ANTICANCER RESEARCH 24: 1817-1822 (2004)

Mevastatin-induced Apoptosis and Growth Suppression in U266 Myeloma Cells

JUDIT JÁNOSI1, ANNA SEBESTYÉN2, JÓZSEF BOCSI2, GÁBOR BARNA2, KATALIN NAGY2, ISTVÁN VÁLYI-NAGY1 and LÁSZLÓ KOPPER2

1Department of Haematology, National Medical Center, Budapest; 2Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

Abstract. Statins have been used successfully in the treatment statins have commonly been used in the last few years to treat of hypercholesterinaemia. Moreover, in vitro studies have shown hypercholesterinaemia and to prevent and treat that statins can trigger apoptosis in a variety of tumor cell lines. atherosclerosis of the coronary arteries (4). Mevastatin, a In the present study we analysed the effect of mevastatin –a novel inhibitor of HMG-COA reductase, the rate-limiting novel inhibitor of HMG-COA reductase, the rate-limiting enzyme of the mevalonate pathway (5) and mevalonic acid is enzyme of the mevalonate pathway– on U266 human myeloma an essential precursor of isoprenoid compounds including cells. Apoptosis induced by mevastatin was associated with farnesyl isoprenoid, cholesterol etc. There are scattered data increased caspase activity and depolarisation of the that statins could have an antiproliferative effect. Lovastatin mitochondrial membrane. Expression of Bcl-2 mRNA and has been shown to arrest both tumor and normal cells in the protein was down-regulated, with no change in Bax or Bcl-XL G1-phase of the cell cycle (6,7). Two clinical studies showed protein production. The mitochondrial program was supported reduction in the number of newly diagnosed colorectal cancer by caspase-8 and cleaved-Bid activity. None of the antibodies during a five-year follow-up period in patients reciving neutralizing the death-ligand/death-receptor pathway –TRAIL- pravastatin and simvastatin (8,9). Moreover, in vitro studies R2Fc, anti-TNF-·, anti- FASL(NOK-1)– influenced the have shown that statins can trigger apoptosis in a variety of mevastatin-induced apoptosis. Mevastatin also stimulated tumor cell lines. shedding of syndecan-1 from the surface of myeloma cells. The Apoptosis is an important mode of cell death (10). The apoptosis inducing effect of mevastatin could be considered as apoptosis program is switched on either by internal inducers a potential participant in a complex antitumor protocol. increasing mitochondrial membrane permeability or by the death ligand/receptor complex. In both cases, the The limited success of cancer chemotherapy calls for new and downstream caspase system is the main executor (11,12). better drugs with more relevance to the regulation of tumor Apoptosis is a critical process in normal B-cell function and, growth and progression. Generally, antiproliferative and in many cases, the abnormalites in the apoptotic pathways proapoptotic functions are still the leading targets, but DNA contribute to the pathogenesis of B-cell malignancies has to share the center stage with other, mainly signal (13,14). This scenario has particular relevance to multiple transducing, molecules. Some proteins (e.g. lamin B and myeloma (MM), which is characterized by the continuous p21RAS) are posttranslationally modified by isoprenylation (1). accumulation of malignant plasma cells in the bone marrow p21RAS is a key regulator of cell survival (2). Since mutant (15). It is possible that protective mechanisms, which inhibit p21RAS function has been considered as an important or suppress apoptosis, may participate in the induction and contributor to tumor development and progression, it is maintenance of a malignant MM clone (16). suggested that the inhibition of RAS-prenylation (e.g. by The aim of the present study was to determine the effect hydroxymethylglutaryl coenzyme A (HMG-COA) reductase of mevastatin on proliferation and apoptosis in U266 inhibitors) may have therapeutic value (2,3). A family of myeloma cells.

Materials and Methods

Correspondence to: Judit Jánosi, M.D., Department of Haematology, Cell culture. Experiments were performed on a human U266 National Medical Center, Szabolcs u. 33-35, Budapest, Hungary. myeloma cell line. The cells were cultured in RPMI-1640 (Sigma- Tel: 00-36-06-20-9-441-501, e-mail: [email protected] Aldrich, St Louis, MO, USA), with 10% fetal bovine serum (Gibco- BRL, Rockville, MD, USA), 0.03% glutamine and pen-strep, at Key Words: Mevastatin, multiple myeloma, apoptosis, caspase. 37ÆC, in 5% CO2 atmosphere. For all experiments cells growing in

0250-7005/2004 $2.00+.40 1817 ANTICANCER RESEARCH 24: 1817-1822 (2004) the exponential phase of the culture were used. Cells were treated Primers: with mevastatin in doses of 0.5-1-2.5 Ìg/ml for 0-96 h in 24-well ‚-actin 538bp: 5,GTG-GGG-CGC-CCC-AGG-CAC-CA3, 5,CTC- plates/flasks at a density of 6 x105 cells/ml. CTT-AAT-GTC-ACG-CAG-GAT-TTC3, Bcl-2 389 bp: 5,CGA-CTT-CGC-CAG-GAT-GTC-CAG-CCA-G3, Chemicals. Mevastatin (Sigma-Aldrich) was dissolved in DMSO (10 5,ACT-TGT-GGC-TCA-GAT-AGG-CAC-CCA-G3, mg/ml) and was stored at –20ÆC. To study the role of caspases Bax 517 bp: 5,CAT-GAA-GAC-AGG-GGC-CCT-T3, 5,CAT-CTT- ZVAD-fmk (Enzym System Product, Dublin, CA, USA), a general CTT-CCA-GAT-GGT3. caspase inhibitor, Z-IETD-fmk (Pharmingen, Franklin Lakes, NJ, USA), a caspase-8 inhibitor, and Z-LEHD-fmk (Pharmingen), a Caspase activity. The cells were washed in PBS and the caspase-3 caspase-9 inhibitor, were given in 50-100 ÌM to mevastatin-treated activity was detected by fluorigenic substrate, DEVD-AMC (Sigma- cultures. In the long mevastatin treatment period (72-96 h), the Aldrich) in PBS–EGTA buffer (5 mM EGTA, 1 mM DTT, 12.5 mM caspase inhibitor was provided every 24 h. DEVD-AMC) with excitation at 380 nm and extension at 460 nm. Each sample contained 5x105/ml cells. Samples were measured in a Nuclear morphology. After cytospin preparation, the cells were fixed Fluoroskan Ascent Fluorimeter (Labsystems, Aachen, Germany). in 80% methanol and stained with HE and examined under light Data was analysed by using Microcal Origin Software (Microcal microscope. Software, Northampton, MA, USA).

Cell cycle analysis and apoptosis measurements. Ethanol-fixed cells Depolarisation of mitochondrial membrane. To detect the were pelleted in centrifuge (Wifuge, 1500 rpm) and suspended in mitochondrial membrane depolarization, 5x105 cells were incubated 500 Ìl alkalic buffer (200 mM disodiumphosphate, pH 7.8, adjusted with DiOC6 (20nM, 3,3-dihexylocarbocyan-iodide, Sigma-Aldrich) with 200 mM citrate, and contained 100 Ìg/ml RNase (Sigma- and propidium iodide for 15 min and the changes of fluorescence Aldrich). The samples were left at room temperature for 30 min were measured by flow cytometry (at 530-620 nm). and 5 Ìl ethidium bromide (Calbiochem, Darmstadt, Germany) was given (final concentration: 10 Ìg/ml). The cells were kept for Detection of Bax translocation. The treated and control cells were another 15 min before flow cytometric measurements to estimate incubated with CMTMRos (Molecular Probes) (5 ÌM) for 15 min. cell cycle and proportion of sub-G1 (apoptotic) cells. The flow After washing with PBS, they were fixed in ice-cold 80% methanol. cytometric measurements were made on a FACSCAN flow We used a polyclonal antibody (1:20) specific for Bax (DAKO) and cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and, for the reaction was developed by Vectastain ABC Kit and each sample, data were collected from 6-10,000 cells. Data were Streptavidin–FITC (DAKO). Samples were examined by confocal analysed using a List software (Verity Software House, Topsham, microscope (Bio-Rad, MRC1024). ME, USA). TRAIL, TNF-alfa, FASL neutralization. TÔ study the role of autocrine Western blot. Treated cells were washed in 0.9% NaCl solution twice, TRAIL, TNF-alfa and FASL in the mevastatin-induced apoptosis, pelleted (1300 g, 5 min, Sigma-Aldrich, 3K10 Laborcentrifuge), the potential effects of these death ligands were neutralized using resuspended in lysis buffer (15 mM NaCl, 5 mM Tris-HCl, 1 mM recombinant TRAIL-R2Fc (Alexis, San Diego, CA, USA, 100ng/ml), NaF, 10% glycerol, 1% NP40, 0.5 mM sodium-vanadate, 1 mM monoclonal anti-human TNF-alfa (Alexis, 1 ng/ml) or NOK-1 (anti- PMSF, 10 Ìl/mg leupeptin) and incubated for 10 min on ice. After FASL antibody, Pharmingen, 1 ng/ml). incubation, samples were centrifuged (20 min, 15000g, 4ÆC) and the protein content was measured from the supernatants by Bradford Syndecan-1 measurement by flow cytometry. Cells were fixed in ice- assay. Thirty Ìg of total protein was used for Western blot on a cold 80% methanol and incubated with anti-syndecan antibody 12.5% polyacrylamide gel and electrotransferred onto PVDF (Serotec, Düsseldorf, Germany, MCA681, 1:200). The reaction was membrane (Bio-Rad, Hercules, CA, USA). Monoclonal antibodies developed with biotinylated anti-mouse antibody for 45 min for Bcl-2 (DAKO, Glostrup, Denmark, 1:1000), Bid (Transduction followed by washing and subsequent addition of avidin-FITC. Cells Laboratories, Lexington, KS, USA 1:1500) and polyclonal antibody were analysed using FACSCAN flow cytometry. for Bax (DAKO, 1:2000) were used for Western blot analysis. Developing was performed by Vectastain ABC Kit and ECL+Plus Statistical analysis. Results are expressed as mean±SEM and were chemiluminescense detection kit (Amersham Pharmacia Biotech, analysed using Student's paired t-test. Buckinghamshire, UK). Results RT-PCR. Total RNA was isolated from the 10 x 106 cells by Qiagen RNeasy kit using standard procedures. Complementary DNA was Effect of mevastatin on U266 cells. Mevastatin inhibited cell synthesized from 100 ng RNA using MMLV reverse transcriptase proliferation and induced apoptosis in U266 cells. and random primers (Gibco-BRL). In semiquantitative PCR, ‚- Proliferation was blocked mainly in G1-phase, indicated by actin was used as a control to monitor amplification efficiency and the increase of cells with G1 DNA content (data not shown). the quality of the cDNA. Parameters of PCR reaction: 65ÆC, 1 The mevastatin-induced apoptosis was time (24-96 h)- and min, 72ÆC, 1 min, 28 cycles. In PCR reactions red-Taq polymerase dose (1-2.5 Ìg/ml)-dependent. The apoptotic response was enzyme (Sigma-Aldrich) was used. PCR products were analysed in 1.5% agarose gel electrophoresis, visualised by ethidium bromide relatively slow, appearing after 48 h and reaching the highest staining and evaluated by Eagle Eye video densitometer values at 72-96 h (Figure 1). These changes were accompanied (Strategene, La Jolla, CA, USA). by the fragmentation of the nuclei.

1818 Jánosi et al: Mevastatin-induced Apoptosis in Myeloma Cells

Figure 1. Time-dependent apoptotic effect of mevastatin detected by FACS analysis. The percentage of apoptotic cells after 0.25 – 1 – 2.5 Ìg/ml mevastatin treatment in U266 myeloma cells.

The mechanism of the induced apoptosis. The role of caspases was proved by the inhibitory effect of Z-VAD-fmk on the mevastatin-induced apoptosis in U266 cells (Figure 2A). Furthermore, caspase-3 is a key protease in the late phase of the apoptosis. Mevastatin treatment was followed by an increase of the cleaved colorimetric substrate of caspase-3, indicating that caspase-3 was activated (Figure 2B). Apoptosis was also inhibited in cells pretreated with caspase-8 inhibitor or caspase-9 inhibitor (Figure 3A,B). Using DiOC6 staining, a dose-dependent depolarization of the mitochondrial membrane was found at 48 h after mevastatin treatment (Figure 4). Among the factors regulating mitochondrial membrane permeability, the expression of Bcl-2 mRNA was down-regulated in U266 cells (Figure 5A), but the expression of Bax and Bcl-XL was not influenced (data not shown). The Western blot analysis Figure 2. A. The effect of Z-VAD-fmk in mevastatin-induced apoptosis. supported the decreased expression of Bcl-2 protein (Figure The percentage of apoptotic cells was measured by flow cytometry after 5B). The expression of proapoptotic Bax protein was not 100ÌM Z-VAD-fmk and 1Ìg/ml mevastatin (Meva). changed, however the Bcl-2/Bax ratio was decreased and the B. Activation of caspase-3 after 1-2.5Ìg/ml mevastatin (Meva) treatment. Time-dependent activity of caspase-3 was measured by DEVD-AMC location of Bax protein was also changed. After 72-h (MFU: microfluorescense unit). mevastatin treatment, confocal microscopy suggested that the homogenous cytoplasmic Bax staining was replaced by a bright mitochondrial staining (data not shown). We studied the cleavage of Bid by Western blot analysis: the effect of mevastatin, suggesting that the change in caspase-8 detectable fragment of Bid decreased at 72 h after activity is independent from death-receptors (Table I). mevastatin treatment (blot not shown). The potential role of the death-ligands (TRAIL, TNF·, Syndecan-1 shedding. There are observations that apoptosis FasL) in the mevastatin- induced apoptosis was checked by of myeloma cells could be accompanied by the shedding of neutralization experiments. None of the neutralizing cell surface syndecan-1. Our results further supported this antibodies (TRAIL-R2Fc, anti-TNF·, Nok1) influenced the finding: flow cytometric analysis showed that the level of cell

1819 ANTICANCER RESEARCH 24: 1817-1822 (2004)

Figure 3. The role of caspase-8 and caspase-9 in 2.5Ìg/ml mevastatin (Meva)-induced apoptosis. The percentage of apoptotic cells was measured by flow cytometry. A. Caspase-8 (C8) was inhibited by Z-IETD-fmk (C8inh) (50ÌM). B. Caspase-9 (C9) was inhibited by Z-LEHD-fmk (C9inh) (50ÌM).

proliferation and induces cell cycle arrest and apoptosis in the human adenocarcinoma cell line CaCo2 (17). The tumor cell types that undergo apoptosis upon exposure to lovastatin, another HMG-CoA reductase inhibitor, include cells from myeloma, prostate cancer, acute myeloid leukemia, pancreatic cancer and colon cancer (18-22). Apoptosis in response to lovastatin is associated with release of cytochrome c, activation of caspase-3, PARP cleavage and down-regulation of Bcl-2 mRNA and protein in AML cells (23,24). In this study, we found that mevastatin induced cell cycle arrest and apoptosis in U266 human myeloma cells. Apoptosis induction was dependent on caspase-3 activity, which is a cricial step in almost all apoptotic response. It Figure 4. Time-dependent mitochondrial membrane depolarization after 1-2.5Ìg/ml mevastatin treatment. seems that both caspase-8 and caspase-9 took part in caspase The mitochondrial membrane depolarization was detected by DiOC6 activation, but the extrinsic (death receptor) and intrinsic (20nM) and the percentage of cells with depolarized mitochondria was (mitochondrial) apoptotic pathways reacted differently. (The evaluated by flow cytometry (530-620nm). involvement of caspases was proved by the use of different caspase inhibitors.) Caspase-8 could be activated either by death receptors or surface syndecan-1 expression and the number of syndecan- by other caspases. In U266 cells, none of the death ligand 1-positive cells was time-dependently reduced during neutralizing antibodies – TRAIL-R2Fc, anti-TNF· and anti- mevastatin-induced cell death (24h: 57.33 %, 48h: 53.4%, FasL(Nok-1) – influenced the mevastatin-induced apoptosis. 72h: 54.4% of syndecan-1-positive cells in control and 24h: Therefore the second option is highly possible, i.e. caspase-8 31.8%, 48h: 28.7%, 72h: 5.91% in treated samples). was activated by other caspases (e.g. caspase-3) through the activity of the mitochondrial pathway. In that case, caspase-8 Discussion serves as a helper to speed up apoptosis by cleaving Bid, which induces the release of cytochrome c from mitochondria. It has recently been found that statins can induce apoptosis Cytochrome c in complex with Apaf-1 activates caspase-9, in a variety of malignant cells. Mevastatin inhibits cell which in turn activates caspase-8 (25).

1820 Jánosi et al: Mevastatin-induced Apoptosis in Myeloma Cells

Figure 5. Expression of Bcl-2 after mevastatin treatment in U266 cells. A. Time-dependent (1., 5.: control; 2., 6.: 24h; 3., 7.: 48h; 4., 8.: 72h) expression of ‚-actin and Bcl-2 mRNA detected by RT-PCR after 2.5 Ìg/ml mevastatin treatment. B. Western blot analysis of Bcl-2 protein expression after 72-h mevastatin treatment (1.: 2.5Ìg/ml mevastatin, 2.: 1Ìg/ml mevastatin, 3.: control).

Many data of our study supported that mevastatin acts Table I. The effect of TRAIL (anti-TRAIL), TNF-· (anti-TNF·) and primarily through the intrinsic apoptotic machinery. Using FASL (NOK1) neutralization in mevastatin-induced apoptosis. DiOC , it was found that the depolarization of mitochondrial The percentage of apoptotic cells was measured by flow cytometry after 6 48h, 72h treatment. membrane increased at 48 h after treatment. Expression of Bcl-2 (both mRNA and protein) decreased, while Bax was 48h 72h translocated from the cytosol to the mitochondria, as shown Control 10.61±0.49% 12.45±1.48% by confocal microscopy. Finally, the caspase-9 inhibitor Mevastatin 49.65±4.17% 64.5±7.77% blocked the mevastatin-induced apoptosis. The time-course Nok1 10.33±0.61% 11.09±1.23% of apoptosis –the relatively slow start and speeding up later– Anti-TNF· 9.75±0.31% 13.18%±1.38% also support the early involvement of the mitochondrial Anti-TRAIL 13±1.41% 15±0% Metastatin+Nok1 39±4.24% 60±4.27% components with gradual help of caspase-8. Metastatin+Anti-TNF· 43.5±3.52% 58±5.65% It has been shown that a transmembrane heparan sulfate Metastatin+Anti-TRAIL 46±2.82% 56±2.86% glycosaminoglycan, syndecan-1, is expressed by plasma cells as well as myeloma cells (26,27) , but the exact role of syndecan-1 expression is still unknown. The shedding of the extracellular part of syndecan-1 was also observed during the apoptosis of patients with gastric adenocarcinoma, and 1 out of 14 patients myeloma cells, though the mechanism again is unknown (28). responded for 16 weeks. Lovastatin was well tolerated, We also found that mevastatin-induced apoptosis was without neurological, hematological, liver or renal toxicity accompanied by a reduced level of cell surface syndecan-1 in (30). An elderly patient with relapsed AML was treated with U266 cells. It is highly possible that syndecan-1 shedding and lovastatin (2mg/kg/day for 54 days). This regimen was its appearance in the circulation could be a clinical marker for effective in managing blast counts during the period of drug apoptosis in myeloma cells. administration (31). Pravastain given to patients with Clinical trials are going on to establish statins as hepatocellular carcinoma (0.5mg/kg/day) in conjuction with chemotherapeutic agents. In a phase I clinical trial, one 5-fluorouracil doubled the median of survival time (32). minor response was observed at 30-35 mg/kg/day in a patient It is unrealistic that statins could be used as single diagnosed with anaplastic astrocytomas (29). There was no antitumor agents. However, the potentiation of antitumor evidence of efficacy in patients with breast, prostate, ovarian activity of the existing chemotherapeutic regimens by statins and primary central nervous system malignancies (29). A could extend the therapeutic efficiency of such combinations phase II study of high-dose lovastatin (35 mg/kg/day orally for by opening up or inducing apoptotic response. This view is a week, supplemented with ubiquinone) was conducted in supported by our findings with mevastatin in myeloma cells.

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Leukemia 16: 1362-1371, 2002. Accepted April 6, 2004

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