Targeting the p53/xCT/GSH Axis with PRIMA-1Met Combined with Sulfasalazine Shows Therapeutic Potential in Chronic Lymphocytic Leukemia

Abstract

In Chronic Lymphocytic Leukemia (CLL), mutations at the TP53 tumor suppressor gene are an important hallmark since they may strongly influence the therapeutic decision. PRIMA-1^Met (also known as APR-246/Eprenetapopt) is a small molecule able to restore the wild-type (wt) p53 conformation to mutant p53 proteins and to stimulate apoptosis in tumor cells; in addition, it can deplete the glutathione reservoir, increasing reactive oxygen species (ROS) production. In this study, we investigated whether combining PRIMA-1^Met with Sulfasalazine (SAS), a SLC7A11/xCT inhibitor, reduces CLL cell viability by targeting mutant p53 and the glutathione pathway. The results demonstrated that, in CLL cells, PRIMA-1^Met did not restore the wt functions in the mutant p53 proteins, but it strongly reduced the antioxidant defense and induced cell death. PRIMA-1^Met and SAS combination synergistically reduced cell survival regardless of p53 status and further impaired antioxidant capacity, especially in mutant p53 cells, linking their cytotoxic effect to redox imbalance. Thus, the association of PRIMA-1^Met with drugs targeting the antioxidant response could represent a valid strategy to kill CLL cells carrying either wt or mutant p53.

Keywords: GSH; P53; PRIMA-1Met; SLC7A11/xCT; antioxidant defenses; oxidative stress.

Figure 1

Protein levels of p53, SLC7A11/xCT, and Bcl-2 proteins in OSU and MEC-1 untreated cells. (A) Western blots are representative of three independent experiments (Supplementary Figure S7). β-actin (β-ACT) is used for loading control; (B) Histogram showing the mean + SEM of p53, xCT, and Bcl-2 protein levels normalized for β-actin from three independent experiments (for original blot, see Supplementary Figure S7).

Figure 2

Apoptosis induction in OSU and MEC-1CLL cell lines. Annexin V-FITC/PI assay was performed in both cell lines treated with 5 μM and 10 μM PRIMA-1^Met (P) alone or in combination with 300 μM Sulfasalazine (S). (A) The histograms show the means + SD of the percentage of Annexin/PI double-positive cells detected by cytofluorimetric assay of three independent experiments. The complete statistical analysis is reported in Supplementary Material (Table S1); (B) Representative Western blots showing the levels of Bcl-2 protein in OSU and MEC-1 cells treated for 24 h with 5 μM or 10 μM PRIMA-1^Met alone or in combination with 300 μM Sulfasalazine (S). β-actin (β-ACT) was used as loading control (for original blots, see Supplementary Figure S8); (C) The histograms showed the means ± SEM of Bcl-2 chemiluminescence, normalized for β-actin, calculated as fold over the levels determined in untreated cells, of at least three independent experiments; ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Cell viability of OSU and MEC-1 cells treated with PRIMA-1^Met alone and in combination with SAS. Cell viability after 24 h and 48 h of PRIMA-1^Met (P) and/or Sulfasalazine (S) treatments in OSU (A) and MEC-1 (B) cell lines. Cell viability was analyzed by MTT assays in cells exposed for 24 h and 48 h to increasing concentrations of PRIMA-1^Met (5, 10, 20 μM) alone or in combination with Sulfasalazine (300 μM). The histograms report the means ± SD of at least three independent experiments. D values, calculated after 48 h of treatment, were obtained for the combinations of PRIMA-1^Met and SAS on OSU (C) and MEC-1 cells (D). PRIMA-1^Met concentrations were: 40 (●), 20 (○), 10 (■), and 5 μM (□). SAS concentrations were: 1200, 600, 300, and 150 μM. The experimental D value for additivity was calculated using combinations of two serial dilutions of the tested compounds. Mean ± SD: 1.01 ± 0.32 (n = 36). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

PRIMA-1^Met and SAS reduce the viability of wt and mutant p53 CLL cells. Wt p53 (A) and mutant p53 (B) carrying CLL cells were stimulated with CpG-ODN/IL15 and treated with PRIMA-1^Met (P) and Sulfasalazine (S) at the indicated concentrations (1, 2.5 and 5 μM PRIMA-1^Met, 300 μM Sulfasalazine) for 24 h, 48 h, and 72 h and viability was calculated as the percentage of double Annexin V and PI negative cells. For each point, the percentage of viable cells was normalized for the percentage of viable cells in the corresponding untreated sample (100%). The histogram reports the average of the values (from 6 CLL patients carrying wt p53; from 8 CLL patients carrying mutant p53) + SEM; ns not significant; * p < 0.05, ** p< 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Modulation of p53, p21, and MDM2 in OSU and MEC-1 cells following PRIMA-1^Met treatments. Representative Western blots showing the expression of p53, p21, and MDM2 proteins in OSU (A) and MEC-1 (B) cells treated for 24 h with 5–20 μM PRIMA-1^Met. β-actin (β-ACT) was used as a loading control. Histograms showing the expression of p53, p21, and MDM2 proteins, normalized for β-actin, in OSU (C) and MEC-1 (D) cells treated for 24 h with 5–20 μM PRIMA-1^Met. Reported are the means ± SEM of chemiluminescence calculated as fold over the same protein levels in untreated cells of four independent experiments: * p < 0.05. (For original blots, see Supplementary Figure S9A,B).

Figure 6

Modulation of p53 by PRIMA-1^Met treatment in the absence or the presence of CpG-ODN/IL15. (A) Representative Western blot of wt p53, MDM2, and p21 protein modulation after PRIMA-1^Met (sample #16, Table S2); β-actin was used for loading control and normalization; the relative histogram on the right represents p53 data from three independent samples (samples #10, #16, #17, Table S2) reported as means + SEM; (B) Representative Western blot of wt p53, MDM2 and p21 protein modulation after PRIMA-1^Met and CpG-ODN/IL15 pre-treatment (sample #16, Table S2); the relative histogram on the right represents p53 data from two independent samples (samples #16, #17, Table S2) reported as means + SEM; (C) Representative Western blot of mutant p53, MDM2 and p21 protein modulation after PRIMA-1^Met (sample #9, Table S2); the relative histogram on the right represents p53 data from three independent samples (samples #3, #4, #9, Table S2) reported as means + SEM; (D) Representative Western blot of mutant p53, MDM2 and p21 protein modulation after PRIMA-1^Met and CpG-ODN/IL15 pre-treatment (sample #9, Table S2); the relative histogram on the right represents p53 data from two independent samples (samples #4, #9) reported as means + SEM; * p < 0.05, ** p < 0.01. (For original blots, see Supplementary Figure S10A,B).

Figure 7

Levels of SLC7A11/xCT in OSU and MEC-1 cells. (A) Representative Western blot showing the levels of SLC7A11/xCT protein in OSU and MEC-1 cells treated with PRIMA-1^Met (5 µM, 10 µM, 20 µM) alone or in combination with 300 µM SAS after 24 h. β-actin (β-ACT) was used as a loading control; (B) Histogram showing the expression of SLC7A11/xCT protein in OSU and MEC-1 cells treated for 24 h with 5 μM–10 μM PRIMA-1^Met alone or in combination with 300 μM SAS. Reported are the means ± SEM of chemiluminescence calculated as fold over the levels of the same proteins in untreated cells of at least three independent experiments. β-actin is the internal control for normalization; * p < 0.05, ** p < 0.01. (For original blots, see Supplementary Figure S11).

Figure 8

Levels of GSH and ROS in OSU and MEC-1 cells following PRIMA-1^Met and SAS treatments. (A) The levels of GSH were measured by HPLC analysis in OSU and MEC-1 cells treated with PRIMA-1^Met (5 µM or 10 µM) alone or combined with 300 µM SAS after 24 h. The histogram reports the mean ± SD of three independent experiments. GSH concentrations were expressed as µE/g of total protein; (B) Levels of ROS in OSU and MEC-1 cells treated with PRIMA-1^Met alone or in combination with SAS after 24 h. Data are reported as mean of fluorescence ± SD obtained with cytofluorimetric analysis and calculated as fold over the levels observed in untreated cells in three independent experiments; ns not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 9

Enzymatic activity involved in redox balance and lipid peroxidation accumulation in OSU and MEC-1 cells pre-treated with H₂O₂. Determination of (A) glutathione reductase (GR) activity, (B) glutathione peroxidase (GPx) activity, (C) NADPH oxidase activity, and (D) malondialdehyde (MDA, a lipid peroxidation marker) intracellular level in H₂O₂ pre-treated OSU and MEC-1 cells following PRIMA-1^Met alone and in combination with SAS. Each panel is representative of three independent experiments; data are reported as means + SD; ns, not significant, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 10

Metabolic markers in CLL patients’ cells. Determination of (A) GSH level, (B) GSH + GSSG content, (C) cellular total antioxidant capacity, (D) malondialdehyde (MDA) intracellular level in CLL patients’ cells following 1 μM PRIMA-1^Met alone and in combination with 300 μM SAS. Each panel is representative of three independent experiments; data are reported as means + SD. Green columns, wt p53 CLL cells; blue columns, mutant p53 CLL cells. ns, not significant, * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.