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Cited by 8Acute Lymphoblastic Leukemia researchCancer, Hypoxia, and MetabolismBiochemical and Molecular Research

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Selective Inhibition of Deamidated Triosephosphate Isomerase by Disulfiram, Curcumin, and Sodium Dichloroacetate: Synergistic Therapeutic Strategies for T-Cell Acute Lymphoblastic Leukemia in Jurkat Cells

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Flores-López, Luis A., De la Mora-De la Mora, Ignacio, Malagón-Reyes, Claudia M., García-Torres, Itzhel, Martínez-Pérez, Yoalli, López-Herrera, Gabriela, Hernández-Alcántara, Gloria, León-Avila, Gloria, López-Velázquez, Gabriel, Olaya-Vargas, Alberto, Gómez-Manzo, Saúl, Enríquez-Flores, Sergio·2024·Biomolecules
DOI 10.3390/biom14101295PMC PMC11506356
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Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is a challenging childhood cancer to treat, with limited therapeutic options and high relapse rates. This study explores deamidated triosephosphate isomerase (dTPI) as a novel therapeutic target. We hypothesized that selectively inhibiting dTPI could reduce T-ALL cell viability without affecting normal T lymphocytes. Computational modeling and recombinant enzyme assays revealed that disulfiram (DS) and curcumin (CU) selectively bind and inhibit dTPI activity without affecting the non-deamidated enzyme. At the cellular level, treatment with DS and CU significantly reduced Jurkat T-ALL cell viability and endogenous TPI enzymatic activity, with no effect on normal T lymphocytes, whereas the combination of sodium dichloroacetate (DCA) with DS or CU showed synergistic effects. Furthermore, we demonstrated that dTPI was present and accumulated only in Jurkat cells, confirming our hypothesis. Finally, flow cytometry confirmed apoptosis in Jurkat cells after treatment with DS and CU or their combination with DCA. These findings strongly suggest that targeting dTPI represents a promising and selective target for T-ALL therapy.

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Figures

Figure 1 Molecular docking analysis of n-dTPI and dTPI crystallographic structures. Conformational representations of DS docked at the interface of n-dTPI (a) and dTPI (b) are shown. The proximity of Cys residues near the interface is highlighted, suggesting potential interaction sites for DS. Conformational representations of CU docked at the interface of n-dTPI (c) and dTPI (d) are shown; the hydrophobic surfaces of proteins are highlighted in red. A deeper penetration of both DS and CU into the interface of the deamidated enzyme (dTPI) compared to the non-deamidated form (n-dTPI) is observed. Figures modeled with PyMOL version 2.5.0 (Schrödinger Inc., New York, NY, USA).
Figure 1 Molecular docking analysis of n-dTPI and dTPI crystallographic structures. Conformational representations of DS docked at the interface of n-dTPI (a) and dTPI (b) are shown. The proximity of Cys residues near the interface is highlighted, suggesting potential interaction sites for DS. Conformational representations of CU docked at the interface of n-dTPI (c) and dTPI (d) are shown; the hydrophobic surfaces of proteins are highlighted in red. A deeper penetration of both DS and CU into the interface of the deamidated enzyme (dTPI) compared to the non-deamidated form (n-dTPI) is observed. Figures modeled with PyMOL version 2.5.0 (Schrödinger Inc., New York, NY, USA).CC BY · PubMed Central
Figure 2 Inactivation assays of recombinant n-dTPI and dTPI enzymes. Both enzymes (0.2 mg/mL) were incubated for 2 h at 37 °C with gradually increasing concentrations of the respective compounds: (a) DS (0 to 1000 μM) and (b) CU (0 to 1500 μM). Following incubation, aliquots were taken for enzyme activity measurement as described in the Methods section. The enzymatic activity was normalized, and 100% corresponds to the assays in the absence of the compound. Filled black squares represent n-dTPI activity, while filled red squares represent dTPI activity. The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments.
Figure 2 Inactivation assays of recombinant n-dTPI and dTPI enzymes. Both enzymes (0.2 mg/mL) were incubated for 2 h at 37 °C with gradually increasing concentrations of the respective compounds: (a) DS (0 to 1000 μM) and (b) CU (0 to 1500 μM). Following incubation, aliquots were taken for enzyme activity measurement as described in the Methods section. The enzymatic activity was normalized, and 100% corresponds to the assays in the absence of the compound. Filled black squares represent n-dTPI activity, while filled red squares represent dTPI activity. The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments.CC BY · PubMed Central
Figure 3 Extrinsic fluorescence spectra of TPIs after incubation with DS and CU. Enzymes (0.2 mg/mL) were incubated without or with 250 μM DS and 1500 μM CU for 2 h at 37 °C. Following incubation, excess compounds were removed, and extrinsic fluorescence was measured in the presence of 100 μM ANS with excitation at 395 nm. (a) shows the spectra in the absence of any compounds (control). (b,c) show the spectra after incubation with DS and CU, respectively. n-dTPI (black line) and dTPI (red line). The results represent the mean of three independent experiments.
Figure 3 Extrinsic fluorescence spectra of TPIs after incubation with DS and CU. Enzymes (0.2 mg/mL) were incubated without or with 250 μM DS and 1500 μM CU for 2 h at 37 °C. Following incubation, excess compounds were removed, and extrinsic fluorescence was measured in the presence of 100 μM ANS with excitation at 395 nm. (a) shows the spectra in the absence of any compounds (control). (b,c) show the spectra after incubation with DS and CU, respectively. n-dTPI (black line) and dTPI (red line). The results represent the mean of three independent experiments.CC BY · PubMed Central
Figure 4 Effects of DS and CU on cell viability and TPI activity in normal T-cell lymphocytes and Jurkat cells. Cells (1 × 105 per well) were incubated with increasing concentrations of DS and CU. Following incubation, cell viability was assessed using MTT assays and endogenous TPI activity was determined by enzymatic activity assays. (a,c) normal T lymphocytes, (b,d) Jurkat cells. Results are expressed as percentages relative to the untreated control set to 100%. The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments. Statistical differences were analyzed using one-way ANOVA with Tukey’s post-hoc test, with a significance level set at p = 0.01 (**).
Figure 4 Effects of DS and CU on cell viability and TPI activity in normal T-cell lymphocytes and Jurkat cells. Cells (1 × 105 per well) were incubated with increasing concentrations of DS and CU. Following incubation, cell viability was assessed using MTT assays and endogenous TPI activity was determined by enzymatic activity assays. (a,c) normal T lymphocytes, (b,d) Jurkat cells. Results are expressed as percentages relative to the untreated control set to 100%. The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments. Statistical differences were analyzed using one-way ANOVA with Tukey’s post-hoc test, with a significance level set at p = 0.01 (**).CC BY · PubMed Central
Figure 5 Effects of combined DCA and CU treatment on cell viability and TPI activity in normal T lymphocytes and Jurkat cells. Cells (1 × 105 per well) were pre-treated with 12 mM DCA for 24 h at 37 °C, followed by exposure to increasing concentrations of DS or CU for an additional 24 h. MTT and enzyme activity assays were then performed to assess cell viability and endogenous TPI activity, respectively. (a,c) normal T lymphocytes and (b,d) Jurkat cells. Results are expressed as percentages relative to the untreated control group (set to 100%). The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments. Statistical differences were analyzed using one-way ANOVA with Tukey’s post-hoc test, with a significance level set at p = 0.01 (**).
Figure 5 Effects of combined DCA and CU treatment on cell viability and TPI activity in normal T lymphocytes and Jurkat cells. Cells (1 × 105 per well) were pre-treated with 12 mM DCA for 24 h at 37 °C, followed by exposure to increasing concentrations of DS or CU for an additional 24 h. MTT and enzyme activity assays were then performed to assess cell viability and endogenous TPI activity, respectively. (a,c) normal T lymphocytes and (b,d) Jurkat cells. Results are expressed as percentages relative to the untreated control group (set to 100%). The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments. Statistical differences were analyzed using one-way ANOVA with Tukey’s post-hoc test, with a significance level set at p = 0.01 (**).CC BY · PubMed Central
Figure 6 Western blot analysis of TPI isoforms in normal T lymphocytes and Jurkat cells. In (a) and (b), lanes 1, 2, and 3, each loaded with 1 μg of protein, serve as migration standards for recombinant n-dTPI, dTPI, and ddTPI, respectively. Lanes 4–6 in (a) contain total protein extracts from normal T lymphocytes, while lanes 7–9 in (a) and lanes 4–10 in (b) correspond to protein extracts from Jurkat cells, with 100 μg of protein loaded per lane. For normal T lymphocytes, lane 4 is the control (untreated), lane 5 is treated with 250 μM DS, and lane 6 is treated with 1500 μM CU. In Jurkat cells, lanes 7, 8, and 9 in (a) represent the control (untreated), treatment with 250 μM DS, and treatment with 1500 μM CU, respectively. In (b), lane 4 is the control condition (untreated), while lanes 6 and 7 were treated with 100 and 250 μM DS, respectively. Finally, lanes 8, 9, and 10 were treated with 500, 1000, and 1500 μM of CU, respectively. The positive and negative poles of the gel are indicated on the left side of each panel. Full-length (uncropped) blots for panels (a,b) are presented in Supplementary Figure S5.
Figure 6 Western blot analysis of TPI isoforms in normal T lymphocytes and Jurkat cells. In (a) and (b), lanes 1, 2, and 3, each loaded with 1 μg of protein, serve as migration standards for recombinant n-dTPI, dTPI, and ddTPI, respectively. Lanes 4–6 in (a) contain total protein extracts from normal T lymphocytes, while lanes 7–9 in (a) and lanes 4–10 in (b) correspond to protein extracts from Jurkat cells, with 100 μg of protein loaded per lane. For normal T lymphocytes, lane 4 is the control (untreated), lane 5 is treated with 250 μM DS, and lane 6 is treated with 1500 μM CU. In Jurkat cells, lanes 7, 8, and 9 in (a) represent the control (untreated), treatment with 250 μM DS, and treatment with 1500 μM CU, respectively. In (b), lane 4 is the control condition (untreated), while lanes 6 and 7 were treated with 100 and 250 μM DS, respectively. Finally, lanes 8, 9, and 10 were treated with 500, 1000, and 1500 μM of CU, respectively. The positive and negative poles of the gel are indicated on the left side of each panel. Full-length (uncropped) blots for panels (a,b) are presented in Supplementary Figure S5.CC BY · PubMed Central
Figure 7 Western blot analysis of TPI isoforms in normal T-cell lymphocytes and Jurkat cells with combined DCA and CU treatment. In both panels, lanes 1–3 represent recombinant n-dTPI, dTPI, and ddTPI enzymes loaded at 1 μg protein per lane, serving as migration standards. In both panels, lanes 4–9 with 100 μg of protein loaded per lane. In a and b, lane 4 shows control Jurkat cells (untreated); lane 5 shows 12 mM DCA. In (a) lane 6 and 7 are Jurkat cells pretreated with 12 mM DCA followed by incubation with 100 and 250 μM DS, respectively. In (b) lane 6 and 7 are Jurkat cells pretreated with 12 mM DCA followed by incubation with 1000 μM and 1500 μM CU, respectively. Finally, in both, a and b, lanes 8–9 show normal T lymphocytes pretreated with 12 mM DCA followed by incubation with 250 μM DS (a) or 1500 μM CU (b). The polarity of the gel is indicated on the left side of the panel. Full-length blots (uncropped blots) are shown in Supplementary Figure S6.
Figure 7 Western blot analysis of TPI isoforms in normal T-cell lymphocytes and Jurkat cells with combined DCA and CU treatment. In both panels, lanes 1–3 represent recombinant n-dTPI, dTPI, and ddTPI enzymes loaded at 1 μg protein per lane, serving as migration standards. In both panels, lanes 4–9 with 100 μg of protein loaded per lane. In a and b, lane 4 shows control Jurkat cells (untreated); lane 5 shows 12 mM DCA. In (a) lane 6 and 7 are Jurkat cells pretreated with 12 mM DCA followed by incubation with 100 and 250 μM DS, respectively. In (b) lane 6 and 7 are Jurkat cells pretreated with 12 mM DCA followed by incubation with 1000 μM and 1500 μM CU, respectively. Finally, in both, a and b, lanes 8–9 show normal T lymphocytes pretreated with 12 mM DCA followed by incubation with 250 μM DS (a) or 1500 μM CU (b). The polarity of the gel is indicated on the left side of the panel. Full-length blots (uncropped blots) are shown in Supplementary Figure S6.CC BY · PubMed Central
Figure 8 MGO and AGEs levels in Jurkat cells following DCA, DS, and CU treatment. Quantification of MGO (a,c) and AGEs (b,d) in Jurkat cells following treatment with DCA, DS, CU, or their combination (*). As observed, increasing concentrations of DS and CU lead to a dose-dependent rise in both MGO and AGEs. Notably, pretreatment with DCA before DS or CU administration results in a further significant increase in MGO and AGEs production. The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments.
Figure 8 MGO and AGEs levels in Jurkat cells following DCA, DS, and CU treatment. Quantification of MGO (a,c) and AGEs (b,d) in Jurkat cells following treatment with DCA, DS, CU, or their combination (*). As observed, increasing concentrations of DS and CU lead to a dose-dependent rise in both MGO and AGEs. Notably, pretreatment with DCA before DS or CU administration results in a further significant increase in MGO and AGEs production. The results represent the average of three independent experiments, with error bars indicating the variation observed in the experiments.CC BY · PubMed Central
Figure 9 Western blot analysis of apoptosis-related proteins in Jurkat cells. (a) Expression of ERK1/2 and its phosphorylated form following treatment with DCA, CU, or their combination. The corresponding bar graphs quantify the decrease in total and phosphorylated ERK1/2 levels relative to the control. (b) Expression of Bcl-2 and Bax proteins under treatment with DCA, CU, or their combination. The bar graphs represent the relative expression levels of these proteins. (c) Procaspase-7 and cleaved caspase-7 levels following treatment with DCA, CU, or their combination. The bar graphs quantify the relative abundance of procaspase-7 and cleaved caspase-7 compared to the control. β-Actin was used as a loading control for all Western blots. Each lane was loaded with 100 μg of total protein extract. Statistical analysis was performed using a one-way ANOVA followed by the Tukey-Kramer test. Significance levels are indicated as follows: * p ≤ 0.01 compared to the control, ¥ p ≤ 0.01 compared to 12 mM DCA treatment, & p ≤ 0.01 compared to 0.5 mM CU treatment, $ p ≤ 0.01 compared to 12 mM DCA + 0.5 mM CU treatment. Full-length blots (uncropped blots) are shown in Supplementary Figure S7b–d for Total ERK 1/2, pERK, and β-Actin, respectively; Supplementary Figure S8b–d for Bcl2, Bax, and β-Actin, respectively, and Supplementary Figure S9b,c for Procaspase-7, cleaved caspase-7, and β-Actin, respectively.
Figure 9 Western blot analysis of apoptosis-related proteins in Jurkat cells. (a) Expression of ERK1/2 and its phosphorylated form following treatment with DCA, CU, or their combination. The corresponding bar graphs quantify the decrease in total and phosphorylated ERK1/2 levels relative to the control. (b) Expression of Bcl-2 and Bax proteins under treatment with DCA, CU, or their combination. The bar graphs represent the relative expression levels of these proteins. (c) Procaspase-7 and cleaved caspase-7 levels following treatment with DCA, CU, or their combination. The bar graphs quantify the relative abundance of procaspase-7 and cleaved caspase-7 compared to the control. β-Actin was used as a loading control for all Western blots. Each lane was loaded with 100 μg of total protein extract. Statistical analysis was performed using a one-way ANOVA followed by the Tukey-Kramer test. Significance levels are indicated as follows: * p ≤ 0.01 compared to the control, ¥ p ≤ 0.01 compared to 12 mM DCA treatment, & p ≤ 0.01 compared to 0.5 mM CU treatment, $ p ≤ 0.01 compared to 12 mM DCA + 0.5 mM CU treatment. Full-length blots (uncropped blots) are shown in Supplementary Figure S7b–d for Total ERK 1/2, pERK, and β-Actin, respectively; Supplementary Figure S8b–d for Bcl2, Bax, and β-Actin, respectively, and Supplementary Figure S9b,c for Procaspase-7, cleaved caspase-7, and β-Actin, respectively.CC BY · PubMed Central
Figure 10 Detection of cell death in normal T lymphocytes and Jurkat cells after treatment with DCA, DS, CU, or their combinations (DCA + DS or DCA + CU). Cells were incubated with 12 mM DCA, 100 µM DS, or 250 µM CU for 24 h at 37 °C. For combination treatments, cells were first exposed to 12 mM DCA for 24 h, followed by an additional 24-h incubation with 100 µM DS or 250 µM CU. At the end of the incubation period, cells were washed, resuspended at a density of 1 × 106/mL, and stained with Annexin V and Propidium Iodide, for flow cytometry analysis. (a) shows untreated control cells. (b–d) represent cells treated with DCA, DS, and CU, respectively. (e,f) display cells pretreated with DCA followed by DS and CU, respectively. Quadrants indicate distinct cell populations: Q1 (necrotic cells), Q2 (late apoptotic cells), and Q4 (early apoptotic cells). The data presented in the figure are representative of 100,000 cells analyzed across two independent experiments.
Figure 10 Detection of cell death in normal T lymphocytes and Jurkat cells after treatment with DCA, DS, CU, or their combinations (DCA + DS or DCA + CU). Cells were incubated with 12 mM DCA, 100 µM DS, or 250 µM CU for 24 h at 37 °C. For combination treatments, cells were first exposed to 12 mM DCA for 24 h, followed by an additional 24-h incubation with 100 µM DS or 250 µM CU. At the end of the incubation period, cells were washed, resuspended at a density of 1 × 106/mL, and stained with Annexin V and Propidium Iodide, for flow cytometry analysis. (a) shows untreated control cells. (b–d) represent cells treated with DCA, DS, and CU, respectively. (e,f) display cells pretreated with DCA followed by DS and CU, respectively. Quadrants indicate distinct cell populations: Q1 (necrotic cells), Q2 (late apoptotic cells), and Q4 (early apoptotic cells). The data presented in the figure are representative of 100,000 cells analyzed across two independent experiments.CC BY · PubMed Central

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Authors
Flores-López, L. A., De la Mora-De la Mora, I., Malagón-Reyes, C. M., García-Torres, I., Martínez-Pérez, Y., López-Herrera, G., Hernández-Alcántara, G., León-Avila, G., López-Velázquez, G., Olaya-Vargas, A., Gómez-Manzo, S., & Enríquez-Flores, S.
Affiliations
  • Instituto Nacional de Pediatria (MX)
  • Laboratorio de Biomoléculas y Salud Infantil, Instituto Nacional de Pediatría, Mexico City 04530, Mexico
  • Universidad Nacional Autónoma de México (MX)
  • Tecnológico de Monterrey (MX)
  • Laboratorio de Inmunodeficiencias, Instituto Nacional de Pediatría, Mexico City 04530, Mexico
  • Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Apartado Postal 70-159, Mexico City 04510, Mexico
  • Departamento de Zoología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala S/N, Casco de Santo Tomás, Ciudad de México 11340, Mexico
  • Trasplante de Células Madre y Terapia Celular, Instituto Nacional de Pediatría, Mexico City 04530, Mexico
  • Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Mexico City 04530, Mexico
Corresponding author
Flores-López, Luis A.
ORCID
Venue
Biomolecules
Publisher
MDPI AG
Published
2024-10-13
Volume
14
Issue
10
Pages
1295
ISSN
2218-273X (ISSN-L)
DOI
10.3390/biom14101295
PMC
PMC11506356
OpenAlex
W4403401779
Cited by
6
References
74
Citations by year
2026: 5 · 2025: 1
FWCI
2.49
Type
journal article
Language
en
Open access
Licence
Locations
  • Biomolecules (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT), Ciencia de Frontera 2023 (CF-2023-I-811, CF-2023-I-2755, 2019/072, 2022/067)
  • E022 Program, National Institute of Pediatrics, Mexico City, Mexico (Recursos Fiscales para la Investigación) (CF-2023-I-811, CF-2023-I-2755, 2019/072, 2022/067)
Topics
  • Health Sciences › Medicine › Hematology › Acute Lymphoblastic Leukemia research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
Keywords
T-cell acute lymphoblastic leukemia, disulfiram, curcumin, dichloroacetate, enzyme inhibition, synergistic drug combination, apoptosis, Jurkat cells, T cells
MeSH terms
Cell Survival, Curcumin, Dichloroacetic Acid, Disulfiram, Drug Synergism, Enzyme Inhibitors, Humans, Triose-Phosphate Isomerase, Apoptosis, Jurkat Cells, Precursor T-Cell Lymphoblastic Leukemia-Lymphoma

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