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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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Effect of DCA on phosphorylation of the PDH-E1α subunit and cell growth and viability in OSCC(A) Western blot of the phospho-S293 E1α PDH and of the total enzyme subunit in OSCC cells following a 24 h incubation with vehicle and 10 mM DCA; GAPDH levels were used as loading control. The blot shown is representative of three independent experiments. Panel on the right: quantitative analysis of phospho-S293 E1 α PDH expression relative to total PDH E1α normalized to GAPDH, carried out by a densitometric analysis (“Image Lab” software, Biorad). The results are the means (± SEM) of three independent experiments. (*) P < 0.05. (B) Dose-dependent effect of DCA treatment on cell growth analysed by using an impedentiometric technique (xCELLigence RTCA MP System, Roche, Germany). Continuous monitoring of cell adhesion and proliferation was carried out for 24 h after incubation of cells with the indicated concentrations of DCA, and expressed as the percentage (%) of the cell index of untreated cells; means (± SEM) of three repeats. (*): P < 0.05. (C) Dose-dependence effect of DCA treatment on cell viability. Cells were exposed for 24 h to the indicated concentrations of DCA and viability determined with the MTS assay. Non-malignant human HEK 293 cells were used as representative of a non tumor cell line. Cell viability is expressed as the percentage (%) of untreated cells. The data shown are means (± SEM) of 6 independent experiments; (*) P < 0.05; (**) P < 0.01.
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Figure 2: Effect of DCA on phosphorylation of the PDH-E1α subunit and cell growth and viability in OSCC(A) Western blot of the phospho-S293 E1α PDH and of the total enzyme subunit in OSCC cells following a 24 h incubation with vehicle and 10 mM DCA; GAPDH levels were used as loading control. The blot shown is representative of three independent experiments. Panel on the right: quantitative analysis of phospho-S293 E1 α PDH expression relative to total PDH E1α normalized to GAPDH, carried out by a densitometric analysis (“Image Lab” software, Biorad). The results are the means (± SEM) of three independent experiments. (*) P < 0.05. (B) Dose-dependent effect of DCA treatment on cell growth analysed by using an impedentiometric technique (xCELLigence RTCA MP System, Roche, Germany). Continuous monitoring of cell adhesion and proliferation was carried out for 24 h after incubation of cells with the indicated concentrations of DCA, and expressed as the percentage (%) of the cell index of untreated cells; means (± SEM) of three repeats. (*): P < 0.05. (C) Dose-dependence effect of DCA treatment on cell viability. Cells were exposed for 24 h to the indicated concentrations of DCA and viability determined with the MTS assay. Non-malignant human HEK 293 cells were used as representative of a non tumor cell line. Cell viability is expressed as the percentage (%) of untreated cells. The data shown are means (± SEM) of 6 independent experiments; (*) P < 0.05; (**) P < 0.01.

Mentions: Before evaluating the effect of DCA on vital parameters of the OSCC cell lines, the efficacy of the compound to inhibit its recognized target pyruvate dehydrogenase kinase (PDK) was assessed by Western blotting using a specifici antibody recognizing the phosphorylation state of the E1α subunit (residue S293) of the PDH complex [23]. Figure 2A shows that the basal level of both PDH and P-PDH-E1 were comparable among the three OSCC cell lines and that following a 24 h-treatment with 10 mM DCA (an effective concentration reported in literature [13–17]) a significant reduction in the normalized level of P-PDH-E1 was observed with this value resulting slightly lower in PE15 as compared with HSC-2 and HSC-3 (45–50% vs. 20% inhibition).


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 phosphorylation of the PDH-E1α subunit and cell growth and viability in OSCC(A) Western blot of the phospho-S293 E1α PDH and of the total enzyme subunit in OSCC cells following a 24 h incubation with vehicle and 10 mM DCA; GAPDH levels were used as loading control. The blot shown is representative of three independent experiments. Panel on the right: quantitative analysis of phospho-S293 E1 α PDH expression relative to total PDH E1α normalized to GAPDH, carried out by a densitometric analysis (“Image Lab” software, Biorad). The results are the means (± SEM) of three independent experiments. (*) P < 0.05. (B) Dose-dependent effect of DCA treatment on cell growth analysed by using an impedentiometric technique (xCELLigence RTCA MP System, Roche, Germany). Continuous monitoring of cell adhesion and proliferation was carried out for 24 h after incubation of cells with the indicated concentrations of DCA, and expressed as the percentage (%) of the cell index of untreated cells; means (± SEM) of three repeats. (*): P < 0.05. (C) Dose-dependence effect of DCA treatment on cell viability. Cells were exposed for 24 h to the indicated concentrations of DCA and viability determined with the MTS assay. Non-malignant human HEK 293 cells were used as representative of a non tumor cell line. Cell viability is expressed as the percentage (%) of untreated cells. The data shown are means (± SEM) of 6 independent experiments; (*) P < 0.05; (**) P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Effect of DCA on phosphorylation of the PDH-E1α subunit and cell growth and viability in OSCC(A) Western blot of the phospho-S293 E1α PDH and of the total enzyme subunit in OSCC cells following a 24 h incubation with vehicle and 10 mM DCA; GAPDH levels were used as loading control. The blot shown is representative of three independent experiments. Panel on the right: quantitative analysis of phospho-S293 E1 α PDH expression relative to total PDH E1α normalized to GAPDH, carried out by a densitometric analysis (“Image Lab” software, Biorad). The results are the means (± SEM) of three independent experiments. (*) P < 0.05. (B) Dose-dependent effect of DCA treatment on cell growth analysed by using an impedentiometric technique (xCELLigence RTCA MP System, Roche, Germany). Continuous monitoring of cell adhesion and proliferation was carried out for 24 h after incubation of cells with the indicated concentrations of DCA, and expressed as the percentage (%) of the cell index of untreated cells; means (± SEM) of three repeats. (*): P < 0.05. (C) Dose-dependence effect of DCA treatment on cell viability. Cells were exposed for 24 h to the indicated concentrations of DCA and viability determined with the MTS assay. Non-malignant human HEK 293 cells were used as representative of a non tumor cell line. Cell viability is expressed as the percentage (%) of untreated cells. The data shown are means (± SEM) of 6 independent experiments; (*) P < 0.05; (**) P < 0.01.
Mentions: Before evaluating the effect of DCA on vital parameters of the OSCC cell lines, the efficacy of the compound to inhibit its recognized target pyruvate dehydrogenase kinase (PDK) was assessed by Western blotting using a specifici antibody recognizing the phosphorylation state of the E1α subunit (residue S293) of the PDH complex [23]. Figure 2A shows that the basal level of both PDH and P-PDH-E1 were comparable among the three OSCC cell lines and that following a 24 h-treatment with 10 mM DCA (an effective concentration reported in literature [13–17]) a significant reduction in the normalized level of P-PDH-E1 was observed with this value resulting slightly lower in PE15 as compared with HSC-2 and HSC-3 (45–50% vs. 20% inhibition).

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