Pitavastatin Is a Highly Potent Inhibitor of T-Cell Proliferation

Abstract

Repositioning of approved drugs is an alternative time- and cost-saving strategy to classical drug development. Statins are 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase inhibitors that are usually used as cholesterol-lowering medication, and they also exhibit anti-inflammatory effects. In the present study, we observed that the addition of Pitavastatin at nanomolar concentrations inhibits the proliferation of CD3/CD28 antibody-stimulated human T cells of healthy donors in a dose-dependent fashion. The 50% inhibition of proliferation (IC50) were 3.6 and 48.5 nM for freshly stimulated and pre-activated T cells, respectively. In addition, Pitavastatin suppressed the IL-10 and IL-17 production of stimulated T cells. Mechanistically, we found that treatment of T cells with doses <1 µM of Pitavastatin induced hyperphosphorylation of ERK1/2, and activation of caspase-9, -3 and -7, thus leading to apoptosis. Mevalonic acid, cholesterol and the MEK1/2 inhibitor U0126 reversed this Pitavastatin-mediated ERK1/2 activation and apoptosis of T cells. In summary, our results suggest that Pitavastatin is a highly potent inhibitor of T-cell proliferation, which induces apoptosis via pro-apoptotic ERK1/2 activation, thus representing a potential repositioning candidate for the treatment of T-cell-mediated autoimmune diseases.

Keywords: ERK1/2 activation; Pitavastatin; apoptosis; drug repositioning; inhibitor of T-cell proliferation.

Figure 1

Pitavastatin inhibited proliferation, as well as IL-10 and IL-17 production of stimulated T cells, but had no influence on T-cell activation. Human resting T cells freshly stimulated with anti-CD3/CD28 antibodies were cultured with increasing concentrations of different statins (A) or 100 nM Pitavastatin in the presence or absence of either 6 mM MVA, 10 µM FPP, 10 µM GGPP or 100 µM cholesterol (chol) (B) for 72 h. DNA synthesis was determined by standard [³H]-thymidine uptake. [³H]-TdR incorporation is shown as mean percentage ± SEM of DNA synthesis in relation to control cultures (vehicle) set to 100%. (C–E) Human T cells were freshly stimulated with anti-CD3/CD28 antibodies and incubated with increasing concentrations of Pitavastatin. Expression levels of the T-cell activation markers CD69 after 16 h (C) and CD25 after 48 h (D) were analyzed by flow cytometry. Representative histograms (left) and mean fluorescence intensity (MFI, right) are displayed. Cell-culture supernatants were harvested 72 h after treatment, and concentrations of IFN-γ, IL-5, IL-17 and IL-10 were determined with specific ELISA (E). Data are presented as the mean ± SEM of n = 4 independent experiments. Statistical analysis was performed with One-Way ANOVA and Dunnett’s Multiple Comparison Analysis Test as post hoc test. (**** p ≤ 0.0001, *** p  ≤  0.001, ** p  ≤  0.01, * p  ≤  0.05).

Figure 2

Pitavastatin blocked cell-cycle progression and induced cell death in freshly stimulated T cells. Human T cells were freshly stimulated with anti-CD3/CD28 antibodies and cultured with increasing concentrations of Pitavastatin for 72 h. DNA content and cell-cycle phase of cells were determined by using propidium iodide staining and flow cytometric analysis. Representative histograms of DNA content are shown in (A) (G₀/G₁ (black), S (light gray) and G₂/M (dark gray)). (B) The bars indicate the percentage of cells in G₀/G₁ phase, S phase and G₂/M phase. Data are presented as the mean + SEM of n = 4 independent experiments. Cells were stained after 72 h with Annexin V-FITC/PI for flow cytometric analysis. (C) Representative flow cytometry dot plots and (D) quantification of early and late apoptotic cells of n = 4 independent experiments are shown. Statistical analysis was performed with One-Way ANOVA and Dunnett’s Multiple Comparison Analysis Test as post hoc test. (**** p ≤ 0.0001, *** p  ≤  0.001, ** p  ≤  0.01, * p  ≤  0.05).

Figure 3

Pitavastatin activated caspases in freshly stimulated T cells. Resting human T cells were freshly stimulated with anti-CD3/CD28 antibodies and cultured with increasing concentrations of Pitavastatin or 100 nM Pitavastatin in the presence or absence of 6 mM MVA or 100 µM cholesterol (chol). Cells were stained after 72 h with active caspase-8-binding FITC-IETD-FMK (A), active caspase 9-binding FITC-LEHD-FMK reagent (B) or CellEvent™ Caspase-3/7 Green detection reagent (C) for flow cytometric analysis. Mean + SEM of relative caspase expression of n = 3 independent experiments are displayed. (D) For kinetic analyses, human T cells were stimulated with anti-CD3/CD28 antibodies and cultured with increasing concentrations of Pitavastatin in the presence of Caspase-3/7 Green detection reagent. Kinetic measures of the number of caspase-3/7 positive cells were recorded by the IncuCyte S3 imaging system at 3 h intervals for 84 h. (E) Resting human T cells were freshly stimulated with anti-CD3/CD28 antibodies and cultured with high concentrations of Pitavastatin. Cells were stained after 72 h with CellEvent™ Caspase-3/7 Green detection reagent. Results of n = 3 independent experiments are presented as mean values of relative caspase expression. Statistical analysis was performed with One-Way ANOVA and Dunnett’s Multiple Comparison Analysis Test as post hoc test. (**** p ≤ 0.0001, *** p  ≤  0.001, ** p  ≤  0.01, * p  ≤  0.05).

Figure 4

Pitavastatin enhanced ERK phosphorylation in freshly stimulated T cells. Resting human T cells were freshly stimulated with anti-CD3/CD28 antibodies and cultured with increasing concentrations of Pitavastatin (A) or 100 nM Pitavastatin in the presence or absence of either 6 mM MVA or 100 µM cholesterol (chol). (B) After 24 h, 48 h or 72 h incubation, cells were lysed and analyzed by Western blot. (A,B) Relative expression of p-ERK1/2 based on densitometric quantification. Quantitative data are presented as mean + SEM from n = 4 independent experiments. (C) Representative Western blot images of p-ERK1/2 and total ERK1/2. (D,E) Resting human T cells were freshly stimulated with anti-CD3/CD28 antibodies and cultured with high concentrations of Pitavastatin. After 48 h of incubation, cells were lysed and analyzed by Western blot. (D) Relative expression of p-ERK1/2 based on densitometric quantification. Quantitative data are presented as the mean + SEM from n = 3 independent experiments. (E) Representative Western blot images of p-ERK1/2 and total ERK1/2. Statistical analysis was performed with One-Way ANOVA and Dunnett’s Multiple Comparison Analysis Test as post hoc test. (**** p ≤ 0.0001, *** p  ≤  0.001, ** p  ≤  0.01, * p  ≤  0.05).

Figure 5

ERK hyperphosphorylation is responsible for Pitavastatin-induced apoptosis. Resting human T cells were freshly stimulated with anti-CD3/CD28 antibodies and cultured with 100 nM Pitavastatin in the presence or absence of 10 µM of the MEK1/2 inhibitor U0126. (A) Cells were stained after 72 h with CellEvent™ Caspase-3/7 Green detection reagent. Mean + SEM of relative caspase expression of n = 3 independent experiments are displayed. (B) Representative Western blot images of p-ERK1/2 and total ERK1/2. Statistical analysis was performed with One-Way ANOVA and Dunnett’s Multiple Comparison Analysis Test as post hoc test. (**** p ≤ 0.0001, *** p  ≤  0.001).

Figure 6

Pitavastatin suppressed proliferation and induced apoptosis in pre-activated T cells. Resting human T cells were pre-activated for 48 h with anti-CD3/CD28 antibodies and cultured with increasing concentrations of different statins (A) or 100 nM Pitavastatin in the presence or absence of 6 mM MVA or 100 µM cholesterol (chol) (B) for 72 h. DNA synthesis was determined by standard [³H]-thymidine uptake. [³H]-TdR incorporation is shown as mean percentage of DNA synthesis in relation to control cultures (vehicle) set to 100%. Cells were stained after 72 h with Annexin V-FITC/PI for flow cytometric analysis. (C) Representative flow cytometry dot plots and (D) quantification of early and late apoptotic cells of n = 3 independent experiments are shown. (E) Resting human T cells were pre-activated for 48 h with anti-CD3/CD28 antibodies and cultured with increasing concentrations of Pitavastatin (left) or 100 nM Pitavastatin in the presence or absence of 6 mM MVA or 100 µM cholesterol (chol) (right). Cells were stained after 72 h with CellEvent™ Caspase-3/7 Green detection reagent. Relative caspase expression of n = 3 independent experiments is presented as mean + SEM. Statistical analysis was performed with One-Way ANOVA and Dunnett’s Multiple Comparison Analysis Test as post hoc test. (**** p ≤ 0.0001, *** p  ≤  0.001, ** p  ≤  0.01, * p  ≤  0.05).

Figure 7

Schematic presentation of the functional effects of Pitavastatin on T cells. (A) Pitavastatin inhibits proliferation in freshly stimulated and pre-activated T cells. Inhibition of proliferation was associated with reduced IL-17 and IL-10 secretion and apoptosis induction. (B) By inhibiting HMG-CoA reductase, the cholesterol concentration in the lipid rafts is reduced, leading to hyperphosphorylation of ERK1/2. Sustained activation of ERK1/2 induces the activation of caspase-9 and caspase-3/7, leading to cell death.