Statins in lymphangioleiomyomatosis. Simvastatin and atorvastatin induce differential effects on tuberous sclerosis complex 2-null cell growth and signaling

Abstract

Mutations of the tumor suppressor genes tuberous sclerosis complex (TSC)1 and TSC2 cause pulmonary lymphangioleiomyomatosis (LAM) and tuberous sclerosis (TS). Current rapamycin-based therapies for TS and LAM have a predominantly cytostatic effect, and disease progression resumes with therapy cessation. Evidence of RhoA GTPase activation in LAM-derived and human TSC2-null cells suggests that 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor statins can be used as potential adjuvant agents. The goal of this study was to determine which statin (simvastatin or atorvastatin) is more effective in suppressing TSC2-null cell growth and signaling. Simvastatin, but not atorvastatin, showed a concentration-dependent (0.5-10 μM) inhibitory effect on mouse TSC2-null and human LAM-derived cell growth. Treatment with 10 μM simvastatin induced dramatic disruption of TSC2-null cell monolayer and cell rounding; in contrast, few changes were observed in cells treated with the same concentration of atorvastatin. Combined treatment of rapamycin with simvastatin but not with atorvastatin showed a synergistic growth-inhibitory effect on TSC2-null cells. Simvastatin, but not atorvastatin, inhibited the activity of prosurvival serine-threonine kinase Akt and induced marked up-regulation of cleaved caspase-3, a marker of cell apoptosis. Simvastatin, but not atorvastatin, also induced concentration-dependent inhibition of p42/p44 Erk and mTORC1. Thus, our data show growth-inhibitory and proapoptotic effects of simvastatin on TSC2-null cells compared with atorvastatin. These findings have translational significance for combinatorial therapeutic strategies of simvastatin to inhibit TSC2-null cell survival in TS and LAM.

Figures

Figure 1.

Simvastatin, but not atorvastatin, induces tuberous sclerosis complex 2 (TSC2)-null cell rounding. A total of 1.5 × 105TSC2-null cells were plated on 6-well plates and treated with 10 μM of atorvastatin or simvastatin added freshly every other day as described in Results. Untreated cell were used as a control. Representative images were taken using a phase-contrast microscope.

Figure 2.

Simvastatin, but not atorvastatin, abrogates TSC2-null cell growth. TSC2-null (A) or lymphangioleiomyomatosis (LAM)-derived cells (B) were treated with atorvastatin (triangles) or simvastatin (squares) over the range of concentrations (0.5, 1.0, 5.0, and 10 μM). Untreated cells were used as control. Cell counts were performed and analyzed with three measurements for each condition. Each point represents the mean of three to eight measurements ± SEM. #P < 0.01 for cells treated with simvastatin or atorvastatin versus untreated cells. *P < 0.01 for cells treated with atorvastatin versus simvastatin by analysis of variance (Bonferroni-Dunn).

Figure 3.

Rapamycin differentially enhances inhibitory effects of simvastatin or atorvastatin on TSC2-null cell growth. Cells were maintained in Dulbecco’s modified Eagle medium with 10% FBS for 6 days in complete media supplemented with freshly made 20 nM of rapamycin (open diamonds); atorvastatin with the range of concentrations 0.5, 1.0, 5.0, and 10 µM (triangles); or a combination of both (solid diamonds) (A). TSC2-null cells were treated with 20 nM of rapamycin (open diamonds); simvastatin with the range of concentrations 0.5, 1.0, 5.0, and 10 µM (squares); or a combination of both (solid diamonds) (B). Control cells were maintained at the same conditions and treated with diluent. Cell counts were performed and analyzed; two measurements were taken for each condition. Each point represents the mean of two measurements ± SEM. #P < 0.01 for cells with treatment versus untreated cells. *P < 0.01 for cells with single treatment simvastatin or atorvastatin versus combination with rapamycin alone by analysis of variance (Bonferroni-Dunn).

Figure 4.

Simvastatin-induced down-regulation of Akt/PKB, p42/p44 Erk, and mTORC1 and up-regulation of cleaved caspase-3. TSC2-null cells were treated with 0, 0.5, 1.0, 5.0, and 10 μM of atorvastatin or simvastatin followed by immunoblot analyses with phospho-Ser473-Akt/PKB and Akt/PKB (A), phospho-S6 and ribosomal protein S6 (B), phospho-p42/p44 Erk and total p42/p44 Erk (C), and with cleaved caspase-3 (D) antibodies.