Statins induce apoptosis through inhibition of Ras signaling pathways and enhancement of Bim and p27 expression in human hematopoietic tumor cells

Abstract

Recently, statins have been demonstrated to improve cancer-related mortality or prognosis in patients of various cancers. However, the details of the apoptosis-inducing mechanisms remain unknown. This study showed that the induction of apoptosis by statins in hematopoietic tumor cells is mediated by mitochondrial apoptotic signaling pathways, which are activated by the suppression of mevalonate or geranylgeranyl pyrophosphate biosynthesis. In addition, statins decreased the levels of phosphorylated extracellular signal–regulated kinase 1/2 and mammalian target of rapamycin through suppressing Ras prenylation. Furthermore, inhibition of extracellular signal–regulated kinase 1/2 and mammalian target of rapamycin by statins induced Bim expression via inhibition of Bim phosphorylation and ubiquitination and cell-cycle arrest at G1 phase via enhancement of p27 expression. Moreover, combined treatment of U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, and rapamycin, a mammalian target of rapamycin inhibitor, induced Bim and p27 expressions. The present results suggested that statins induce apoptosis by decreasing the mitochondrial transmembrane potential, increasing the activation of caspase-9 and caspase-3, enhancing Bim expression, and inducing cell-cycle arrest at G1 phase through inhibition of Ras/extracellular signal–regulated kinase and Ras/mammalian target of rapamycin pathways. Therefore, our findings support the use of statins as potential anticancer agents or concomitant drugs of adjuvant therapy.

Keywords

Statin Bim Ras extracellular signal–regulated kinase 1/2 mammalian target of rapamycin

Introduction

Statins, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are widely known and used drug agents for reducing cholesterol levels; thus, they are used in the treatment of hyperlipidemia and cardiovascular disease.^1,2 Recently, it has been reported that statins improve cancer-specific mortality or prognosis in patients of various cancers, such as endocrine-related gynecologic cancers, esophageal cancer, breast cancer, multiple myeloma, colorectal cancer, and lung cancer.^3–8 Hence, statins may be used as potential anticancer drugs or concomitant drugs of adjuvant therapy.

Statins suppress the production of mevalonate (MVA) through inhibiting HMG-CoA reductase, thereby reducing production of farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), ubiquinone, dolichol, and squalene, which are intermediate products of the MVA pathway. These intermediate productions regulate various cellular functions, such as cell signaling pathways, cell respiration, production of glycoprotein, and components of the cell membrane.^9,10 It has been reported that lovastatin reduced the volume fraction of liver nodules and cell proliferation within the liver nodules via suppression of ubiquinone production in a rat model for chemically induced hepatocarcinogenesis.¹¹ In addition, dolichol attenuated simvastatin-induced apoptosis and disrupted the involvement of unfolded protein response in human neuroblastoma SH-SY5Y cells.¹² It has also been indicated that lovastatin inhibited cell proliferation and induced cell-cycle arrest in human hematopoietic tumor cell lines.¹³ FPP or GGPP are involved in the prenylation of small GTPase, such as Ras. Statins have been demonstrated to inhibit Ras prenylation and Ras signaling downstream effectors, including extracellular signal–regulated kinase 1/2 (ERK1/2), Akt, and mammalian target of rapamycin (mTOR), thereby inducing apoptosis through suppression of GGPP biosynthesis.^14–19 Moreover, lactone structure of statins suppressed proteasome activity and induced apoptosis.²⁰ However, the detailed mechanisms by which statins induce apoptosis, either by blocking the MVA pathway or proteasome inhibition, remain unclear. In this study, we investigated the mechanism by which statins induce apoptosis in human hematopoietic tumor cell lines.

Materials and methods

Materials

Fluvastatin, simvastatin, and MG132 were purchased from Calbiochem (San Diego, CA, USA). Mevalonic acid lactone (MVA), FPP, GGPP, squalene, ubiquinone, isopentenyladenine, and dolichol were purchased from Sigma (St. Louis, MO, USA). U0126 was purchased from Promega (Madison, WI, USA). Rapamycin, z-VAD-fmk, z-LEHD-fmk, and z-DEVE-fmk were purchased form Wako (Osaka, Japan). These reagents were dissolved in dimethyl sulfoxide. And then, these dissolved reagents were resuspended in phosphate-buffered saline (PBS; 0.05 M, pH 7.4) and filtrated through syringe filters (0.45 μm; IWAKI GLASS, Tokyo, Japan) before use.

Cell culture

HL-60 (human acute promyelocytic leukemia) and U937 (human histcytic lymphoma) cells were obtained from Riken Cell Bank (Ibaraki, Japan). IM9 (human multiple myeloma) cells were obtained from Health Science Research Resources Bank (Osaka, Japan). ARH77 (human multiple myeloma) cells were obtained from DS Pharma Biomedical (Osaka, Japan). These cells were cultured in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 μg/mL penicillin (Gibco), 100 U/mL streptomycin (Gibco), and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.4; Wako) in an atmosphere containing 5% CO₂.

Trypan blue dye exclusion assay

The effect of fluvastatin and simvastatin on cell survival/proliferation was determined using the trypan blue dye exclusion assay. Prior to each experiment, cells (2 × 10³ cells/well) were plated onto 96-well plates. After culturing for 24 h, the cells were exposed to fluvastatin and simvastatin for various times. Equal volumes of cell suspension and 0.4% trypan blue solution were mixed gently, loaded into a hemocytometer, and the viable cells (unstained) and dead cells (stained blue) were counted. Each experiment was performed in triplicate. Results are reported from an average of at least five independent experiments.

Annexin V apoptosis assay

Apoptosis was measured using an Annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Becton Dickinson, Bedford, MA, USA) as per the manufacturer’s protocol. Briefly, cells were washed twice in PBS and then resuspended in binding buffer with Annexin V-FITC. The cells were allowed to incubate for 15 min at room temperature and then analyzed using a BD LSRFortessa (Becton Dickinson) flow cytometer.

Measurement of caspase-9 and caspase-3 proteolytic activities

Caspase-9 and caspase-3 enzyme activities were measured by proteolytic cleavage of the fluorogenic substrate LEHD-AFC and DEVD-AFC using the Caspase-9 Fluorometric Assay Kit and Caspase-3 Fluorometric Assay Kit (BioVision Inc., Milpitas, CA, USA). HL-60 cells were treated with fluvastatin or simvastatin for 48 h (control cells were incubated without statins). The cells were collected, washed in PBS, and lysed in lysis buffer provided by the kit. For the assay, a solution of cell lysates containing 50 μM substrate was incubated at 37°C for 1 h. The release of AFC from the substrate was measured fluorimetrically using a fluorescence spectrophotometer (F-4010; Hitachi, Tokyo, Japan) with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The results were corrected for protein content of the lysates and are expressed as the change in proteolytic cleavage of the substrate (pM) for 1 h/mg protein. The protein content of the cell lysate was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA).

Cytofluorometric determination of mitochondrial transmembrane potential ∆Ψ_m

To evaluate mitochondrial transmembrane potential ∆Ψ_m, cells (5 × 10⁵ mL⁻¹) were incubated with 3,3-dihexyloxacarbocyanine iodide (40 nM DiOC6 in PBS; Molecular Probes, Eugene, OR, USA) for 5 min at 37°C followed by flow cytometer analysis (BD LSRFortessa). The cells were washed with PBS and analyzed at an excitation wavelength of 482 nM and emission was measured at 504 nM.

Proteasome activity assay

To measure the kinetics of proteasome caspase, trypsin, or chymotrypsin activities, HL-60 cells were treated with fluvastatin, simvastatin, or MG132 for 24 h and then washed sequentially with cold 1× PBS and lysis buffer (HEPES (pH 8), 1 mM dithiothreitol (DTT)). Cells were scraped off with a cell lifter followed by sonication and centrifugation at 14,000 r/min for 5 min at 4°C. Soluble extract (25 μg) was diluted in a reaction buffer (20 mM HEPES, 0.05 mM ethylenediaminetetraacetic acid (EDTA; pH 8), 0.035% sodium dodecyl sulfate (SDS)). The extract, containing a mixture of 26S and 20S proteasomes, was incubated in 10 μM of the fluorogenic substrate Z-Leu-Leu-Glu-AMC (caspase like), Bz-Val-Gly-Arg-AMC (trypsin like), or Suc-Leu-Leu-Val-Tyr-AMC (chymotrypsin like) for 1.5 h. The release of AMC from the substrate was measured fluorimetrically using a fluorescence spectrophotometer (F-4010; Hitachi) with an excitation wavelength of 360 nm and emission wavelength of 465 nm.

Western blotting

HL-60 cells treated under various conditions were lysed with a lysis buffer (20 mM Tris-HCl pH 8.0 (Wako), 150 mM NaCl (Wako), 2 mM EDTA (Wako), 100 mM NaF (Wako), 1% NP40 (Wako), 1 μg/mL leupeptin (Sigma), 1 μg/mL antipain (Sigma), and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma)), and the protein concentrations of the resulting cell lysates were determined using a BCA protein assay kit (Pierce). An aliquot of each extract (40 μg protein) was fractionated by electrophoresis in an SDS-polyacrylamide gel and transferred to a Hypbond-P polyvinylidene difluoride (PVDF) membrane (Amersham Life Science, Arlington Heights, IL, USA). Membranes were blocked with a solution containing 3% skim milk and then incubated overnight at 4°C with each of the following antibodies: anti-Ras (C-4) antibody, anti-Bim (H-191) antibody, anti-p53 (FL-393) antibody, anti-p21 (H-164) antibody, anti-p27 (F-8) antibody, anti-ubiquitin (FL-76) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; ERK1/2) (Thr²⁰²/Tyr²⁰⁴) antibody, anti-phospho-mTOR (Ser²⁴⁴⁸) antibody, anti-phospho-Bim (Ser⁶⁹) antibody, anti-p44/42 MAPK (ERK1/2) antibody, anti-mTOR antibody (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (Sigma). Subsequently, the membranes were incubated for 1 h at room temperature with anti-rabbit IgG sheep antibody or anti-mouse IgG sheep antibody coupled to horseradish peroxidase (Amersham). Reactive proteins were visualized using a chemiluminescence (Luminata Forte) kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.

Immunoprecipitation

HL-60 cells treated under various conditions were lysed with lysis buffer containing CHAPS (Wako). Whole-cell lysates were obtained, precleared with protein A-sepharose (Amersham), and incubated overnight with 5 μg of anti-ubiquitin antibody (Santa Cruz Biotechnology). Subsequently, immunocomplexes were captured with protein A. The beads were pelleted, washed three times, and boiled in SDS sample buffer. The presence of immunocomplexes was determined by western blotting analysis.

Cell-cycle analysis

Cell cycle was analyzed using propidium iodide (Sigma). Briefly, cells were washed twice in PBS and then resuspended in 70% ethanol. After 24 h, cells were washed twice in PBS and then resuspended in DNAase-free RNAase A (Sigma) and propidium iodide. The cells were allowed to incubate for 15 min at room temperature and then analyzed using a BD LSRFortessa (Becton Dickinson) flow cytometer.

Statistical analysis

All results are expressed as means and SD of several independent experiments. Multiple comparisons of the data were performed by analysis of variance (ANOVA) with Dunnett’s test. The p values less than 5% were regarded as significant.

Results

Statins induce apoptosis through the mitochondrial pathway

Fluvastatin and simvastatin were found to induce cell death in HL-60, IM9, ARH77, and U937 cells in a concentration- and time-dependent manner, as shown in Figure 1. Caspase-9 and caspase-3 activities, as well as Annexin V-positive cells measured in HL-60 cells 48 h after the addition of 5 μM fluvastatin or 10 μM simvastatin, showed a marked increase as compared to the control (PBS-treated cells; Figure 2(a)–(c)). To further confirm the role of caspase-9 and caspase-3 activation in statins-mediated apoptosis, HL-60 cells were preincubated with a pan-caspase inhibitor (z-VAD-fmk), caspase-9-selective inhibitor (z-LEHD-fmk), or a caspase-3-selective inhibitor (z-DEVD-fmk) 3 h prior to the addition of statins. After 72 h of incubation, cell viability was measured by the trypan blue dye method, as described above. The results showed that the caspase-9 and caspase-3-selective inhibitors provided full protection against statin-induced cell death (Figure 2(d)).

Figure 1.

Statins induce cell death in hematopoietic tumor cell lines. Cell viability of fluvastatin or simvastatin-treated (a) HL-60, (b) IM9, (c) ARH77, and (d) U937 cells as measured by the trypan blue dye exclusion assay. The cells were treated with various concentrations of fluvastatin or simvastatin for 1, 2, and 3 days. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test).

Figure 2.

Increase in Annexin V–positive cells and caspase-3 activities are associated with statin-induced cell death. (a and b) HL-60 cells were exposed to the indicated concentrations of fluvastatin and simvastatin for 48 h. (a) Caspase-9 and (b) caspase-3 activities are expressed as the amount of the caspase-9 substrate LEHD-AFC and caspase-3 substrate DEVD-AFC proteolytically cleaved in picomoles per hour per milligram of protein. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (c) HL-60 cells were exposed to the indicated concentrations of fluvastatin and simvastatin for 48 h and then stained with an Annexin V apoptosis assay kit. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (d) HL-60 cells were treated with fluvastatin and simvastatin for 72 h in the presence or absence of 50 μM z-VAD-fmk, 50 μM z-LEHD-fmk, or 50 μM z-DEVE-fmk, and the cell viability was measured. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (e) Mitochondrial transmembrane potential is decreased in cells treated with fluvastatin or simvastatin for 48 h.

Next, the effect of statins on the mitochondrial transmembrane potential was determined. After exposure to fluvastatin and simvastatin for 48 h, a decrease in the mitochondrial transmembrane potential was observed in HL-60 cells (Figure 2(e)).

The apoptosis-inducing effect of statins is mediated by defective prenylation

A previous study has indicated that statins, including lactone structure, induced apoptosis via inhibition of proteasome activity, but not suppression of isoprenylation of small GTPases.²⁰ We examined whether statins inhibit proteasome activity in HL-60 cells. Statins did not suppress proteasome caspase activity, such as trypsin-like and chymotrypsin-like activity, but MG132 inhibited these enzyme activities (Figure 3(a)–(c)).

Figure 3.

Statin-induced apoptosis by suppressing GGPP biosynthesis. (a–c) Non-inhibition of the proteasome activity by fluvastatin and simvastatin. After treatment of 5 μM fluvastatin, 10 μM simvastatin, and 10 μM MG132 for 24 h at 37°C, HL-60 cells were lysated. Cell extracts were incubated for 1.5 h, at which point the fluorogenic peptide substrate for the (a) caspase-like activity, (b) trypsin-like activity, and (c) chymotrypsin-like activity of the proteasome 7-AMC was added to the extracts. The fluorescence assays (excitation = 360 nm; emission = 465 nm) were conducted at room temperature. These results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (d and e) HL-60 cells were pretreated with 1 mM mevalonate (MVA), 10 μM farnesyl pyrophosphate (FPP), 10 μM geranylgeranyl pyrophosphate (GGPP), 30 μM squalene, 100 μM isopentenyladenine, 300 μM dolichol, or 30 μM ubiquinone for 4 h and then with (d) fluvastatin or (e) simvastatin for 72 h. These results are representative of five independent experiments.*p < 0.01 versus control (ANOVA with Dunnett’s test).

The most prominent effect of statins is the inhibition of HMG-CoA reductase in the MVA pathway. MVA metabolism yields a series of isoprenoid compounds that are incorporated into cholesterol and prenylated proteins essential for cell growth and metabolism. To assess whether different isoprenoids could attenuate statins-mediated apoptosis, the effects of co-treatment with MVA, FPP, GGPP, squalene, ubiquinone, isopentenyladenine, and dolichol on statin-induced apoptosis were determined. HL-60 cells were pretreated with 1 mM MVA, 10 μM FPP, 10 μM GGPP, 30 μM squalene, 100 μM isopentenyladenine, 300 μM dolichol, and 30 μM ubiquinone. Fluvastatin or simvastatin were added to the cell suspensions to obtain a final concentration of 5 or 10 μM, respectively. After 72 h, cell viability was measured by the trypan blue dye method described above. No significant differences in cell viability were observed when cells were treated with statins in the presence of FPP, squalene, isopentenyladenine, dolichol, and ubiquinone. However, pre-treatment with MVA and GGPP significantly inhibited statin-induced apoptosis (Figure 3(d)–(e)).

Statins enhance the expression of Bim and p27 through inhibition of the Ras signaling pathway

To identify the molecules involved in statin-induced apoptosis, Ras protein prenylation and the activity of its downstream effectors were examined. Fluvastatin and simvastatin suppressed Ras prenylation in HL-60 cells (Figure 4(a)) and inhibited the expression of phosphorylated ERK1/2 and mTOR (Figure 4(a)). No significant changes in the level of phosphorylated Akt and p38MAPK were detected in statin-treated cells compared to control cells (data not shown).

Figure 4.

Statins specifically increase the expression of Bim by suppressing the activation of the Ras/ERK and Ras/mTOR pathways in HL-60 cells. (a) HL-60 cells were treated with fluvastatin or simvastatin for 1, 3, 6, 12, or 24 h. Control cells were treated with PBS and cultured in serum-containing medium for 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against Ras, phosphorylated ERK1/2 (phospho-ERK1/2), phosphorylated mTOR (phospho-mTOR), ERK1/2, and mTOR. (b) Whole-cell lysates were generated and immunoblotted with antibodies against phosphorylated Bim (phospho-Bim), Bim_EL, and β-actin (internal standard). (c) Ubiquitylation of Bim in HL-60 cells. HL-60 cells were incubated with fluvastatin and simvastatin for 24 h. Control cells were treated with PBS and cultured in serum-containing medium for 24 h. Proteins immunoprecipitated with anti-ubiquitin antibody were immunoblotted with anti-Bim antibody. Ubiquitylated Bim was detected as upper shifted bands in anti-Bim blotting. (d) Effect of MG132 on statin-induced downregulation of Bim ubiquitylation. HL-60 cells were incubated with MG132. After 4 h, cells were treated with fluvastatin and simvastatin for 24 h. Proteins immunoprecipitated with the anti-ubiquitin antibody were immunoblotted with the anti-Bim antibody. Ubiquitylated Bim was detected as an upper shifted band in anti-Bim blotting.

Proteins from the Bcl-2 family have been identified as essential components of mitochondrial apoptotic signaling pathways. Our previous studies have indicated that statins or nitrogen-containing bisphosphonates suppressed the Ras/ERK and Ras/mTOR pathway and enhanced Bim expression in head and neck carcinoma and hematopoietic tumors.^18,19 Activation of ERK1/2 promotes the phosphorylation of Bim_EL and leads to a substantial increase in the turnover of Bim_EL.²¹ mTOR also regulates the Bim protein levels.²² Phosphorylation of Bim_EL induces its ubiquitylation and degradation by the proteasome.^21,23 To determine the possible involvement of Bim_EL in statin-induced apoptosis, the expression of Bim_EL was examined. As shown in Figure 4(b), statins significantly induced the expression of Bim_EL and suppressed the phosphorylated Bim. We also found that statins inhibited Bim ubiquitylation via suppression of phosphorylated Bim (Figure 4(c)). In addition, MG132 enhanced the expression of poly-ubiquitinated Bim, but did not affect the statin-induced downregulation of Bim ubiquitylation (Figure 4(d)). These results indicate that statin-induced apoptosis is mediated by an increase of Bim_EL expression via suppression of the Ras/ERK and Ras/mTOR pathways.

Activation of ERK1/2 and mTOR promotes cell-cycle progression via regulating cyclin-dependent kinase inhibitors, such as suppressing the expression of p21 and p27.^24–26 We next investigated cell-cycle distribution of HL-60 cells when treated with 5 μM fluvastatin and 10 μM simvastatin. Fluvastatin and simvastatin induced cell-cycle arrest in G1 phase and enhanced p27 expression, a key regulator responsible for G1 to S phase checkpoint, but not affected p53 and p21 expressions in HL-60 cells (Figure 5). Moreover, co-treatment of HL-60 cells with U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, and rapamycin, a mTOR inhibitor, induced apoptosis and cell-cycle arrest in G1 phase, suppressing phosphorylated and ubiquitinated Bim, and increased Bim_EL and p27 expression at levels similar to those in response to ERK1/2 and mTOR suppression by statins (Figure 6), confirming the participation of ERK1/2 and mTOR pathways.

Figure 5.

Induction of cell-cycle arrest at G1 phase and p27 expression by statins on HL-60 cells. (a) HL-60 cells were treated with fluvastatin or simvastatin for 24 h. The cell-cycle distribution changes were monitored by flow cytometry. Relative percentages of cells in each phase of the cell cycle as indicated. (b) HL-60 cells were treated with fluvastatin or simvastatin for 1, 3, 6, 12, or 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against p53, p21, p27, and β-actin.

Figure 6.

U0126 and rapamycin induce cell death via Bim and p27 expression. (a) HL-60 cells were treated with 5 μM U0126, 10 μM rapamycin, 5 μM fluvastatin, and 10 μM simvastatin for 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against phosphorylated ERK1/2 (phospho-ERK1/2), phosphorylated mTOR (phospho-mTOR), and mTOR. (b) HL-60 cells were treated with 5 μM U0126, 10 μM rapamycin, 5 μM fluvastatin, and 10 μM simvastatin for 72 h. Cell viability was measured by the trypan blue dye exclusion assay. The results are representative of five independent experiments. *p < 0.01 versus control (ANOVA with Dunnett’s test). (c and d) HL-60 cells were treated with U0126 and rapamycin for 24 h. (c) Whole-cell lysates were generated and immunoblotted with antibodies against phospho-Bim, Bim_EL, and β-actin. (d) Proteins immunoprecipitated with anti-ubiquitin antibody were immunoblotted with anti-Bim antibody. Ubiquitylated Bim was detected as upper shifted bands in anti-Bim blotting. (e) HL-60 cells were treated with U0126 and rapamycin for 24 h. The cell-cycle distribution changes were monitored by flow cytometry. Relative percentages of cells in each phase of the cell cycle as indicated. (f) HL-60 cells were treated with U0126 and rapamycin for 1, 3, 6, 12, or 24 h. Whole-cell lysates were generated and immunoblotted with antibodies against p53, p21, p27, and β-actin.

Discussion

In this study, we demonstrated that statins induce apoptosis by decreasing mitochondrial membrane permeability, increasing caspase-9 and caspase-3 activity, and inducing cell-cycle arrest at G1 phase via suppression of MVA and GGPP biosynthesis; however, no correlation with proteasome inhibition was observed. A previous report has indicated that lovastatin induces apoptosis via enhancing p21 and p27 expressions based on inhibition of proteasome activity, but does not affect suppression of MVA pathway intermediates in breast cancer cells.²⁷ However, it has been reported that simvastatin induces cell death by decreasing GGPP biosynthesis, without the involvement of proteasome inhibition in Burkitt’s lymphoma cells.²⁸ Moreover, in a prior study, we have reported that mevastatin inhibited the activity of the MVA and MAPK pathways resulting in the induction of apoptosis; the underlying mechanism of action was found to be the inhibition of GGPP biosynthesis.¹⁷ These findings suggest that the effect of statins is mediated by the mitochondrial apoptotic signaling pathway and cell-cycle arrest at G1 phase via the suppression of GGPP biosynthesis in hematopoietic tumors.

It has been suggested that statins inducing cell death were associated with suppression of small GTPase prenylation.^29–31 GGPP is an important factor for Ras membrane-anchoring, and inhibition of GGPP biosynthesis facilitates the dissociation of Ras from the cell membrane, thereby suppressing Ras-mediated signaling.^32,33 Our results clearly demonstrated that statins decrease the prenylation of Ras and inhibit the activation of ERK1/2 and mTOR. We have previously reported that statins induce a decrease in phosphorylated ERK1/2 and mTOR in head and neck carcinoma cell lines.¹⁹ It has also been reported that nitrogen-containing bisphosphonates suppressed Ras prenylation and ERK1/2 and mTOR activation through the inhibition of GGPP biosynthesis.¹⁸ These findings support our experimental model that statins induce apoptosis through suppression of the Ras/ERK and Ras/mTOR pathways in hematopoietic tumors.

Mitochondrial pathway-mediated apoptosis involves Bcl-2 family proteins, such as Bcl-2 and Bim. Our previous studies have suggested that the inhibition of ERK1/2 and mTOR activation enhanced Bim expression and induced apoptosis via mitochondrial and caspase-dependent pathways.^18,19 In this study, statins enhanced Bim_EL expression and inhibited Bim ubiquitylation via suppression of Bim phosphorylation. In addition, combined treatment of U0126 and rapamycin induced apoptosis through suppressing ubiquitylation and phosphorylation of Bim and enhancing Bim_EL expression. Moreover, MG132 did not affect the statin-induced downregulation of Bim ubiquitylation. Phosphorylation of Bim_EL by ERK1/2 promotes Bim ubiquitylation and degradation via the proteasome pathway,²¹ and activation of mTOR regulates Bim expression.³⁴ In addition, PD184352, a selective MEK1/2 inhibitor, reduces the MG132-induced Bim ubiquitylation by inhibiting ERK1/2 and Bim phosphorylation.²¹ These findings indicate that statins enhance Bim_EL expression through the suppression of Bim phosphorylation and ubiquitylation via inhibiting the Ras/ERK and Ras/mTOR pathways; thus, increased Bim_EL expression is partly responsible for the induction of apoptosis by statins.

In this study, we found that statins induced cell-cycle arrest of G1 phase via enhanced p27 expression based on the inhibition of ERK1/2 and mTOR activation. Co-treatment of U0126 and rapamycin also inhibited cell-cycle progression from G1 to S phase and promoted the expression of p27. It has been reported that lovastatin promoted cell-cycle arrest of G1 phase via inhibition of Ras prenylation in human hematopoietic tumors.³⁵ It has also been demonstrated that cell-cycle inhibition of G1 checkpoints by atorvastatin was associated with suppression of Ras signaling in CD4-positive T cells.³⁶ Moreover, activation of ERK1/2 and mTOR is regulated with p27 expression level via activation of Skp2, known as E3 ubiquitin ligase of p27.^37,38 These findings suggest that inhibiting Ras/ERK and Ras/mTOR pathways by statins induces cell-cycle arrest of G1 phase.

In conclusion, this study provides evidence that statins induce apoptosis and cell-cycle arrest by decreasing mitochondrial transmembrane potential, increasing the activation of caspase-9 and caspase-3 and enhancing Bim and p27 expressions through inhibition of the Ras/MEK/ERK and Ras/mTOR pathways; therefore, statins may act more effectively on tumors with variability of Ras. These findings suggest that statins might be a potential anticancer agent and could be used in combination with other anticancer drugs for treatment of hematopoietic tumors.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by a Grant-in-Aid for Scientific Research (C; grant no. 15K08116) and Grant-in-Aid for Young Scientists (B; grant no. 16K18965) from the Japan Society for the Promotion of Science (JSPS) and by Ministry of Education, Culture, Sports, Science, and Technology (MEXT)-Supported Program for the Strategic Research Foundation at Private Universities, 2014–2018 (grant no. S1411037).

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Statins induce apoptosis through inhibition of Ras signaling pathways and enhancement of Bim and p27 expression in human hematopoietic tumor cells

Abstract

Keywords

Introduction

Materials and methods

Materials

Cell culture

Trypan blue dye exclusion assay

Annexin V apoptosis assay

Measurement of caspase-9 and caspase-3 proteolytic activities

Cytofluorometric determination of mitochondrial transmembrane potential ∆Ψm

Proteasome activity assay

Western blotting

Immunoprecipitation

Cell-cycle analysis

Statistical analysis

Results

Statins induce apoptosis through the mitochondrial pathway

The apoptosis-inducing effect of statins is mediated by defective prenylation

Statins enhance the expression of Bim and p27 through inhibition of the Ras signaling pathway

Discussion

Footnotes

Declaration of conflicting interests

Funding

References

Cytofluorometric determination of mitochondrial transmembrane potential ∆Ψ_m