Aspirin Suppresses the Growth and Metastasis of ... · Osteosarcoma, an extremely aggressive form...

Cancer Therapy: Preclinical

Aspirin Suppresses the Growth and Metastasis ofOsteosarcoma through the NF-kB PathwayDan Liao, Li Zhong, Tingmei Duan, Ru-Hua Zhang, Xin Wang, Gang Wang,Kaishun Hu, Xiaobin Lv, and Tiebang Kang

Abstract

Purpose: Aspirin has recently been reported to reduce boththe incidence and the risk of metastasis in colon cancer.However, there is no evidence at the cellular levels or in theanimal models for such an effect of aspirin on cancermetastasis.

Experimental Design:MTT assay, colony formation assay, andapoptosis assaywere employed to analyze the effects of aspirin onthe osteosarcoma cell viability in vitro. The NF-kB activity wasmeasured by the NF-kB p65 luciferase reporter. Western blottingwas used to analyze the proteins in cells. The migration andinvasion abilities of osteosarcoma cells in vitro were measuredby the Transwell assay. Xenograft-bearingmicewere used to assessthe roles of aspirin in both tumor growth and metastasis ofosteosarcoma in vivo (n¼ 5–8mice/group). An unpaired Student

t test or ANOVAwith the Bonferroni post hoc test were used for thestatistical comparisons.

Results: Aspirin reduced cell viability in a dose- and time-dependent manner in osteosarcoma cell lines, and aspirin syn-ergistically sensitized osteosarcoma cells to cisplatin (DDP)in vitro and in vivo (P < 0.001). Moreover, aspirin markedlyrepressed the migration and invasion of osteosarcoma cellsin vitro (P < 0.001), and dramatically diminished the occurrenceof osteosarcoma xenograft metastases to the lungs in vivo(P < 0.001). Mechanistically, aspirin diminishes osteosarcomamigration, invasion, and metastasis through the NF-kB pathway.

Conclusions: Aspirin suppresses both the growth and metastasisof osteosarcoma through theNF-kBpathway at the cellular level andin the animal models. Clin Cancer Res; 21(23); 5349–59. �2015 AACR.

IntroductionOsteosarcoma, which has a peak incidence in adolescence, is an

extremely aggressive primarymalignant bone tumor of childhood(1). Currently, the 5-year overall survival is 75% to 77% for theprimary nonmetastatic disease, however, no more than 20% inmetastatic osteosarcoma (2, 3). Patients with metastasis or recur-rence present a formidable challenge despitemodernmultimodaltherapy (4, 5). Therefore, finding new strategies for the patientswith osteosarcoma are necessary and urgent.

Aspirin, an NSAID, was used primarily for anti-inflammationand analgesic (6). Interestingly, a recent population studyreported that acetylsalicylic acid (aspirin) reduces both the inci-dence and the risk of metastasis in colon cancer (7). However,there is no evidence at the cellular level or in animal models forsuch an effect of aspirin on cancer metastasis, even though aspirinis widely used for both the prevention and treatment of manydiseases, including cancers in clinical settings (7–14). In thisreport, we tried to provide comprehensive evidence at the cellular

level and in the animal models to argue that aspirin may be anexcellent auxiliary drug for treating patients with osteosarcoma.

Materials and MethodsReagents and cell culture

U2OS and MG63 cells were cultured according to the instruc-tions from the ATCC. ZOS and U2OS/MTX300 cells weredescribed previously (15). HEK-293T cells were purchased fromthe ATCC, cultured in DMEM (Invitrogen) with 10% FBS(GIBCO), 1 mmol/L glutamine, and 100 U/mL each of penicillinand streptomycin. All cell lines used in this study were authen-ticated using short-tandem repeat profiling less than 6 monthsago when this project was initiated, and the cells have not been inculture for more than 2 months.

Aspirin was purchased from Sigma-Aldrich. Antibodies againstp65(C22B4), XIAP(3B6), CIAP1(D5G9), CIAP2(58C7), BCL2(50E3), Survivin(6E4), Livin(D61D1), and a-Tublin(2148S)were purchased from Cell Signaling Technology. An antibodyagainst GAPDH(SC-166574) was purchased from Santa CruzBiotechnology.

PlasmidsThe full-length p65 cDNA was cloned into the pBABE vector

from human 293T cells. P65 was generated by PCR amplification,subcloned into the pBABE and pCDNA3.1 vectors, and verified byDNA sequencing. The p65 shRNA was purchased from Sigma-Aldrich. The CMV-luciferase plasmid was a gift from Prof. Xiaom-ing Xie (Sun Yat-Sen University, Guangzhou, China; ref. 16).

Cell viability assayU2OS, MG63, or ZOS cells were seeded in 96-well plates at a

density of 3,000 cells per well. They were treated with different

State Key Laboratory of Oncology in South China, CollaborativeInnovationCenter for CancerMedicine, SunYat-SenUniversityCancerCenter, Guangzhou, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Tiebang Kang, State Key Laboratory of Oncology inSouth China, Sun Yat-Sen University Cancer Center, 651 Dongfeng East Road,Guangzhou 510060, China. Phone: 86-20-8734-3183; Fax: 86-20-8734-3170;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0198

�2015 American Association for Cancer Research.

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concentrations of Aspirin (0, 1, 2.5, 5, 10, or 20 mmol/L) and/orchemotherapy drugs (DDP, 10mmol/L) for the indicated times,and the cell viability was measured by MTT assays as describedpreviously (15, 17).

Clone formation assayColony formation assays were performed as described previ-

ously (15, 18). ZOS or U2OS cells plated in triplicate at 500cells per well in 6-well plates were treated with vehicle (DMSO),10 mmol/L DDP, 5 mmol/L aspirin, or 10mmol/L DDP plus 5mmol/L aspirin for 48 hours, and then cultured for 10 days. Thecell clones were washedwith PBS, and then fixed inmethanol anddyed with 0.1% crystal violet, and the colonies that containedmore than 50 cells were counted.

Apoptosis flow-cytometry assayDrug-induced apoptosis was detected using an Annexin V-

EGFP apoptosis detection kit (KeyGEN). Treated ZOS, MG63, orU2OS cells (negative control, 10 mmol/L DDP, 5mmol/L aspirin,or 10 mmol/L DDP plus 5 mmol/L aspirin for 48 hours) wereharvested, the assay were performed according to the manufac-turer's instruction. The stained cells were analyzed using aCytomics FC500 flow cytometer (Beckman Coulter).

The effect of combination of DDP and aspirinThis effect of the combination of DDP and aspirin was assessed

by the Chou-Talalay method (19, 20). Briefly, the cells weretreated with serial dilutions of DDP and aspirin separately orsimultaneously for 48 hours, and the viability of the cells wasquantified using MTT assays. A combination index (CI) wascalculated using CalcuSyn software following the equation:(CI) ¼ (D)1/(Dx)1 þ (D)2/(Dx)2, where (D)1 and (D)2 are thedoses of drugs 1 and 2 that have x effect when used in combina-tion; and (Dx)1 and (Dx)2 are the doses of drugs 1 and 2 that havethe same x effect when used alone. Themolar ratio ofDDP:aspirinis 1:1. Adrug combination is additive, synergistic and antagonisticwhen CI ¼ 1.0, < 1.0, and > 1.0, respectively.

Transfection experimentsp65 siRNAs were synthesized by GenePharma. Transfections

were performed according to themanufacturer's instructions usingthe LipofectamineRNAiMAX transfection reagent (Invitrogen) and

50 nmol/L siRNA. The transfections were performed as describedpreviously (15). Briefly, asynchronously growing cells seededat 2.5� 105 cells per well in a 6-well plate or at 1 � 106 cells in a 10-cmplate were transfected with 2 or 12 mg plasmid DNA, respectively,using Lipofectamine 2000 (Invitrogen). Cell lines that stablyexpressed the NC or p65 shRNAs were established by the SigmashRNA system according to the manufacturer's instructions.

Dual-luciferase reporter assayTransfection was performed as described previously (15). The

transfected p65-siRNA cells incubated for 24 hours or U2OS cellsstably overexpressing p65 were transfected with NF-kB p65 lucif-erase reporter and pRL-TK Renilla luciferase construct (Promega)per well using Lipofectamine 2000 (Invitrogen). After 24 hours,the cells were treated with aspirin (5mmol/L). The cells were thenanalyzed after an additional 24 hours according to the Dual-Luciferase Assay System protocol (Promega).

Boyden chamber assaysThe migration and invasion of osteosarcoma cells were exam-

ined using 24-well Boyden chambers with 8-mm inserts coatedwithout (migration) or with Matrigel (invasion) as previouslydescribed (21). A total of 5� 104 cells per well were plated on theinserts and cultured at 37 oC in the upper chambers withoutserum and supplemented with Aspirin (5 mmol/L). After 24hours, the cells that crossed the inserts were stained with crystalviolet (0.005%, sigma) and counted as the number of cells perfield of view under phase-contrast microscopy.

Western blottingWestern blotting was performed as previously described (18).

Briefly, the cells were lysed in RIPA buffer containing proteaseinhibitor andphosphatase inhibitor cocktails (ThermoScientific).The nuclear protein was isolated according to the protocol pro-vided by the Nuclear Protein Extraction Kit (Thermo Scientific).The protein extracts were resolved using SDS-PAGE and trans-ferred to polyvinylidene difluoride membranes for Western blot-ting using ECL detection reagents (Beyotime Co. Haimen).

Mouse xenograftAnimal experiments were approved by the Animal Research

Committee of Sun Yat-sen University Cancer Center and wereperformed in accordance with established guidelines. For osteo-sarcoma xenograft growth of orthotopic animal model, U2OS/MTX300 cells were used as previously described (15, 22). Twoweeks after the cells were injected, the mice were randomlyseparated into four groups (n ¼ 6). The mice were treated withDMSO, aspirin (100 mg/kg) by intragastric administration everyday, and/or DDP (3mg/kg) by i.p. injection every week. Themicewere monitored every 2 days for tumor formation and sacrificedwhen the tumors reached approximately 1.5 cm in diameter, andthe tumor xenografts were harvested and weighted. The tumorvolume was calculated using the formula V ¼ 4/3 p[1/4(D1þD2)2, as described previously (22).

For the spontaneous metastasis model, human osteosarcomacells were transplanted orthotopically into the bones of miceas previously described (15, 22). We used 143B cells stablyexpressing luciferase (143B-luci) or U2OS/MTX300 cells stablyexpressing luciferase (U2OS/MTX300-luci) and U2OS/MTX300-luci–derived cells, including U2OS/MTX300-p65sh#1, -p65sh#2,-vector and -p65–bearing stable knockdowns of p65,

Translational Relevance

Osteosarcoma, an extremely aggressive form of primarymalignant bone tumor in childhood, has a high rate of lungmetastasis. A recent population study reported that aspirinreduces both the incidence and the risk of metastasis in coloncancer. Our results showed that aspirin inhibits osteosarcomametastasis and enhances the chemotherapy of osteosarcomain vitro and in vivo, which is thefirst evidence at the cellular leveland in an animalmodel for the inhibition of aspirin on cancermetastasis. This strongly argues that aspirin may be an excel-lent auxiliary drug for treating patients with osteosarcoma,suggesting that it is worthy to design a clinical trial using thecombination of aspirin with the current standard therapies forpatients with osteosarcoma.

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Clin Cancer Res; 21(23) December 1, 2015 Clinical Cancer Research5350




overexpressing vector, and p65, respectively. One week after thecells were injected, the mice were randomly separated into twogroups (n ¼ 6). The mice were treated with DMSO or aspirin(100 mg/kg) by intragastric administration every day. When thexenografts reached 1.5 cm in diameter, the lungs of mice bearingthe osteosarcoma tumor xenografts that stably expressed lucifer-ase were analyzed using an IVIS Lumina Imaging System (Xeno-gen). The mice were then sacrificed, the lungs of the mice wereweighed, and the number of metastatic nodules in the lungs wascounted. The harvested lungs were fixed in formalin (4 mmol/L)and stained by hematoxylin and eosin (H&E) for histologicassessment, and total RNA was extracted for quantitative real-time reverse transcription-PCR (qRT-PCR) analysis of humanHPRT mRNA expression.

qRT-PCRqRT-PCR was performed using a LightCycler 480 instrument

(Roche Diagnostics) and SYBR Premix Ex Taq (TaKaRa) accord-ing to the manufacturer's instructions. All reactions were carriedout in a 10 mL reaction volume in triplicate. The primers forGAPDH were obtained from Invitrogen. Standard curves weregenerated, and the relative amount of target gene mRNA wasnormalized to that of GAPDH. The specificity was verified bymelting curve analysis. To quantify cancer metastasis in mouselungs, qRT-PCR for human hypoxanthine-guanine-phosphor-ibosyl transferase (hHPRT) was performed on TRizol (Invitro-gen)-isolated total RNA using primers for hHPRT and 18SrRNA. The oligo nucleotide sequences of the qRT-PCR primersare as follows:

Gene primer (F: forward, R: reverse)

P65 F: 50-AGCTCAAGATCTGCCGAGTG-30

R: 50-ACATCAGCTTGCGAAAAGGA-30

GAPDH F: 50-ATCACCATCTTCCAGGAGCGA-30

R: 50-CCTTCTCCATGGTGGTGAAGAC-30

hPRT F: 50-TTCCTTGGTCAGGCAGTATAATCC-30

R: 50-AGTCTGGCTTATATCCAACACTTCG-30

18sRNA F: 50-CGGCTACCACATCCAAGGAA-30

R: 50-GCTGGAATTACCGCGGCT-30

Statistical analysisThedata are presented as themean� SD. The error bars indicate

the SD. Two-sided ANOVA with the Bonferroni post hoc test, two-tailed Student t test, or Mann–Whitney test was used to comparethe differences between subgroups. �, P < 0.05 was considered tobe significant; ��, P < 0.01 and ��, P < 0.001 was considered to bestrongly significant.

ResultsAspirin inhibits the osteosarcoma growth in vitro and in vivo

We recently showed that the inhibition of the NF-kB pathwayimpeded the growth of osteosarcoma and increased the sensitivityof osteosarcoma to chemotherapies in vitro and in vivo (15). It iswell known that aspirin inhibits the activity ofNF-kB (23, 24) andthe aspirin induces apoptosis in many cancers (25–28), wereasoned that aspirin would improve the efficiency of osteosar-coma to chemotherapies. Using the osteosarcoma cell lines,including U2OS, ZOS, and MG63, we observed that aspirinreduced cell viability in a dose- and time-dependent manner

(Fig. 1A). Because patients with osteosarcoma often developchemoresistence in clinic, we sought to determinewhether aspirinsensitizes osteosarcoma cells to DDP, the common used chemo-therapeutic drug in osteosarcoma patients. As shown in Fig. 1Band C; Supplementary Fig. S1, the combination of DDP withaspirin was more efficiently decreasing the osteosarcoma cellviability and the clone formation ability (P < 0.05). Furthermore,Aspirin sensitized osteosarcoma cells to the DDP-induced apo-ptosis in vitro (Fig. 1D; Supplementary Fig. S2). In addition, thecombination of DDP with aspirin was synergistic at low concen-trations showed by Fa-CI plots (Fig. 1E). Collectively, these resultsindicate that aspirin inhibits the proliferation of osteosarcomacells and promotes the apoptosis of osteosarcoma cells inducedby DDP in vitro.

Next, we investigated whether aspirin could inhibit theosteosarcoma growth using the osteosarcoma orthotropic ani-mal model in vivo. As shown in Fig. 2B, sacrificed the tumor-bearing mice at the 24th day, we found the combinationof aspirin and DDP showed more significant inhibition oftumor growth, compared with either aspirin or DDP alone(P < 0.001). As shown in Fig. 2A and C, consistent with thetumor growth curve, the tumor weights and volumes weremore decreased in the group of combining aspirin with DDP,compared with aspirin or DDP alone (P < 0.01). More impor-tantly, the combination of aspirin and DDP did not increasetheir toxicities in vivo, as indicated by mouse body weight(Fig. 2D). Taken together, these results reveal that aspirinenhances the sensitivity of osteosarcoma to chemotherapiesin vitro and in vivo.

Aspirin inhibits the osteosarcoma metastasis in vivoAspirin has recently been reported to reduce both the inci-

dence and the risk of metastasis in colon cancer (7), andosteosarcoma is often resistant to the conventional chemother-apy drugs because of high rate of lung metastasis (29), wesought to determine whether aspirin has inhibition on osteo-sarcoma metastasis in vivo. As shown in Fig. 3A; SupplementaryFig. S3, aspirin markedly suppressed the migration and inva-sion of osteosarcoma cells as measured by Boyden chamberassays in vitro (P < 0.01). More strikingly, using a spontaneousin vivo metastasis model of human osteosarcoma cells (Sup-plementary Fig. S4), and evaluated cancer metastasis to thelungs when xenografts reached 1.5 cm in diameter. Lumines-cence imaging for the lungs identified the severity of lungmetastasis was inhibited by the aspirin (Fig. 3B). Aspirindramatically diminished the occurrence of osteosarcoma xeno-graft metastases to the lungs, as indicated by the number ofmetastatic nodules in the lung (Fig. 3C, P < 0.05) and the wetlung weight (Fig. 3E, P < 0.01). H&E staining also showed thataspirin reduced the metastasis in the lungs of the mice bearingU2OS/MTX300 or 143B xenografts compared with DMSOtreatment (Fig. 3D). In addition, the human HPRT mRNAlevels detected by real-time PCR were decreased by 89% inU2OS/MTX300 xenografts and 77% in 143B xenografts model,respectively (Fig. 3E). Collectively, these results demonstratethat aspirin inhibits osteosarcoma metastasis in vivo.

Aspirin inhibits osteosarcoma metastasis through the NF-kBpathway

Aspirin has been shown to inhibit the activity of NF-kB,which plays key roles in diverse physiologic and pathologic

Aspirin Suppresses Osteosarcoma through the NF-kB Pathway

www.aacrjournals.org Clin Cancer Res; 21(23) December 1, 2015 5351




Figure 1.Aspirin inhibits the proliferation and promotes apoptosis of osteosarcoma cells. A, aspirin inhibits osteosarcoma cell (U2OS, MG63, and ZOS) viability in a time-and dose-dependent manner. Osteosarcoma cells were treated with different concentrations of aspirin for the indicated times, and the viability of thesecells wasmeasured usingMTT assays; ��, P <0.01. A two-sided ANOVAwith a Bonferroni post hoc test was used for statistical analysis. B and C, aspirin augments thechemosensitivity of osteosarcoma cells to DDP. B, the indicated cells were treated with 10 mmol/L DDP with or without 7.5 mmol/L aspirin for 48 hours asindicated, and the viability of these cells was measured by MTT assays; n ¼ 3; �� , P < 0.01. (Continued on the following page.)

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processes, including apoptosis, proliferation, migration, inva-sion, and metastasis (30–32). In addition, we recently showedthat the inhibition of the NF-kB pathway impaired the growthof osteosarcoma and increased the sensitivity of osteosarcomato chemotherapies in vitro and in vivo (15). Therefore, we werevery curious to determine whether aspirin also had an inhib-itory effect on the osteosarcoma metastasis by modulating theNF-kB pathway. Osteosarcoma cells transfected with NF-kBluciferase plasmid, and then incubated with aspirin for24 hours. The p65 NF-kB luciferase reporter activity wasdecreased by 50% after treated with aspirin in osteosarcomacells (Fig. 4A, P < 0.01). Consistently, the nuclear localizationof p65 protein, the indicator of NF-kB transcription activity,was also decreased in osteosarcoma cells treated with aspirin(Fig 4B). Consequently, the NF-kB–targeted genes, such asCIAP, XIAP, BCL2, and Survivin, were inhibited by aspirin ina dose- and time-dependent manner in both ZOS and U2OScells (Fig. 4C).

Next, we explored whether the inhibition of aspirin on oste-osarcoma metastasis depends on the NF-kB pathway. First, asshown in Fig. 5A–C; Supplementary Fig. S5, aspirin had no effecton the NF-kB luciferase reporter activity, the migration andinvasion of osteosarcoma cells knockdown of p65 by siRNA inthese cells (P > 0.05). Conversely, as shown in Fig. 5D–F; Sup-

plementary Fig. S6, the impairment of aspirin on these eventswerecompletely rescued by in these osteosarcoma cells overexpressingp65. Second, we stably knocked down and overexpressed the p65in U2OS/MTX300-luci cells, which were identified by Westernblotting (Fig. 6A). Using a spontaneous metastasis model in vivoand the assays mentioned above, as shown in Fig. 6B–E, theinhibition of osteosarcomametastasis to the lungs by aspirin wasnot further enhanced in the cells bearing a stable knockdown ofp65 by shRNA, but was abolished in the cells stably overexpres-sing p65. These results demonstrate that Aspirin diminishes boththe growth and metastasis of osteosarcoma through the NF-kBpathway.

DiscussionPatients with metastatic or relapsed osteosarcoma have a

poor prognosis and new therapies are needed. Aspirin hasrecently been reported to reduce the incidence and risk ofmetastasis in colon cancer patients (7). In the present study,we demonstrated that aspirin diminishes the growth andmetastasis of osteosarcoma through the NF-kB pathway in vitroand in vivo.

Aspirin can inhibit the activity of NF-kB (23, 24), and theinhibition of the NF-kB pathway repressed the growth of

Figure 2.Aspirin impedes the osteosarcomagrowth in vivo. A to D, U2OS/MTX300cells were implanted in the proximal tibiaand allowed to grow for 24 days(n¼ 6/group). Starting on day 14, themicewere given 100 mg/kg aspirin every dayand/or DDP (3 mg/kg) once a week i.p.The tumor volumes and mice weight weremonitored on day 24 and then every 2days, as indicated (B and D), and thexenografts were excised and weighed onday 24 (A andC); �� ,P <0.01; �� , P <0.001.A two-sided ANOVA with a Bonferronipost hoc test was used for statisticalanalysis.

(Continued.) C, ZOS or U2OS cells plated in triplicate at 500 cells per well in 6-well plates were treated with vehicle (DMSO), 10 mmol/L DDP, 5 mmol/L aspirin, or10 mmol/L DDP plus 5 mmol/L aspirin for 48 hours, cultured for 10 days and then subjected to clone formation assays; n ¼ 3; �� , P < 0.01. A two-sided ANOVAwith a Bonferroni post hoc test was used for statistical analysis. D, aspirin promotes DDP-induced apoptosis in osteosarcoma cell lines. The indicatedcells were treated with 10 mmol/L DDP with or without 5 mmol/L aspirin for 48 hours as indicated, and then subjected to Annexin V assays, and the percentages ofapoptosis in the indicated cells were shown as the histogram; n ¼ 3. Bars, SD; �� , P < 0.01. A two-sided ANOVA with a Bonferroni post hoc test was used forstatistical analysis. E, the combination of DDP with aspirin is synergistic at low concentrations. U2OS cells were treated with different concentrations of bothDDP and aspirin for 48 hours, and the viability of the cells was analyzed by MTT assays. The molar ratio of DDP:aspirin is 1:1; n ¼ 3. A drug combination is additivewhen CI ¼ 1.0, antagonistic when CI > 1.0 and synergistic when CI < 1.0, respectively.






osteosarcoma and increased the sensitivity of osteosarcoma tochemotherapies in vitro and in vivo (15). Thus, it was reason-able to speculate that aspirin may sensitize osteosarcoma cellsto DDP. Indeed, this was the case, as our results indicated thatthe combination of DDP and aspirin was more efficient inkilling osteosarcoma cells in vitro and in vivo than either DDPor aspirin alone. This is consistent with the fact that regularand long-time intake of aspirin with a significant reduction inthe prevention and treatment of colorectal cancer (7, 33, 34),as inflammation has key roles in tumor initiation, promotion,and metastasis (35). In fact, SDX-308, another NSAID agent,has been shown to impair the multiple myeloma cell prolif-eration and osteoclast activity by inhibiting the NF-kB activitysignaling (31). On the other hand, De Luna-Bertos found thattherapeutic doses of NSAIDs (dexketoprofen, ketorolac, meta-mizle, and aspirin) modulated differentiation and antigenicprofile activity of MG63 cells, whereas both dexketoprofen and

ketorolac inhibited osteoblast growth by arresting the cell-cycle and inducing apoptosis, which are influenced by thedose, duration of treatment, and cells used (36–39). Theseresults indicate that the effects of these different NSAIDs arecomplicated in bone biology and/or osteosarcoma, and thedifferences in these data may be explained by the dose,treatment duration, and different NSAIDs used (40).

Strikingly, recent population studies support that aspirinreduces the risk of metastasis in colon cancer (7, 41). However,there is no evidence at the cellular levels or in the animalmodels for such an effect of aspirin on cancer metastasis, eventhough aspirin is widely used for both the prevention andtreatment of many diseases, including cancers in clinical set-tings (8, 10–14, 9). In this report, we demonstrated thataspirin markedly suppressed the migration and invasion ofosteosarcoma cells in vitro, and that Aspirin inhibited themetastasis of osteosarcoma in vivo. Mechanistically, this

Figure 3.Aspirin suppresses the osteosarcomamigration, invasion, and metastasis.A, aspirin inhibits the invasion andmigration abilities of osteosarcomacells. The indicated cells were treatedwith or without aspirin (5 mmol/L), andcell migration and invasion weredetermined as described in Materialsand Methods; n ¼ 3. ��P < 0.01. A two-tailed Student t test was used forstatistical analysis. B to F, aspirininhibits osteosarcomametastasis to thelungs in nude mice in vivo. Both U2OS/MTX300 cells and 143B cells stablyexpressing luciferase were used toanalyze in vivo spontaneousmetastasisto the lungs of nude mice treated withor without aspirin (100 mg/kg/d) in anorthotopic osteosarcoma model asdescribed in Materials and Methods;n ¼ 6 for each group. B showsrepresentative images, whereas Cillustrates the statistical results forthe number of metastatic nodulesin the lungs of nudemice using aMann–Whitney test. Dots, scores. Bars, SD;�� , P < 0.01. The expression of humanHPRT mRNA relative to mouse 18SrRNA in the lungs of tumor-bearingnude mice is indicated in F. Theaverages and error bars correspond tothe means� SDs. D and E, illustrate thewet lung weights and H&E staining oflungs from the tumor-bearing nudemice, respectively. Bars, SD; � , P < 0.05and �� , P < 0.01. A two-tailed Student ttest was used for statistical analysis.

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inhibition of aspirin on migration, invasion, and metastasis ofosteosarcoma is dependent on the NF-kB pathway. This is thefirst comprehensive evidence at the cellular levels and in theanimal models to argue that aspirin may be benefit for treatingcancer patients with potential metastasis. Consistently, duringthe preparation of our article, Ogawa and colleagues reportedthat aspirin reduced lung cancer metastasis to regional lymphnodes in vivo (42). Notably, the antitumor of aspirin is indef-inite, as aspirin is protective in some cancer types, such ascolorectal, gastric, liver, and prostate cancers (7, 10, 43),whereas aspirin is useless for some other cancer types (44–46). For randomized trials of aspirin, low dose and high doseis defined as 75 to 300 mg daily and 300 to 1,500 mg daily,respectively (47). In this report, the dose of aspirin we used

was 100 mg/kg daily in mice, which is highly efficient todiminish the tumor growth and metastasis using the osteo-sarcoma orthotopic mouse model. The dose of 100 mg/kgdaily in mice is equal to 8.13 mg/kg daily in humans, meaningthat the aspirin level we tested is about 400 to 700 mg daily forhumans weighing 50 to 80 kg. This dose of aspirin fits thedefinition of high-dose (300–1,500 mg daily) aspirin studiedin previous randomized trials (47) and should provide ade-quate systemic exposure. At the same time, it should be notedthat aspirin is used at 100 or 300 mg daily in the ongoingadjuvant aspirin trials for colorectal, breast, gastrooesopha-geal, and prostate cancers (47).

In summary, we herein provide comprehensive evidence for aninhibitory effect of aspirin on osteosarcoma metastasis and

Figure 4.Aspirin inhibits the NF-kB pathway. A,inhibition of the luciferase activityof NF-kB by aspirin. The indicated cellswere transfected with a luciferasereporter for NF-kB, treated with orwithout aspirin (5 mmol/L) for 48 hours,and subjected to luciferase assays asdescribed in Materials and Methods;�� , P < 0.01. A two-tailed Student t testwas used for statistical analysis. B,inhibition of p65 nuclear translocation byaspirin. The indicated cells were treatedwith or without aspirin (5mmol/L) for 48hours and then subjected to cellfractionation and analyzed by Westernblotting. C, downstream targetsof NF-kB, such as BCL2, CIAP2, Survivin,Livin, and XIAP, were downregulated byaspirin in a dose- and time-dependentmanner in both U2OS and ZOS cells. Theindicated cells were treated withdifferent concentrations of aspirin forthe indicated times and then analyzed byWestern blot analysis. GAPDH was usedas a loading control; n ¼ 3.






Figure 5.Aspirin represses the osteosarcomamigration and invasion via the NF-kB pathway in vitro. A and B, aspirin has aminimal effect on the NF-kB transcriptional activityin both U2OS and ZOS cells knockdown of p65. The indicated cells were transfected with negative control or p65-siRNA for 24 hours and then transfectedwith an NF-kB luciferase reporter for 24 hours. The cells were treated with or without aspirin (5 mmol/L) for 24 hours, and then subjected to luciferase assays (B) asdescribed in Materials and Methods. The efficiency of p65-siRNA was determined by Western blotting (A); n ¼ 3. Bars, SD; �� , P < 0.01. A two-sidedANOVA with a Bonferroni post hoc test was used for statistical analysis. C, aspirin has no effect on the invasion and migration abilities of osteosarcoma cellsknockdown of p65. The indicated cells transfected with or without p65-siRNA (as indicated) for 24 hours were treated with or without aspirin (5 mmol/L) for24 hours, and then the migration and invasion assays (as indicated) were subsequently performed as described in Materials and Methods; n ¼ 3; �� , P < 0.01.A two-tailed Student t test was used for statistical analysis. D and E, the effect of aspirin on the NF-kB transcriptional activity is rescued in both U2OS andZOS cells stably overexpressing p65. The indicated stable transfectants (D) were transfected with an NF-kB luciferase reporter for 24 hours. The cells weretreated with or without aspirin (5 mmol/L) for 24 hours, and then subjected to luciferase assays (E) as described in Materials and Methods; n ¼ 3. Bars, SD;�� , P < 0.01. A two-sided ANOVAwith a Bonferroni post hoc test was used for statistical analysis. F, the impairment of aspirin on migration and invasion are rescuedin both U2OS and ZOS cells stably overexpressing p65. The indicated stable transfectants were treated with or without aspirin (5 mmol/L) for 24 hoursand then subjected to migration and invasion assays as described in Materials and Methods; n ¼ 3. Bars, SD, �� , P < 0.01. A two-sided ANOVA with Bonferronipost hoc test was used for statistical analysis.


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demonstrate that aspirin enhances osteosarcoma chemotherapy.Because osteosarcoma is an extremely aggressive cancer type thatlacks any targeted therapies, our findings strongly suggest thataspirin may be an excellent auxiliary drug for treating patientswith osteosarcoma.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D. Liao, T. KangDevelopment of methodology: D. Liao, L. Zhong

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Liao, L. Zhong, T. Duan, X. Wang, G. WangAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): D. Liao, L. Zhong, T. Duan, X. Wang,G. WangWriting, review, and/or revision of the manuscript: D. Liao, T. KangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.-H. Zhang, K. Hu, X. LvStudy supervision: T. Kang

AcknowledgmentsThe authors thank the members of the laboratory for helpful comments on

the article.

Figure 6.Aspirin suppresses osteosarcomametastasis via the NF-kB pathwayin vivo. The effect of aspirin onosteosarcoma metastasis to the lungsdepends on p65 in vivo. The indicatedU2OS/MTX300 cells stably expressingluciferase were used to determine thepresence of in vivo spontaneousmetastases to lungs of nude mice in anorthotopic model of osteosarcoma asdescribed in Materials and Methods.A, the indicated proteinswere analyzedby Western blot analysis in theindicated stable cells. B, the lungs of themice bearing osteosarcoma tumorxenografts that stably expressedluciferase were analyzed using an IVISLumina Imaging System (Xenogen);n¼ 6. C, H&E staining of lungs from thetumor-bearing nude mice in B. D, theexpression of human HPRT mRNArelative to mouse 18S rRNA in the lungsof tumor-bearing nude mice in B. Theaverages and error bars correspond tothe means � SDs. E, The number ofmetastatic nodules in the lungs of nudemice in B using a Mann–Whitney test.Dots, scores. Bars, SD; �� , P <0.01. F, thewet lung weights from the tumor-bearing nude mice in B. Bars,SD; � , P < 0.05 and �� , P < 0.01.A two-tailed Student t test wasused for statistical analysis.






Grant SupportThis work was supported by the key project (2013ZX10002008005; to

T. Kang), the National Nature Science Foundation in China (81125015; toT. Kang), the 973 project (2012CB967000; to T. Kang).

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 January 29, 2015; revised June 18, 2015; accepted July 6, 2015;published OnlineFirst July 22, 2015.

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2015;21:5349-5359. Published OnlineFirst July 22, 2015.Clin Cancer Res Dan Liao, Li Zhong, Tingmei Duan, et al.

B Pathwayκthrough the NF-Aspirin Suppresses the Growth and Metastasis of Osteosarcoma

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