Immune regulatory effects of simvastatin on regulatory T cell-mediated tumour immune tolerance

Abstract

Statins are potent inhibitors of hydroxyl-3-methylglutaryl co-enzyme A (HMG-CoA) reductase, and have emerged as potential anti-cancer agents based on preclinical evidence. In particular, compelling evidence suggests that statins have a wide range of immunomodulatory properties. However, little is known about the role of statins in tumour immune tolerance. Tumour immune tolerance involves the production of immunosuppressive molecules, such as interleukin (IL)-10, transforming growth factor (TGF)-β and indoleamine-2,3-dioxygenase (IDO) by tumours, which induce a regulatory T cell (T_reg) response. In this study, we investigated the effect of simvastatin on the production of IL-10, TGF-β and IDO production and the proliferation of T_regs using several cancer cell lines, and Lewis lung cancer (3LL) cells-inoculated mouse tumour model. Simvastatin treatment resulted in a decrease in the number of cancer cells (3LL, A549 and NCI-H292). The production of the immune regulatory markers IL-10, TGF-β in 3LL and NCI-H292 cells increased after treatment with simvastatin. The expression of IDO and forkhead box P3 (FoxP3) transcription factor was also increased in the presence of simvastatin. In a murine 3LL model, there were no significant differences in tumour growth rate between untreated and simvastatin-treated mice groups. Therefore, while simvastatin had an anti-proliferative effect, it also exhibited immune tolerance-promoting properties during tumour development. Thus, due to these opposing actions, simvastatin had no net effect on tumour growth.

Introduction

Hydroxyl-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors (or statins) are in widespread use due to their low-density lipoprotein (LDL) cholesterol-reducing properties. A number of multi-centre clinical trials have demonstrated the efficacy of HMG-CoA reductase inhibitors in reducing morbidity and mortality associated with coronary artery disease [1,2]. A growing body of evidence also suggests that statins might have chemopreventative properties against cancer [3,4]. There is preclinical evidence of the anti-proliferative, pro-apoptotic, anti-invasive and radiosensitizing properties of statins [5–8]; however, based on some lines of meta-analysis, the association between statins and cancer development remains controversial. Considerable evidence suggests that immune-mediated mechanisms play a dominant role in the effects of statins. For example, statins attenuate T cell activation and proliferation, thereby inhibiting the secretion of proinflammatory cytokines and enhancing the secretion of anti-inflammatory cytokines [9,10]. According to recent data, statins appear to play a significant role in the regulation of regulatory T cells (T_regs) [11,12]. Statins decrease the syntheses of the isopernoid intermediates farnesyl pyrophosphate and geranylgeranyl pyrophosphate, which results in decreased activation of the small G-proteins, Ras and Rho. This pathway may be a potential mechanism for the induction of T_regs by statins [13,14]. T_regs, or CD4⁺CD25⁺ T cells, play a key role in tumour-related immune tolerance by suppressing immune responses. Because T_regs are related to cancer development and progression, it is important to evaluate the relationship between statins and T_regs with regard to the immune tolerance of the tumour microenvironment [15]. Although the immunomodulatory potential of statins has been suggested, there are currently no reports on the effects of statins on tumour immune tolerance.

To evaluate the role of statins in tumour immune tolerance, we investigated the effects of simvastatin on indoleamine-2,3-dioxygenase (IDO) production and T_reg-mediated tumour immune tolerance.

Materials and methods

Mice and cell lines

Seven-week-old C57Bl/6 mice were purchased from the Orient Co., Ltd (Gapyeong, Korea). Animals were housed in climate-controlled quarters (24 ± 1°C at 50% humidity) under a 12-h light/12-h dark cycle and were maintained under specific pathogen-free conditions. We performed all experiments according to international guidelines on the use of laboratory animals. The Lewis lung cancer (3LL) cell line was derived originally from a C57Bl/6 mouse, and was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). 3LL cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin. Study mice were inoculated with 3LL cells [2 × 10⁵ in 0·1 ml phosphate-buffered saline (PBS)] by subcutaneous injection into the right forelimb. Forty mice were then randomized into four groups as follows: non-inoculated control group, 3LL-inoculated cancer control, low-dose statin and high-dose statin (10 or 50 mg/kg/day simvastatin, respectively; CJ Pharmacia, Seoul, Korea) groups. The drug was administered by oral gavage q.i.d. in a solution of 0·5% methylcellulose (Sigma-Aldrich, St Louis, MO, USA) containing 0·025% Tween 20 (Sigma-Aldrich). Vehicle control mice (control and cancer control) were gavaged with this same solution. Treatment was initiated 7 days after 3LL inoculation and continued for 21 days. Total tumour volume was measured by a blinded observer using a caliper to measure perpendicular diameters every 4 days. Tumour volume was calculated from the shortest and longest diameters of the tumour mass, according to the following formula [16]: volume (mm³) = (shortest diameter)² × (longest diameter) × 0·5. Mice that developed tumours were killed on day 28, after which the lungs were removed, rinsed with saline and then fixed in 4% paraformaldehyde.

Cell viability assays

Viability assays were based on the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-trazolium bromide (MTT; Amresco, Solon, OH, USA). Cells (control or drug-treated) were rinsed twice with PBS, and then seeded into a 96-well flat-bottomes plate in fetal bovine serum (FBS)-free medium (5 × 10⁴/well in 200 µl). After 24 h incubation, MTT solution was added to each well and incubated at 37°C 2 h, and then the MTT solution was removed; 200 µl dimethylsulphoxide (DMSO; Sigma-Aldrich) was added and incubated at room temperature for 30 min in a shaker. Finally, the density of each well was detected at 540 nm using a microplate reader (CODA; Bio-Rad, San Diego, CA, USA). Cell viability was expressed as a relative viability of tumour cells (% of control cultures incubated with medium only) and was calculated using the following formula: (1 − average absorbance of the experimental group/average absorbance of the control group) × 100%.

Cell cycle analysis

Cell cycle analysis was performed following propidium iodide staining [17]. Cells in culture were treated with simvastatin for 24–48 h. Detached and attached cells were harvested and incubated with CycleTEST™ PLUS DNA Reagent (Becton Dickinson, San Jose, CA, USA). DNA content of the samples was analysed by fluorescence activated cell sorter (FACScan) flow cytometry (Beckman Coulter, Fullerton, CA, USA), and cell cycle distribution was analysed using Multicycle AV (Phoenix Flow System, San Diego, CA, USA).

Enzyme-linked immunosorbent assay (ELISA)

3LL and NCI-H292 cells were cultured in FBS-free media alone or media plus simvastatin (5, 10 or 15 µM) for 24 h. Supernatants from 3LL, NCI-H292 cell cultures (1 × 10⁶ cells/ml) were collected and the concentrations of interleukin (IL)-10 and transforming growth factor (TGF)-β were assessed by ELISA (Biosource, Camarillo, CA, USA), according to the manufacturer's instructions. Simvastatin was obtained from CJ Pharmacia.

FACS analysis

We used a mouse T_reg staining kit (eBioscience, San Diego, CA, USA) for FACS analysis. Splenocytes of mice from each group were isolated using a CD4⁺ T cell isolation kit (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany), and then CD4⁺ T cells were isolated by depleting non-CD4⁺ T cells. The isolated CD4⁺ T cell population was incubated with the following monoclonal antibodies (mAbs) for 30 min (min) at 4°C: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (RM4-5), phycoerythrin (PE)-conjugated anti-mouse CD25 (PC61·5) and PE-Cy5-conjugated anti-mouse/rat forkhead box P3 (FoxP3). FACS analysis was performed using an Epics XL system (Beckman Coulter).

Real-time polymerase chain reaction (PCR)

FoxP3 gene expression in splenocytes of mice from each group was evaluated by real-time PCR. Following the isolation of splenocytes, RNA was isolated using a total RNA isolation kit (iNtRON Biotechnology, Seongnam, Korea) and cDNA was prepared using a kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. FoxP3 gene expression was quantified using the SYBR Premix Ex Tag quantitative PCR kit (Takara, Otsu, Japan) and an ABI PRISM 7000 system, with glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) expression as a control. Amplification was performed in a total volume of 25 µl for 40 cycles of 5 s at 95°C and 31 s at 60°C. Samples were analysed in triplicate, and relative expression was determined by normalizing the expression of the target gene to that of GAPDH, and then comparing the normalized value to a normalized reference control sample to calculate fold-change. The primers that were used for amplification spanned intron/exon boundaries to minimize the amplification of genomic DNA. The primer sequences were as follows: GAPDH 5′-TGAAGCAGGCATGGAGGG-3′ and 5′-CGAAGGTGGAAGAGTGGGAG-3′; FoxP3 5′-CAGCTGCCTACAGTGCCCCTA-3′ and 5′-CATTTGCCAGCAGTGGGTAG-3′; IDO 5′-CGGACTGAGAGGACACAGGTTAC-3′ and 5′-ACACATACGCCATGGTGATGTAC-3′.

Western blot analysis

Tumours were homogenized by sonification for 30 s on ice at 100 W, and then subjected to centrifugation at 12 000 g for 10 min at room temperature. Total protein was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% acrylamide gel, and then electrotransferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was incubated in a solution of 5% fat-free skimmed milk in Tris-buffered saline (TBS)/0·05% Tween 20 for 1 h at room temperature, followed by an anti-IDO mAb (1 : 1000; Upstate, Charlottesville, VA, USA) or an anti-FoxP3 antibody (1 : 1000; BioLegend, San Diego, CA, USA). The membranes were stripped and re-probed with an anti-β-actin mAb (Sigma Aldrich) to verify equal loading of protein in each lane. To detect immunoreactive proteins, the membrane was incubated with horseradish peroxidase (HRP)-linked secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Secondary antibody was detected using WEST-one (iNtRON Biotechnology), according to the manufacturer's instructions. Immunoreactive bands were quantified by scanning the X-ray films, followed by analysis using SigmaScan-Pro, version 5·01 (SPSS Inc., Chicago, IL, USA).

Statistics

All data are expressed as means ± standard error of the mean (s.e.). One-way analysis of variance (anova) was used to determine statistically significant differences between groups. A P-value of less than 0·05 was considered significant.

Results

Simvastatin inhibits the growth of cancer cells

To evaluate the effect of statins on the growth of tumour cells, we treated human lung cancer cell lines (A549, NCI-H292), C57Bl/6 mouse originated 3LL cells with simvastatin, and then measured cell growth by MTT assay after 24 and 48 h (Fig. 1). Simvastatin decreased the number of human and mouse lung cancer cells in a dose-dependent manner. To determine the mechanism of simvastatin-induced growth inhibition, we analysed the cell cycle distribution of simvastatin-treated tumour cells using propidium iodide staining and FACS in A549 cells and 3LL cells. There was a time-dependent decrease in the percentage of cells in S phase and an increase in G₀/G₁ phase cells following treatment with simvastatin (Fig. 2). In mammalian cells, cell cycle progression through G₁ phase is regulated by cyclin D₁, in association with cyclin-dependent kinase (cdk) 4 and cdk2. Because simvastatin induced the accumulation of cells in the G₀/G₁ phase, we examined the effect of simvastatin on cyclin D₁ expression in 3LL cells. Treatment of cells with simvastatin for up to 48 h resulted in a reduction in cyclin D₁ expression (Fig. 3). These results were consistent with cell cycle analysis and suggested that simvastatin induces G₁ arrest by inhibiting the expression of cyclin D₁.

Fig. 1.

Simvastatin decreased the number of cancer cells. Cells were incubated at an initial density of 1 × 10⁶ cell/ml in RPMI-1640 containing 10% fetal bovine serum in the presence or absence of increasing concentrations of simvastatin, and were then cultured for 48 h. Samples were removed at 24 and 48 h, and cell proliferation was analysed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-trazolium bromide (MTT) assay. Compared to the 24-h numbers, the proliferations of Lewis lung carcinoma (3LL), A549 and NCI-H292 cells at 48 h were decreased in a dose-dependent manner by treatment with simvastatin. The data represent the means ± standard error (s.e.) of three independent experiments (*P < 0·05).

Fig. 2.

Effect of simvastatin on cell cycle progression. Simvastatin treatment results in a decrease in the percentage of cells in the S phase and an increase in the number of cells in G_0/1. Cell cycle progression was analysed at 24 and 48 h after simvastatin treatment in A549 cells and Lewis lung carcinoma (3LL) cells. There was a time-dependent decrease in the percentage of cells in the S phase and an increase in the percentage of cells in the G_0/1 phase following simvastatin treatment (*P < 0·05).

Fig. 3.

Simvastatin down-regulates cyclin D₁ expression in Lewis lung carcinoma (3LL) cells. Cells were treated with simvastatin for 48 h, then the media was removed and cells were incubated in control or treatment media. Cells were collected at 24 and 48 h, and then whole cell lysate was analysed by Western blot using an anti-cyclin D₁ antibody.

Effects of simvastatin on the production of immunomodulatory cytokines in vitro

Simvastatin increases the production of IL-10 and TGF-β in cancer cell lines

IL-10 and TGF-β are associated with tumour immune tolerance and T_reg activation. To determine whether simvastatin regulated IL-10 and TGF-β production in vitro, we treated 3LL and NCI-H292 cells with increasing concentrations of simvastatin. Treatment with simvastatin increased IL-10 and TGF-β production (Fig. 4).

Fig. 4.

Simvastatin increases interleukin (IL)-10 and transforming growth factor (TGF)-β production in lung cancer cells in vitro. Cells were incubated in fetal bovine serum-free culture media alone, or in culture media plus simvastatin (5, 10 or 15 µM) for 48 h, and then IL-10 and TGF-β production was analysed by enzyme-linked immunosorbent assay (ELISA). (a) Simvastatin increased IL-10 production in a dose-dependent manner; (b) simvastatin also increased TGF-β production. The data represent the means ± standard error (s.e.) of at least three independent experiments (*P < 0·01 compared to the control group, **P < 0·05 compared to the control group).

Simvastatin increases FoxP3 and IDO expression

To determine whether simvastatin regulated the gene expression of FoxP3, isolated tumour-infiltrating lymphocytes (TILs) were treated with simvastatin and then FoxP3 expression was analysed by real-time PCR. FoxP3 is a transcription factor that serves as a reliable intracellular marker for T_regs, and high numbers of CD4⁺CD25⁺FoxP3⁺ cells can be found in circulation as well as in tumours [18]. FoxP3 expression was higher in simvastatin-treated TILs (Fig. 5a). To confirm the results of real-time PCR analysis of isolated TILs, we analysed FoxP3 protein levels in tumours from simvastatin-treated and untreated control mice by Western blotting. FoxP3 protein levels were higher in tumours from simvastatin-treated mice compared to untreated control mice (Fig. 5b). There was also a corresponding increase in IDO levels in the simvastatin groups. Thus, real-time PCR and Western blot analysis demonstrated that the expression of FoxP3, as well as IDO expression in tumours, increases with simvastatin treatment.

Fig. 5.

Simvastatin increases the expressions of forkhead box P3 (FoxP3) and indoleamine-2,3-dioxygenase (IDO). (a) Tumour-infiltrating lymphocytes of Lewis lung carcinoma (3LL) control mice were isolated, and then FoxP3 gene expression was analysed by real-time polymerase chain reaction (PCR). FoxP3 gene expression was higher in the simvastatin-treated groups than in the control group (10 µM and 15 µM, *P < 0·05 compared to the control group). (b) Tumours were removed from mice from the indicated treatment groups, and IDO and FoxP3 levels were analysed by Western blot. Sample loading was normalized using a probe for beta-actin. The expression of IDO and FoxP3 within the tumour mass was increased by simvastatin treatment.

Simvastatin increases the percentage of T_regs

To assess the relationship between simvastatin and T_reg proliferation, splenocytes were isolated from non-inoculated control mice and 3LL-inoculated cancer control, low-dose and high-dose simvastatin mice. The percentage of CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ cells in each group was analysed by FACS using the Epics XL system. There was an increase in the percentage of CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ cells in the simvastatin groups compared to the 3LL-inoculated cancer control group (Fig. 6a). A representative dot plot showing the increased percentage of T_regs following simvastatin treatment is shown in Fig. 6b. These results indicated that simvastatin enhances the T_regsin vivo.

Fig. 6.

Simvastatin increases the percentage of regulatory T cells (T_reg) cells in vivo. C57Bl/6 mice were inoculated with Lewis lung carcinoma (3LL) cells, and then either untreated or treated with simvastatin (10 or 50 mg/kg daily). After 4 weeks, mice were killed and spleens were extracted. The percentage of CD4⁺CD25⁺ and CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ cells in the spleens of each group was analysed by fluorescence activated cell sorter (FACS). The percentage of CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ cells was significantly higher in the high-dose (50 mg/kg) simvastatin group compared to the control group (*P < 0·01 compared to the 3LL control group).

Simvastatin has no effect on the growth of tumours

To determine whether simvastatin affect tumour growth, we measured tumour size in mice treated with simvastatin and in untreated 3LL cancer control mice. We did not observe a significant inhibitory effect of simvastatin on tumour growth. Furthermore, there was no significant difference in tumour mass between the low-dose (10 mg/kg) and high-dose (50 mg/kg) statin groups (Fig. 7).

Fig. 7.

Tumour-bearing mice (10 mice per group) were treated with vehicle or simvastatin (10 mg/kg or 50 mg/kg) starting at day 7 after inoculation until day 28. Tumour volume was measured on the indicated days. Data represent the means ± standard deviation. There were no significant differences in tumour size between the untreated Lewis lung carcinoma (3LL) group and the simvastatin-treated groups.

Discussion

In the current study, we provide evidence of a novel mechanism by which statins regulate T_reg-mediated tumour immune tolerance. Simvastatin decreased the number of tumour cells in vitro through the induction of a G₁/S phase arrest. Simvastatin also activated and enhanced T_regsin vivo. These opposing actions of simvastatin resulted in no net effect on the inhibition of tumour growth in a murine 3LL model.

Statins comprise a therapeutic class of agents that reduce plasma cholesterol levels by inhibiting HMG-CoA reductase, the rate-controlling enzyme in cholesterol production. A growing body of evidence suggests that statins may also have chemopreventative properties against cancer [3]. However, there is a long-standing debate concerning the association between statins and cancer. In a review of rodent carcinogenicity tests, statins initiated or promoted cancer in rats and mice [19]. The putative growth-suppressive properties of statins are believed to be mediated through the inhibition of HMG-CoA reductase, but other mechanisms have also been suggested [5,20]. HMG-CoA reductase inhibitors can synchronize tumour cells, and thereby exert an anti-proliferative effect, by blocking the G₁–S cell cycle transition. However, while statins can inhibit cell proliferation, they also appear to enhance tumour progression; the mechanism responsible for this phenomenon was unknown. There have been several reports of the immunomodulatory effects of statin [21,22]. Statins attenuate T cell activation and proliferation, thereby inhibiting the secretion of proinflammatory cytokines and enhancing the secretion of anti-inflammatory cytokines [9,10]. For example, statins have been shown to reduce IL-1, IL-6, IL-8, IL-12 and interferon (IFN)-γ levels, and consequently reduce T cell activation. Recent evidence suggests that statins play a role in the modulation of immune responses through the regulation of T_regs, which are involved in tumour immune tolerance [11,12,23]. In the current study, we demonstrated that simvastatin treatment induces FoxP3 expression, and that this increase in FoxP3 expression is accompanied by an expansion of T_regs, as determined by FACS analysis of CD4⁺CD25⁺FoxP3⁺ cells. The X chromosome-encoded forkhead transcription factor, FoxP3, was identified recently as a key player in T_reg biology [24]. This is the most specific marker for T_regs and is expressed specifically in CD4⁺CD25⁺ T cells in the thymus and the periphery [25]. FoxP3 was not observed in tumour cells except pancreatic carcinoma cells [26], highly restricted to the subset of T cells and correlating with suppressor activity [27,28]. The induction of T_regs by simvastatin correlated with the production of IL-10 and TGF-β, which are T_reg inducers [29].

IDO is a rate-limiting enzyme in tryptophan catabolism. It, too, has emerged as an important immunoregulatory enzyme. A localized reduction in tryptophan, which is an essential amino acid, and the concomitant production of immunomodulatory tryptophan metabolites contributes to the immunosuppressive effects of IDO [30,31]. The up-regulation of IDO at inflammatory sites leads to the depletion of tryptophan and the generation of intermediate catabolites (kynurenines), which exert inhibitory effects on T lymphocytes. IDO is also expressed by tumour cells. Immunogenic tumours that are engineered to overexpress IDO grow more aggressively, and this effect correlates with a decrease in activated T cells at the tumour site [31]. Furthermore, the expression of IDO in cancer cells has been associated with the induction of T_regsin vitro and in vivo[32,33]. In the current study, we evaluated the relationship between simvastatin and IDO expression. In a murine 3LL model, mice that were treated with simvastatin exhibited higher levels of IDO protein expressions than untreated cancer control mice (Fig. 5). Because IDO influences T_reg proliferation, we also examined the effect of simvastatin on T_reg-mediated immune regulation. The percentage of T_regs in mice was higher in simvastatin-treated mice than in control mice (Fig. 6). T_regs can be generated from conventional CD4⁺CD25^- T cells following exposure to antigens and IL-10, and the production of IL-10 or TGF-β by the induced T_regs appears to mediate their inhibitory properties [34]. The presence of these regulatory cells in cancer patients might be important in the suppression of T cells and subsequent tumour growth. Our results demonstrate that simvastatin increases IL-10 production and IDO expression in cancer cells, and induces the expansion of T_regs.

In conclusion, simvastatin inhibits cancer cell proliferation through the induction of G₁/S phase arrest. However, simvastatin also induces the immune suppressive cytokines and the expansion of functionally active T_regs. Increased numbers of T_regs induced by simvastatin in the cancer microenvironment would enable tumour cells to escape immune surveillance and proliferate. These opposing effects of simvastatin would result in no net effect on tumour growth.

Disclosure

None.