Inhibitors of the mevalonate pathway as potential therapeutic agents in multiple myeloma

Leukemia Research 31 (2007) 341–352

Inhibitors of the mevalonate pathway as potential therapeutic agents in multiple myeloma Cindy Baulch-Brown a , Timothy J. Molloy b , Sung Lin Yeh a , David Ma b , Andrew Spencer a,c,∗ a

c

Myeloma Research Group, Department of Clinical Haematology and Bone Marrow Transplantation, Ground Floor, South Block, Alfred Hospital, Commercial Road, Melbourne, Vic. 3004, Australia b Haematology Research Unit, St Vincent’s Hospital, Sydney, NSW 2010, Australia Department of Medicine, Monash University, Monash University Medical School, Alfred Hospital, Melbourne, Vic. 3004, Australia Received 20 March 2006; received in revised form 13 July 2006; accepted 14 July 2006 Available online 22 September 2006

Abstract Clinical studies have suggested that bisphosphonates may prolong the survival of sub-sets of myeloma patients. Newer nitrogen containing bisphosphonates such as zoledronate act, at least in part, by inhibiting farnesyl diphosphate synthase and subsequent protein prenylation, furthermore, limited data suggests that zoledronate exerts a direct anti-tumour effect against human myeloma cell lines. We therefore investigated the anti-myeloma potential of zoledronate in comparison to, and in combination with, two other inhibitors of the mevalonate pathway: the HMGCoA reductase inhibitor fluvastatin and the farnesyl transferase inhibitor SCH66336. We found that fluvastatin was able to inhibit the proliferation of myeloma cells more effectively than zoledronate or SCH66336 and that combinations of zoledronate and fluvastatin, but not zoledronate and SCH66336 acted synergistically. Our data indicated that the anti-proliferative effect of mevalonate pathway inhibitors is mediated principally via prevention of geranylgeranylation and is the result of both cell cycle arrest and apoptosis induction. Microarray and quantitative real-time PCR analyses further demonstrated that genes related to apoptosis, cell cycle control, and the mevalonate pathway were particularly affected by zoledronate and fluvastatin, and that some of these genetic effects were synergistic. We conclude that the mechanisms of geranylgeranylation inhibition mediated anti-myeloma effects warrant further evaluation and may provide novel targets for future therapeutic development. © 2006 Elsevier Ltd. All rights reserved. Keywords: Myeloma; Apoptosis; Prenylation; Bisphosphonates; HMGCoA reductase inhibitors

1. Introduction Multiple myeloma (MM) is an incurable malignancy characterised by the infiltration of malignant plasma cells in the bone marrow, hypersecretion of monoclonal antibodies and osteolytic bone lesions [1]. Although initially responsive to therapy, the majority of MM patients relapse and die as ∗ Corresponding author at: Myeloma Research Group, Department of Clinical Haematology and Bone Marrow Transplantation, Ground Floor, South Block, Alfred Hospital, Commercial Road, Melbourne, Vic. 3004, Australia. Tel.: +61 3 9276 3392; fax: +61 3 9276 2298. E-mail address: [email protected] (A. Spencer).

0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.07.018

a result of acquired drug resistance [2]. Novel therapeutic approaches are therefore required. Long-term administration of the bisphosphonate (BP) pamidronate has been shown to reduce skeletal complications and improve quality of life in patients with MM [3]. Furthermore, MM patients undergoing second line chemotherapeutic treatment in combination with pamidronate have a survival advantage compared to similar patients not receiving pamidronate (21 months versus 14 months, p = 0.04) [4] suggesting a direct or indirect, via modulation of the bone marrow micro-environment, antimyeloma effect. Of particular interest is the anti-tumour potential of newer generation bisphosphonates such as zoledronate. Evidence from in vitro studies by a number of

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groups indicates that nitrogen-containing BPs such as zoledronate interfere with osteoclast recruitment, differentiation and action, and induce apoptotic cell death of these cells by inhibition of farnesyl diphosphate synthase and subsequent disruption of the mevalonate pathway [5]. The mevalonate pathway plays an important role in cell growth and survival. Mevalonate is synthesised intracellularly from 3 -hydroxy-3-methylglutaryl coenzyme A (HMGCoA) in a process catalysed by HMGCoA reductase, the ratelimiting enzyme in this pathway [6]. Mevalonate metabolism yields a series of isoprenoid compounds that are incorporated into cholesterol, isopentenyl adenine, prenylated proteins and other end products essential for cell growth. Statins are competitive inhibitors of HMGCoA reductase and have been shown to not only block synthesis of mevalonate but to inhibit the growth and proliferation of both normal and tumour cells [7–9]. Furthermore, in some experimental systems, inhibition of HMGCoA reductase has been shown to induce cell death by apoptosis induction [8,9]. Disruption of the mevalonate pathway prevents synthesis of the isoprenoids farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GPP). These isoprenoids are used for posttranslational modification of a variety of proteins including Ras and Ras-related GTP binding proteins; with isoprenylation being essential for membrane attachment and participation of the modified proteins in signal transduction processes [9]. Ras signalling cascades play an important role in MM. Binding of IL-6 to its receptor activates the JAK-STAT and Ras-MAPK pathways, resulting in proliferation of MM cells and protection from apoptosis [10]. Similarly, activating Ras mutations enable IL-6 independent growth and dexamethasone and doxorubicin-resistant phenotypes [11]. Based on these data we have investigated the anti-MM potential of the mevalonate pathway inhibitors fluvastatin and zoledronate and the farnesyl transferase inhibitor SCH66336.

2. Materials and methods 2.1. Cell lines Human multiple myeloma cell lines (HMCL) RPMI 8226, U266 and NCI-H929 were purchased from the American Type Culture Collection (Rockville, USA). OPM2 and LP-1 were purchased from Deutsche SammLung von Mikroorganismen und Zellkulturen (Braunshwieig, Germany). Cells were maintained in RPMI-1640 supplemented with 10% (v/v) foetal bovine serum (FBS) at 37 ◦ C with 5% CO2 .

converted to MVA), farnesol (FOH) and geranylgeraniol (GGOH) were purchased from Sigma, Australia. Cell titer 96 aqueous one solution cell proliferation assay reagent was purchased from Promega, Australia. Propidium iodide/Rnase solution was purchased from Becton Dickinson, USA. Supersignal West Pico Chemiluminescent Substrate was purchased from Pierce, USA. All other chemicals were purchased from Sigma, Australia unless stated otherwise. 2.3. Antibodies Fluorescein isothiocyanate (FITC) conjugated AnnexinV was purchased from Biosource, USA. Polyclonal antibodies to Rap1A, lamin A/C, Mcl-1 and Bcl-XS/L were purchased from Santa Cruz Biotechnology, USA; as were monoclonal antibodies to Bad, Bax, STAT and pSTAT. Monoclonal antibodies to pRb and Bcl2-␣ were purchased from Becton Dickinson and Chemicon, USA, respectively, while monoclonal antibody to tubulin was purchased from Sigma, Australia. Polyclonal antibodies to ERK 1/2 and active MAPK (ERK 1/2) were purchased from Promega, USA, as was donkey anti-rabbit (H + L) horseradish peroxidase (HRP) conjugated antibody. HRP conjugated donkey anti-goat antibodies were purchased from Santa Cruz Biotechnology and sheep antimouse HRP conjugated antibodies were purchased from Amersham Pharmacia, USA. 2.4. Cell proliferation HMCL were seeded in 96 well plates at a concentration of 2 × 105 ml−1 and incubated for 24 and 72 h with various concentrations of mevalonate pathway inhibitors fluvastatin (2.5–50 ␮M), zoledronate (0.01–100 ␮M) and SCH66336 (0.05–5 ␮M) (6× 100 ␮l replicates at each concentration) prior to the addition of 20 ␮l of Promega’s Celltiter 96 Aqueous One Solution Cell Proliferation reagent. Following 3 h incubation 490 nm absorbance was determined using a 96 well plate reader. The role of farnesylated and geranylgeranylated proteins in HMCL survival was assessed by treating the HMCL with each of the mevalonate pathway inhibitors in the presence of 100 ␮M mevalonic acid lactone (MVA), 10 ␮M farnesol (FOH) or 10 ␮M geranylgeraniol (GGOH); and determining viability at 24 and 72 h as described above. Results from the tetrazolium reduction assays were normalised to untreated controls. Results were expressed as mean ± S.E.M. Differences in HMCL viability were analysed using a Student’s paired t-test. 2.5. Analysis of cell cycle and detection of apoptosis using fluorescence-activated cell sorted (FACS) analysis

2.2. Reagents Zoledronate and fluvastatin were gifts from Novartis Pharma, Switzerland. Farnesyl transferase inhibitor SCH66336 was a gift from Schering Plough, Australia. Dexamethasone, mevalonic acid lactone (which is readily

HMCL were treated with 100 ␮M zoledronate, 10 ␮M SCH66336 or 25 ␮M fluvastatin for 24 or 72 h prior to fixing for cell cycle analysis or staining with AnnexinV FITC to detect apoptosis. For cell cycle analysis 1 × 106 cells were resuspended in 5 ml of 1% (w/v) paraformaldehyde in

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PBS and incubated on ice for 15 min. Following two PBS washes of 5 min at 300 × g, cells were resuspended in 5 ml of ice cold 70% (v/v) ethanol and stored for at least 24 h at −20 ◦ C. For analysis, fixed cells were washed twice in PBS as above and resuspended in 0.5 ml of PI/Rnase A solution and incubated for 30 min. Cells were analysed with a FACScan flow cytometer and CELLQuest software (Becton Dickinson, USA). For AnnexinV FITC staining cells were washed once in an excess volume of 1 × AnnexinV binding buffer (2.5 mM CaCl2 , 0.14 M NaCl, 10 mM HEPES pH 7.4). Following centrifugation, 2 × 105 cells were resuspended in 100 ␮l of 1 × AnnexinV binding buffer and incubated with 1 ␮l of FITC-conjugated AnnexinV (Biosource, USA) for 15 min at room temperature. Cells were washed once in 1 × AnnexinV binding buffer and resuspended in a final volume of 200 ␮l. Immediately prior to analysis 0.125 ␮g/ml propidium iodide was added in order to distinguish necrotic cells. The proportion of live cells was enumerated on a FACScan flow cytometer with CELLQuest software (Becton Dickinson, USA). 2.6. Immunoblot analysis To obtain whole cell extracts for immunoblot analysis, 1 × 107 treated or untreated HMCL were washed once in ice cold PBS, pelleted by centrifugation at 500 × g for 5 min and resuspended in 200 ␮l of lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 1× protease inhibitor cocktail (Roche, USA) and 1 mM PMSF). Cell lysate was clarified by centrifugation at 7500 × g for 10 min at 4 ◦ C. Protein concentration was determined using the DCTM protein assay (Bio-Rad, Hercules, CA). Proteins were analysed by SDS-PAGE using the discontinuous buffer system described by Laemmli [12]. Protein samples were prepared in 1× SDS sample buffer, boiled for 5 min and 50 ␮g whole cell extract electrophoresed through a 4% stacking gel and an 8 or 12% acrylamide separating gel. Separated proteins were transferred to nitrocellulose membranes which were subsequently blocked for 1 h at room temperature or 4 ◦ C overnight in 5% (w/v) non-fat dry milk (NFDM) in PBST (PBS containing 0.1% (v/v) Tween 20). After blocking, the membrane was incubated for 1 or 2 h at room temperature with primary antibody diluted to appropriate concentration in 1% (w/v) NFDM-PBST. Membranes were washed 3× for 10 min in PBS-T and incubated for 1 h at room temperature with appropriate HRP-conjugated secondary antibody diluted in 1% (w/v) NFDM-PBST. For optimal detection of phosphorylated proteins, NFDM was replaced with 1% (w/v) bovine serum albumin for blocking and 0.5% (w/v) bovine serum albumin for antibody incubations. Following incubation with secondary antibody the membrane was washed 3× as above and immunoreactive bands detected using SuperSignal West Pico Substrate (Pierce, USA) and visualized by autoradiography. Equal protein loading was confirmed by reprobing membranes with monoclonal antibody to tubulin.

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2.7. Isobologram analysis Isobologram analysis was used to assess whether synergistic anti-HMCL effects were seen when the mevalonate pathway inhibitors were used in combination with each other or the common therapeutic compound dexamethasone. This method allows the identification of interactions between two drugs, regardless of the mechanism of action of the individual drug [13,14]. Dose response curves were plotted for the effect of each drug alone or in combination on the HMCL at 72 h. From these, an appropriate combined inhibition constant (i.e. IC80 is 80% inhibition of cell proliferation) was determined for each curve. The ratio of the combined drug IC to the equivalent IC value of each drug alone was calculated and plotted as an isobologram. 2.8. Microarray analyses Custom microarrays consisting of 19,000 human oligonucleotide features were provided by the Adelaide Microarray Facility (University of Adelaide, Adelaide, Australia). Two micrograms of total RNA extracted from NCI-H929 cells treated with 25 ␮M fluvastatin and/or 100 ␮M zoledronate at 24 and 72 h post-treatment was used to synthesise cDNA into which Cy3 or Cy5 fluorescent dyes (Amersham Pharmacia Biotech, North Ryde, New South Wales, Australia) were incorporated, using an indirect labelling method. Microarrays were scanned using an Axon GenePix 4000B microarray scanner (Molecular Devices, Union City, CA, USA) and the resulting image was analysed using GenePix Pro software Version 3.0 (Axon Instruments, Foster City, CA, USA). Each time-point consisted of two microarrays onto which were hybridised probes synthesised from two separate cell treatments. GeneSpring (Silicon Genetics, Redwood City, CA, USA) was used normalise the data using the LOWESS method [15], and also for subsequent analysis. A gene was considered upregulated or downregulated if it had at least a two-fold difference in expression relative to the control channel. 2.9. Quantitative real-time PCR Quantitative real-time PCR reactions were performed using the Rotorgene 3000 (Corbett Research, Mt. Waverly, Australia) using the dsDNA binding fluorescent dye, SYBRTM Green I. PCR reactions were performed in 0.1 ml thin-walled PCR tubes (Corbett Research). All amplifications were performed at a primer concentration of 200 nM, and Mg2+ concentration of 2 mM. Platinum QPCR SYBR Green Mix (SYBR Green I fluorescent dye, 60 U/ml Platinum Taq DNA polymerase, 40 mM Tris–HCl (pH 8.4), 100 mM KCl, 6 mM MgCl2 , 400 ␮M dGTP, 400 ␮M dATP, 400 ␮M dCTP, 400 ␮M dUTP, 40 U/ml UDG, stabilisers; Invitrogen Life Technologies) was used together with primers and 50–50 ng of template. Thermal cycling programme consisted of denaturation at 95 ◦ C for 1:15 s, followed by 50 cycles of 95 ◦ C

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At 72 h, 25 ␮M fluvastatin significantly inhibited all three HMCL (p < 0.05 by paired Student’s t-test) with inhibition ranging from >50% for U266 to >95% for NCI-H929. Zoledronate (100 ␮M) significantly inhibited NCI-H929 and LP-1 (p < 0.05 by paired Student’s t-test) with inhibition ranging from 35 to 85%. SCH66336 at a concentration of 10 ␮M inhibited NCI-H929 by 45% (p < 0.05 by paired Student’s ttest) but had little effect on the other HMCL (Fig. 1). Three HMCL—LP-1, NCI-H929 and U266 were selected for more detailed analyses based on their differing sensitivity to the test compounds. Apoptosis induction and modulation of cell cycle status was investigated further (Table 1). In all three HMCL, 24 h treatment with SCH66336 or fluvastatin resulted in an increased number of cells in the G0/G1 phases, however, by 72 h only the fluvastatin treated U266 and LP-1 had increased numbers of cells in the G0/G1 phase when compared to an untreated control. NCI-H929 cells at 72 h treated with fluvastatin showed decreased number of cells in the G0/G1 phase and an increased apoptotic sub-G0 population whereas SCH66336 treatment resulted in a decreased S phase and a slightly increased sub-G0 population. Zoledronate did

for 45 s, a 30 s annealing step at varying temperatures, and a 30 s extension step at 73 ◦ C. Fluorescent readings were taken during the extension step of each cycle. Melting curve analysis was also performed to ensure the amplification of a single PCR product. Reactions with no RT and no template were included as negative controls. Samples were run in duplicate for four to six independent cell treatment experiments for every gene. All samples were calculated relative to an average of the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1) genes, which were also amplified for each sample.

3. Results Results from the tetrazolium reduction assays indicated that the effect of fluvastatin, zoledronate and SCH66336 on cell viability varied for each of the HMCL tested. In the majority of HMCL none of the compounds was particularly effective at inducing apoptosis within 24 h (data not shown).

Table 1 Summary of cell cycle data following treatment of HMCL with mevalonate pathway inhibitors SubG0a

pb

G0/G1

p

S

p

G2/M

p

NCI 24 h U F S Z

2.1 ± 0.7 2.3 ± 0.8 2.0 ± 0.6 2.2 ± 1.0

0.24 0.58 0.67

53.3 ± 4.7 77.4 ± 3.6 66.4 ± 8.4 54.5 ± 6.0

0.00 0.07 0.45

16.9 ± 1.2 5.0 ± 1.8 11.2 ± 4.3 15.5 ± 2.4

0.00 0.22 0.36

28.7 ± 4.0 15.7 ± 1.9 21.0 ± 4.3 28.8 ± 4.1

0.03 0.00 0.53

NCI 72 h U F S Z

1.9 ± 0.3 25.0 ± 1.9 4.7 ± 1.4 2.3 ± 0.7

0.01 0.14 0.46

67.1 ± 3.1 62.7 ± 2.4 66.2 ± 3.2 67.5 ± 0.8

0.02 0.58 0.88

11.0 ± 0.9 6.7 ± 1.5 6.8 ± 0.7 9.5 ± 0.5

0.02 0.01 0.22

20.8 ± 2.6 7.5 ± 0.7 23.2 ± 2.1 21.6 ± 0.6

0.02 0.40 0.71

LP-1 24 h U F S Z

2.0 ± 0.3 2.8 ± 0.6 2.9 ± 0.7 3.1 ± 0.9

0.13 0.17 0.21

66.5 ± 7.3 70.5 ± 6.9 72.8 ± 7.0 63.0 ± 8.2

0.28 0.32 0.43

15.5 ± 2.8 13.5 ± 3.8 12.4 ± 4.3 19.9 ± 5.4

0.22 0.19 0.22

17.1 ± 5.5 13.8 ± 4.2 12.3 ± 2.5 15.5 ± 3.1

0.16 0.29 0.60

LP-1 72 h U F S Z

2.4 ± 0.4 6.6 ± 2.6 3.5 ± 0.5 2.2 ± 0.2

0.26 0.04 0.59

53.6 ± 3.3 66.6 ± 8.1 55.5 ± 7.9 57.0 ± 3.8

0.15 0.76 0.42

25.0 ± 3.7 10.3 ± 4.7 17.7 ± 4.8 24.0 ± 4.6

0.01 0.15 0.78

20.3 ± 0.4 13.9 ± 6.0 24.5 ± 3.7 33.8 ± 15.4

0.39 0.33 0.48

U266 24 h U F S Z

2.3 ± 0.5 2.6 ± 1.3 2.6 ± 1.0 2.1 ± 0.5

0.79 0.75 0.02

60.8 ± 1.2 67.6 ± 3.6 65.5 ± 1.7 63.9 ± 1.9

0.17 0.08 0.07

13.9 ± 3.0 8.0 ± 1.5 9.0 ± 2.6 12.4 ± 3.4

0.06 0.01 0.32

23.2 ± 3.6 22.3 ± 5.8 23.4 ± 4.9 22.7 ± 4.5

0.76 0.89 0.72

U266 72 h U F S Z

2.2 ± 0.4 3.8 ± 1.1 1.7 ± 0.0 1.5 ± 0.1

0.15 0.66 0.26

62.0 ± 2.0 72.4 ± 4.2 56.3 ± 7.1 60.8 ± 2.5

0.07 0.52 0.17

10.4 ± 2.1 3.4 ± 0.4 8.4 ± 1.5 11.2 ± 2.5

0.06 0.12 0.41

25.4 ± 2.9 20.6 ± 5.1 26.9 ± 4.1 27.0 ± 3.2

0.16 0.07 0.10

a b

All values are mean ± S.E.M. Treated samples vs. untreated controls—Student’s test; U: untreated; F: fluvastatin 25 ␮M; S: SCH66336; Z: zoledronate 100 ␮M.

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Table 2 Summary of AnnexinV/PI flow cytometry experiments following treatment with mevalonate pathway inhibitors for 72 h Viable (%)a

pb

NCI-H929 Untreated Fluvastatin SCH66336 Zoledronate

82.3 ± 5.8 23.8 ± 7.0 77.3 ± 5.3 81.7 ± 2.0

0.02 0.52 0.91

LP-1 Untreated Fluvastatin SCH66336 Zoledronate

88.2 ± 1.2 62.1 ± 9.0 75.4 ± 6.2 86.1 ± 2.0

0.08 0.12 0.13

U266 Untreated Fluvastatin SCH66336 Zoledronate

84.5 ± 3.1 71.9 ± 7.0 75.2 ± 5.3 86.4 ± 2.0

0.02 0.02 0.12

All values are mean ± S.E.M. Treated samples vs. untreated controls–Student’s test; fluvastatin 25 ␮M; SCH66336; Zoledronate 100 ␮M. a

b

Fig. 1. Mevalonate pathway inhibitor induced apoptosis. (a) Zoledronate (100 ␮M), (b) fluvastatin (25 ␮M) and (c) SCH66336 (10 ␮M) induced varying levels of apoptosis in five HMCL treated for 72 h. Percentage apoptosis is relative to untreated control, data is mean ± S.E. of the mean from three experiments (* p < 0.05 by paired Student’s t-test).

not effect the cycling of LP-1 or U266 cells at any time point but at 72 h NCI-H929 showed a decreased S phase. AnnexinV FITC staining to discriminate between apoptotic and live cells indicated that none of the mevalonate pathway inhibitors induced apoptosis by 24 h (Table 2). After 72 h fluvastatin and SCH66336 reduced the percentage of viable LP-1 and NCIH929 cells, however, neither of these compounds affected the percentage of viable U266 cells. Zoledronate decreased the

percentage of viable NCI-H929 cells after 72 h but had little or no effect on LP-1 or U266 cell lines. Immunoblot analysis indicated that treatment of HMCL with inhibitors of the mevalonate pathway did not inhibit expression of the apoptosis related proteins Bcl-XS/L , Bcl-2, Bad or Bax. In contrast expression of anti-apoptotic Mcl1 and phospho-Rb was inhibited in NCI-H929 or NCI-H29 and LP-1, respectively, following 72 h treatment with fluvastatin (data not shown). The expression of STAT and ERK 1/2 was not effected by the mevalonate pathway inhibitors apart from active ERK that was increased at 72 h following treatment with zoledronate (data not show). Antibodies to Rap1A and lamin A/C were used to determine the protein prenylation status of treated and untreated cells. Rap1A is modified by geranylgeranylation, while an accumulation of pre-lamin A is indicative of decreased farnesylation. Accumulation of unprenylated Rap1A was observed in all HMCL following treatment with fluvastatin or zoledronate (Fig. 2a) at both 24 and 72 h. Accumulation of pre-lamin A was observed for LP1 (Fig. 2b) and NCI-H929 cells treated with fluvastatin, and to a lesser when treated with SCH66336 or zoledronate, at 72 h. Tetrazolium reduction assays carried out in the presence of mevalonate pathway intermediates MVA, FOH or GGOH demonstrated that protein prenylation was crucial for HMCL survival. The effect of zoledronate was not reversed by MVA whereas addition of FOH provided partial protection in three of four HMCL and the addition of GGOH protected three of four HMCL (Fig. 3a). The effect of fluvastatin was prevented in three of five HMCL by the addition of MVA. FOH partially protected two HMCL from the effect of fluvastatin while GGOH prevented apoptosis in three of five HMCL and partially protected the other two (Fig. 3b). As expected none of the mevalonate pathway intermediates reversed the effects of SCH66336 (data not shown).

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Fig. 2. Inhibition of prenylation. LP-1, NCI-H929 and U266 were treated with 100 ␮M zoledronate (Z), 25 ␮M fluvastatin (F) or 10 ␮M SCH66336 (S) for 72 h before preparing cell lysates. (U) untreated. Lysates were analysed by immunoblotting to detect (a) unprenylated Rap1A, indicative of decreased geranylgeranylation, or (b) pre-lamin A, indicative of decreased farnesylation (representative blot of LP-1 shown). (a) Composite of three experiments with a representative example (LP-1 blot) of tubulin staining to demonstrate equal protein loading.

Isobolograms were constructed using dose response curves plotted as the mean ± S.E.M. of three separate experiments (results summarised in Table 3). Zoledronate and fluvastatin acted synergistically to induce cell death in all five HMCL as did either zoledronate or fluvastatin when combined with dexamethasone. Combinations of SCH66336 and zoledronate, or SCH66336 and fluvastatin varied in effect on each cell lines from antagonistic to additive to synergistic depending on the concentration of each agent. The combination of SCH66336 and dexamethasone acted synergistically in all cell lines studied with the exception of U266, which var-

Table 3 Summary of potential synergistic interactions between inhibitors of the mevalonate pathway

SCH + Zol Zol + Fluv Fluv + SCH Zol + Dex Fluv + Dex

RPMI-8226

LP-1

OPM-2

NCI-H929

U266

0 + +/− + +

− + + +/− +/−

− + − + +

+/− +/0 + + +

+ + −/0 + +/−

Interactions were classed as antagonistic (−), additive (0) or synergistic (+). More than one symbol indicates that the interaction varied depending on the concentration of each of the compounds.

Fig. 3. Abrogation of apoptosis with mevalonate pathway intermediary addback. Mevalonate pathway intermediates mevalonic acid (MVA) 100 ␮M, farnesol (FOL) 10 ␮M and geranylgeraniol (GGOL) 10 ␮M were added to five HMCL following treatment with zoledronate (a) or fluvastatin (b). Percentage apoptosis is relative to untreated control, data is mean ± S.E. of the mean from three experiments.

ied from antagonistic to synergistic as the concentration of dexamethasone increased. Representative examples of plotted isobologram results are provided in Fig. 4. Microarray analysis was used to further investigate gene expression in zoledronate and/or fluvastatin treated NCIH929 cells. Of the 19,000 independent features on the microarrays, 256 were found to be upregulated by more than two-fold at 24 h, and 197 downregulated by more than twofold. At 72-h post-treatment, 145 genes were upregulated, and 165 downregulated. Genes likely to be most significant to the anti-MM action of zoledronate and fluvastatin are shown in Table 4 . Although not among the most significantly up- and down-regulated genes, geranyl- and farnesyl-related genes were also included. Of the several groups of genes identified as being differentially regulated by microarray analysis, apoptotic genes were particularly over-represented. Subsequently the modulation of apoptosis-related genes was confirmed by quantitative real-time PCR analyses (Fig. 5a–f). Zoledronate and fluvastatin, both singly and in combination, significantly increased the expression of almost all apop-

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Table 4 Genes identified as being differentially regulated in response to a combination of zoledronate and fluvastatin, 24 and 72 h after treatment Gene

Accession number

(a) Geranyl/farnesyl-related genes Rab geranylgeranyltransferase, alpha subunit Rab geranylgeranyltransferase, beta subunit Geranylgeranyl diphosphate synthase 1 Protein geranylgeranyltransferase type I, beta subunit Farnesyltransferase, CAAX box, alpha Farnesyl-diphosphate farnesyltransferase 1 Farnesyl diphosphate synthase Farnesyltransferase, CAAX box, beta Isopentenyl-diphosphate delta isomerase

NM 004581 NM 004582 NM 004837 NM 005023 NM 002027 NM 004462 NM 002004 L00635 NM 004508

1.1 0.6 0.4 0.2 5.6 3.3 1.6 1.4 0.8

1.5 0.5 1.7 0.1 0.9 0.4 0.7 0.7 0.2

(b) Genes of interest most significantly differentially expressed at 24 h post-treatment Apoptosis Optineurin Death-associated protein B-cell receptor-associated protein 29 Caspase-1 Ras homolog gene family, member B BCL2-like 11 Spindlin family, member 2 Excision repair cross-complementing rodent repair deficiency, complementation group 3 Tumor protein p53 inducible nuclear protein 1 Excision repair cross-complementing rodent repair deficiency, complementation group 5

AF061034 AF056436 NM 018844 NM 001223 D16875 NM 006538 NM 019003 AK024185 AL133074 NM 000123

11.2 10.2 9.2 8.9 8.9 7.6 7.1 2.7 3.2 2.3

1.8 2.1 0.8 1.5 5.2 1.1 0.8 5.8 5.1 2.2

GTPase signalling RAB GTPase activating protein 1-like Ras and Rab interactor 2 PTPL1-associated RhoGAP 1 RAB5B RAB3A-interacting molecule 2 SLIT-ROBO Rho GTPase activating protein 3 Rho guanine nucleotide exchange factor (GEF) 3 Son of sevenless homolog 2 (Drosophila) G protein-coupled receptor 126 RAB32 Regulator of G-protein signalling 11

AF279778 M37190 NM 004815 NM 002868 NM 014677 AK021822 NM 019555 L20686 AL110111 NM 006834 NM 003834

13.2 12.9 12.8 11.3 11.3 10.2 9.4 8.6 6.5 5.4 0.1

1.3 2.3 0.9 1.2 3.8 1.1 1.3 0.9 0.8 1.6 2.1

X98259 NM 004060 AK024078 AB023215

13.2 0.1 0.1 0.1

1.0 0.8 1.1 1.7

NM 001558 AF055033 AJ278348 NM 003884 NM 001425 NM 004626 NM 005461 M14584 NM 018423

12.2 9.6 8.5 7.5 6.7 6.2 0.1 0.1 0.1

1.4 0.7 0.7 1.2 1.2 1.8 1.3 1.7 0.9

NM 004449 NM 005851 NM 004830 AK022677 AF123659 NM 007359 U78628 NM 001699

19.3 16.2 12.5 12.4 12.1 0.1 0.1 0.1

1.1 1.5 4.9 0.7 0.7 1.2 1.6 1.7

Cell cycle M-phase phosphoprotein, mpp8 Cyclin G1 Ataxia telangiectasia and Rad3-related KIAA0998 protein Cell growth/proliferation Interleukin 10 receptor, alpha Insulin-like growth factor binding protein 5 Pappalysin 2 P300/CBP-associated factor Epithelial membrane protein 3 Wingless-type MMTV integration site family, member 11 (Wnt11) V-maf musculoaponeurotic fibrosarcoma oncogene homolog B Interleukin 6 Serine/threonine/tyrosine kinase 1 Oncogene-related V-ets erythroblastosis virus E26 oncogene-like Tumor suppressor deleted in oral cancer-related 1 Cofactor required for Sp1 transcriptional activation, subunit 3 Membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5) Leucine zipper, putative tumor suppressor 1 Cancer susceptibility candidate 3 Leukemia inhibitory factor receptor AXL receptor tyrosine kinase

Fold change at 24 h

Fold change at 72 h

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Table 4 (Continued ) Gene

Accession number

(c) Genes of interest most significantly differentially expressed at 72 h post-treatment Apoptosis TRAIL-R1 NM 003844 Peroxiredoxin 3 NM 006793 Serine/threonine kinase 17a NM 004760 Poly(ADP-ribose) polymerase family, member 14 AB033094 Growth arrest and DNA-damage-inducible, beta NM 015675 Protein phosphatase 1, regulatory (inhibitor) subunit 15A NM 014330

Fold change at 24 h

Fold change at 72 h

0.2 1.0 2.7 1.1 2.3 2.2

7.1 5.1 4.3 0.3 0.1 0.1

AF090906 AF279778 D16875 NM 018460 NM 001665 NM 004163

0.1 13.2 12.7 1.5 0.3 3.1

15.7 2.8 1.6 0.2 0.2 0.1

NM 004336 NM 006101 NM 003670 NM 001827 AK022385 NM 001760

0.4 1.4 2.9 0.4 0.3 0.4

9.3 8.3 5.2 4.9 0.3 0.3

Cell growth/proliferation Insulin-like growth factor 1 Kruppel-like factor 6 Serine protease inhibitor, Kunitz type 1 Transforming growth factor, beta 2 Fibroblast growth factor 8 Interleukin 3 receptor, alpha Interleukin 2 receptor, gamma

M27544 NM 001300 NM 003710 AK021874 NM 006119 M74782 NM 000206

0.3 1.5 0.5 1.4 0.6 0.3 13.2

4.5 0.2 0.2 0.2 0.2 0.1 0.1

Oncogene-related WAP four-disulfide core domain 1 Growth arrest-specific 8

AF302109 NM 001481

1.5 1.6

0.2 0.2

GTPase signalling Rho guanine nucleotide exchange factor (GEF) 12 RAB GTPase activating protein 1-like Ras homolog gene family, member B Rho GTPase activating protein 15 Ras homolog gene family, member G RAB27B, member RAS oncogene family Cell cycle BUB1 budding uninhibited by benzimidazoles 1 homolog Kinetochore associated 2 Basic helix-loop-helix domain containing, class B, 2 CDC28 protein kinase regulatory subunit 2 Cyclin B2 Cyclin D3

totic genes analysed compared to the methanol-only carrier (p < 0.05). Caspase-1, -4, and -7 were induced by 13.4, 6.6, and 5.9-fold, respectively, by combination treatment at 24 h, and remained upregulated by 6.1-, 4.5-, and 1.8-fold, respectively, at 72 h. p53 and p53-interacting protein showed a 19.6- and 9.1-fold upregulation at 24 h, and 5.3- and 3.8fold upregulation at 72 h, respectively. The expression of p53 and ERCC3 was considerably higher in cells treated with a combination of fluvastatin and zoledronate than with either compound alone.

4. Discussion Evidence from in vitro studies by a number of groups indicates that nitrogen-containing BPs such as zoledronate interfere with osteoclast recruitment, differentiation and action, and induce apoptotic cell death of these cells by disrupting the mevalonate pathway [16]. Several studies have established that zoledronate effectively induces apoptosis of HMCL [17–19]. The purpose of this study was to determine whether other inhibitors of the mevalonate pathway had similar poten-

tial. Using tetrazolium reduction assays we found that the HMGCoA reductase inhibitor fluvastatin inhibited the proliferation of HMCL more effectively than the zoledronate or SCH66336. AnnexinV/PI flow cytometry indicated lower levels of cell death than those observed using the tetrazolium reduction assays. Given that the tetrazolium reduction assay relies on metabolic activity, it was hypothesized that treatment with mevalonate pathway inhibitors induced cell cycle arrest prior to inducing apoptosis and this was subsequently confirmed by cell cycle analyses (Table 1). Published data demonstrate that treatment of HMCL with the nitrogen containing bisphosphonates YM125, YM529 and zoledronate results in S phase cell cycle arrest [17,18,20,21] as does treatment with the mevalonate pathway inhibitors mevastatin and lovastatin [22,23]. We hypothesize that the discrepancy in our results is due to the particular inhibitors and concentrations chosen. Studies in other cell lines have reported that lower concentrations of nitrogen containing bisphosphonates induce S phase cell cycle arrest while higher concentrations similar to those that we used induce G0/G1 arrest [24]. Similarly, high concentrations of the mevalonate pathway inhibitor lovastatin induce G0/G1 cell cycle arrest, while

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Fig. 4. Representative examples of isobologram analyses. The line of additivity indicates where data points will sit if the interaction of two inhibitors is purely additive, while curves to the left indicate synergy and curves to the right indicate antagonism. Examples (a–d) all illustrate experimental data for HMCL RPMI-8226. (a) Fluvastatin and zoledronate; (b) fluvastatin and SCH66336; (c) fluvastatin and dexamethasone; (d) zoledronate and dexamethasone.

low concentration induce G2/M arrest [25,26]. As entry into S phase depends on phosphorylation of the retinoblastoma gene product pRb [27], arrest in the G0/G1 phase suggested hypophosphorylation of pRb, and this conclusion was confirmed by our immunoblot data. Similar observations have been made following treatment of keratinocytes with lovastatin and nitrogen containing bisphosphonates [24]. It has recently been reported that mevalonate pathway inhibitioninduced apoptosis of HMCL was associated with a decrease in Mcl-1 expression [23]. Our results support this observation as Mcl-1 decreased in NCI-H929 cells at 72 h following treatment with fluvastatin, however, no detectable decrease in Mcl-1 levels was observed in the other HMCL or following other treatments during the 72 h period. We confirmed that treatment with various mevalonate pathway inhibitors altered the isoprenylation status of HMCL [11,23,28]. Immunoblot analysis demonstrated that treatment with the bisphosphonate zoledronate resulted in an accumulation of unprenylated Rap1A indicating inhibition of geranylgeranylation. Treatment with the HMGCoA reductase inhibitor fluvastatin decreased both farnesylation and geranylgeranylation. As isoprenylation of GTP-binding proteins is essential for membrane attachment and participation in signal transduction processes, we examined the effect of mevalonate pathway inhibitors on the JAK-STAT and MAPK/ERK pathways, both of which have been identified as growth/survival pathways in myeloma cells [29]. We found that the mevalonate pathway inhibitors generally had no effect on either pathway, with the exception of zoledronate

which increased the level of active ERK after 72 h. This has been reported in another cell type prompting the hypothesis that increased ERK activity maintains hypophosphorylated pRb levels which in turn mediates growth inhibition [28]. Addback experiments using MVA, FOH and GGOH indicated that geranylgeranylated proteins rather than farnesylated proteins are required for suppression of apoptosis in HMCL, a finding supported by recent published studies [21,23,30]. Interestingly, addition of MVA was unable to completely reverse apoptosis induced by fluvastatin in all HMCL studied, suggesting that in some cases the growth inhibitory effect of fluvastatin may be partially mediated by other mechanisms, possibly inhibition of cholesterol synthesis. We therefore hypothesize that inhibition of both geranylgeranylation and cholesterol synthesis in HMCL may have additive effects on growth arrest, but that inhibition of geranylgeranylation is more important. This hypothesis is supported by a study investigating the effect of lovastatin on cell cycle progression in HL60 and MOLT-4 cells which reported distinct roles for mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, with both being required for normal cycling [25]. Microarray analysis was subsequently used to examine the effect of fluvastatin and zoledronate on genes involved in GTPase signal transduction, cell cycle control, growth and proliferation, and apoptosis (Table 4). Both statins and zoledronate are known to inhibit the activity of GTPases such as Ras and Rho in a variety of cells types as a consequence of their prevention of GTPase prenylation [31,32].

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Fig. 5. Evaluation of apoptosis-related genes with micro-array. Six apoptotic genes found to be differentially regulated by microarray analysis following zoledronate and fluvastatin treatment were measured by quantitative real-time PCR. (a) Caspase-1, (b) caspase-4, (c) caspase-7, (d) ERCC3, (e) p53, and (f) p53-inducible nuclear protein 1. (* p < 0.05, ** p < 0.001 by paired Student’s t-test). Values correspond to mean ± S.E. (n = 4–6).

The microarray results demonstrate an increase in expression of a number of GTPase-related genes (Rab5b, Rab27b, Rab32, and Rab3a-interacting molecule 2) at 24 h postcombination treatment. Similarly upregulated were several guanine nucleotide exchange factors (GEFs), which are responsible for activating GTPase proteins. These results suggest that cells respond to inhibition of GTPase signalling by an immediate upregulation of GTPase and accessory genes as a compensatory response. The activities of several GTPases

also provide a close link between the melavonate pathway and cell cycle control. Mevalonate pathway disruption has been shown, for example, to prevent the geranylgeranylation of small Rho GTPases and to in turn inhibit the degradation of the Cdk inhibitor protein p27kip1 , which is required for G1 to S phase transition [33]. The results presented here suggest that the cell cycle control is also disrupted at the genetic level, with a marked down-regulation of several cell-cycle genes related genes including cyclins B2, D3, and G1. p53 demonstrated

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a marked upregulation at 24 h, particularly when HMCL received combined zoledronate–fluvastatin treatment. Similarly, quantitative real-time PCR showed that several other pro-apoptotic genes were also upregulated, particularly the down-stream apoptotic effector Caspase-7. p53-inducible nuclear protein 1 (TP53INP1), which is thought to regulate p53-dependent apoptosis through phosphorylation of p53 [34], was also significantly induced, displaying a 9.1fold upregulation at 24 h. Finally, the ‘excision repair crosscomplementing rodent repair deficiency, complementation group 3’ (ERCC3) gene, expressed in response to DNA strand breakage and which in turn activates caspases [35] was also found to be upregulated at both time points. Previous studies have highlighted nitrogen containing bisphosphonates, farnesyl transferase inhibitors and HMGCoA reductase inhibitors as potential therapeutic agents for MM [5,11,21,23,28,30,36–39]. The cellular and gene expression studies described here not only demonstrate synergistic antiMM interactions between fluvastatin, zoledronate and dexamethasone but also that zoledronate and fluvastatin disrupt a variety of cellular processes that contribute to their anti-MM activity. These effects may take the form of a cascade, effecting GTPase expression and signalling initially, followed by cell cycle inhibition and then the stimulation of apoptosis. p53 could be considered a common link between many of these pathways.

Acknowledgments We would like to thank Novartis Pharmaceuticals Australia Pty Ltd. for providing fluvastatin and zoledronate and Schering-Plough Pty Ltd. for providing SCH66336. This work was funded in part by The Anderson Trust. CindyBaulch Brown and Timothy Molloy performed experimental work and contributed to the writing of the manuscript, Sung Lin Yeh performed experimental work, David Ma contributed to the writing of the manuscript and experimental design, Andrew Spencer provided the study concept and experimental design and contributed to the writing of the manuscript.

References [1] Kastrinakis NG, Gorgoulis VG, Foukas PG, Dimopoulos MA, Kittas C. Molecular aspects of multiple myeloma. Ann Oncol 2000;11(10):1217–28. [2] Dalton WS, Jove R. Drug resistance in multiple myeloma: approaches to circumvention. Semin Oncol 1999;26(5 Suppl 13):23–7. [3] Berenson JR, Lichtenstein A, Porter L, Dimopoulos MA, Bordoni R, George S, et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. Myeloma Aredia Study Group. N Engl J Med 1996;334(8):488–93. [4] Berenson JR, Lichtenstein A, Porter L, Dimopoulos MA, Bordoni R, George S, et al. Long-term pamidronate treatment of advanced multiple myeloma patients reduces skeletal events. Myeloma Aredia Study Group. J Clin Oncol 1998;16(2):593–602.

351

[5] Green JR. Anti-tumor potential of bisphosphonates. Med Klin (Munich) 2000;95(Suppl 2):23–8. [6] Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990;343(6257):425–30. [7] Kusama T, Mukai M, Iwasaki T, Tatsuta M, Matsumoto Y, Akedo H, et al. Inhibition of epidermal growth factor-induced RhoA translocation and invasion of human pancreatic cancer cells by 3hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors. Cancer Res 2001;61(12):4885–91. [8] Xia Z, Tan MM, Wong WW, Dimitroulakos J, Minden MD, Penn LZ. Blocking protein geranylgeranylation is essential for lovastatininduced apoptosis of human acute myeloid leukemia cells. Leukemia 2001;15(9):1398–407. [9] Ghosh PM, Mott GE, Ghosh-Choudhury N, Radnik RA, Stapleton ML, Ghidoni JJ, et al. Lovastatin induces apoptosis by inhibiting mitotic and post-mitotic events in cultured mesangial cells. Biochim Biophys Acta 1997;1359(1):13–24. [10] Chatterjee M, Honemann D, Lentzsch S, Bommert K, Sers C, Herrmann P, et al. In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6/gp130/STAT3 pathway. Blood 2002;100(9):3311–8. [11] van de Donk NW, Kamphuis MM, Lokhorst HM, Bloem AC. The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells. Leukemia 2002;16(7):1362–71. [12] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(259):680–5. [13] Berenbaum MC. What is synergy? Pharmacol Rev 1989;41(2): 93–141. [14] Tallarida RJ, Porreca F, Cowan A. Statistical analysis of drug-drug and site–site interactions with isobolograms. Life Sci 1989;45(11): 947–61. [15] Cleveland WS. Robust locally weighted regression and smoothing scatterplots. J Am Stat Assoc 1974;74:829–36. [16] Shipman CM, Rogers MJ, Vanderkerken K, Van Camp B, Graham R, Russell G, et al. Bisphosphonates–mechanisms of action in multiple myeloma. Acta Oncol 2000;39(7):829–35. [17] Aparicio A, Gardner A, Tu Y, Savage A, Berenson J, Lichtenstein A. In vitro cytoreductive effects on multiple myeloma cells induced by bisphosphonates. Leukemia 1998;12(2):220–9. [18] Derenne S, Amiot M, Barille S, Collette M, Robillard N, Berthaud P, et al. Zoledronate is a potent inhibitor of myeloma cell growth and secretion of IL-6 and MMP-1 by the tumoral environment. J Bone Miner Res 1999;14(12):2048–56. [19] Tassone P, Forciniti S, Galea E, Morrone G, Turco MC, Martinelli V, et al. Growth inhibition and synergistic induction of apoptosis by zoledronate and dexamethasone in human myeloma cell lines. Leukemia 2000;14(5):841–4. [20] Shipman CM, Rogers MJ, Apperley JF, Russell RG, Croucher PI. Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumour activity. Br J Haematol 1997;98(3):665–72. [21] Shipman CM, Croucher PI, Russell RG, Helfrich MH, Rogers MJ. The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway. Cancer Res 1998;58(23):5294–7. [22] Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTPbinding proteins, including Ras. J Bone Miner Res 1998;13(4): 581–9. [23] van de Donk NW, Kamphuis MM, van Kessel B, Lokhorst HM, Bloem AC. Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels. Blood 2003;102(9):3354–62. [24] Reszka AA, Halasy-Nagy J, Rodan GA. Nitrogen-bisphosphonates block retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation. Mol Pharmacol 2001;59(2):193–202.

352

C. Baulch-Brown et al. / Leukemia Research 31 (2007) 341–352

[25] Martinez-Botas J, Ferruelo AJ, Suarez Y, Fernandez C, GomezCoronado D, Lasuncion MA. Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives. Biochim Biophys Acta 2001;1532(3):185–94. [26] Naderi S, Blomhoff R, Myklebust J, Smeland EB, Erikstein B, Norum KR, et al. Lovastatin inhibits G1/S transition of normal human B-lymphocytes independent of apoptosis. Exp Cell Res 1999;252(1):144–53. [27] Bartek J, Bartkova J, Lukas J. The retinoblastoma protein pathway and the restriction point. Curr Opin Cell Biol 1996;8(6):805–14. [28] Iguchi T, Miyakawa Y, Yamamoto K, Kizaki M, Ikeda Y. Nitrogencontaining bisphosphonates induce S-phase cell cycle arrest and apoptosis of myeloma cells by activating MAPK pathway and inhibiting mevalonate pathway. Cell Signal 2003;15(7):719–27. [29] Hideshima T, Richardson P, Anderson KC. Novel therapeutic approaches for multiple myeloma. Immunol Rev 2003;194:164–76. [30] Gordon S, Helfrich MH, Sati HI, Greaves M, Ralston SH, Culligan DJ, et al. Pamidronate causes apoptosis of plasma cells in vivo in patients with multiple myeloma. Br J Haematol 2002;119(2):475–83. [31] Madonna R, Di Napoli P, Massaro M, Grilli A, Felaco M, De Caterina A, et al. Simvastatin attenuates expression of cytokine-inducible nitricoxide synthase in embryonic cardiac myoblasts. J Biol Chem 2005 Apr 8;280(14):13503–11. [32] Russell RG, Rogers MJ. Bisphosphonates: from the laboratory to the clinic and back again. Bone 1999;25:97–106.

[33] Hirai A, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Tatsuno I, et al. Geranylgeranylated rho small GTPase(s) are essential for the degradation of p27Kip1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL-5 cells. J Biol Chem 1997;272(1): 13–6. [34] Okamura S, Arakawa H, Tanaka T, Nakanishi H, Ng CC, Taya Y, et al. p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis. Mol Cell 2001;8:85–94. [35] Dunkern TR, Fritz G, Kaina B. Cisplatin-induced apoptosis in 433B and 27-1 cells defective in nucleotide excision repair. Mutat Res 2001;486(4):249–58. [36] Bolick SC, Landowski TH, Boulware D, Oshiro MM, Ohkanda J, Hamilton AD, et al. The farnesyl transferase inhibitor, FTI-277, inhibits growth and induces apoptosis in drug-resistant myeloma tumor cells. Leukemia 2003;17(2):451–7. [37] Beaupre DM, Cepero E, Obeng EA, Boise LH, Lichtenheld MG. R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways. Mol Cancer Ther 2004;3(2): 179–86. [38] Ochiai N, Uchida R, Fuchida S, Okano A, Okamoto M, Ashihara E, et al. Effect of farnesyl transferase inhibitor R115777 on the growth of fresh and cloned myeloma cells in vitro. Blood 2003;102(9): 3349–53. [39] Shi Y, Gera J, Hsu JH, Van Ness B, Lichtenstein A. Cytoreductive effects of farnesyl transferase inhibitors on multiple myeloma tumor cells. Mol Cancer Ther 2003;2(6):563–72.