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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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Role of p21 and ILF3 pathway in mediating the anti-leukemic activity of DCA in p53 leukemic cellsIn A-B, proteomic profiling in HL-60 cells upon DCA treatment revealed significant different (at least 3 folds) levels for 17 proteins in DCA treated cultures compared to the untreated cells (Supplementary Table 2). In A, the top ten GO processes, prioritized in according to p-value, in response to DCA treatment is shown. In B, the protein network generated by the shortest path algorithm using the list of the differently expressed proteins (more than 3 fold induction/reduction) is reported. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles evidence protein identified in our proteomic analysis. Lines connecting the nodes indicate indirect interactions (activation, induction, modification) or direct binding; the arrowheads indicate the direction of the interaction. Blue broken line circles highlight network hubs as indicated by network statistics. In C-D, HL-60 cells were transfected with either control scrambled (scr) siRNA, or p21 siRNA (C) or ILF3 siRNA (D) before treatment with DCA (30 mM). After transfection, levels of p21 mRNA were analyzed by quantitative RT-PCR (C-D). Data are expressed as arbitrary units (a.u.). In parallel transfected cultures were analyzed for cell viability upon exposure to DCA; results are expressed as percentage of viable cells with respect to the control cultures (set to 100%). Data are reported as means±SD of results from three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisk, p<0.05). In E, a schematic representation of the potential p53-independent pathway mediating the anti-leukemic activity of DCA is shown.
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Figure 4: Role of p21 and ILF3 pathway in mediating the anti-leukemic activity of DCA in p53 leukemic cellsIn A-B, proteomic profiling in HL-60 cells upon DCA treatment revealed significant different (at least 3 folds) levels for 17 proteins in DCA treated cultures compared to the untreated cells (Supplementary Table 2). In A, the top ten GO processes, prioritized in according to p-value, in response to DCA treatment is shown. In B, the protein network generated by the shortest path algorithm using the list of the differently expressed proteins (more than 3 fold induction/reduction) is reported. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles evidence protein identified in our proteomic analysis. Lines connecting the nodes indicate indirect interactions (activation, induction, modification) or direct binding; the arrowheads indicate the direction of the interaction. Blue broken line circles highlight network hubs as indicated by network statistics. In C-D, HL-60 cells were transfected with either control scrambled (scr) siRNA, or p21 siRNA (C) or ILF3 siRNA (D) before treatment with DCA (30 mM). After transfection, levels of p21 mRNA were analyzed by quantitative RT-PCR (C-D). Data are expressed as arbitrary units (a.u.). In parallel transfected cultures were analyzed for cell viability upon exposure to DCA; results are expressed as percentage of viable cells with respect to the control cultures (set to 100%). Data are reported as means±SD of results from three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisk, p<0.05). In E, a schematic representation of the potential p53-independent pathway mediating the anti-leukemic activity of DCA is shown.

Mentions: To gain insight into the p53-independent induction of p21 by DCA, we adopted a proteomic approach using the HL-60 p53 leukemic model in order to rule out any potential interference by mutated p53 protein. For this purpose, we used a 1-DE gel approach for protein pre-fractionation integrated into a typical LC-MS/MS workflow for protein identification, performed as previously described [22]. A label-free approach (spectral counting) was then used for relative protein abundance quantification. Overall, our proteomic analysis identified 727 proteins in the total lysate of untreated and DCA treated HL-60 cells. According to statistical tests, HL-60 exposed to DCA for 24 hours exhibited a significant difference (at least 3-fold) in the abundance of 17 proteins compared to the untreated counterpart, with 10 up-regulated proteins and 7 down-regulated proteins (Supplementary Tables 1 and 2). Network analysis, using MetaCore bioinformatic tools of the 17 significantly deregulated proteins, revealed the involvement of Cellular metabolic processes as the main GO processes altered by DCA treatment (Figure 4A). In addition, when MetaCore software was used to map the shortest paths of interactions among the differentially expressed proteins, 12 out of 17 proteins were brought together with p21 as the hub of the identified network (Figure 4B). Interestingly p21 was directly connected with interleukin enhancer-binding factor 3 (ILF3, also known as NFAT-90).


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)

Role of p21 and ILF3 pathway in mediating the anti-leukemic activity of DCA in p53 leukemic cellsIn A-B, proteomic profiling in HL-60 cells upon DCA treatment revealed significant different (at least 3 folds) levels for 17 proteins in DCA treated cultures compared to the untreated cells (Supplementary Table 2). In A, the top ten GO processes, prioritized in according to p-value, in response to DCA treatment is shown. In B, the protein network generated by the shortest path algorithm using the list of the differently expressed proteins (more than 3 fold induction/reduction) is reported. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles evidence protein identified in our proteomic analysis. Lines connecting the nodes indicate indirect interactions (activation, induction, modification) or direct binding; the arrowheads indicate the direction of the interaction. Blue broken line circles highlight network hubs as indicated by network statistics. In C-D, HL-60 cells were transfected with either control scrambled (scr) siRNA, or p21 siRNA (C) or ILF3 siRNA (D) before treatment with DCA (30 mM). After transfection, levels of p21 mRNA were analyzed by quantitative RT-PCR (C-D). Data are expressed as arbitrary units (a.u.). In parallel transfected cultures were analyzed for cell viability upon exposure to DCA; results are expressed as percentage of viable cells with respect to the control cultures (set to 100%). Data are reported as means±SD of results from three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisk, p<0.05). In E, a schematic representation of the potential p53-independent pathway mediating the anti-leukemic activity of DCA is shown.
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Figure 4: Role of p21 and ILF3 pathway in mediating the anti-leukemic activity of DCA in p53 leukemic cellsIn A-B, proteomic profiling in HL-60 cells upon DCA treatment revealed significant different (at least 3 folds) levels for 17 proteins in DCA treated cultures compared to the untreated cells (Supplementary Table 2). In A, the top ten GO processes, prioritized in according to p-value, in response to DCA treatment is shown. In B, the protein network generated by the shortest path algorithm using the list of the differently expressed proteins (more than 3 fold induction/reduction) is reported. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles evidence protein identified in our proteomic analysis. Lines connecting the nodes indicate indirect interactions (activation, induction, modification) or direct binding; the arrowheads indicate the direction of the interaction. Blue broken line circles highlight network hubs as indicated by network statistics. In C-D, HL-60 cells were transfected with either control scrambled (scr) siRNA, or p21 siRNA (C) or ILF3 siRNA (D) before treatment with DCA (30 mM). After transfection, levels of p21 mRNA were analyzed by quantitative RT-PCR (C-D). Data are expressed as arbitrary units (a.u.). In parallel transfected cultures were analyzed for cell viability upon exposure to DCA; results are expressed as percentage of viable cells with respect to the control cultures (set to 100%). Data are reported as means±SD of results from three independent experiments, each performed in duplicate (Mann-Whitney rank-sum test; asterisk, p<0.05). In E, a schematic representation of the potential p53-independent pathway mediating the anti-leukemic activity of DCA is shown.
Mentions: To gain insight into the p53-independent induction of p21 by DCA, we adopted a proteomic approach using the HL-60 p53 leukemic model in order to rule out any potential interference by mutated p53 protein. For this purpose, we used a 1-DE gel approach for protein pre-fractionation integrated into a typical LC-MS/MS workflow for protein identification, performed as previously described [22]. A label-free approach (spectral counting) was then used for relative protein abundance quantification. Overall, our proteomic analysis identified 727 proteins in the total lysate of untreated and DCA treated HL-60 cells. According to statistical tests, HL-60 exposed to DCA for 24 hours exhibited a significant difference (at least 3-fold) in the abundance of 17 proteins compared to the untreated counterpart, with 10 up-regulated proteins and 7 down-regulated proteins (Supplementary Tables 1 and 2). Network analysis, using MetaCore bioinformatic tools of the 17 significantly deregulated proteins, revealed the involvement of Cellular metabolic processes as the main GO processes altered by DCA treatment (Figure 4A). In addition, when MetaCore software was used to map the shortest paths of interactions among the differentially expressed proteins, 12 out of 17 proteins were brought together with p21 as the hub of the identified network (Figure 4B). Interestingly p21 was directly connected with interleukin enhancer-binding factor 3 (ILF3, also known as NFAT-90).

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