Simvastatin impairs growth hormone-activated signal transducer and activator of transcription (STAT) signaling pathway in UMR-106 osteosarcoma cells

Abstract

Recent studies have demonstrated that statins reduce cell viability and induce apoptosis in various types of cancer cells. The molecular mechanisms underlying these effects are poorly understood. The JAK/STAT pathway plays an important role in the regulation of proliferation and apoptosis in many tissues, and its deregulation is believed to be involved in tumorigenesis and cancer. The physiological activation of STAT proteins by GH is rapid but transient in nature and its inactivation is regulated mainly by the expression of SOCS proteins. UMR-106 osteosarcoma cells express a GH-responsive JAK2/STAT5 signaling pathway, providing an experimental model to study the influence of statins on this system. In this study we investigated the actions of simvastatin on cell proliferation, migration, and invasion on UMR-106 cells and examined whether alterations in GH-stimulated JAK/STAT/SOCS signaling may be observed. Results showed that treatment of osteosarcoma cells with simvastatin at 3 to 10 µM doses decreases cell proliferation, migration, and invasion in a time- and dose-dependent manner. At the molecular level, although the mechanisms used by simvastatin are not entirely clear, the effect of the statin on the reduction of JAK2 and STAT5 phosphorylation levels may partially explain the decrease in the GH-stimulated STAT5 transcriptional activity. This effect correlated with a time- and dose-dependent increase of SOCS-3 expression levels in cells treated with simvastatin, a regulatory role that has not been previously described. Furthermore, the finding that simvastatin is capable of inducing SOCS-3 and CIS genes expression shows the potential of the JAK/STAT pathway as a therapeutic target, reinforcing the efficacy of simvastatin as chemotherapeutic drug for the treatment of osteosarcoma.

Figure 1. Effects of simvastatin treatment on proliferation and cytotoxicity of UMR-106 osteosarcoma cells.

Cells were treated with increasing doses of simvastatin (SIM) as indicated and cell proliferation was measured using a bromodeoxyuridine (BrdU) incorporation assay. Proliferation levels were measured at 24, 48 and 72 hours and results were expressed as percentage to control cells (100%) (A). Statistical differences were observed after 24 h at 10 µM (p<0.01). IC₅₀ values obtained at 24 (B) and 48 hours (C) after simvastatin treatment. Values are the mean ± SEM. Cell index was measured by a dynamic monitoring response (RT-CES assay) to increasing simvastatin doses for the indicated time period. The arrow indicates the time when simvastatin was added (D). IC₅₀ value was calculated at 48 hours (E).

Figure 2. Effects of simvastatin on cell viability in BRL-4 and MCF-7 cells.

BRL-4 cells (A) and MCF-7 cells (B) were treated with increasing doses of simvastatin for 24, 48 and 72 hours as indicated. Cell viability was measured using the MTT method and expressed as percentage to control cells (100%). IC₅₀ values were calculated after 72 hours of simvastatin treatment for BRL-4 (C) and MCF-7 (D) cells. Values are the mean of three replicates ±SEM.

Figure 3. Migration and invasion levels of Simvastatin UMR-106 treated cells.

UMR-106 monolayer were scraped with a pipette tip and incubated in the absence or presence of simvastatin. Wounds were photographed at 48 h after treatment and a representative result of three experiments is shown to schematize the inhibitory effect of simvastatin on migration rate (A). Cells were seeded in the upper level of 8 µm-Boyden chamber uncoated (B) or coated with matrigel (C), attracted by simvastatin and 10% FBS supplemented medium in the lower chamber. After 48 h migrating cells were stained with crystal violet methanol solution, the 8 µm-pore membranes were cut off and crystals were dissolved in 10% acetic acid. Absorbance solution was measured at 540 nm. Quantitative response was expressed as percentage of the control ± SEM of two replicates. a means p<0.001, b means p<0.05 vs control, after a one-way ANOVA analysis.

Figure 4. Effect of simvastatin on JAK/STAT phosphorylation in UMR-106 cells.

Cells were pretreated with simvastatin as indicated, and then stimulated with 50(A) or 10 h (B) of simvastatin incubation. β-actin levels were used as a load control protein.

Figure 5. Effect of simvastatin-pretreated UMR-106 cells on GH- or FBS-induced transcriptional activity.

After transfection, cells were incubated for 8-free medium with 10 µM simvastatin or vehicle for 16 h. Then, cells were stimulated with 50 nM GH or 10%FBS –as indicated- for 16 hours. Cells were transiently transfected with pSPI-GLE-Luc reporter plasmid activated by STAT5 (A) and co-transfected with pSTAT5 S730A (B); pISRE-Luc reporter plasmid activated by STAT1/3 (C), pSTAT3-Luc reporter plasmid activated by STAT3 (D) or pSOCS-1, -2, -3 gene promoter plasmids (E). Treatments are as follows: C  =  Non-stimulated transfected control cells; SIM  =  Simvastatin. Luciferase expression results are expressed relative to non-treated cells. a means p<0.001, vs control cells; b p<0.05, vs control cells; c p<0.05 vs GH-treated cells. Values are mean ± SEM, after a one-way ANOVA analysis.

Figure 6. Effect of simvastatin and GH treatment of UMR-106 cells on SOCS gene expression.

(A) mRNA expression after GH treatment in the time points indicated for SOCS-2, SOCS-3 and CIS. Gene expression of SOCS-3 (B) and CIS (C) after 10 µM simvastatin treatment in the time points indicated. Gene expression of SOCS-3 (D) and CIS (E) in cells treated with simvastatin for 8 hours in increasing doses prior to stimulation with GH 50 nM for 60 minutes. Treatments are as follows: C  =  Non-stimulated control cells; SIM  =  Simvastatin. Values are the mean ± SD (n = 3) and normalized to the level of the reference gene cyclophiline. a p<0.05, b p<0.005, c p<0.001, after a two-way ANOVA analysis.