Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a...

Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinomamodel: a p53-independent mitochondrial-mediated apoptotic mechanism

Masa-Aki Shibata1,2,4, Yuko Ito1, Junji Morimoto3

and Yoshinori Otsuki1,2,4

1Department of Anatomy and Biology, 2High-Tech Research Center and3Laboratory Animal Center, Osaka Medical College, 2--7 Daigaku-machi,Takatsuki, Osaka 569-8686, Japan

4To whom correspondence should be addressedEmail: [email protected] or [email protected]

The effects of lovastatin, a potent inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, were studied in amouse model of metastatic mammary cancer carrying ap53 mutation. Mice bearing mammary tumors, induced byinoculation of syngeneic BALB/c mice with BJMC3879cells, were treated with lovastatin at 0, 25 and 50 mg/kgthree times a week. Tumor volumes were significantlyreduced in a dose-dependent manner throughout the 6week study and were associated with both a decrease inDNA synthesis and an increase in apoptosis. The high doseof lovastatin also inhibited lung metastasis. In a corollaryin vitro study, flow cytometric analyses of lovastatin-treated mammary cancer cells additionally showed cellcycle arrest at G1 phase and decreases in S and G2/Mphases. Laser scanning cytometric analyses further demon-strated that cancer cells in S and G2/M were particularlysusceptible to the effects of lovastatin. Transmissionelectron microscopic evaluation of TUNEL-confirmedapoptotic bodies in lovastatin-treated mammary carci-noma cells revealed many free 30-OH ends of DNA incondensed chromatin within fragmented nuclei thatoccasionally assumed a characteristic half-moon shape.Consistent with initiation of apoptosis, cellular caspase-8,caspase-9 and caspase-3 activities were elevated in lovasta-tin-treated cells. The mitochondrial membrane potentialwas also decreased, with subsequent release of cytochrome c.However, lovastatin-induced cell death was significantlyreduced by the broad spectrum caspase inhibitorz-VAD-fmk, as well as the caspase-9 inhibitor z-LEHD-fmk and the caspase-3 inhibitor z-DEVD-fmk, but not bythe specific caspase-8 inhibitor z-IETD-fmk. Since immu-noelectron microscopy showed translocation of Bax to themitochondria in lovastatin-treated cells, lovastatin-inducedapoptosis may, therefore, be ultimately dependent on Baxinduction of cytochrome c release. These results suggestthat lovastatin may be useful as an adjuvant therapy inbreast cancers containing p53mutations due to its ability toboth suppress DNA synthesis and induce p53-independentmitochondria-mediated apoptosis.

Introduction

Breast cancer is one of the most frequent female cancers andthe incidence has risen dramatically world wide in the pastdecade (1); in the USA alone the disease has become thesecond leading cause of death in women (1). Although breastcancer still ranks fifth as a cause of female mortality in Japan,the number of deaths attributed to breast cancer has risen2.6-fold between 1975 and 1998 in this country as well (2).Lovastatin is a competitive inhibitor of hydroxymethylglu-

taryl-coenzyme A (HMG-CoA) reductase, the rate-limitingenzyme in cholesterol biosynthesis, and is generally regardedas a safe and effective compound used extensively for thetreatment of hypercholesterolemia (3). Other properties oflovastatin have been identified, however, that suggest it mayalso have chemotherapeutic effects. In breast cancer cell cul-tures lovastatin induces two cell cycle inhibitors, p21Waf1 andp27Kip1, and leads to cell cycle arrest in the G1 phase (4--7). Inother in vitro studies apoptosis has been induced by lovastatinin cancer cells derived from various organs, including thebreast (7--10).Statins, including lovastatin itself, have been shown to var-

iously inhibit tumor growth and metastasis in vivo in severaldifferent implantable rodent tumorigenesis models, e.g. inmammary carcinoma (7,9--11), fibrosarcoma (12), colon ade-nocarcinoma (13), melanoma (14) and pancreatic neoplasias(15). However, data specific to the effect of statins on mam-mary cancers, particularly on their anti-metastatic properties,is limited; only one group, using lovastatin on a murine sarcoidmammary carcinoma cell line (F3II) established andinoculated into syngeneic mice, has investigated the anti-metastatic effects induced by statins in any depth (9,10). Inthis study we show that lovastatin suppressed tumor growthand metastasis in vivo in a mouse mammary cancer model and,as a result of data produced in correlated in vitro studies, webelieve that we can identify at least part of the suppressionmechanism.

Materials and methods

Experimental compound

The sodium salt of lovastatin was used for the in vivo study (Merk & Co.,Rathway, NJ). The lovastatin was sonicated into suspension in saline foranimal experiments. For the in vitro studies lovastatin was purchased fromWako Pure Chemical Industries Ltd (Osaka, Japan). For in vitro use theinactive lactone form of lovastatin was converted to the active dihydroxyopen acid form by dissolution in ethanol, followed by incubation in 0.4 MNaOH in ethanol at 50�C for 2 h and adjusting the pH to 7.2 with 1 M HCl.Aliquots of this stock solution (10 mM) were stored at �20�C.

In vivo studies

Animals. A total of 42 female 6-week-old BALB/c mice were used in thisstudy (Japan SLC, Hamamatsu, Japan). The animals were housed at 4 per cagein the preliminary dosage--tolerance study and 5 per plastic cage in the tumorgrowth phase on wood chip bedding with free access to water and food undercontrolled temperature (21� 2�C), humidity (50� 10%) and lighting (12--12 hlight--dark cycle). All animals were held for a 1 week acclimatization period

Abbreviations: BrdU, 5-bromo-20-deoxyuridine; fmk, fluoromethyl ketone;H&E, hematoxylin and eosin; HMG-CoA, hydroxymethylglutaryl-coenzymeA; MMTV, mouse mammary tumor virus; PBS, phosphate-buffered saline;RFU, relative fluorescence units; TEM, transmission electron microscopic;TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nickend-labeling.

Carcinogenesis vol.25 no.10 # Oxford University Press 2004; all rights reserved. 1887

Carcinogenesis vol.25 no.10 pp.1887--1898, 2004doi:10.1093/carcin/bgh201

by guest on April 13, 2016

http://carcin.oxfordjournals.org/D

ownloaded from

http://carcin.oxfordjournals.org/

before study commencement. All manipulations of mice were performed inaccordance with the procedures outlined in the Guide for the Care and Use ofLaboratory Animals of Osaka Medical College.

Preliminary dosage--tolerance study. To ensure that toxicity would not be afactor, we conducted a preliminary dosage--tolerance study for the proposed 0(saline), 25 and 50 mg/kg dosages of lovastatin using 4 mice for each dose. Theanimals were injected i.p. with the appropriate dose 3 times a week for 9consecutive weeks. No deaths occurred in any of the groups and only the50 mg/kg group showed any weight loss (10%) at week 9 comparedwith controls, indicating that no dose adjustments were necessary.

Tumor growth study. The BJMC3879 mammary adenocarcinoma cell lineused was derived from a metastatic focus within a lymph node from a femaleBALB/c mouse that had had mouse mammary tumor virus (MMTV) injectedinto the inguinal mammary glands (16). The mammary tumors resulting fromMMTV inoculation showed a high metastatic propensity to lungs and lymphnodes (17), a trait retained through culture.

For the current study BJMC3879 cells [5 � 106 cells/0.3 ml phosphate-buffered saline (PBS)] were inoculated s.c. into the right inguinal region of 30female BALB/c mice. Two weeks later, when tumors had developed to~0.2 cm diameter, groups of 10 mice each were injected i.p. with either vehiclealone or with 25 or 50 mg/kg lovastatin 3 times a week for 6 weeks. Individualbody weights were recorded weekly; using calipers, each mammary tumor wasalso measured weekly and tumor volumes calculated using the formula max-imum diameter � (minimum diameter)2 � 0.4 (18). Three hours after the lastlovastatin or vehicle treatment all animals were injected i.p. with 100 mg/kg5-bromo-20-deoxyuridine (BrdU) (Sigma Chemical Co., St Louis, MO). Onehour after BrdU injection the mice were killed with diethyl ether and exsan-guinated.

Histopathology. At necropsy tumors and lymph nodes, routinely those fromthe axillary and femoral regions as well as those appearing abnormal, wereremoved, fixed in 4% formaldehyde solution in phosphate buffer and pro-cessed through to paraffin embedding. Lungs were inflated with the formalde-hyde solution prior to excision and immersion in fixative. The individual lobeswere subsequently removed from the bronchial tree, trimmed into seven piecesand examined for metastatic foci before being similarly processed to paraffinembedding. All paraffin-embedded tissues were cut at 4 mm, with sequentialsections stained with hematoxylin and eosin (H&E) for histopathologicalexamination and reserved unstained for immunohistochemistry.

p53 immunohistochemistry. The avidin--biotin complex method was used forp53 immunohistochemistry. Unstained sections were immersed in distilledwater and heated by microwave irradiation for antigen retrieval prior toincubation with an anti-p53 mouse monoclonal antibody (Clone Pab240;Santa Cruz Biotechnology, Santa Cruz, CA) that reacts with the mutantprotein in fixed specimens.

DNA synthesis. As previously mentioned, all animals received 100 mg/kgBrdU (Sigma Chemical Co.) i.p. 1 h prior to killing with ether and the tumorsfrom five animals selected from each treatment group were subsequentlyevaluated for DNA synthesis rate as inferred by BrdU incorporation. Usingunstained paraffin-embedded tissue sections, DNA was denatured in situ byincubation in 4 N HCl solution for 20 min at 37�C. The incorporated BrdU wasvisualized after exposure to an anti-BrdU mouse monoclonal antibody (CloneBu20a; Dako, Glostrup, Denmark). The numbers of BrdU-positive S phasecells per 5000 cells were counted in five random high power (�400) fields ofviable tissue and the BrdU labeling indices were expressed as a percentage oftotal cells counted. Sections of small intestine served as positive controls.

TUNEL assay for apoptosis. For the quantitative analyses of apoptosis, sec-tions from paraffin-embedded tumors were assayed using the terminal deoxy-nucleotidyl transferase-mediated dUTP-FITC nick end-labeling (TUNEL)method in conjunction with an apoptosis in situ detection kit (Wako PureChemical Industries) with minor modifications to the manufacturer’s protocol.TUNEL-positive cells were counted in viable regions peripheral to areas ofnecrosis in tumor sections. The numbers of TUNEL-positive cells per5000 cells were counted in five random high power (�400) fields andexpressed as a percentage of the total cells counted. Sections from testeswere used as positive controls.

In vitro studies

Cell lines. BJMC3879 cells, derived from a metastatic focus of murine mam-mary adenocarcinoma (16,17), were maintained in Dulbecco’s modifiedEagle’s medium or RPMI-1640 containing 10% fetal bovine serum withstreptomycin/penicillin in an incubator under 5% CO2. Cell cultures of thehuman breast cancer line MDA-MB231 were also maintained under the sameconditions and served as a corollary test system.

Cell growth. BJMC3879 cells were plated the day before lovastatin treatmentat 1 � 104 cells/well in 6-well plates. They were subsequently incubated for48 h with culture medium containing vehicle alone or with medium containinglovastatin at various concentrations up to 80 mM and counted. In addition, theresponse to lovastatin over time was determined by examining the effects of10 and 20 mM lovastatin on cell growth for 24 and 48 h.

Flow cytometric analysis of cell cycle. Flow cytometric analysis was con-ducted on trypsinized BJMC3879 cell suspensions harvested after 24 h treat-ment with 20 mM lovastatin and fixed in cold 70% ethanol. The cells werestained with a 50 mg/ml propidium iodide solution containing 100 mg/mlRNase A for 30 min on ice just prior to analysis with a flow cytometer(EPICS Elite ESP; Coulter Co., Miami, FL) and the percentage of cells ineach phase of the cell cycle was determined using a Multicycle Cell CycleAnalysis program (Coulter).

TUNEL/light microscopy evaluation. BJMC3879 cells, grown in 2-wellchamber slides and treated with 20 mM lovastatin for 24 h, were fixed in 4%formaldehyde solution in phosphate buffer and the TUNEL staining procedureperformed as described in the in vivo study. The numbers of TUNEL-positivecells per 1000 counted in five random high power (�400) fields by conven-tional light microscopy were expressed as a percentage of the total cellscounted.

TUNEL/transmission electron microscopy evaluation. BJMC3879 cells,grown in 2-well chamber slides and treated with 0 or 20 mM lovastatin for24 h, were fixed in a solution of 2.5% glutaraldehyde/2% paraformaldehyde in0.1 M phosphate buffer. These cells were then post-fixed with 1% osmiumtetroxide in phosphate buffer, dehydrated, embedded in epoxy resin andcollected on nickel grids that were subjected to an etching step using saturatedsodiummetaperiodate. A sheep anti-digoxigenin antibody conjugated to 10 nmcolloidal gold (British BioCell International, Golden Gate, UK) was applied inplace of a peroxidase-conjugated antibody (Apop Tag; Intergen Co., Purchase,NY) (19,20); cells previously exposed to the TUNEL procedure were stainedwith uranyl acetate and lead. All cell preparations were scanned under a modelH-7100 electron microscope (Hitachi Co., Tokyo, Japan). Transmission elec-tron microscopic (TEM) images of cell nuclei (control group normal lookingnuclei; treated group apoptotic bodies) were captured and image analysis ofDNA fragmentation performed using the NIH Image program (21). The label-ing density of free 30-OH DNA ends was evaluated based on the number ofimmunogold particles per nuclear area of 10 randomly selected cells from eachof control and lovastatin-treated cultures.

Laser scanning cytometric analysis. Human mammary carcinoma MDA-MB231 cells were grown in 2-well chamber slides (Lab-Tek II: Nalgen NuncInternational, Naperville, IL), treated with 20 mM lovastatin for 32 h and fixedin 4% paraformaldehyde solution. The fluorescein-TUNEL method was thenapplied for detection of apoptotic cells (Intergen Co., New York, NY). Afterthe TUNEL procedure, nuclear DNA was stained with a 50 mg/ml propidiumiodide solution containing 100 mg/ml RNase A for 30 min at 37�C for cellcycle analysis. The numbers of fluorescein-TUNEL-positive cells weremeasured and their cell cycle phases determined with a microscope-basedmultiparameter laser scanning cytometer (LSC2; Olympus Optical Co.,Tokyo, Japan) and the resulting data analyzed with WinCyte software(Compucyte Co., MA).

BrdU and p53 immunohistochemistry. Mouse BJMC3879 cells treated withlovastatin for 24 h were incubated for 30 min in culture medium containing50 mM BrdU (Sigma Chemical Co.) and trypsinized. The cells were thencounted, fixed with 70% ethanol and distributed at 1�104 cells/well on96-well opaque plates. The plates were dried using a hair dryer and the driedcells within each well covered with 1 N HCl for 20 min at 37�C, carefullyrinsed with PBS and exposed to an anti-BrdU antibody--peroxidase conjugate(Fab fragment; Roche Diagnostics, Indianapolis, IN) for 60 min. Each well wasthen rinsed and covered with a luminol substrate (Chemiluminescence; RocheDiagnostics) for 5 min. Light emissions of the samples were measured using aLuminoskan Acent (ThermoElectron Co., Helsinki, Finland). Additionallovastatin-treated BJMC3879 cells grown on 2-well chamber slides were alsoformalin fixed, microwave irradiated and immunohistochemically stained forp53 as described above.

Caspase activities. The activities of caspase-8, caspase-9 and caspase-3 weremeasured in cells treated with 20 mM lovastatin for 24 h using a fluorometricprotease assay kit (MBL Inc., Nagoya, Japan) in which cells were lysed with0.1% Triton X-100 lysis buffer and the protein concentration adjusted to25 mg in each sample. Caspase activity was measured in terms of fluorescenceintensity using a VersaFluor fluorometer (Bio-Rad, Hercules, CA).

Specific caspase inhibition. Two hours prior to lovastin exposure, BJMC3879cells, plated at 1 � 104 cells/well in 96-well culture plates overnight, were

M.-A.Shibata et al.

1888



ownloaded from


treated variously with the pan-caspase inhibitor z-VAD-fmk (fluoromethylketone), caspase-8-specific z-IETD-fmk, caspase-9-specific z-LEHD-fmk orcaspase-3-specific z-DEVD-fmk; control cells were exposed to the 1% DMSOvehicle in which all inhibitors were dissolved. All caspase inhibitors wereobtained from MBL Inc. and applied at concentrations of 10 and 100 mM.After 24 h, cell viability was measured using the MTT assay (RocheDiagnostics) and absorbance was measured spectrophotometrically at a dualwave length of 540 and 680 nm (ImmunoReader; Nippon InterMed Co.,Tokyo, Japan).

Mitochondrial membrane potential (Dcm). Changes in cellular Dcm weredetermined using a fluorescent cationic dye, 5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-benzamidazolocarbocyanin iodide (JC-1) (Mit-E-C MitochondrialPermeability Detection Kit; Biomol Research Laboratories, PlymouthMeeting, PA), which easily penetrates cells and aggregates in negativelycharged healthy mitochondria, giving them a red fluorescence. When Dcmcollapses, the dye can no longer accumulate inside the mitochondria (22) anddistributes throughout the cell. The dissipated dye assumes a monomeric formthat fluoresces green, rather than red, resulting in an overall decrease influorescence intensity. The changes in Dcm in lovastatin-treated and controlcells measured 24 h after exposure were expressed in terms of relative fluor-escence units (RFU) using a VersaFluor fluorometer (Bio-Rad) with a 485--495nm excitation filter and a 585--595 nm emission filter.

Release of cytochrome c. After incubation in culture medium with and with-out 20 mM lovastatin for 12 and 24 h, both floating and attached cells wereharvested, rinsed once in PBS, immersed in a cold buffer solution (10 mMTris--HCl, pH 7.5, 0.3 M sucrose, 1 mM aprotinin, 10 mM pepstatin, 10 mMleupeptin, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), homo-genized and centrifuged at 700 g for 10 min at 4�C. The supernatants werecentrifuged further at 10 000 g for 30 min at 4�C. Supernatants containing thecytosolic fraction were collected separately and protein concentrations deter-mined by the Bradford assay (Bio-Rad). ELISA was performed using theCytochrome C ELISA Kit (Chemicon International Inc., Temecula, CA) todetermine cytochrome c release into the cytosol.

Western blotting for cytochrome c. The presence of cytosolic cytochrome cwas also confirmed by western blots using both 20 mg protein from thecytosolic fraction used for ELISA as well as protein derived from whole celllysates of cells treated with 20 mM lovastatin for 24 h. Cell lysates wereadditionally used for analysis of Bcl-2, Bax and Bid expression. Aliquots of10 and 20 mg protein were fractionated in 10 and 14% Tris--glycine gels underreducing conditions and transferred to nitrocellulose membranes. Primaryantibodies used in this study, which included anti-cytochrome c mouse mono-clonal antibody (clone 7H8.2C12; Pharmingen, San Diego, CA) and anti-Bax,anti-Bid and anti-Bcl-2 rabbit and anti-actin goat polyclonal antibodies (clonesN-20, D-19, N-19 and I-19, respectively; Santa Cruz Biotechnology) wereapplied to the membranes and the resulting conjugates visualized on X-rayfilms using enhanced chemiluminescence (Perkin Elmer Life Sciences Inc.,Boston, MA).

Immunoelectron microscopy for Bax. BJMC3879 cells, grown in 2-wellchamber slides for 24 h in medium with or without 20 mM lovastatin,were fixed in a 0.05% glutaraldehyde/4% paraformaldehyde solution in0.1 M phosphate buffer. Immunoelectron microscopy was performed aspreviously described (23). Briefly, fixed cells were sequentially immersedin 0.1 M phosphate buffer containing 5% sucrose for 1 h, in 10% sucrosefor 1 h and in 20% sucrose for 1 h. The indirect immunoperoxidasetechnique was then employed, using an anti-Bax rabbit polyclonal anti-body (N-20; Santa Cruz Biotechnology). The cells were ultimately fixed in1% osmium tetroxide in 0.1 M PBS for 1 h, stained again with 1% uranylacetate in 70% ethanol, dehydrated and embedded in epoxy resin.Samples were examined under a model H-7100 electron microscope(Hitachi Co.).

Statistical analysis

Data containing dose--response effects were subjected to analysis of variance(ANOVA), while Scheffe’s t-test was employed for differences between themeans. Data from cytometric studies, Dcm analyses, caspase activity andinhibition studies and cytochrome c release evaluations were compiled andcompared between control and lovastatin-treated groups using a two-sidedStudent’s t-test. The two-sided Fisher’s exact probability test was used toevaluate the significance of histopathological findings.

Results

In vivo studies

Body weights and tumor growth. Body weights of control andlovastatin-treated mice bearing mammary tumors are compiledand shown in Figure 1A. As can be seen, mice treated with50 mg/kg lovastatin were significantly lighter than were con-trols by experimental week 2 and thereafter, but, even at week6, the weight difference between high dose and control animalswas only 10%. At 25 mg/kg lovastatin, no statistical differ-ences in body weights between control and treated mice wereobserved. This experiment was terminated at experimentalweek 6 when the first mortality occurred in the control groupdue to liver metastasis from a mammary carcinoma.Tumor volumes are presented in Figure 1B. Tumor growth,

as inferred by computed volume, was significantly inhibited inmice receiving both 25 and 50 mg/kg lovastatin from week 2 ina dose-dependent manner when compared with controls. Bythe end of the experiment the average tumor volume in controlanimals was 926� 414 mm3, while those of mice receiving 25and 50 mg/kg were 586 � 211 mm3 and 414 � 66 mm3,respectively.

Histopathology. Histopathologically, the mammary carcinomasinduced by BJMC3879 cell inoculation proved to be moder-ately differentiated adenocarcinomas that, by immunohisto-chemistry, contain the same p53 mutation carried by the cellline (Figure 2A). Lung metastasis occurred in 100% of controlanimals and in 80% of the animals in both lovastatin-treatedgroups, but the metastatic foci tended to be markedly smallerin the 50 mg/kg group (Figure 2C) than were those observed inboth the mice receiving 25 mg/kg and the control animals(Figure 2B). For quantitative analysis, metastatic foci werecharacterized into two categories: microscopic and macro-scopic foci consisting of 430 cells and macroscopic lesionsmeasuring 41 mm. In the high dose (50 mg/kg) group, therewas a significant decrease in the numbers of foci per mouse41mm (P 5 0.05 as compared with control), as well as atendency toward fewer metastases of any size (Figure 3A).The number of metastases-positive lymph nodes per mousetended to decrease in a dose-dependent manner as well, buta statistical significance could not be demonstrated (Figure3B). Liver metastasis was observed in the single controlcase that died at week 6.

Levels of DNA synthesis and apoptosis. DNA synthesis levelsin mammary carcinomas of lovastatin-treated mice, as inferredby BrdU labeling indices, are illustrated in Figure 3C. DNAsynthesis in tumors apparently decreased in a dose-dependentmanner, reaching statistically significant values in micereceiving 50 mg/kg (Figure 3C). Levels of apoptosis in lesions,as assessed by the TUNEL assay, are shown in Figure 3D;apoptosis was significantly increased in tumors from the50 mg/kg group as compared with levels seen in tumors fromcontrol mice.

In vitro studies

Effects of lovastatin on cell growth and cell cycle. Inhibitionof cell growth was evident with as little as 5--10 mM lovastatinand occurred in a dose-dependent manner; concentrations of420 mM induced a strong inhibitory effect. We thereforechose exposures of 10 and 20 mM for further in vitro study.As demonstrated in Figure 4A, significant inhibition of cell

growth occurred within 24 h of exposure to both 10 and 20 mM

Lovastatin suppresses cancer growth and metastasis

1889



ownloaded from


lovastatin. After 48 h treatment, 20 mM induced a 64% inhibi-tion of cell growth (Figure 4B), inducing a rounded cellmorphology and reduced plate adherence without apparentcytotoxicity.As measured by flow cytometry, 24 h exposure to 20 mM

lovastatin induced a significant elevation of the numbers ofcells in G1 phase as compared with untreated cells (Figure 4C).There were also significant reductions in S and G2/M fractionsin lovastatin-treated cell suspensions (Figure 4C). DNA synth-esis in treated BJMC3879 cells, as assessed by BrdU incor-poration, was also decreased in a dose-dependent manner(Figure 4D).

Effects of lovastatin on apoptosis. Quantitative analysis ofTUNEL staining by light microscopy revealed a significantincrease in the numbers of TUNEL-positive cells after24 h lovastatin treatment as compared with control cells(Figure 5A). Laser scanning cytometry of human mammarycarcinoma MDA-MB231 cells stained by the fluorescentTUNEL procedure with propidium iodide indicated that lovas-tatin both increased the number of cells in G1 arrest andsuppressed the number entering the S and G2/M phases (Figure5B and C). The relative intensity of fluorescein TUNEL-positive cells is represented in Figure 5D; only 14% of control

cells demonstrated weak (i.e. nearly equivalent to background)TUNEL staining, while 60.6% of lovastatin-treated cells werestrongly TUNEL positive. Figure 5E shows the cell cycledistribution of lovastatin-treated cells; ~50% of cells in the Sand G2/M phases were TUNEL positive, whereas TUNEL-positive cells accounted for only 10.5% of cells in G1.

Fig. 1. Body weights (A) and tumor volumes (B) in mammary carcinomasfrom female BALB/c control mice and mice treated with 25 and 50 mg/kglovastatin. Each group consisted of 10 mice. Data presented are means� SDvalues. (A) Body weights of mice treated with 50 mg/kg were significantlysuppressed (��P 5 0.01) as compared with those observed in the controlgroup, but only by 10%. (B) Tumor volume was significantly reduced byweek 2 with 50 mg/kg lovastatin and at weeks 5 and 6 with 25 mg/kg(�P 5 0.05; ��P 5 0.01).

Fig. 2. (A) p53 immunohistochemistry of mouse mammary carcinomainduced by BJMC3879 cell inoculation into BALB/c mice. Note nuclearstaining for abnormal p53 protein, indicating that these cells carrymutant p53. p53 imunohistochemistry, �200. (B) Metastatic lesion froma mouse treated with vehicle alone. (C) Metastatic lesion (arrowhead)from a mouse treated with 50 mg/kg lovastatin. Note the differences insize of metastatic foci between control and treated animals. (B) and(C), H&E, �40.

M.-A.Shibata et al.

1890



ownloaded from


Apoptosiswas further confirmedbyultrastructural analysis ofuntreated (Figure 6A) and treated BJMC3879 cells (Figure 6B),the latter demonstrating fragmented nuclei with charac-teristic half-moons of condensed chromatin. In quantitativeanalyses of TUNEL by TEM (Figure 6C), a small number(8.3 � 1.7/mm2) of free 30-OH DNA ends, as visualized byadhered immunogold particles, were observed in nuclear chro-matin of untreated mammary carcinoma cells (Figure 6A,inset), while the number of immunogold particles was signifi-cantly elevated (129.5 � 75.8/mm2) in the chromatin oflovastatin-treated cells (Figure 6B, inset).

Apoptosis signaling pathway. Significantly elevated activitiesof not only caspase-3 but also of caspase-8 and caspase-9 wereobserved in BJMC3879 cells treated with 20 mM lovastatin for24 h (Figure 7A). To determine whether caspase activation isnecessary to induce lovastatin-induced apoptosis, BJMC3879cells were treated with caspase inhibitors. Combination treat-ment with 20 mM lovastatin and 100 mM z-VAD-fmk (a broadspectrum inhibitor of caspases), z-LEHD-fmk (an inhibitorspecific to caspase-9) or z-DEVD-fmk (a specific inhibitor ofcaspase-3) significantly increased cell viability as comparedwith lovastatin alone (Figure 7B). However, z-IETD-fmk(a specific inhibitor of caspase-8) did not prevent lovastatin-induced cell death (Figure 7B). This strongly suggeststhe engagement of the mitochondria-mediated apoptoticpathway.

Dcm decreased upon exposure to lovastatin, as shown inFigure 7C, and ELISA further determined that cytochrome cprotein levels in cytosolic fractions were significantlyincreased over levels detected in control cells after 12 and 24 htreatment with 20 mM lovastatin (Figure 7D). The westernblots shown in Figure 7E also demonstrate cytochrome crelease into the cytosol of cells treated with 20 mM lovastatinfor 24 h. Expression of Bax and Bid was very weak both incytosolic fraction and in whole cell lysates of lovastatin-trea-ted cells (Figure 7E). Expression of Bcl-2 was not detected ineither control or lovastatin-treated cells (data not shown) norwas cleavage of Bid observed.

Immunoelectron microscopy for Bax. Since the translocationof Bax to mitochondria is an important step in the induction ofapoptosis (24), immunoelectron microscopy was employed todetect Bax protein in the mitochondria of treated and controlcells. Immunoelectron microscopy demonstrated that Bax pro-tein was present in and along the outer membrane of mitochon-dria in lovastatin-treated cells (Figure 7G), but not in controlcells (Figure 7F).

Discussion

Several large clinical trials have demonstrated that statins, ingeneral, have the ability to greatly reduce cardiovascular-related morbidity and mortality in patients with and without

Fig. 3. (A) Lung metastases were categorized as microscopic and/or macroscopic foci composed of 430 cells and macroscopic nodules 41 mm diameter.Multiplicity of all lung metastatic foci (microscopic þ macroscopic foci) was decreased in a dose-dependent manner in lovastatin-treated mice, but the decreasewas not statistically significant. Note, however, that the number of macroscopic foci41 mm in diameter was significantly decreased with 50 mg/kg lovastatin ascompared with controls (�P 5 0.05). (B) The number of lymph nodes with metastasis/mouse decreased in a non-significant dose-dependent manner. (C) BrdUlabeling indices in mammary tumors decreased in a dose-dependent manner with 50 mg/kg lovastatin (��P 5 0.01). (D) The number of TUNEL-positive(apoptotic) cells in mammary carcinomas was significantly increased in mice treated with 50 mg/kg lovastatin (��P 5 0.01). Data presented aremeans � SD values.


1891



ownloaded from


coronary disease. In two additional clinical trials evaluatingcoronary events in patients, there was a 43 and 19% decreasein the number of new cases of colon cancer diagnosed over a 5year follow-up period in cohorts taking pravastatin and sim-vastatin, respectively (25,26). Lovastatin, an inhibitor ofHMG-CoA reductase, is one of the statins in therapeutic usetoday and is widely prescribed to reduce hypercholesterolemia.Additional data from one 5 year clinical safety and efficacystudy of lovastain also revealed a reduction in cancer deathswithin the treated group (27).In several animal studies specifically of mammary disease,

chemopreventive effects of lovastatin and simvastatin onradiation-induced mammary tumorigenesis have been demon-strated in rats (11), in syngeneic mice inoculated with mam-mary carcinoma cells (9,10) and in a transgenic mousemammary cancer model (7). There are further reports of effic-acy against cancers in other organ systems as well (28--31).The lethal metastatic proclivity of mammary cancer is

possibly the characteristic that makes it of such clinicalimportance. Patients presenting with metastatic disease arefrequently incurable. It is reported that once breast cancersreach �4 cm, the chances of tumor recurrence or metastasesincrease dramatically (32), therefore, treatments that offersuppression of both tumor growth and metastasis have signific-ant clinical implications. There is limited information withrespect to the anti-metastatic properties of statins in mammarycancers. In fact, it appears that only one group reported anyanti-metastatic effects, specifically in studies of lovastatin on

syngeneic mice inoculated with cells from a proprietarymurine F3II sarcoid mammary carcinoma culture (9,10).Lovastatin induces apoptosis (7,8,33). Lovastatin has also

been shown to inhibit cell growth with arrest at G1 and reducetransition to the S and G2/M phases of the cell cycle (4--7). Thismay be related to elevation of two key cell cycle inhibitors,p21Waf1 and p27Kip1 (5--7), that have been reported to suppresscell cycle progression, resulting in G1 arrest in both normal andtumor cell lines derived from mammary glands (6). However,it was recently reported that, at least in prostate cancer celllines, transcriptional regulation and proteosomal degradationof E2F-1 may be critical regulatory events in growth inhibitionand that p21Waf1=Cip1 induction and G1 arrest are not generalmechanisms in lovastain-mediated cell death (34).The reduction in DNA synthesis and induction of apoptosis

are of particular interest in neoplastic diseases. Bearing inmind that lovastatin is a potent HMG-CoA antagonist, it hasbeen reported that HMG-CoA reductase activity is elevatedjust before DNA synthesis (35). Elevated DNA synthesis andincreased cell proliferation are generally regarded as hallmarksof aggressive cancers and current radiation therapies are mosteffective against the more susceptible proliferating cell. Gen-erally, cells located in the late G1 and G2/M phases of the cellcycle are most sensitive to ionizing radiation-induced celldeath, whereas cells in S phase are most resistant (36). TheG1 arrest effect of statins potentially sensitizes cells to radi-ation (36). In fact, it was previously reported that a Ras-associated increase in radiation resistance in osteosarcoma

Fig. 4. (A) The dose--response characteristics of 0--80 mM lovastatin on mouse mammary carcinoma-derived BJMC3879 cells. The dosages selected for thein vitro experiments (10 and 20 mM lovastatin) were those that elicited significant cell death without cytotoxicity. (B) Inhibition of cell growth was sequentiallyexamined after treatment with 10 and 20 mM lovastatin, which induced significant cell death 24 and 48 h after treatment. (C) Cell cycle distribution (percentage ofcells in a specified phase) was measured in mammary carcinoma cells treated with 20 mM lovastatin using flow cytometry. Lovastatin induced arrest in G1 phaseand inhibition of both the S and G2/M phases. (D) DNA synthesis, assessed by BrdU incorporation, was measured in cells treated with 10 and 20 mM lovastatin.DNA synthesis was significantly decreased in lovastatin-treated cells in a dose-dependent manner. Data were calculated from three repeated experiments incontrol and lovastatin-treated cells. Data presented are means � SD values. ��Statistical significance determined at P 5 0.01.

M.-A.Shibata et al.

1892



ownloaded from


Fig. 5. (A) BJMC3879 cells (mouse mammary carcinoma cell line), treated or not with 20 mM lovastatin for 24 h, were analyzed for apoptotic cells usingthe TUNEL procedure with light microscopy. The numbers of TUNEL-positive (apoptotic) cells significantly increased with lovastatin treatment.Four samples from both control and lovastatin-treated cells were examined. Data presented are means � SD values. ��Statistical significance determined atP 5 0.01. (B--E) MDA-MB231 cells (human mammary carcinoma cell line) treated or not with 20 mM lovastatin for 32 h, stained with fluorescein-TUNELand propidium iodide and analyzed for cell cycle phase using laser scanning cytometry. (B) A DNA histogram of treated and control cells shows decreases in S andG2/M fractions induced by lovastatin. (C) Cell distribution (percentage of cells in each phase) demonstrates significant increases in G1 fractions and decreases inG2/M fractions induced by lovastatin (��P 5 0.01). (D) This histogram shows increased fluorescein-TUNEL-positive cells (apoptotic cells) with lovastatinexposure as compared with controls. (E) With lovastatin treatment ~50% of S and G2/M phase cells were TUNEL-positive (apoptotic), suggesting that cancercells in these phases are highly susceptible to lovastatin. Four samples each of control and lovastatin-treated cells were examined. Data presented aremeans � SD values. ��Statistical significance determined at P 5 0.01.


1893



ownloaded from


cells can be reversed by lovastatin treatment (37). In contrast,our laser scanning cytometric analysis demonstrated thathuman mammary carcinoma MDA-MB231 cells cyclingthrough both the S and G2/M phases were highly susceptibleto lovastatin-induced apoptosis. This may be a significantclinical benefit if lovastain induces apoptotic cell death in thetypically radiation-resistant S phase.There are two pathways currently proposed to play major

roles in regulating apoptosis in mammalian cells: an extrinsicpathway mediated by one or more death receptors and anintrinsic pathway mediated by mitochondria (38). In the extrin-sic death receptor/ligands pathway, caspase activation occursas a direct consequence of death receptor ligation, withupstream caspase-8 cleaving and activating downstream pro-teases such as caspase-9 and caspase-3. In the intrinsic mito-chondrial pathway, Bax, a member of the Bcl-2 family, playsthe leading role. Bax normally resides in the cytosol in aquiescent state. After an apoptotic stimulus, Bax is translocatedinto the mitochondria and promotes the release of cytochrome c(24), possibly by forming a pore (39) or a voltage-dependentanion channel (40) in the outer mitochondrial membrane. Boththe death receptor and the mitochondrial pathways are linkedby cleavage of Bid by caspase-8. It is known that cleaved Bidalso translocates to mitochondria and induces Bax-dependentrelease of cytochrome c (41). Once in the cytosol, cytochrome cactivates Apaf-1, which then activates procaspase-9, which, inturn, activates caspase-3, triggering apoptosis. We initiallyconfirmed that lovastatin-induced cell death is by apoptosisrather than necrosis using both TUNEL/light microscopyand TUNEL/TEM techniques. Although elevated caspase-8,caspase-9 and caspase-3 activities were observed in lovastatin-treated cells, the fact that lovastatin-induced cell death wasreduced by both the broad spectrum caspase inhibitor z-VAD-fmk and inhibitors specific for caspase-9 and caspase-3(z-LEHD-fmk and z-DEVD-fmk, respectively), but that celldeath was not reduced by exposure to z-IETD-fmk, an inhibitorspecific for caspase-8, strongly suggests that the intrinsic mito-chondrial pathway is engaged in lovastatin-induced apoptosis.This is further indicated by the decrease in Dcm and release ofcytochrome c into the cytosol and correlates well with a recentreport that in human promyelocytic leukemia HL-60 cellslovastatin also induces activation of caspase-3, release of cyto-chrome c into the cytosol and a decrease in Dcm (42).As previously discussed, mitochondria-mediated apoptosis

is regulated by Bcl-2 family proteins (43,44), with Bcl-2protecting against apoptosis by forming heterodimers withpro-apoptotic Bax, thus preventing the collapse of Dcmand release of cytochrome c from mitochondria. We wereunable, however, to detect expression of Bcl-2 in either controlor lovastatin-treated BJMC3879 cells under our test condi-tions. We also saw no increase in Bax expression in lovasta-tin-treated carcinoma cells, a finding in agreement with arecent paper (45). Because Bax translocation is a critical stepfor reduction of Dcm, we decided to look at the location ofBax in relation to mitochondria using immunoelectron micro-scopy. We found that Bax protein aggregated along the outermitochondrial membrane only in cells exposed to lovastatin,indicating that lovastatin-induced apoptosis is, in all likeli-hood, due to Bax-dependent release of cytochrome c, despitea lack of increased protein expression. Further, caspase-8activity was elevated in lovastatin-treated mammary carci-noma cells in vitro without apparent Fas ligand stimulation.It could be argued that, since caspase-8 has been shown to be

C

B

A

Fig. 6. (A and B) Electron microscopic studies using immunogold labelingwith TUNEL/TEM (transmission electron microscopy). (A) A lovastatin-untreated BJMC3879 cell showing fairly randomly dispersed nuclearchromatin with only a few immunogold particles (inset) indicating the sitesof free 30-OH DNA ends. (B) In a lovastatin-treated BJMC3879 cell,lovastatin-induced features typical of apoptosis as characterized by nuclearfragments containing a half-moon of condensed chromatin and many moreimmunogold particles, indicating frequent free 30-OH ends (inset). �4300;inset, �16 000. Scale bar ¼ 1 mm. (C) The number of 30-OH ends of DNAstrand breaks per mm2 nucleus was also significantly increased in lovastatin-treated cells as compared with controls. Ten nuclei from both control andlovastatin-treated cells were examined. Data presented are means � SDvalues. ��Statistical significance determined at P 5 0.01.

M.-A.Shibata et al.

1894



ownloaded from


capable of autoactivation by ligand and is DISC independent(46), lovastatin itself may directly or indirectly activate cas-pase-8 and initiate apoptosis. However, because we did notfind evidence of Bid cleavage and because the caspase-8-specific inhibitor z-IETD-fmk did not prevent lovastatin-induced cell death, it appears that within the parameters of

this investigation neither caspase-8 activation nor involve-ment are factors in lovastatin-induced apoptosis. The precisemechanism by which lovastatin decreases Dcm is not known.Cholesterol is a major component of the lipoproteins compris-ing both the cellular and mitochondrial membranes. It is pos-sible that inhibition of cholesterol biosynthesis by lovastatin

Fig. 7. Investigation of the apoptotic signaling pathway in BJMC3879 cells with or without 20 mM lovastatin for 24 h. We evaluated four or five samples each ofcontrol and lovastatin-treated cells. Data presented are mean � SD values. (A) Caspase activities were evaluated according to fluorometric assays. Activities ofcaspase-8, caspase-9 and caspase-3 were significantly elevated in the mammary carcinoma cells treated with lovastatin (��P 5 0.01). (B) Cell viability wasdetermined by the MTT assay in mammary carcinoma cells treated with 20 mM lovastatin with or without exposure to either 10 or 100 mM caspase inhibitors. Cellviability (%) was significantly increased by the broad spectrum caspase inhibitor z-VAD-fmk (��P5 0.01), by the caspase-9 inhibitor z-LEHD-fmk (�P5 0.05)and by the caspase-3 inhibitor z-DEVD-fmk (�P 5 0.05) at the 100 mM concentration only. However, cell viability was unaffected by addition of anyconcentration of the caspase-8 inhibitor z-IETD-fmk. Data are expressed as percentages of the vehicle control. (C) The mitochondrial membrane potential (Dcm)was measured with a fluorescent cationic dye and expressed in terms of intensity. Lovastatin induced a significant decrease in Dcm (�P 5 0.05). RFU, relativefluoresecence unit. (D) The levels of cytosolic cytochrome c, as determined by ELISA, were significantly elevated in cells after both 12 and 24 h lovastatintreatment (��P5 0.01). (E) Comparison of western blots of cytosol fractions versus whole cell lysates of BJMC3879 cells treated with 20 mM lovastatin for 24 h.Elevated levels of cytochrome c were confirmed in cytosolic fractions of lovastatin-treated cells (upper). Lovastatin did not increase the expression of either Bax orBid in whole cell lysates (lower). b-actin served as an internal control. (F andG) Bax immunoelectron microscopy of BJMC3879 cells treated or not with 20 mMlovastatin. (F) Ultrastructural study of an untreated cell, showing weak detection of Bax protein in mitochondria (arrow heads). (G) Bax protein can clearly beseen along the mitochondrial membrane (arrow heads) in a lovastatin-treated BJMC3879 cell. �39 000. Scale bar ¼ 0.5 mm.


1895



ownloaded from


may alter the integrity of these membranes (47) or may inhibitthe formation of new membranes in proliferating tumor cellsthat require high levels of cholesterol.It was obvious to us that lovastatin-induced apoptosis in

BJMC3879 cells, which carry a p53 mutation, must occurthrough a p53-independent mechanism or, perhaps, the p53homolog p73 substitutes in inducing apoptosis induction incells with non-functional p53. Whatever the case, lovastatinhas been shown to induce apoptosis in a wide variety ofcancer cells, irrespective of their p53 or pRb status. Since50% of human cancers have mutations in p53 (48), thefact that lovastatin induces a p53-independent apoptoticresponse may be highly relevant to treating many humanneoplasms.Preclinical studies in different animal species have indicated

that dosages up to 200 mg/kg/day yield drug concentrations inthe range 2--20 mM (49) and that circulating concentrations of2--4 mM were well tolerated for months in all animal modelstested. In a human phase I study, 25--45mg/kg/day were used incancer treatment (50). These dosages produced drug concen-trations up to 3.9 mM and are associated with anti-proliferativeeffects in vitro (50).We similarly saw anti-proliferative activityin vitrowith ~5 mM and more in our present study series. Thus,the dosages used in our in vivo study are considered to be closeto the clinical dosages used for cancer treatment.

In conclusion, we report that lovastatin significantly inducedapoptosis and inhibited cell proliferation in vivo in p53-mutatedmouse mammary tumors and in vitro in our study of bothmurine and human mammary cancer cells. Possibly of moreimportance, we also saw a reduction in the number and size oflung metastases in the mammary model. Although the inhibi-tion of metastasis may simply be a reflection of suppression oftumor growth and decreased DNA synthesis, the therapeuticbenefit remains. This and other studies suggest the usefulnessof lovastatin as an adjuvant to current radio- and chemothera-pies for breast cancer, particularly those carrying p53mutations, and possibly as a chemotherapeutic/chemopreven-tative in and of itself.

Acknowledgements

We thank Deborah E.Devor-Henneman for critical review of the manuscript.We also thank to Mr Kazuhiko Nakayama (Olympus Optical Co., Tokyo) forassistance with the LSC analysis, Mr Teruo Ueno (Central Research Center,Osaka Medical College) for assistance with the flow cytometric analysis,Ms Eiko Shibata (High-Tech Research Center, Osaka Medical College) forexcellent technical assistance and Ms Hidemi Hiyama for excellent secretarialassistance. This investigation was supported by a Development of Character-istic Education grant, funded by the Japan Private School Promotion Founda-tion, and by the High-Tech Research Program of Osaka Medical College,Osaka, Japan, with additional support from the Ministry of Education, Culture,Sports, Science and Technology of Japan (no.15591368 to M.A.S.).

Fig. 7. Continued.

M.-A.Shibata et al.

1896



ownloaded from


References

1.Parker,S.L., Tong,T.A., Bolden,S. and Wingo,P.A. (1996) Cancerstatistics. CA Cancer J. Clin., 46, 5--27 [Comment, CA Cancer J. Clin.,1996, 46, 3--4].

2.Kuroishi,T. and Tominaga,S. (2001) Epidemiology of breast cancer.Jpn. J. Cancer Chemother., 28, 168--173.

3.Goldstein,J.L. and Brown,M.S. (1990) Regulation of the mevalonatepathway. Nature, 343, 425--430.

4.Keyomarsi,K., Sandoval,L., Band,V. and Pardee,B. (1991)Synchronization of tumor and normal cells from G1 to multiple cellcycles by lovastatin. Cancer Res., 51, 3602--3609.

5.Gray-Bablin,J., Rao,S. and Keyomarsi,K. (1997) Lovastatin induction ofcyclin-dependent kinase inhibitors in human breast cells occurs in a cellcycle-independent fashion. Cancer Res., 57, 604--609.

6.Rao,S., Lowe,M., Herliczek,T.W. and Keyomarsi,K. (1998) Lovastatin-mediated G1 arrest in normal and tumor breast cells is through inhibitionof CDK2 activity and redistribution of p21 and p27, independent of p53.Oncogene, 17, 2393--2402.

7.Shibata,M.A., Kavanaugh,C., Shibata,E., Abe,H., Nguyen,P., Otsuki,Y.,Trepel,J.B. and Green,J.E. (2003) Comparative effects of lovastatinon mammary and prostate oncogenesis in transgenic mouse models.Carcinogenesis, 24, 453--459.

8.Borner,M.M., Myers,C.E., Sartor,O., Sei,Y., Toko,T., Trepel,J.B. andSchneider,E. (1995) Drug-induced apoptosis is not necessarily dependenton macromolecular synthesis or proliferation in the p53-negative humanprostate cancer cell line PC-3. Cancer Res., 55, 2122--2128.

9.Alonso,D.F., Farina,H.G., Skilton, G., Gabri,M.R., de Lorenzo,M.S. andGomez,D.E. (1998) Reduction of mouse mammary tumor formationand metastasis by lovastatin, an inhibitor of the mevalonate pathway ofcholesterol synthesis. Breast Cancer Res. Treat., 50, 83--93.

10.Farina,H.G., Bublik,D.R., Alonso,D.F. and Gomez,D.E. (2002) Lovastatinalters cytoskeleton organization and inhibits experimental metastasis ofmammary carcinoma cells. Clin. Exp. Metastasis, 19, 551--559.

11. Inano,H., Suzuki,K., Onoda,M. andWakabayashi,K. (1997) Anti-carcinogenicactivity of simvastatin during the promotion phase of radiation-inducedmammary tumorigenesis of rats. Carcinogenesis, 18, 1723--1727.

12.Matar,P., Rozados,V.R., Roggero,E.A. and Scharovsky,O.G. (1998)Lovastatin inhibits tumor growth and metastasis development of a ratfibrosarcoma. Cancer Biother. Radiopharm., 13, 387--393.

13.Broitman,S.A., Wilkinson,J.T., Cerda,S. and Branch,S.K. (1996) Effectsof monoterpenes and mevinolin on murine colon tumor CT-26 in vitro andits hepatic ‘‘metastases’’ in vivo. Adv. Exp. Med. Biol., 401, 111--130.

14. Jani,J.P., Specht,S., Stemmler,N., Singh,S.V., Gupta,V. and Katoh,A.(1993) Metastasis of B16F10 mouse melanoma inhibited by lovastatin, aninhibitor of cholesterol biosynthesis. Invasion Metastasis, 13, 314--324.

15.Kusama,T., Mukai,M., Iwasaki,T., Tatsuta,M., Matsumoto,Y., Akedo,H.,Inoue,M. and Nakamura,H. (2002) 3-Hydroxy-3-methylglutaryl-coenzymea reductase inhibitors reduce human pancreatic cancer cell invasion andmetastasis. Gastroenterology, 122, 308--317.

16.Morimoto,J., Imai,S., Haga,S., Iwai,Y., Iwai,M., Hiroishi,S., Miyashita,N.,Moriwaki,K. and Hosick,H.L. (1991) New murine mammary tumor celllines. In Vitro Cell Dev. Biol., 27A, 349--351.

17.Shibata,M.A., Morimoto,J. and Otsuki,Y. (2002) Suppression of murinemammary carcinoma growth and metastasis by HSVtk/GCV gene therapyusing in vivo electroporation. Cancer Gene Ther., 9, 16--27.

18.Shibata,M.A., Liu,M.-L., Knudson,M.C., Shibata,E., Yoshidome,K.,Bandy,T., Korsmeyer,S.J. and Green,J.E. (1999) Haploid loss of baxleads to accelerated mammary tumor development in C3(1)/SV40-TAgtransgenic mice: reduction in protective apoptotic response at thepreneoplastic stage. EMBO J., 18, 2692--2701.

19. Inoki,C., Ito,Y., Yamashita,H. et al. (1997) Image analysis andultrastructural detection of DNA strand breaks in human endometriumby in situ end-labeling techniques. J. Histotechnol., 20, 321--328.

20.Okamura,N., Ito,Y., Shibata,M.A., Ikeda,T. and Otsuki,Y. (2002) Fas-mediated apoptosis in human lens epithelial cells of cataracts associatedwith diabetic retinopathy. Med. Electron. Microsc., 35, 234--241.

21.Hayashi,R., Ito,Y., Matsumoto,K., Fujino,Y. and Otsuki,Y. (1998)Quantitative differentiation of both free 30-OH and 50-OH DNA endsbetween heat-induced apoptosis and necrosis. J. Histochem. Cytochem.,46, 1051--1059.

22.Cossarizza,A., Baccarani-Contri,M., Kalashnikova,G. and Franceschi,C.(1993) A new method for the cytofluorimetric analysis of mitochondrialmembrane potential using the J-aggregate forming lipophilic cation5,50,6,60 tetrachloro-1,10,3,30 tetraethylbenzimidazolcarbocyanine iodide(JC-1). Biochem. Biophys. Res. Commun., 197, 40--45.

23.Akao,Y., Otsuki,Y., Kataoka,S., Ito,Y. and Tsujimoto,Y. (1994) Multiplesubcellular localization of bcl-2: detection in nuclear outer membrane,endoplasmic reticulum membrane and mitochondrial membranes. CancerRes., 54, 2468--2471.

24. Jurgensmeier,J., Xie,Z., Deveraux,Q., Ellerby,L., Bredesen,D. and Reed,J.(1998) Bax directly induces release of cytochrome c from isolatedmitochondria. Proc. Natl Acad. Sci. USA, 95, 4997--5002.

25.Sacks,F.M., Pfeffer,M.A., Moye,L.A. et al. (1996) The effect ofpravastatin on coronary events after myocardial infarction in patientswith average cholesterol levels. Cholesterol and Recurrent Events Trialinvestigators. N. Engl. J. Med., 335, 1001--1009.

26.Pedersen,T.R., Berg,K., Cook,T.J. et al. (1996) Safety and tolerability ofcholesterol lowering with simvastatin during 5 years in the ScandinavianSimvastatin Survival Study. Arch. Intern. Med., 156, 2085--2092.

27.Groups,T.L.S. (1993) Lovastatin 5-year safety and efficacy study. Arch.Intern. Med., 153, 1079--1087.

28.Clutterbuck,R.D., Millar,B.C., Powles,R.L., Newman,A., Catovsky,D.,Jarman,M. and Millar,J.L. (1998) Inhibitory effect of simvastatin on theproliferation of human myeloid leukaemia cells in severe combinedimmunodeficient (SCID) mice. Br. J. Haematol., 102, 522--7.

29.Soma,M.R., Baetta,R., de Renzis,M.R. et al. (1995) In vivo enhancedantitumor activity of carmustine [N,N0-bis (2-chloroethyl)-N-nitrosourea]by simvastatin. Cancer Res., 55, 597--602.

30.Hawk,M.A., Cesen,K.T., Siglin,J.C., Stoner,G.D. and Ruch,R.J. (1996)Inhibition of lung tumor cell growth in vitro and mouse lung tumorformation by lovastatin. Cancer Lett., 109, 217--222.

31.Narisawa,T., Fukaura,Y., Terada,K., Umezawa,A., Tanida,N., Yazawa,K.and Ishikawa,C. (1994) Prevention of 1,2-dimethylhydrazine-inducedcolon tumorigenesis by HMG-CoA reductase inhibitors, pravastatin andsimvastatin, in ICR mice. Carcinogenesis, 15, 2045--2048.

32.Carter,C.L., Allen,C. and Henson,D.E. (1989) Relation of tumor size,lymph node status and survival in 24,740 breast cancer cases. Cancer, 63,181--187.

33.Padayatty,S.J., Marcelli,M., Shao,T.C. and Cunningham,G.R. (1997)Lovastatin-induced apoptosis in prostate stromal cells. J. Clin.Endocrinol. Metab., 82, 1434--1439.

34.Park,C., Lee,I. and Kang,W.K. (2001) Lovastatin-induced E2F-1 modula-tion and its effect on prostate cancer cell death. Carcinogenesis, 22,1727--1731.

35.Quesney-Huneeus,V., Wiley,M.H. and Siperstein,M.D. (1979) Essentialrole for mevalonate synthesis in DNA replication. Proc. Natl Acad. Sci.USA, 76, 5056--5060.

36.Chan,K.K., Oza,A.M. and Siu,L.L. (2003) The statins as anticancer agents.Clin. Cancer Res., 9, 10--19.

37.Miller,A.C., Kariko,K., Myers,C.E., Clark,E.P. and Samid,D. (1993)Increased radioresistance of EJras-transformed human osteosarcoma cellsand its modulation by lovastatin, an inhibitor of p21ras isoprenylation.Int. J. Cancer, 53, 302--307.

38.Hengartner,M.O. (2000) The biochemistry of apoptosis. Nature, 407,770--776.

39.Saito,M., Korsmeyer,S.J. and Schlesinger,P.H. (2000) BAX-dependenttransport of cytochrome c reconstituted in pure liposomes. Nature CellBiol., 2, 553--555.

40.Shimizu,S., Narita,M. and Tsujimoto,Y. (1999) Bcl-2 family proteinsregulate the release of apoptogenic cytochrome c by the mitochondrialchannel VDAC. Nature, 399, 483--487.

41.Luo,X., Budihardjo,I., Zuo,H., Slaughter,C. and Wang,X. (1998) Bid, aBcl2 interacting protein, mediates cytochrome c release from mitochon-dria in response to activation of cell surface death receptor. Cell, 94,481--490.

42.Wang,I.K., Lin-Shiau,S.Y. and Lin,J.K. (2000) Induction of apoptosis bylovastatin through activation of caspase-3 and DNase II in leukaemiaHL-60 cells. Pharmacol. Toxicol., 86, 83--91.

43.Gross,A., McDonnell,J.M. and Korsmeyer,S.J. (1999) BCL-2 familymembers and the mitochondria in apoptosis. Genes Dev., 13, 1899--1911.

44.Kroemer,G. and Reed,J.C. (2000) Mitochondrial control of cell death.Nature Med., 6, 513--519.

45.Di Matola,T., D’Ascoli,F., Luongo,C., Bifulco,M., Rossi,G., Fenzi,G.and Vitale,M. (2001) Lovastatin-induced apoptosis in thyroid cells:involvement of cytochrome c and lamin B. Eur. J. Endocrinol., 145,645--650.

46.Martin,D.A., Siegel,R.M., Zheng,L. and Lenardo,M.J. (1998) Membraneoligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signal. J. Biol. Chem., 273, 4345--4349.

47.Swamy,M.V., Cooma,I., Reddy,B.S. and Rao,C.V. (2002) Lamin B,caspase-3 activity and apoptosis induction by a combination of


1897



ownloaded from


HMG-CoA reductase inhibitor and COX-2 inhibitors: a novel approachin developing effective chemopreventive regimens. Int. J. Oncol., 20,753--759.

48.Greenblatt,M.S., Bennett,W.P., Hollstein,M. and Harris,C.C. (1994)Mutations in the p53 tumor suppressor gene: clues to cancer etiologyand molecular pathogenesis. Cancer Res., 54, 4855--4878.

49.Kornbrust,D.J., MacDonald,J.S., Peter,C.P., Duchai,D.M., Stubbs,R.J.,Germershausen,J.I. and Alberts,A.W. (1989) Toxicity of the

HMG-coenzyme A reductase inhibitor, lovastatin, to rabbits.J. Pharmacol. Exp. Ther., 248, 498--505.

50.Thibault,A., Samid,D., Tompkins,A.C. et al. (1996) Phase I study oflovastatin, an inhibitor of the mevalonate pathway, in patients with cancer.Clin. Cancer Res., 2, 483--491.

Received November 19, 2003; revised April 19, 2004; accepted May 27, 2004

M.-A.Shibata et al.

1898



ownloaded from


Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a...

Documents

Transcript of Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a...