P < 0.05. The inset shows the citrate synthase (CS) activity measured on the same samples. (G) Protein expression levels of the five OxPhos complexes (CI to CV), determined by immunoblot assay on total cell lysates using a cocktail of specific antibodies; β-actin was used as loading control. The blotting is representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359228&req=5

Figure 1: Comparative analysis of the metabolic profiles of different OSSC-derived cell lines(A) Graphical representation of OCR values in intact cells measured by respirometric assay: light green columns represent the OCRLEAK values, obtained after addition of the FoF1-ATP synthase inhibitor oligomycin to intact cells; dark green columns indicate the OCRATP values calculated as the difference between the overall endogenous OCR and the oligomycin-insensitive OCR. The values reported are mean (± SEM) of six independent experiments with the OCRs normalized to the protein content. Statistical significance: (*) P < 0.05, (**) P < 0.01 for OCRLEAK; (#), P < 0.05, (##) P < 0.01 for OCRATP. (B) Cellular content of NAD+ (empty columns), NADH (black columns) and of their ratio (grey columns) normalized to the protein content of each sample, calculated from three independent experiments. (*) P < 0.05 as total NAD content. (C) Flow cytometric analysis of ΔΨm in OSCC stained with the specific probe TMRE; 10,000 events for each sample were acquired and analyzed with the CellQuest software. (D) Measurement of lactate in culture medium; 2 × 106 cells were plated and, after 24 h of incubation, the lactate released were determined as indicated in Material and Methods and normalized to the cellular proteins. The data reported means (±SEM) of three independent experiments. (*) P < 0.05, (**) P < 0.01. (E) Analysis of the OxPhos/Glycolysis metabolic flux ratio calculated as the ratio between the OCRATP (see panel A) and the lactate amounts (see panel D). Statistical significance, (*) P < 0.05, (**) P < 0.005. (F) NADH dehydrogenase (CI) and cytochrome c oxidase (CIV) enzymatic activities measured spectrophotometrically as detailed in Materials and Methods; the results are means (± SEM) of three independent experiments, (*), P < 0.05. The inset shows the citrate synthase (CS) activity measured on the same samples. (G) Protein expression levels of the five OxPhos complexes (CI to CV), determined by immunoblot assay on total cell lysates using a cocktail of specific antibodies; β-actin was used as loading control. The blotting is representative of three independent experiments.
Mentions: Figure 1A shows the results of a comparative analysis of the rates of endogenous respiration in intact OSCC cells performed by high-performance oxymetry. The oxygen consumption rates (OCRs) were normalized to the cellular protein content and corrected for the residual oxygen consumption after the addition of the Complex I inhibitor rotenone, and therefore attributable to the mitochondrial respiratory chain activity. The HSC-2 cell line displayed the lowest OCR when compared to the HSC-3 and PE15 cell lines. In the presence of the FoF1-ATP synthase inhibitor oligomycin, the endogenous OCR was depressed but at different extent in the three OSCC samples (lighter colored columns in Fig. 1A). The difference between the overall endogenous OCR and that in the presence of oligomycin (darker colored columns in Fig. 1A) provides a measure of the oxygen consumption linked to the oxidative phosphorylation (OxPhos) (OCRATP) and indicate a different OxPhos efficiency among the three OSCC cell lines in the sequence HSC-2<HSC-3<PE15. The oligomycin-insensitive OCR is a measure of the respiratory chain activity not linked to the synthesis of ATP and controlled by dissipative proton fluxes across the membrane also known as proton leaks (OCRLEAK). To notice, the residual OCRLEAK displayed a 2–3 fold larger value in HSC-2 as compared with HSC-3 and P15 and the lowest OCRATP.

Bottom Line: In this study we tested comparatively the effects of DCA on three different OSCC-derived cell lines, HSC-2, HSC-3, PE15.DCA treatment of the three OSCC cell lines, at pharmacological concentrations, resulted in stimulation of the respiratory activity and caused a remarkably distinctive pro-apoptotic/cytostatic effect on HSC-2 and HSC-3.This was accompanied with a large remodeling of the mitochondrial network, never documented before, leading to organelle fragmentation and with enhanced production of reactive oxygen species.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pre-Clinical and Translational Research, IRCCS, CROB, Rionero in Vulture, Potenza, Italy.

ABSTRACT
Reprogramming of metabolism is a well-established property of cancer cells that is receiving growing attention as potential therapeutic target. Oral squamous cell carcinomas (OSCC) are aggressive and drugs-resistant human tumours displaying wide metabolic heterogeneity depending on their malignant genotype and stage of development. Dichloroacetate (DCA) is a specific inhibitor of the PDH-regulator PDK proved to foster mitochondrial oxidation of pyruvate. In this study we tested comparatively the effects of DCA on three different OSCC-derived cell lines, HSC-2, HSC-3, PE15. Characterization of the three cell lines unveiled for HSC-2 and HSC-3 a glycolysis-reliant metabolism whereas PE15 accomplished an efficient mitochondrial oxidative phosphorylation. DCA treatment of the three OSCC cell lines, at pharmacological concentrations, resulted in stimulation of the respiratory activity and caused a remarkably distinctive pro-apoptotic/cytostatic effect on HSC-2 and HSC-3. This was accompanied with a large remodeling of the mitochondrial network, never documented before, leading to organelle fragmentation and with enhanced production of reactive oxygen species. The data here presented indicate that the therapeutic efficacy of DCA may depend on the specific metabolic profile adopted by the cancer cells with those exhibiting a deficient mitochondrial oxidative phosphorylation resulting more sensitive to the drug treatment.

Show MeSH
Related in: MedlinePlus