Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of DCA on intracellular ROS generation and apoptosis(A) Cellular ROS production assayed by flow-cytometry fluorescent probe DCF (upper panel). Cells were incubated for 24 h with 10 mM DCA ± 10 mM of the ROS scavenger NAC added 4 h before the analysis. The bar histogram on the right shows the mean intensity of the DCF-related fluorescence (MFI) expressed as fold-change of the untreated cells and are means ± SEM of three independent experiments. (*) P < 0.05; (#) < 0.05 vs DCA-untreated. (B) Representative LSCM imaging of ROS production in living OSCC cells treated with DCA as in panel (A) and assessed by DCF. Magnification of selected areas (indicated by the white frame) in DCA-treated cells are shown at the top of each panel. The images are representative of three different preparations yielding similar results. The histogram below the images shows the quantitative analysis of the DCF-related fluorescence/cell; the values are means ± SEM of three independent experiments under each condition wherein the digitalized fluorescence images from at least five randomly selected optical fields (each containing about 25 cells) were analyzed. (C) Measurement of apoptotic and necrotic cells performed by flow-cytometry after staining cells with annexin-V and propidium iodide. Cells were incubated with 20 mM DCA alone or co-incubated with DCA and 10 mM NAC for 48 h. Data, expressed as percentage of total events analysed, are the means ± SEM of three independent experiments. (*) P < 0.05. (D) Protein expression levels of the anti-apoptotic factors, Bcl-xL and survivin, assayed by Western blotting in untreated and 10 mM DCA-treated cells for 24 h (left panel); β-actin served as loading control. Graph bars on the right show the average (± SEM) of data resulting from densitometric analysis of three independent blots. (*) P < 0.05, (**) P < 0.01 vs DCA-untreated; (#) P < 0.001 vs HSC-2 and HSC-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359228&req=5

Figure 5: Effect of DCA on intracellular ROS generation and apoptosis(A) Cellular ROS production assayed by flow-cytometry fluorescent probe DCF (upper panel). Cells were incubated for 24 h with 10 mM DCA ± 10 mM of the ROS scavenger NAC added 4 h before the analysis. The bar histogram on the right shows the mean intensity of the DCF-related fluorescence (MFI) expressed as fold-change of the untreated cells and are means ± SEM of three independent experiments. (*) P < 0.05; (#) < 0.05 vs DCA-untreated. (B) Representative LSCM imaging of ROS production in living OSCC cells treated with DCA as in panel (A) and assessed by DCF. Magnification of selected areas (indicated by the white frame) in DCA-treated cells are shown at the top of each panel. The images are representative of three different preparations yielding similar results. The histogram below the images shows the quantitative analysis of the DCF-related fluorescence/cell; the values are means ± SEM of three independent experiments under each condition wherein the digitalized fluorescence images from at least five randomly selected optical fields (each containing about 25 cells) were analyzed. (C) Measurement of apoptotic and necrotic cells performed by flow-cytometry after staining cells with annexin-V and propidium iodide. Cells were incubated with 20 mM DCA alone or co-incubated with DCA and 10 mM NAC for 48 h. Data, expressed as percentage of total events analysed, are the means ± SEM of three independent experiments. (*) P < 0.05. (D) Protein expression levels of the anti-apoptotic factors, Bcl-xL and survivin, assayed by Western blotting in untreated and 10 mM DCA-treated cells for 24 h (left panel); β-actin served as loading control. Graph bars on the right show the average (± SEM) of data resulting from densitometric analysis of three independent blots. (*) P < 0.05, (**) P < 0.01 vs DCA-untreated; (#) P < 0.001 vs HSC-2 and HSC-3.

Mentions: Data from the literature suggest that the cytotoxic effect of DCA on tumor cells might be caused by unbalance of the cellular redox homeostasis [25]. To verify this possibility we assessed the intracellular reactive oxygen species (ROS) with the peroxide-oxidizable fluorescent probe DCF by flow-cytometry. The results presented in Fig. 5A show that 10 mM DCA treatment for 24 h caused a N-acetyl cysteine (NAC)-sensitive increase of the mean fluorescence intensity in HSC-2 and HSC-3 cell lines (2–3 fold change as compared with the basal ROS level). Conversely, DCA-treatment of the PE15 cell line resulted in a less significant change of the DCF-related fluorescence. These observations were confirmed at the single cell level by confocal microscopy imaging using the same probe (Fig. 5B).


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)

Effect of DCA on intracellular ROS generation and apoptosis(A) Cellular ROS production assayed by flow-cytometry fluorescent probe DCF (upper panel). Cells were incubated for 24 h with 10 mM DCA ± 10 mM of the ROS scavenger NAC added 4 h before the analysis. The bar histogram on the right shows the mean intensity of the DCF-related fluorescence (MFI) expressed as fold-change of the untreated cells and are means ± SEM of three independent experiments. (*) P < 0.05; (#) < 0.05 vs DCA-untreated. (B) Representative LSCM imaging of ROS production in living OSCC cells treated with DCA as in panel (A) and assessed by DCF. Magnification of selected areas (indicated by the white frame) in DCA-treated cells are shown at the top of each panel. The images are representative of three different preparations yielding similar results. The histogram below the images shows the quantitative analysis of the DCF-related fluorescence/cell; the values are means ± SEM of three independent experiments under each condition wherein the digitalized fluorescence images from at least five randomly selected optical fields (each containing about 25 cells) were analyzed. (C) Measurement of apoptotic and necrotic cells performed by flow-cytometry after staining cells with annexin-V and propidium iodide. Cells were incubated with 20 mM DCA alone or co-incubated with DCA and 10 mM NAC for 48 h. Data, expressed as percentage of total events analysed, are the means ± SEM of three independent experiments. (*) P < 0.05. (D) Protein expression levels of the anti-apoptotic factors, Bcl-xL and survivin, assayed by Western blotting in untreated and 10 mM DCA-treated cells for 24 h (left panel); β-actin served as loading control. Graph bars on the right show the average (± SEM) of data resulting from densitometric analysis of three independent blots. (*) P < 0.05, (**) P < 0.01 vs DCA-untreated; (#) P < 0.001 vs HSC-2 and HSC-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of DCA on intracellular ROS generation and apoptosis(A) Cellular ROS production assayed by flow-cytometry fluorescent probe DCF (upper panel). Cells were incubated for 24 h with 10 mM DCA ± 10 mM of the ROS scavenger NAC added 4 h before the analysis. The bar histogram on the right shows the mean intensity of the DCF-related fluorescence (MFI) expressed as fold-change of the untreated cells and are means ± SEM of three independent experiments. (*) P < 0.05; (#) < 0.05 vs DCA-untreated. (B) Representative LSCM imaging of ROS production in living OSCC cells treated with DCA as in panel (A) and assessed by DCF. Magnification of selected areas (indicated by the white frame) in DCA-treated cells are shown at the top of each panel. The images are representative of three different preparations yielding similar results. The histogram below the images shows the quantitative analysis of the DCF-related fluorescence/cell; the values are means ± SEM of three independent experiments under each condition wherein the digitalized fluorescence images from at least five randomly selected optical fields (each containing about 25 cells) were analyzed. (C) Measurement of apoptotic and necrotic cells performed by flow-cytometry after staining cells with annexin-V and propidium iodide. Cells were incubated with 20 mM DCA alone or co-incubated with DCA and 10 mM NAC for 48 h. Data, expressed as percentage of total events analysed, are the means ± SEM of three independent experiments. (*) P < 0.05. (D) Protein expression levels of the anti-apoptotic factors, Bcl-xL and survivin, assayed by Western blotting in untreated and 10 mM DCA-treated cells for 24 h (left panel); β-actin served as loading control. Graph bars on the right show the average (± SEM) of data resulting from densitometric analysis of three independent blots. (*) P < 0.05, (**) P < 0.01 vs DCA-untreated; (#) P < 0.001 vs HSC-2 and HSC-3.
Mentions: Data from the literature suggest that the cytotoxic effect of DCA on tumor cells might be caused by unbalance of the cellular redox homeostasis [25]. To verify this possibility we assessed the intracellular reactive oxygen species (ROS) with the peroxide-oxidizable fluorescent probe DCF by flow-cytometry. The results presented in Fig. 5A show that 10 mM DCA treatment for 24 h caused a N-acetyl cysteine (NAC)-sensitive increase of the mean fluorescence intensity in HSC-2 and HSC-3 cell lines (2–3 fold change as compared with the basal ROS level). Conversely, DCA-treatment of the PE15 cell line resulted in a less significant change of the DCF-related fluorescence. These observations were confirmed at the single cell level by confocal microscopy imaging using the same probe (Fig. 5B).

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