Fluvastatin Induces Apoptosis in Primary and Transformed Mast Cells

Abstract

Statin drugs are widely employed in the clinic to reduce serum cholesterol. Because of their hydroxymethylglutaryl coenzyme A reductase antagonism, statins also reduce isoprenyl lipids necessary for the membrane anchorage and signaling of small G-proteins in the Ras superfamily. We previously found that statins suppress immunoglobulin E (IgE)-mediated mast cell activation, suggesting these drugs might be useful in treating allergic disease. Although IgE-induced function is critical to allergic inflammation, mast cell proliferation and survival also impact atopic disease and mast cell neoplasia. In this study, we describe fluvastatin-mediated apoptosis in primary and transformed mast cells. An IC₅₀ was achieved between 0.8 and 3.5 μM in both cell types, concentrations similar to the reported fluvastatin serum C_max value. Apoptosis was correlated with reduced stem cell factor (SCF)-mediated signal transduction, mitochondrial dysfunction, and caspase activation. Complementing these data, we found that p53 deficiency or Bcl-2 overexpression reduced fluvastatin-induced apoptosis. We also noted evidence of cytoprotective autophagy in primary mast cells treated with fluvastatin. Finally, we found that intraperitoneal fluvastatin treatment reduced peritoneal mast cell numbers in vivo These findings offer insight into the mechanisms of mast cell survival and support the possible utility of statins in mast cell-associated allergic and neoplastic diseases. SIGNIFICANCE STATEMENT: Fluvastatin, a statin drug used to lower cholesterol, induces apoptosis in primary and transformed mast cells by antagonizing protein isoprenylation, effectively inhibiting stem cell factor (SCF)-induced survival signals. This drug may be an effective means of suppressing mast cell survival.

Fig. 1.

Fluvastatin induces apoptosis in mouse BMMC and PMC. (A) C57BL/6 BMMC were cultured for the indicated times in 1 μM of fluvastatin, and apoptosis was assessed by PI-DNA staining of subdiploid DNA, as measured by flow cytometry. P values were calculated by comparing fluvastatin vs. DMSO paired time points by two-way ANOVA. (B) BMMC were cultured for 4 days in the indicated fluvastatin concentrations. Apoptosis was measured by PI-DNA staining. P values were calculated by comparing fluvastatin to the DMSO control by two-way ANOVA. (C) BMMC were cultured as in (A) for 4 days, and Caspase-3/7 activity was assessed by cleavage of fluorescent substrate and flow cytometry. (D) BMMC from the indicated strains were cultured in increasing concentrations of fluvastatin for 4 days, and IC₅₀ values were determined. (E) Primary PMC were cultured for 4 days in the indicated fluvastatin concentrations, and apoptosis was assessed by PI-DNA staining. (F) Human skin-derived mast cells were cultured for 4 days in the indicated concentrations of fluvastatin, and apoptosis was assessed by PI-DNA staining. Concentrations of “0” indicate DMSO controls in each figure, which were matched to the highest DMSO concentration present in fluvastatin-treated cells. Data shown for all figures are from six samples/point from one of at least three experiments. Not significant (NS), P ≤ 0.05 *, P ≤ 0.01 **, P ≤ 0.001 ***, and P ≤ 0.0001**** by ANOVA.

Fig. 2.

Fluvastatin-induced apoptosis is linked to the geranylgeranyl pyrophosphate (GGPP) pathway. (A) Simplified version of cholesterol and isoprenoid synthesis pathways, with inhibitors indicated in gray font. (B) BMMC were cultured for 4 days with DMSO, fluvastatin, or ZA (10 μM). Apoptosis was assessed by PI-DNA staining and flow cytometry. (C) BMMC were cultured for 4 days with fluvastatin ± MVA (1 mM), GGPP, or FPP (10 μM each). Apoptosis was assessed as in (A). (D) BMMC were cultured for 4 days with DMSO, fluvastatin, farnesyltransferase inhibitor II (10 μM), or GGT inhibitor 286 (20 μM). Apoptosis was assessed as in (A). Data shown are from 12 samples/point (A and B) or nine samples/point (C) from one of at least two experiments that gave similar results. **P < 0.01; ****P < 0.0001; NS, not significant when comparing samples to fluvastatin (FLU) or DMSO controls as indicated, using one-way ANOVA. MVA, mevalonic acid.

Fig. 3.

Fluvastatin inhibits SCF-mediated ERK phosphorylation. (A and B) BMMC were cultured for 24 hours with DMSO or fluvastatin (1 μM) prior to stimulation with 200 ng/ml SCF for the indicated times. Image in (A) shows example Western blot data; (B) shows results from three populations. (C) BMMC were cultured for 4 days with fluvastatin (2.5 μM) or the indicated ERK inhibitors (10, 10, and 5 μM, respectively). Data are from three populations. ****P < 0.0001 by ANOVA.

Fig. 4.

Fluvastatin induces Bcl-2– and p53-dependent apoptosis. BMMC from C57BL/6, H-2^k-p53 KO (A) or Bcl2 Tg (B) mice were cultured with Fluvastatin (10 μM) for the indicated times. Apoptosis was determined by subdiploid DNA content measured by PI-DNA stain using flow cytometry. Data shown are mean ± S.D. from six to nine samples from one of two separate experiments. Some error bars are too small to be shown. **P < 0.01; ****P < 0.0001 by ANOVA.

Fig. 5.

Fluvastatin (Fluva) induces LC3 II and p62 accumulation in BMMC. (A) Example Western blot of BMMC cultured for 24 hours with DMSO or Fluva (10 μM) ± chloroquine (CQ), used to enhance LC3 II and p62 detection. (B) Summary data from three BMMC populations treated as in (A), using 5 or 10 μM Fluva compared with matched DMSO samples. *P < 0.05 by paired t test comparing each Fluva-treated sample to the DMSO control. (C) C57BL/6 BMMC were cultured with vehicle (DMSO) or Fluva ± CQ or bafilamycin A (BafA) for 48 hours, and apoptosis was measured by PI-DNA staining and flow cytometry. Data shown are from six to nine samples/point. ****P < 0.0001 by ANOVA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 6.

Fluvastatin reduces peritoneal mast cell number in vivo. C57BL/6 mice (four to six per group) were injected with 10 mg/kg i.p. fluvastatin once daily for 4 days. Peritoneal lavage fluid was analyzed by flow cytometry to identify mast cells Figure A-C (FcεRI+, c-Kit+). Not significant (NS), P ≤ 0.05 *, P ≤ 0.01 **, P ≤ 0.001 ***, and P ≤ 0.0001**** by t test.

Fig. 7.

Fluvastatin induces apoptosis in transformed mast cells. (A) The indicated mastocytoma cell lines (PDMC-1, P815, RBL-2H3) were cultured for 4 days in fluvastatin, and IC₅₀ values were calculated as in Fig. 1. Data are means and S.D. of eight samples, with P values calculated by one-way ANOVA. (B) P815 cells were cultured for 24 hours in fluvastatin (10 μM), and cell cycle was measured using PI-DNA staining and flow cytometry. Data are means and S.D. of seven to nine samples from one of three representative experiments, with P values calculated by t test. (C and D) Representative histogram and summary data of P815 cells stained with Di(OC₆)₃ after 48 hours of culture with fluvastatin (10 μM). (D) shows means and S.D. of six samples, with P value calculated by t test. (E) P815 cells were cultured for 48 hours, and Caspase-9 activity was assessed by cleavage of fluorescent substrate and flow cytometry. Data are means and S.D. of 12 samples from one of two representative experiments, with P value calculated by t test. Fluvastatin, (Fluva); S, synthesis. P ≤ 0.05 *, P ≤ 0.01 **, P ≤ 0.001 ***, and P ≤ 0.0001****

Fig. 8.

Fluvastatin increases c-Kit expression but inhibits signaling in mastocytoma cells. (A) P815 cells were cultured for the indicated times in DMSO or fluvastatin, and c-Kit expression was measured by flow cytometry. (B) P815 cells were cultured for 24 hours in DMSO or fluvastatin (10 μM), and lysates were probed for phosphorylated (p) and total ERK or AKT expression by Western blot. Bar charts show summary data from six to seven samples/point. (C) P815 cells were cultured for 4 days with DMSO, fluvastatin (10 μM), or the indicated ERK (10, 5, 10 μM, respectively) or AKT (10 μM) inhibitors, and apoptosis was measured by PI-DNA staining and flow cytometry Not significant (NS), P ≤ 0.05 *, P ≤ 0.01 **, P ≤ 0.001 ***, and P ≤ 0.0001**** by ANOVA (A and C) or t test (B).