Simvastatin potently induces calcium-dependent apoptosis of human leiomyoma cells

Abstract

Statins are drugs commonly used for the treatment of high plasma cholesterol levels. Beyond these well known lipid-lowering properties, they possess broad-reaching effects in vivo, including antitumor effects. Statins inhibit the growth of multiple tumors. However, the mechanisms remain incompletely understood. Here we show that simvastatin inhibits the proliferation of human leiomyoma cells. This was associated with decreased mitogen-activated protein kinase signaling and multiple changes in cell cycle progression. Simvastatin potently stimulated leiomyoma cell apoptosis in a manner mechanistically dependent upon apoptotic calcium release from voltage-gated calcium channels. Therefore, simvastatin possesses antitumor effects that are dependent upon the apoptotic calcium release machinery.

Keywords: Apoptosis; Calcium; Calcium Channel; Cell Proliferation; Inositol Trisphosphate Receptor (InsP3R); Leiomyoma; Statin; Tumor; Tumor Cell Biology.

FIGURE 1.

Antiproliferative effects of simvastatin on human leiomyoma cells. A and B, morphologic effects of simvastatin (SIMV) treatment for 48 h on HuLM cells (A) and primary human leiomyoma cells (B). C and D, MTT proliferation assay results for cells treated with 0–10 μm SIMV with treatment end points at 24, 48, and 72 h showing a dose-response curve (C) and a time-response curve (D). a.u., arbitrary units. In D, the significance signs in the lower part of the graph apply to both 48 and 72 h. E and F, Western blotting showing the effects of SIMV treatment for 48 h on proliferating cell nuclear antigen (PCNA, E) and phosphorylated and total ERK expression (F) along with quantification. α-Tubulin was used as a loading control. *, p < 0.05; **, p < 0.01 versus 0 μm.

FIGURE 2.

Effects of simvastatin on human leiomyoma cell cycle progression. Cells were synchronized by overnight serum starvation and then treated with 0–10 μm SIMV for 48 h. Cells were then stained with propidium iodide, and flow cytometry was performed. A–F, cell cycle analysis results at 0–10 μm simvastatin concentrations. Top panels, two-dimensional frequency pseudocolor plots. Rectangles represent gating of events to exclude cellular aggregates. Bottom panels, analyzed data using FlowJo software, where each cell cycle phase population is presented in the colors shown in the legend. em, emission; ex, excitation. G and H, histograms showing the percentage of cells at S phase of the cell cycle (G) and the sub-G₀/G₁ (apoptotic) population (H) at each simvastatin concentration. *, p < 0.05; **, p < 0.01 versus 0 μm.

FIGURE 3.

Apoptosis-inducing effects of simvastatin on human leiomyoma cells. A, blinded quantification of percentage of propidium iodide (PI)-positive cells showing dose-dependent apoptosis induction after simvastatin treatment for 48 h. B, caspase-3 enzymatic activity showing that simvastatin treatment for 48 h dose-dependently increases apoptotic signaling in human leiomyoma cells. C, Western blotting showing effects of SIMV treatment for 48 h on Bim expression, along with quantification. Actin was used as a loading control. *, p < 0.05; **, p < 0.01 versus 0 μm.

FIGURE 4.

Effects of simvastatin on cytosolic calcium release in human leiomyoma cells. A, cytosolic calcium levels in control (DMSO, black) cells and 0.1 μm simvastatin treated (red) cells for 24 h. Right panel, representative 340-nm, 380-nm, and pseudocolored Fura-2 ratio images from control (DMSO) and simvastatin-treated cells. Low calcium is represented by “cool” colors such as blue, and higher calcium is indicated by “warmer” colors such as green. B, mitochondrial calcium as measured by Rhod-2 fluorescence after treatment with vehicle (DMSO) or 0.1 μm simvastatin for 24 h. Representative Rhod-2 fluorescent images acquired under identical acquisition parameters are shown in the top panel, and quantified data are shown in the bottom panel (see “Experimental Procedures”). C, mitochondrial polarization as determined by the ratio of JC-1 fluorescence after treatment with DMSO or 0.1 μm simvastatin for 24 h. A decrease in the ratio is indicative of depolarization. D–G, dynamic changes in cytosolic calcium after the first 5 h of exposure to simvastatin as determined by GCaMP6s fluorescence. Concentrations are indicated in the top panels. Graphed are the responses of all cells on a single coverslip. Also included as an inset is an expanded time scale from 150–200 min to visualize the kinetics of individual calcium release events. H, quantification of the number of cells with calcium transients in response to simvastatin treatment pooled from three separate experiments. **, p < 0.02 versus DMSO (vehicle). I, simvastatin-induced morphologic changes in human leiomyoma cells are calcium-dependent. Preloading cells with the calcium chelator BAPTA-AM (bottom panels) prevents simvastatin-induced morphologic changes in a dose-dependent fashion. J and K, simvastatin-induced apoptosis in human leiomyoma cells is calcium-dependent. Preloading cells with the calcium chelator BAPTA-AM prevents simvastatin-induced apoptosis, as demonstrated by decreased caspase-3 activity (J) and the percentage of propidium iodide (PI)-positive cells (K). Effects are evident at 10–20 μm BAPTA-AM concentrations. **, p < 0.01 versus 10 μm SIMV.

FIGURE 5.

Simvastatin-induced apoptosis in human leiomyoma cells is not dependent on ER calcium release. A and B, pretreating cells with the IP₃R inhibitor xestospongin C (XesC) (A) or the cell membrane-permeable phosphoinositide-specific PLC inhibitor U73122 (B) does not prevent simvastatin-induced apoptosis, as measured by caspase-3 activity.

FIGURE 6.

Simvastatin-induced apoptosis is inhibited by non-selective voltage-gated calcium channel inhibitors. A and B, pretreating cells with the voltage-gated calcium channels blockers mibefradil (A) or SKF96365 (SKF) (B) prevents simvastatin-induced apoptosis, as measured by caspase-3 activity. C, pretreatment with the highly selective L-type voltage-gated calcium channel inhibitor nimodipine prevents simvastatin-induced apoptosis, as measured by caspase-3 activity. *, p < 0.05; **, p < 0.01 versus 10 μm SIMV. D, Western blotting for expression of L-type (L-Ca_V, top blot) and T-type (T-Ca_V, bottom blot) voltage-gated calcium channels in human leiomyoma cells. We observed no significant changes in expression levels after simvastatin treatment.

FIGURE 7.

Schematic cartoon showing the proposed mechanism of simvastatin inhibition of proliferation and induction of calcium-dependent apoptosis in human leiomyoma cells. Proximal effects include inhibition of growth factor signaling. Downstream effects include increased expression of the proapoptotic Bcl-2 family member protein Bim through decreased ERK-mediated degradation. This leads to increased Bim_EL activity and mitochondrial leakage of apoptosis-initiating proteins, including cytochrome c. Permeabilization of mitochondria also requires activation of l-type voltage-gated calcium channels and calcium influx into the mitochondria. RTK, receptor tyrosine kinase; CytC, cytochrome c; L-Ca_V, L-type voltage-gated calcium channels.