simvastatin-induced apoptosis in osteosarcoma cells: a key...

Cancer Biology and Signal Transduction

Simvastatin-Induced Apoptosis in OsteosarcomaCells: A Key Role of RhoA-AMPK/p38 MAPKSignaling in Antitumor ActivityWalied A. Kamel1,2,3,4, Eiji Sugihara1, Hiroyuki Nobusue1, Sayaka Yamaguchi-Iwai1,5,Nobuyuki Onishi1, Kenta Maki3, Yumi Fukuchi3, Koichi Matsuo2, Akihiro Muto3,Hideyuki Saya1, and Takatsune Shimizu1,3

Abstract

Osteosarcoma is the most common type of primary bonetumor, novel therapeutic agents for which are urgently needed.To identify such agents, we screened a panel of approved drugswith a mouse model of osteosarcoma. The screen identifiedsimvastatin, which inhibited the proliferation and migrationof osteosarcoma cells in vitro. Simvastatin also induced apo-ptosis in osteosarcoma cells in a manner dependent on inhi-bition of the mevalonate biosynthetic pathway. It also dis-rupted the function of the small GTPase RhoA and inducedactivation of AMP-activated protein kinase (AMPK) and p38MAPK, with AMPK functioning upstream of p38 MAPK. Inhi-

bitors of AMPK or p38 MAPK attenuated the induction ofapoptosis by simvastatin, whereas metformin enhanced thiseffect of simvastatin by further activation of AMPK. Althoughtreatment with simvastatin alone did not inhibit osteosarcomatumor growth in vivo, its combination with a fat-free dietinduced a significant antitumor effect that was enhancedfurther by metformin administration. Our findings suggestthat simvastatin induces apoptosis in osteosarcoma cells viaactivation of AMPK and p38 MAPK, and that, in combinationwith other approaches, it holds therapeutic potential for oste-osarcoma. Mol Cancer Ther; 16(1); 182–92. �2016 AACR.

IntroductionOsteosarcoma is the most common primary malignant bone

tumor in childhood and adolescence (1). Individuals with oste-osarcoma are currently treated with surgery, radiotherapy, andadjuvant or neoadjuvant chemotherapy, including administra-tion of methotrexate, doxorubicin, cisplatin, or ifosphamide(1, 2). Although chemotherapy regimens have improved thesurvival rate of such patients by up to 60% to 70% (3), there isan urgent need for new therapeutic agents, given that many casesof osteosarcoma are initially not sensitive or acquire resistance tocurrent chemotherapeutic drugs (4). We previously generated amousemodel of osteosarcoma on the basis of overexpression of c-MYC in bone marrow stromal cells isolated from Ink4a/Arfknockout mice (5). Injection of the resulting highly tumorigenicosteosarcoma cells (designatedAXT cells) into syngeneic C57BL/6

mice results in the rapid formation of lethal tumors with meta-static lesions that mimic human osteoblastic osteosarcoma.

Drug repositioning, the identification and development of newuses for existing drugs (6), has many advantages over de novo drugdevelopment including a shortened timeline as well as reducedcost and risk. It is therefore an ideal approach to the developmentof new therapies for relatively rare and refractory diseases such asosteosarcoma.

Statins are 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors that exert pleiotropic biologic effectsby preventing the synthesis of mevalonic acid, the precursor ofnonsteroidal isoprenoid compounds that mediate various cellu-lar processes (7). Previous investigations have revealed that statinsexhibit antineoplastic effects in a variety of cancer types, includingosteosarcoma (8–14). The precise molecular mechanism bywhich statins induce apoptosis in osteosarcoma cells hasremained unknown, however. The antitumor potential of statinsfor the treatment of osteosarcoma in animal models has alsoremained largely unexplored.

We have now screened more than 1,100 existing drugs fortherapeutic potential in vitro, with statins being identified as suchdrug candidates. We clarified the effects of simvastatin on osteo-sarcoma cells in vitro and in vivo with the use of our syngeneicmodel, andwe explored themolecularmechanismsof these effects.Onthebasisof themechanistic insight,wepropose that simvastatinholds potential for application to the treatment of osteosarcomain the clinical setting by appropriate drug combination.

Materials and MethodsCell culture

Themouse osteosarcomaAXT cells were established in our labo-ratory as described previously in 2010 (5, 15) and resuscitated and

1Division of Gene Regulation, Institute for Advanced Medical Research, KeioUniversity School of Medicine, Shinjuku-ku, Tokyo, Japan. 2Laboratory of Celland Tissue Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo,Japan. 3Department of Pathophysiology, School of Pharmacy and Pharmaceu-tical Sciences, Hoshi University, Shinagawa-ku, Tokyo Japan. 4Faculty of Sci-ence, Mansoura University, Mansoura, Egypt. 5Department of Orthopedic sur-gery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Takatsune Shimizu, Department of Pathophysiology,School of Pharmacy and Pharmaceutical Sciences, Hoshi University, 2-4-41Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. Phone: 81-3-5498-5309. Fax:81-3-5498-5916. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0499

�2016 American Association for Cancer Research.

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maintained under 5% CO2 at 37�C in IMDM (Thermo FischerScientific) supplemented with 10% FBS. Saos2 and U2OS humanosteosarcoma cell lines were purchased from ATCC in September2016, resuscitated and used for assays within 2 months afterdelivery. Both lines were maintained under 5% CO2 at 37�C inMcCoy 5a medium (Thermo Fischer Scientific) supplementedwith 15% FBS.

ReagentsSimvastatin was obtained from Combi-Blocks, metformin

from LKT Laboratories, rosuvastatin and atorvastatin fromWako,fluvastatin from Tocris Bioscience, and 5-aminoimidazole-4-car-boxamide ribonucleotide (AICAR) from Cell Signaling. Mevalo-nate, geranylgeranyl diphosphate (GGPP), or farnesyl diphos-phate (FPP; Sigma Aldrich) were used at 200, 10, or 10 mmol/L,respectively, unless indicated otherwise. 5-(Tetradecyloxy)-2-furoic acid (TOFA) and a Rho inhibitor (C3 transferase) werefrom Santa Cruz and Cytoskeleton, respectively.

Cell proliferation assayCell viability was measured as previously described (15). For

rescue experiments, the cells were exposed to p38MAPK inhibitorX [BIRB796 (BIRB); Millipore] or 5-iodotubercidin (iodo; Sigma)for 1.5 hours or to compound C (Millipore) for 1 hour beforeincubation with simvastatin or AICAR.

Tumor xenograft modelAll animal care and procedures were performed in accordance

with the guidelines of Hoshi University (Tokyo, Japan) and KeioUniversity (Tokyo, Japan). For establishment of tumor xenografts,AXT cells (1 � 106) suspended in PBS were injected subcutane-ously and bilaterally into the flank of 8-week-old female synge-neic C57BL/6 mice (SLC). Administration of simvastatin wasperformed according to 3 different protocols: (i) mice wereinjected intraperitoneally with simvastatin at 40 mg/kg once aday for 5 days a week over 2 weeks beginning 7 days after cellinjection; (ii) simvastatin suspended in double-distilled watercontaining 0.05% carboxymethyl cellulose was administeredorally at 80 mg/kg once per day for 5 days a week over 3 weeksbeginning 7 days after cell injection; and (iii)mice were fedwith afat-free diet (Research Diets, #D04112303) beginning from theday of cell injection. Two days after cell injection, mice wereinjected intraperitoneally with simvastatin (50 mg/kg), metfor-min (100 mg/kg), or simvastatin (50 mg/kg) plus metformin(50 or 100 mg/kg) once per day for 5 days a week over 3 weeks.Drugs for injection were suspended in normal saline.

Cell migration assayAXT cells were transferred to the upper chamber in 200 mL of

serum-free IMDM containing test agents, and 800 mL of IMDMsupplemented with 10% FBS was placed in the lower chamber.After incubation for 12 hours under 5% CO2 at 37�C, the cellson the lower surface of the filter were counted as describedpreviously (16).

Immunoblot analysisCells were lysed with 2� Laemmli sample buffer (Bio-Rad)

supplemented with b-mercaptoethanol. Immunoblot analysiswas performed according to standard procedures (15), withprimary antibodies to cleaved caspase-3, to phosphorylated ortotal forms of p38 MAPK, AMP-activated protein kinase (AMPK),

AKT, and acetyl coenzyme A carboxylase (ACC), to RhoA, tointegrin-b1, to Rho-GDP dissociation inhibitor (Rho-GDI; allfrom Cell Signaling), and to actin (Santa Cruz Biotechnology).

Detection of Rho-GDI and RhoA interactionAXT cells were lysed with a lysis buffer containing 0.5% Non-

idet P-40 (17), and the resulting supernatant was incubated withrotation for 10 minutes at room temperature with antibodies toRho-GDI (Millipore) and protein G–coupled magnetic Dyna-beads (Life Technologies) as mentioned previously (17).

Detection of apoptotic cells and analysis of cell-cycle profileby flow cytometry

Cells were collected, washed with ice-cold PBS, suspended inAnnexin V–binding buffer (BD Biosciences), and stained withpropidium iodide (Sigma-Aldrich) and allophycocyanin-con-jugated Annexin V (BD Biosciences). After incubation on ice inthe dark for 15 minutes, the cells were assayed for apoptosis byflow cytometry. For cell-cycle analysis, cells were collected, fixedwith 70% ethanol for 48 hours at �20�C, washed 3 times withice-cold PBS, and stained with propidium iodide for 30 min-utes on ice. Data were analyzed with the use of FlowJo software(Tree Star).

Detection of activated RhoACells were lysed with a magnesium-containing buffer

(Millipore) and the RhoA status was evaluated as describedpreviously (18).

Determination of the subcellular localization of RhoAAXT cells were fractionated with the use of a membrane

fractionation kit (Abcam). Cytosolic and membrane fractionswere subjected to immunoblot analysis with antibodies toRhoA to determine the relative amounts of RhoA in eachcompartment.

Reverse transcription and real-time PCR analysisTotal RNA extraction, reverse transcription, and real-time PCR

analysis were performed as previously described (15, 19). Thesequences of PCR primers (forward and reverse, respectively) are50-CAACCGTGAAAAGATGACCC-30 and 50-TACGACCAGAGG-CATACAG-30 for Actb as well as 50-GTGCCCACGGTGTTTGAA-AAC-30 and 50-GGTGTCTGGATAAGAGAGAG-30 for Rhoa.

Statistical analysisAll assays were performed in triplicate, and quantitative data

are expressed as means � SD relative to the control value unlessindicated otherwise. Data were analyzed with the Student t test,and a P < 0.05 was considered statistically significant. �, P < 0.05;��, P < 0.005; ��, P < 0.0005; NS, not significant.

ResultsSimvastatin induces morphologic changes as well as inhibitsthe proliferation and migration of osteosarcoma cells

To discover a safe and effective therapeutic agent for osteosar-coma,we screenedmore than 1,100 FDA-approveddrugswith theuse of a novel humanoid robot (Mahoro, Robotic Biology Insti-tute). Three statins—lovastatin, simvastatin, and fluvastatin—among the 6 statins included in the library were found to inhibitthe proliferation of AXT cells (Fig. 1A andB). This antiproliferative

Simvastatin-Induced Apoptosis in Osteosarcoma

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effect of simvastatin was potent and concentration-dependent,and it was accompanied by the adoption of a spherical morphol-ogy by AXT cells and their irreversible detachment from theculture dish within 12 hours (Fig. 1C and D). To determinewhether these effects of simvastatin were due to inhibition ofthe mevalonate pathway, we examined whether they might beinfluenced by the addition of intermediate metabolites of thispathway includingmevalonate, GGPP, and FPP (20).Mevalonateand GGPP eachmarkedly attenuated the effects of simvastatin onboth cell morphology and proliferation, whereas the actions ofFPP in this regard were less pronounced (Fig. 1E; SupplementaryFig. S1A). Simvastatin also inhibited the proliferation of Saos2andU2OShumanosteosarcoma cells by targeting themevalonatepathway (Fig. 1F). In addition, simvastatin attenuated the migra-tion of AXT cells in a concentration-dependent manner (Fig. 1G;Supplementary Fig. S1B), and this effect was largely preventedby mevalonate or GGPP and was partially attenuated by FPP(Fig. 1H and I). Together, these results suggested that simvastatininhibits the viability and migration of osteosarcoma cells bytargeting the mevalonate pathway.

Simvastatin induces apoptosis in AXT cellsTo examine whether the suppression of osteosarcoma cell

proliferation by simvastatin was due to the induction of apoptoticcell death, we determined the proportion of apoptotic cells afterexposure of AXT cells to simvastatin for 24 hours. Flow cytometricanalysis of cells stained with Annexin V and propidium iodiderevealed that the numbers of both early (Annexin Vþ, propidiumiodide�) and late (Annexin Vþ, propidium iodideþ) apoptoticcells were increased by simvastatin treatment. Furthermore, con-sistent with their effects on the antiproliferative action of simva-statin, the addition of mevalonate or GGPP essentially preventedsimvastatin-induced apoptosis whereas that of FPP had a moremodest inhibitory effect (Fig. 2A–D). The cleaved formof caspase-3, another indicator of apoptosis (21), was also detected in AXTcells treated with simvastatin for 24 hours (Fig. 2E), and thesimvastatin-induced cleavage of caspase-3 was blocked by meva-lonate or GGPP (Fig. 2F). Finally, the induction of AXT cellapoptosis by simvastatin was confirmed by cell-cycle analysis asan increase in the size of the sub-G1 cell fraction (SupplementaryFig. S1C and S1D).

Figure 1.

Effects of simvastatin on the proliferation, morphology, and migration of osteosarcoma cells. A, Screening of 1164 compounds at 1.92 mmol/L for their effectson AXT cell viability after incubation for 2 days. Data are expressed relative to the value for no treatment. The positions of 6 statins are indicated. B,Viability of AXT cells treated with statins at the indicated concentrations for 2 days. C, Phase-contrast microscopy of AXT cell morphology aftersimvastatin treatment for 24 hours. D, Viability of AXT cells treated with simvastatin for 2 days. E, Viability of AXT cells treated with 10 mmol/L simvastatinfor 2 days in the absence or presence of mevalonate (Meva), GGPP, or FPP. F, Viability of Saos2 and U2OS cells treated with 10 mmol/L simvastatin for2 days in the absence or presence of mevalonate. G, Migration of AXT cells (50,000 cells per well) treated with simvastatin for 12 hours. H and I, Migrationof AXT cells (100,000 cells per well) treated with 10 mmol/L simvastatin (Simv) for 12 hours in the absence or presence of mevalonate, GGPP, or FPP.Representative migratory cells stained with crystal violet and quantitative data are shown in H and I, respectively.

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Simvastatin changes the expression and localization of RhoAin AXT cells

FPP and GGPP are required for posttranslational prenylationof various proteins including small GTPases of the Ras and Rho(Rac, Rho, and Cdc42) families (22, 23). The covalent attach-ment of FPP or GGPP to Ras or Rho family members, respec-tively, results in the translocation of these proteins from thecytosol to the plasma membrane, where they participate in theregulation of various cellular processes (22, 23). Whereas RhoAis detected in the membrane fraction of cells under normalconditions (24), we found that most RhoA was present in thecytosolic fraction of AXT cells after treatment with simvastatin(Fig. 3A). In addition, simvastatin increased the expression ofRhoA in AXT cells and the active, GTP-bound form of RhoA(RhoA-GTP) in a manner sensitive to inhibition by mevalonateor GGPP but to a lesser extent by FPP (Fig. 3B–D). Rho-GDInegatively regulates the activity of Rho GTPases in the cytosolby preventing the release of GDP (25). An immunoprecipita-tion assay revealed that simvastatin attenuated the formationof a complex between RhoA and Rho-GDI (Fig. 3E), which issupposed to be a main cause of the accumulation of RhoA-GTPin cytosol. Together, these results suggested that simvastatin

induces the accumulation of RhoA in the cytosol as well asdisrupts the interaction between RhoA-GDP and Rho-GDI inAXT cells, indicating that, despite its increased amount, thefunction of RhoA-GTP is likely impaired.

Simvastatin induces apoptosis via activation of AMPK/p38MAPK signaling

Statins have been found to induce apoptosis in cancer cellsvia activation of AMPK (26) or p38 MAPK (27). Furthermore,activation of AMPK and p38 MAPK shows antitumor potentialin human osteosarcoma (28). We therefore examined the effectsof simvastatin on AMPK and p38 MAPK activation status in AXTcells. Simvastatin induced activation of both AMPKa (phos-phorylation at Thr172) and p38 MAPK (phosphorylation atThr180 and Tyr182) in a concentration- and time-dependentmanner (Fig. 4A and B). Phosphorylation of AMPKa at Ser485

was not affected by simvastatin in AXT cells (SupplementaryFig. S2A).

Activation of AMPK and p38 MAPK by simvastatin was pre-vented by mevalonate or GGPP as well as inhibited to a lesserextent by FPP (Fig. 4C), indicating that the activation of bothkinases is mediated via inhibition of the mevalonate pathway.

Figure 2.

Simvastatin-induced apoptosis in AXT cells. A–D, Flow cytometric analysis of AXT cells treated with 10 mmol/L simvastatin for 24 hours in the absenceor presence of 500 mmol/L mevalonate (A) or of GGPP or FPP (C) and then stained with Annexin V and propidium iodide. The correspondingpercentages of Annexin Vþ cells were also determined (B and D). E and F, Immunoblot analysis of cleaved caspase-3 in AXT cells treated with theindicated concentrations of simvastatin (E) or with 10 mmol/L simvastatin in the absence or presence of mevalonate, GGPP, or FPP for 24 hours (F).






Exposure of AXT cells to the AMPK inhibitor 5-iodotubercidin orto the p38 MAPK inhibitor BIRB796 before treatment with sim-vastatin attenuated both the statin-induced change in cell mor-phology (Supplementary Fig. S2B) and its inhibition of cellviability (Fig. 4D). Such treatment with 5-iodotubercidin orBIRB796 also markedly inhibited the induction of apoptosis bysimvastatin in AXT cells as revealed by flow cytometric analysis ofAnnexin V and propidium iodide staining (Fig. 4E and F) as wellas by immunoblot analysis of cleaved caspase-3 (SupplementaryFig. S2C and S2D). Thesefindings thus indicated that activation ofAMPK and p38MAPKmediates simvastatin-induced apoptosis inAXT cells.

The activation of p38 MAPK by simvastatin was inhibitedby 5-iodotubercidin (Fig. 4G) whereas that of AMPK was notinhibited by BIRB796 (Fig. 4H), indicating that AMPK func-tions as an upstream regulator of p38 MAPK in simvastatin-induced AXT cell death. Consistent with these findings, theAMPK activator AICAR induced the phosphorylation of bothp38 MAPK and ACC (Supplementary Fig. S2E), the latter ofwhich is a downstream substrate of AMPK (29). In addition,AICAR markedly attenuated the proliferation of AXT cells in a

manner sensitive to prior exposure of the cells to the AMPKinhibitor compound C (Supplementary Fig. S2F), indicatingthat activation of AMPK inhibits cell proliferation. Notably,inhibition of Rho activity by C3 transferase triggered the activa-tion of AMPK and p38 MAPK as well as morphologic changesmimicking those caused by simvastatin treatment (Fig. 4I;Supplementary Fig. S2G). Collectively, these data indicatedthat simvastatin induces apoptosis in AXT cells via dysregula-tion of Rho GTPases accompanied by activation of the AMPK/p38 MAPK pathway in a manner dependent on inhibition ofthe mevalonate pathway.

Combined antiproliferative effect of simvastatin andmetformin in AXT cells

Given that simvastatin activates AMPK in AXT cells, we exam-ined whether the combination of simvastatin and metformin, awell-known inducer of AMPK activation (30), could furtherenhance the cytotoxicity of AXT cells. The combination of sim-vastatin and metformin manifested synergistic antitumor activityin prostate cancer previously (31). The combination of both drugsinhibited AXT cell proliferation to an extent greater than that

Figure 3.

Simvastatin disturbs the activation and localization of RhoA in AXT cells. A, Immunoblot analysis of RhoA in membrane and cytosolic fractions ofAXT cells treated with 10 mmol/L simvastatin for 12 hours. Integrin-b1 and actin were examined as loading controls for membrane and cytosolicfractions, respectively. B, RT and real-time PCR analysis of RhoamRNA in AXT cells treated with 5 mmol/L simvastatin for 13 hours. Data are normalized by theamount of Actb mRNA and are means � SD of triplicates from a representative experiment. C, Immunoblot analysis of RhoA in AXT cells treated withsimvastatin for 12 hours. D, Immunoblot analysis of RhoA and RhoA-GTP in AXT cells treated with 10 mmol/L simvastatin for 12 hours in the absence orpresence of mevalonate, GGPP, or FPP. RhoA-GTP was isolated from cell lysates by precipitation with beads conjugated with a GST fusion proteincontaining the Rho binding domain of Rhotekin. E, AXT cells treated with 10 mmol/L simvastatin for 12 hours in the absence or presence of mevalonatewere subjected to immunoprecipitation (IP) with antibodies to Rho-GDI, and the resulting precipitates as well as the input lysates were subjected toimmunoblot analysis with the indicated antibodies.

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apparent with either agent alone (Fig. 5A). This combined effectwas attenuated only, in part, by mevalonate (Fig. 5B), suggestingthat the antiproliferative effect of metformin is independent ofinhibition of the mevalonate pathway. The combination of bothdrugs also induced the accumulation of RhoA-GTP to a greaterextent than did either agent alone,with this combined effect beingabolished in the presence ofmevalonate and attenuated in part bythat of GGPP or FPP (Fig. 5C). In addition, the combinationtreatment induced a shift in the localization of RhoA from themembrane fraction to the cytosol even at lower concentration ofsimvastatin (Fig. 3A, 5D). Furthermore, simvastatin and metfor-min together induced AMPK and p38 MAPK activation to agreater extent than did either drug alone (Fig. 5E and F). Together,these in vitro observations indicated that the combination ofsimvastatin and metformin might possess antitumor activityagainst osteosarcoma.

Combined antitumor effect of simvastatin and a fat-free dietwith or without metformin in vivo

To determine whether simvastatin might possess antitumoractivity against AXT cells in vivo, we injected syngeneic C57BL/6

mice with these cells and then treated the animals with simva-statin by intraperitoneal injection or oral gavage. Although eachtreatment protocol with high dosage of simvastatin induced aslight decrease in tumor weight, these effects were not statisticallysignificant (Supplementary Fig. S3A and S3B). Allmice were alive,fine, and not exhausted during the treatment.

Statins inactivate ACC, an enzyme that carboxylates AcetylCoA and mediates de novo fatty acid biosynthesis and lipogen-esis (32). ACC is also a major downstream target of AMPK, andinactivation of ACC by phosphorylation is thought to triggerapoptosis in cancer cells (33). Simvastatin induced phosphor-ylation of ACC (on Ser79) in a concentration-dependent mannerin AXT cells, and this effect was prevented by mevalonate orGGPP but not by FPP (Fig. 6A and B). These findings suggestedthat simvastatin inactivates ACC via inhibition of the mevalo-nate pathway. The simvastatin-induced inactivation of ACC wasalso inhibited by 5-iodotubercidin (Fig. 6C), suggesting that thiseffect of the statin is also mediated by AMPK activation. Treat-ment of AXT cells with the ACC inhibitor TOFA attenuated cellproliferation in a concentration-dependent manner (Supple-mentary Fig. S3C). Furthermore, the combination of simvastatin

Figure 4.

Simvastatin-induced apoptosis mediated by activation of AMPK and p38 MAPK in a manner dependent on mevalonate pathway inhibition. A and B,Immunoblot analysis of phosphorylated (p-) and total forms of AMPK (A) and p38 MAPK (B) in AXT cells treated with the indicated concentrations ofsimvastatin. C, Immunoblot analysis of AMPK and p38 MAPK phosphorylation in AXT cells treated with 10 mmol/L simvastatin for 20 hours in theabsence or presence of mevalonate, GGPP, or FPP. D, Viability of AXT cells exposed to 10 mmol/L BIRB796 (BIRB) or 3 mmol/L 5-iodotubercidin (Iodo) inthe additional presence of simvastatin for 1 day. E and F, Flow cytometric analysis of AXT cells exposed to 2 mmol/L 5-iodotubercidin or 20 mmol/LBIRB796 in the additional presence of 5 mmol/L simvastatin for 20 hours. Representative profiles (E) and the percentages of Annexin Vþ cells (F) areshown. G and H, Immunoblot analysis of AMPK and p38 MAPK phosphorylation in AXT cells exposed to the indicated concentrations of 5-iodotubercidin(G) or BIRB796 (H) in the additional presence of 5 mmol/L simvastatin for 14 hours. I, Immunoblot analysis of AMPK and p38 MAPK phosphorylation inAXT cells exposed to C3 transferase for 24 hours.






and TOFA inhibited cell proliferation to a greater extent than dideither agent alone (Fig. 6D). Together, these data suggested thatsimvastatin targets fatty acid synthesis via inactivation of ACC.

Then, on the basis of the mechanistic insight above, toenhance the antitumor effect of simvastatin in vivo by further

activation of AMPK and its downstream effect, we examinedcombination of metformin and a fat-free diet. We applied50 mg/kg of simvastatin which was assumed to be safelyadministrable by the former single treatment (SupplementaryFig. S3A and S3B).

Figure 5.

Additive effects of simvastatin and metformin on AXT cell proliferation as well as on RhoA, AMPK, and p38 MAPK signaling. A and B, Viability of AXTcells treated with the indicated concentrations of simvastatin and metformin (Met; A) or with simvastatin, metformin, and mevalonate (B) for 2 days.C, Immunoblot analysis of RhoA and RhoA-GTP in AXT cells treated with the indicated reagents for 12 hours. D, Immunoblot analysis of RhoA in membraneand cytosolic fractions of AXT cells treated with simvastatin and metformin for 12 hours. E and F, Immunoblot analysis of AMPK and p38 MAPKphosphorylation as well as of caspase-3 cleavage in AXT cells treated with 0.5 mmol/L simvastatin and the indicated concentrations of metformin for12 hours (E) or 24 hours (F). The arrow indicates p-p38 MAPK.

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Whereas a fat-free diet alone did not affect the growth of AXTcell tumors in mice (Supplementary Fig. S3D), intraperitonealinjection of simvastatin in mice fed such a diet resulted in asignificant inhibition of tumor growth (Fig. 6E and F). Further-

more, whereasmetformin alone did not significantly affect tumorgrowth inmice fed a fat-free diet, it enhanced the antitumor effectof simvastatin in such animals (Fig. 6E and F). In fact, toxicityappeared to be a problem. In total, 5 of 6 mice in each group

Figure 6.

Antitumor effect of simvastatin in vivo. A and B, Immunoblot analysis of ACC phosphorylation in AXT cells treated with the indicated concentrations ofsimvastatin for 24 hours (A) or with 10 mmol/L simvastatin in the absence or presence of mevalonate, GGPP, or FPP for 20 hours (B). C, Immunoblotanalysis of AMPK and ACC phosphorylation in AXT cells treated with 5 mmol/L simvastatin either alone or together with the indicated concentrations of5-iodotubercidin for 20 hours. D, Viability of AXT cells treated with simvastatin or TOFA as indicated. E and F, Weight of AXT cell–derived tumorsformed in mice fed a fat-free diet and injected intraperitoneally with saline (control), simvastatin (50 mg/kg), metformin (100 mg/kg), or both simvastatin(50 mg/kg) and metformin (50 mg/kg). Data are means � SD (n ¼ 10), with the P values being for comparison with control (Student t test). Theexcised tumors are also shown (F). G, Immunoblot analysis of AMPK and p38 MAPK phosphorylation in 5 randomly selected tumors of each group in E.






died; 1 mouse treated with metformin at 100 mg/kg, 1 withsimvastatin at 50 mg/kg plus metformin at 50 mg/kg, and 3 withsimvastatin at 50 mg/kg plus metformin at 100 mg/kg, respec-tively, albeit without any apparent organ damage (data notshown). In addition, mice treated with simvastatin (50 mg/kg)alone or with simvastatin (50mg/kg) plusmetformin (50mg/kg)showed a body weight loss of 6.2% and 9.7%, respectively,compared with control mice. Finally, we found that the phos-phorylation of AMPK was enhanced in tumors from the micetreated with simvastatin either alone or together with metformincompared with that apparent in control tumors, whereas suchchanges in the phosphorylation of p38 MAPK were less obvious(Fig. 6G).

DiscussionTo identify new and effective therapeutic options for osteosar-

coma, we performed amultidrug screen, which revealed statins ascandidate agents. We further characterized the antitumor activityof simvastatin in vitro, finding that such activity was essentiallyabolished in the presence of mevalonate or GGPP and attenuatedto a lesser extent by FPP. These results thus indicate that theantitumor activity of simvastatin is mediated via inhibition of themevalonate pathway for biosynthesis of isoprenoids. Previousstudies have also shown that statins exert antitumor effects byinhibiting the production of mevalonate and GGPP (34). Ourresults thus suggest that inhibition of protein geranylgeranylationhas a greater impact on the viability of osteosarcoma cells com-pared with that of farnesylation.

We found that simvastatin upregulated the amount ofRhoA-GTP, induced a shift in the localization of RhoA frommembrane to cytosol, and inhibited the interaction betweenRhoA and Rho-GDI, with these effects also appearing to bemediated via inhibition of the mevalonate pathway. Isopre-noids are lipid moieties that are essential for posttranslationalmodification, membrane anchoring, and transforming activityof Ras and Rho proteins (23, 35). Statins were previouslyshown to induce apoptosis in osteosarcoma cells via inacti-vation of RhoA (12, 13). Indeed, a lack of isoprenylation ofRhoA-GTP is likely responsible for the changes in cell mor-phology and inhibition of migration induced by simvastatin inthe present study.

Simvastatin induced the activation of AMPK and p38 MAPKin AXT cells, consistent with previous findings in other cancercell type (36). Again, these effects of simvastatin were found tobe mediated through inhibition of the mevalonate pathway.Moreover, we found that this activation of both AMPK and p38MAPK directly contributes to the induction of apoptosis bysimvastatin in AXT cells. Intriguingly, as the activation of AMPKby simvastatin treatment was fully reversed by supplement ofmevalonate or GGPP in AXT cells, activation of AMPK can beregulated depending on the amount of intermediate metabo-lites of the mevalonate pathway. Conversely, activation ofAMPK was previously shown to result in inhibition of themevalonate pathway through inactivation of HMG-CoA reduc-tase, the rate-limiting enzyme of this pathway (37–39). Phos-phorylation of p38 MAPK was previously shown to be relatedto the induction of cell death by various stimuli includingstatins, with the generation of reactive oxygen species alsohaving been found to mediate this process (27, 40, 41).However, we found that simvastatin did not increase the level

of reactive oxygen species (data not shown). Notably, thedisruption of Rho activity alone can induce activation of AMPK(Fig. 4I) and a similar finding was shown in other organ system(42). Collectively, we proposed the mode of action of simva-statin, treatment of which inactivates Rho kinases accompaniedby the sequential activation of AMPK and p38 MAPK that leadsto induction of apoptosis in osteosarcoma cells (Supplemen-tary Fig. S3E).

Although simvastatin alonemanifested potent cytotoxic effectsin osteosarcoma cells in vitro, it did not exhibit a significantantitumor effect in vivo. We therefore attempted to increase suchin vivo activity of simvastatin by enhancing AMPK activationindependently in the mevalonate pathway. Then, we chose com-bination with metformin which previously exhibited furtherantitumor effect in other cancers (31). Mechanistically, simva-statin and metformin showed additive effects on the accumula-tion of RhoA-GTP and the phosphorylation of AMPK and p38MAPK. In addition, given that AMPK regulates fatty acid synthesisthrough phosphorylation of ACC (29), we examined the antitu-mor effect of simvastatin in animals fed a fat-free diet to furtherenhance this downstream effect of AMPK signaling. Simvastatinalone inhibited the growth of osteosarcoma tumors in mice fed afat-free diet and that this effect was enhanced by metformin.Accumulating evidence suggests that statins sensitize malignantcells to various chemotherapeutic agents and strengthen theantitumor activity (43, 44). Therefore, simvastatin also mightbecome a promising agent that improves chemosensitivity inosteosarcoma.

Collectively, our results have demonstrated the potential ofsimvastatin as a new therapeutic agent for osteosarcoma. Theadoption of simvastatin for such treatment, however, will requirefurther refinement of the optimal conditions for it to exert itsantitumor action and to reduce the toxicity.

Disclosure of Potential Conflicts of InterestH. Saya reports receiving commercial research grant from Daiichi Sankyo

Inc., Eisai Co Ltd. andNihonNohyaku Co Ltd. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: W.A. Kamel, T. ShimizuDevelopment of methodology: W.A. Kamel, S. Yamaguchi-Iwai, T. ShimizuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W.A. Kamel, E. Sugihara, K. Maki, Y. Fukuchi,T. ShimizuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):W.A. Kamel, S. Yamaguchi-Iwai, K. Maki, Y. Fukuchi,T. ShimizuWriting, review, and/or revision of the manuscript: W.A. Kamel, K. Matsuo,T. ShimizuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):W.A. Kamel, H. Nobusue, N. Onishi, T. ShimizuStudy supervision: W.A. Kamel, S. Yamaguchi-Iwai, K. Matsuo, A. Muto,H. Saya, T. Shimizu

AcknowledgmentsWe thank I. Ishimatsu for technical assistance and M. Sato for help with

preparation of this article.

Grant SupportThis work was supported by funding from the Project for development of

Innovative Research on Cancer Therapeutics (P-Direct, to H. Saya) of TheJapan Agency for Medical Research and Development, by Kakenhi grantfrom Japan Society for the Promotion of Science (JSPS; to T. Shimizu;

Kamel et al.





#15K06845), by the Supported Program for the Strategic Research Founda-tion at Private Universities of the Ministry of Education, Culture, Sports,Science, and Technology of Japan (MEXT; T. Shimizu), and by TakedaScience Foundation (T. Shimizu).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 28, 2016; revisedOctober 20, 2016; acceptedOctober 21, 2016;published OnlineFirst October 31, 2016.

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2017;16:182-192. Published OnlineFirst October 31, 2016.Mol Cancer Ther Walied A. Kamel, Eiji Sugihara, Hiroyuki Nobusue, et al. of RhoA-AMPK/p38 MAPK Signaling in Antitumor ActivitySimvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role

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