Anti-proliferative and cytotoxic activity of rosuvastatin against melanoma cells.

INTRODUCTION: Statins are considered potential candidate agents for melanoma chemoprevention. Statin-induced mevalonate pathway inhibition leads to reduction of cholesterol synthesis and also to decreased cellular levels of non-steroidal isoprenoids, geranylgeranyl pyrophosphate and farnesyl pyrophosphate. This results in the impairment of protein prenylation which affects carcinogenesis. AIM: To analyze anti-proliferative and cytotoxic activity of rosuvastatin against melanoma cells.
MATERIAL AND METHODS: Melanoma cell lines (A375 and WM1552C) and normal fibroblasts (BJ) were used as the primary research material. Cells were treated with rosuvastatin at concentrations ranging from 0.01 µM to 10 µM. Cell viability was analyzed with the use of an MTT assay. Expression of proliferation marker Ki67 was assessed on the basis of immunofluorescence staining.
RESULTS: Rosuvastatin reduced A375 and BJ cell viability in a time- and dose-dependent manner. After 72 h incubation, the IC50, half maximal inhibitory concentration, was 2.3 µM for melanoma cells and 7.4 µM for normal fibroblasts. In turn, rosuvastatin exhibited relatively lower activity against WM1552C cells. A significant reduction of Ki67 expression was also noted for BJ fibroblasts after prolonged incubation with the tested drug.
CONCLUSIONS: The results indicate that the anti-melanoma properties of rosuvastatin are highly dependent on the tumor cell line assessed. However, the concentrations required to decrease melanoma cell viability in vitro exceed the plasma concentrations reached in patients treated with rosuvastatin at well-tolerated doses. What is more disturbing, reduction of proliferation and viability observed in BJ fibroblasts indicated that rosuvastatin at high doses may be toxic for normal cells.

Introduction

Melanoma remains the leading cause of deaths from skin cancer. For this reason, growing attention has recently been paid to the inhibition of carcinogenesis at all three stages, initiation, promotion and progression, with the use of synthetic or naturally occurring chemical compounds, which is collectively termed chemoprevention [1, 2]. Given their widespread availability and documented long-term safe use, particular attention has been drawn to statins that act through the inhibition of the mevalonate pathway. Reduced levels of non-steroidal intermediate products of the cholesterol synthesis pathway, e.g. farnesyl pyrophosphate and geranylgeranyl pyrophosphate, impairs prenylation of various signaling proteins involved in the regulation of cell proliferation, differentiation and apoptosis which may modulate cancer cells biology [3-5].

Several reports have indicated that statins possess anticancer properties under in vitro conditions. Time- and dose-dependent viability reduction has been observed in a number of cancer cells treated with different statins. In human melanoma cell lines, lovastatin has been shown to reduce viability/proliferation and induce caspase-dependent apoptosis through a geranylation-specific mechanism [6]. Similar results have been obtained using simvastatin. Viability reduction, DNA fragmentation, cell cycle arrest and subsequent increase in the mRNA levels of p21 and p27 have been observed after prolonged incubation with the tested drug. However, the level of sensitivity to simvastatin is different in various cell lines used in this study [7]. In turn, atorvastatin has been reported to inhibit rho geranyl-geranylation and thus reduce the metastatic potential of human melanoma cells in vitro [8].

To our knowledge, rosuvastatin activity against melanoma cells has not been assessed to date. Its anti-proliferative and cytotoxic activity has been demonstrated in the case of thyroid cancer cells in vitro. Rosuvastatin treatment caused an increase in caspase-3 activity and apoptosis confirmed by DNA fragmentation analysis [9]. Viability reduction has also been noted in hepatic, breast and cervical cancer cell lines [10]. Rosuvastatin has also been reported to reduce the cellular proliferation, colony formation and invasive potential of prostate cancer cells [11].

Aim

A growing body of literature reports indicates that statins may possess chemopreventive activity against melanoma cells through pleiotropic activity. Rosuvastatin is the only drug of this group that has not had its activity against melanoma cells assessed to date. For this reason, the analysis of anti-proliferative and cytotoxic activity of rosuvastatin under in vitro conditions was the aim of this work.

Material and methods

Cell culture

Human melanoma cell lines (A375 and WM1552C) and normal fibroblasts (BJ) were obtained from the American Type Culture Collection. Cells were routinely cultured in DMEM/Ham's F-12 supplemented with 10% fetal bovine serum, 5 µg/ml amphotericin B, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, Germany). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

Viability measurement

Cells were seeded on 24-well plates (BD Biosciences, USA) at a density of 1 × 104 per well for A375 cell line, 3 × 104 for BJ cell line, and 5 × 104 for WM1552C cell line. Differences in cell seeding density were due to the different growth characteristics of cultured cells that, at the time of use for a test, should be in a logarithmic growth phase. After 48 h of preincubation, cells were treated for 72 h with rosuvastatin at concentrations ranging from 0.01 µM to 10 µM. Cell viability was assessed with a tetrazolium-based colorimetric MTT assay. After 2 h of incubation with MTT solution (500 µg/ml, Sigma-Aldrich, Germany), formazan crystals were dissolved in dimethyl sulfoxide (POCH, Poland) and absorbance was measured at 570 nm using a UV-VIS spectrophotometer (Varian CARY 1E UV-Vis, Agilent Technologies, USA). Cell viability was expressed as a percentage of the untreated control.

Proliferation analysis

Cells were seeded on 12 mm diameter coverslips (WITKO, Poland) at 5 × 103 for A375 cell line, 15 × 103 for BJ cell line and 25 × 103 for WM1552C cell line. After 48 h of preincubation, cells were treated with rosuvastatin at concentrations ranging from 0.1 µM to 5 µM for 72 h. Cell proliferation was measured on the basis of Ki67 expression. Cells seeded on coverslips were washed with PBS (Sigma-Aldrich, Germany) and fixed in 2% formaldehyde (Sigma-Aldrich, Germany). Before permeabilization with 0.1% Triton X-100 (Sigma-Aldrich, Germany), non-specific binding sites were blocked with 2% BSA (Sigma-Aldrich, Germany). Cells were then incubated with primary rabbit antibody against Ki67 (dilution 1: 100, 1.5 h, RT) (Novusbio, USA). After washing with Triton X-100 and a second incubation with BSA, cells were incubated with goat anti-rabbit FITC conjugated secondary antibody (dilution 1: 1500, 1 h, RT) (Sigma-Aldrich, Germany). Before counterstaining with DAPI (0.1 µg/ml) (Sigma-Aldrich, Germany), coverslips were washed in PBS and finally mounted in Aqua-Poly/Mount (Polysciences, USA). Ki67 expression was analyzed using NIS-Elements 4.0 software in an Eclipse E800 fluorescence microscope (Nikon, Japan). The number of stained cells per 10× microscope field was counted. Cell proliferation was expressed as a percentage of the untreated control.

Statistical analysis

Experiments were performed in triplicate. All data were presented as means ± SD. The means were compared using one-way ANOVA, followed by Tukey's post-hoc test using Graph Pad Prism 6.0 (Demo Version). Values of p lower than 0.05 were considered statistically significant.

Results

Cells viability assessed on the basis of MTT assay

Rosuvastatin reduced the viability of A375 melanoma cells and BJ fibroblasts in a dose-dependent manner (Figure 1). After 72 h treatment with rosuvastatin at a concentration of 5 µM, the metabolic activity of A375 cancer cells was reduced by 79.2% and the calculated half maximal inhibitory concentration (IC50) was 2.3 µM (Table 1). Rosuvastatin-treated cells were rounded and detached from the growth surface (Figure 2). In turn, WM1552C melanoma cells were insensitive to rosuvastatin at concentrations ranging from 0.01 µM to 2.5 µM. Slightly reduced metabolic activity was observed when cancer cells were incubated with the tested drug at a concentration of 5 µM (Figure 1). Rosuvastatin significantly reduced the viability of normal BJ fibroblasts. At a concentration of 5 µM, cells displayed a more than 60% decrease in viability. Calculated IC50 was 7.4 µM (Table 1). After treatment with increasing concentrations of rosuvastatin, fibroblasts became shrunken and partly lost their adherent phenotype (Figure 2).

Figure 1

Cells viability after 72 h treatment with rosuvastatin assessed on the basis of MTT assay

Table 1

Half maximal inhibitory concentration (IC50) values for rosuvastatin (± SD)

Cell line	IC50 [µM]
A375	2.3 ±0.3
WM1552C	43.9 ±2.8
BJ	7.4 ±0.7

Figure 2

Cells morphology after 72 h treatment with rosuvastatin. Images were taken on a Nikon phase-contrast microscope (magnification 10×)

Cell proliferation assessed on the basis of Ki67 expression

Incubation with rosuvastatin reduced the expression of Ki67 proliferation markers in both melanoma cell lines, but only at a concentration of 5 µM. After 72 h treatment, Ki67 expression in A375 cells was reduced by 29.6% in comparison to the control. In the same conditions, the number of Ki67-positive WM1552C cells was reduced by 14.2% (Figure 3 A). In turn, a significant and dose-dependent reduction of Ki67 expression was observed in normal BJ fibroblasts (Figure 3 B). At a concentration of 0.1 µM, the number of positive cells was reduced by 20.1%, and at 5 µM more than 88.2% of cells were Ki67-negative (Figure 3 A).

Figure 3

Cells proliferation after 72 h treatment with rosuvastatin assessed on the basis of Ki67 expression. A – Proliferation expressed as a percentage of the untreated control. B – BJ cells stained for Ki67. Images were taken on a Nikon fluorescence microscope (magnification 100×). Arrow indicates Ki67-negative cell. Scale bar, 200 µm

Discussion

In recent years, growing interest has been focused on cancer chemoprevention that is defined as the use of natural or synthetic compounds to prevent, inhibit or reverse the multi-step process of carcinogenesis and its secondary prevention (early diagnosis of skin melanoma) [12, 13]. The use of statins as melanoma chemopreventive agents has been based on epidemiological data suggesting their effect on melanoma incidence [14]. Numerous reports from preclinical studies have confirmed the cytotoxic and anti-proliferative effects of statins on melanoma cell lines [6, 7, 15] and animal models [16, 17]. Meta-analysis of clinical trials has not, however, been able to demonstrate any correlation between statin use and melanoma incidence [18-20]. The most recent literature report showed that, instead of preventing melanoma incidence, statins at well-tolerated doses might reduce the growth and metastatic spread of melanoma cells and improve survival [21].

Inhibition of the mevalonate pathway depresses the synthesis of non-steroidal isoprenoids that, through post-translational modification, activate e.g. small G proteins involved in various cellular processes such as proliferation, differentiation and apoptosis. Thus, by influencing protein prenylation statins may alter the biology of cancer cells [22]. Studies on melanoma cell lines by Shellmann et al. demonstrated that the inhibition of protein prenylation mediated by geranylgeranyl pyrophosphate reduces cell viability and induces apoptosis via a geranylation specific mechanism. After 72 h treatment with lovastatin at a concentration of 4 µM, cell viability was reduced by 30% to 80%, depending on the melanoma cell line. Similar to our observations, lovastatin-treated cells were rounded in shape and detached from the growth surface [6].

The varying sensitivity of melanoma cell lines to simvastatin was demonstrated by Saito et al. Simvastatin showed anti-proliferative activity against melanoma cells through induction of apoptosis and cell cycle arrest. Time- and dose-dependent cytotoxicity was also observed in normal human fibroblasts, which is consistent with our results. The addition of geranylgeranyl pyrophosphate to the culture medium completely reversed simvastatin-induced inhibition of cell growth, indicating that protein prenylation is implicated in the decrease in cell viability [7]. The inhibitory effects of statins on melanoma cell proliferation and viability have been confirmed by other research groups [14, 23, 24].

Studies on a murine B16F10 melanoma cell line confirmed the cytotoxic activity of statins in vitro [25]. However, in an animal model fluvastatins failed to reduce tumor growth [26]. In turn, atrovastatin and fluvastatin significantly inhibited lung metastasis. The observed inhibitory effect was due to reduced expression of matrix metalloproteinases, integrin α2, integrin α4, integrin α5 and reduced adhesion to extracellular matrix proteins, i.e. type I collagen, type IV collagen, fibronectin, and laminin [15]. These results indicate a prophylactic potential of statins against metastasis that should be further explored.

Conclusions

The results of our study showed significant differences in the sensitivity of melanoma cell lines to rosuvastatin. What is more concerning, rosuvastatin used at the same concentration range exhibited cytotoxic and anti-proliferative activity against normal human fibroblasts. Investigation of both mechanisms involved in the anti-proliferative and anti-metastatic activity of rosuvastatin and whether these effects are reversible with the addition of geranylgeranyl pyrophosphate is necessary to evaluate the anti-melanoma activity of rosuvastatin.