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The anti-leukemic activity of sodium dichloroacetate in p53mutated/ cells is mediated by a p53-independent ILF3/p21 pathway.

Agnoletto C, Brunelli L, Melloni E, Pastorelli R, Casciano F, Rimondi E, Rigolin GM, Cuneo A, Secchiero P, Zauli G - Oncotarget (2015)

Bottom Line: By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression.The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments.Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy.

ABSTRACT
B-chronic lymphocytic leukemia (B-CLL) patients harboring p53 mutations are invariably refractory to therapies based on purine analogues and have limited treatment options and poor survival. Having recently demonstrated that the mitochondria-targeting small molecule sodium dichloroacetate (DCA) exhibits anti-leukemic activity in p53wild-type B-CLL cells, the aim of this study was to evaluate the effect of DCA in p53mutated B-CLL cells and in p53mutated/ leukemic cell lines. DCA exhibited comparable cytotoxicity in p53wild-type and p53mutated B-CLL patient cell cultures, as well as in p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2). At the molecular level, DCA promoted the transcriptional induction of p21 in all leukemic cell types investigated, including p53 HL-60. By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression. The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments. Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity. Taken together, our results emphasize that DCA is a small molecule that merits further evaluation as a therapeutic agent also for p53mutated leukemic cells, by acting through the induction of a p53-independent pathway.

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Effects of DCA treatment on p53mutated/ leukemic cell linesThe p53mutated B leukemic cell lines MAVER, MEC-1 and MEC-2, as well as the p53 HL-60 cells, were exposed to DCA (30 mM). In A, cell distribution in the different phases of the cell cycle was calculated from the flow cytometry dot plots after BrdU/PI staining and expressed as percentage of the total population. In the upper panel, data are reported as the means of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In the lower panel, representative cell cycle profiles of cultures, either left untreated or treated with DCA, analyzed by flow cytometry are shown. For each cytofluorimetric analysis, the rectangles represent the cells in G0/G1, S, G2/M phases of the cell cycle. In B, cell senescence was visualized by SA-β-gal staining. Representative fields observed under light microscope are shown (arrows, senescent cells); cells with positive staining for SA-β-gal were quantified by counting a total of 1000 cells for each culture condition. Data are expressed as means±SD of six different fields in three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In C, assessment of mitochondrial alterations was carried out by morphological and functional analyses. Transmission electron microscopy images of cytosolic regions show mitochondria (asterisks; original magnification 31,500X) with either normal morphology or cristae remodeling, in untreated or DCA treated cultures, respectively. Representative fields of three independent experiments are shown. In the lower panel, cellular ATP levels were quantified in cell lysates at the indicated time points. Data are reported as percentage with respect to the untreated cultures (set to 100%) and are means±SD of three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisks, p<0.05). In D, the percentage of apoptotic cells was determined by flow cytometry after Annexin V/PI staining. Representative experiments are shown. In the graph, data are reported as the means±SD of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05).
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Figure 2: Effects of DCA treatment on p53mutated/ leukemic cell linesThe p53mutated B leukemic cell lines MAVER, MEC-1 and MEC-2, as well as the p53 HL-60 cells, were exposed to DCA (30 mM). In A, cell distribution in the different phases of the cell cycle was calculated from the flow cytometry dot plots after BrdU/PI staining and expressed as percentage of the total population. In the upper panel, data are reported as the means of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In the lower panel, representative cell cycle profiles of cultures, either left untreated or treated with DCA, analyzed by flow cytometry are shown. For each cytofluorimetric analysis, the rectangles represent the cells in G0/G1, S, G2/M phases of the cell cycle. In B, cell senescence was visualized by SA-β-gal staining. Representative fields observed under light microscope are shown (arrows, senescent cells); cells with positive staining for SA-β-gal were quantified by counting a total of 1000 cells for each culture condition. Data are expressed as means±SD of six different fields in three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In C, assessment of mitochondrial alterations was carried out by morphological and functional analyses. Transmission electron microscopy images of cytosolic regions show mitochondria (asterisks; original magnification 31,500X) with either normal morphology or cristae remodeling, in untreated or DCA treated cultures, respectively. Representative fields of three independent experiments are shown. In the lower panel, cellular ATP levels were quantified in cell lysates at the indicated time points. Data are reported as percentage with respect to the untreated cultures (set to 100%) and are means±SD of three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisks, p<0.05). In D, the percentage of apoptotic cells was determined by flow cytometry after Annexin V/PI staining. Representative experiments are shown. In the graph, data are reported as the means±SD of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05).

Mentions: Although we have previously shown that DCA activates the p53 pathway in p53wild-type B leukemic cells [15], the current set of data suggested that DCA can promote cytotoxicity also independently of functional p53. Thus, to investigate the molecular mechanisms underlining DCA cytotoxicity in leukemic cells with dysfunctional p53, we selected three p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2), which exhibited a dose- and time-dependent cytotoxic response to DCA (Figure 1A) with IC50 values comparable to those assessed for primary p53wild-type and p53mutated B-CLL cells (Table 2). The ability of DCA to promote p53-independent cytotoxicity in leukemic cells was further supported by experiments performed on the p53 HL-60 cell line (Figure 1B). The cytotoxicity induced by DCA in p53mutated/ leukemic cell lines was the result of (i) a cytostatic effect with the accumulation in G1 phase of the cell cycle (Figure 2A), (ii) the promotion of cellular senescence (Figure 2B), (iii) the induction of mitochondrial damage (Figure 2C), coupled to (iv) apoptosis (Figure 2D).


The anti-leukemic activity of sodium dichloroacetate in p53mutated/ cells is mediated by a p53-independent ILF3/p21 pathway.

Agnoletto C, Brunelli L, Melloni E, Pastorelli R, Casciano F, Rimondi E, Rigolin GM, Cuneo A, Secchiero P, Zauli G - Oncotarget (2015)

Effects of DCA treatment on p53mutated/ leukemic cell linesThe p53mutated B leukemic cell lines MAVER, MEC-1 and MEC-2, as well as the p53 HL-60 cells, were exposed to DCA (30 mM). In A, cell distribution in the different phases of the cell cycle was calculated from the flow cytometry dot plots after BrdU/PI staining and expressed as percentage of the total population. In the upper panel, data are reported as the means of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In the lower panel, representative cell cycle profiles of cultures, either left untreated or treated with DCA, analyzed by flow cytometry are shown. For each cytofluorimetric analysis, the rectangles represent the cells in G0/G1, S, G2/M phases of the cell cycle. In B, cell senescence was visualized by SA-β-gal staining. Representative fields observed under light microscope are shown (arrows, senescent cells); cells with positive staining for SA-β-gal were quantified by counting a total of 1000 cells for each culture condition. Data are expressed as means±SD of six different fields in three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In C, assessment of mitochondrial alterations was carried out by morphological and functional analyses. Transmission electron microscopy images of cytosolic regions show mitochondria (asterisks; original magnification 31,500X) with either normal morphology or cristae remodeling, in untreated or DCA treated cultures, respectively. Representative fields of three independent experiments are shown. In the lower panel, cellular ATP levels were quantified in cell lysates at the indicated time points. Data are reported as percentage with respect to the untreated cultures (set to 100%) and are means±SD of three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisks, p<0.05). In D, the percentage of apoptotic cells was determined by flow cytometry after Annexin V/PI staining. Representative experiments are shown. In the graph, data are reported as the means±SD of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05).
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Figure 2: Effects of DCA treatment on p53mutated/ leukemic cell linesThe p53mutated B leukemic cell lines MAVER, MEC-1 and MEC-2, as well as the p53 HL-60 cells, were exposed to DCA (30 mM). In A, cell distribution in the different phases of the cell cycle was calculated from the flow cytometry dot plots after BrdU/PI staining and expressed as percentage of the total population. In the upper panel, data are reported as the means of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In the lower panel, representative cell cycle profiles of cultures, either left untreated or treated with DCA, analyzed by flow cytometry are shown. For each cytofluorimetric analysis, the rectangles represent the cells in G0/G1, S, G2/M phases of the cell cycle. In B, cell senescence was visualized by SA-β-gal staining. Representative fields observed under light microscope are shown (arrows, senescent cells); cells with positive staining for SA-β-gal were quantified by counting a total of 1000 cells for each culture condition. Data are expressed as means±SD of six different fields in three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05). In C, assessment of mitochondrial alterations was carried out by morphological and functional analyses. Transmission electron microscopy images of cytosolic regions show mitochondria (asterisks; original magnification 31,500X) with either normal morphology or cristae remodeling, in untreated or DCA treated cultures, respectively. Representative fields of three independent experiments are shown. In the lower panel, cellular ATP levels were quantified in cell lysates at the indicated time points. Data are reported as percentage with respect to the untreated cultures (set to 100%) and are means±SD of three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisks, p<0.05). In D, the percentage of apoptotic cells was determined by flow cytometry after Annexin V/PI staining. Representative experiments are shown. In the graph, data are reported as the means±SD of results from at least three independent experiments (Mann-Whitney rank-sum test; asterisks, p<0.05).
Mentions: Although we have previously shown that DCA activates the p53 pathway in p53wild-type B leukemic cells [15], the current set of data suggested that DCA can promote cytotoxicity also independently of functional p53. Thus, to investigate the molecular mechanisms underlining DCA cytotoxicity in leukemic cells with dysfunctional p53, we selected three p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2), which exhibited a dose- and time-dependent cytotoxic response to DCA (Figure 1A) with IC50 values comparable to those assessed for primary p53wild-type and p53mutated B-CLL cells (Table 2). The ability of DCA to promote p53-independent cytotoxicity in leukemic cells was further supported by experiments performed on the p53 HL-60 cell line (Figure 1B). The cytotoxicity induced by DCA in p53mutated/ leukemic cell lines was the result of (i) a cytostatic effect with the accumulation in G1 phase of the cell cycle (Figure 2A), (ii) the promotion of cellular senescence (Figure 2B), (iii) the induction of mitochondrial damage (Figure 2C), coupled to (iv) apoptosis (Figure 2D).

Bottom Line: By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression.The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments.Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, University of Ferrara, Ferrara, Italy.

ABSTRACT
B-chronic lymphocytic leukemia (B-CLL) patients harboring p53 mutations are invariably refractory to therapies based on purine analogues and have limited treatment options and poor survival. Having recently demonstrated that the mitochondria-targeting small molecule sodium dichloroacetate (DCA) exhibits anti-leukemic activity in p53wild-type B-CLL cells, the aim of this study was to evaluate the effect of DCA in p53mutated B-CLL cells and in p53mutated/ leukemic cell lines. DCA exhibited comparable cytotoxicity in p53wild-type and p53mutated B-CLL patient cell cultures, as well as in p53mutated B leukemic cell lines (MAVER, MEC-1, MEC-2). At the molecular level, DCA promoted the transcriptional induction of p21 in all leukemic cell types investigated, including p53 HL-60. By using a proteomic approach, we demonstrated that DCA up-regulated the ILF3 transcription factor, which is a known regulator of p21 expression. The role of the ILF3/p21 axis in mediating the DCA anti-leukemic activity was underscored by knocking-down experiments. Indeed, transfection with ILF3 and p21 siRNAs significantly decreased both the DCA-induced p21 expression and the DCA-mediated cytotoxicity. Taken together, our results emphasize that DCA is a small molecule that merits further evaluation as a therapeutic agent also for p53mutated leukemic cells, by acting through the induction of a p53-independent pathway.

Show MeSH
Related in: MedlinePlus