parthenolide, a natural inhibitor of nuclear factor- kb, inhibits

Parthenolide, a Natural Inhibitor of Nuclear Factor-KB, InhibitsLung Colonization of Murine Osteosarcoma CellsYuki Kishida,1,2 Hideki Yoshikawa,2 and Akira Myoui2,3

Abstract Purpose:The transcription factor nuclear factor-nB (NF-nB) regulates the expression of severalgenes important for tumor metastasis and is constitutively active in the highly metastatic murineosteosarcoma cell line LM8. Parthenolide, a sesquiterpene lactone, was reported to inhibit theDNA binding of NF-nB.The purpose of this study is to investigate the usefulness of parthenolideas target for antimetastatic therapies.Experimental Design:We examined the effect of parthenolide on metastasis-associated phe-notypes in vitro and in murine experimental lung metastasis models by s.c. and i.v. inoculation ofLM8 cells.Results:We found that parthenolide strongly induced apoptosis and inhibited cell proliferationand the expression of vascular endothelial growth factor in vitro. In the in vivo metastasismodels,parthenolide treatment suppressed lung metastasis when treatment was initiated concurrentlywith s.c. or i.v. inoculationof tumor cells, whereas lungmetastasis was not reducedwhenparthe-nolide was given after the homing of tumor cells. The growth of s.c. tumors that developed atthe inoculation site was not suppressed by parthenolide.We also found that the genetic inhibitionof NF-nB activity by expressing mutant InBa suppressed lung metastasis in vivo but not s.c.tumor growth. This supports our notion that the metastasis-preventing effect of parthenolide ismediated at least in part by inhibition of NF-nB activity.Conclusions:These findings suggested that NF-nB is a potential molecular target for designingspecific prophylactic interventions against distant metastasis and that parthenolide is a hopefulcandidate for an antimetastatic drug.

Osteosarcoma is the most common primary bone malignan-cy affecting children and young adults. The biggest difficultywith osteosarcoma is its high propensity for pulmonarymetastasis. Patients with extremity lesions in whom lungmetastases are detected at the time of diagnosis have a verypoor prognosis. Modern multidisciplinary therapy has im-proved their outlook; however, up to half of the patients withosteosarcoma are still not cured. Thus, clarification of themolecular mechanism of pulmonary metastasis of osteosarco-ma and introduction of a new intervention method are stillneeded (1).

Previously, we showed that LM8, a highly metastaticsubclone of the Dunn murine osteosarcoma cell line, overex-pressed valosin-containing protein (also known as p97) andexhibited increased nuclear factor-nB (NF-nB) activity (2). Thetransfection of valosin-containing protein in Dunn osteosarco-ma cells could activate NF-nB and its metastatic potential. Wealso reported that the expression of vascular endothelial growthfactor (VEGF) and matrix metalloproteinase, which wereregulated by NF-nB, was up-regulated in LM8 and in Dunncells transfected with valosin-containing protein compared withoriginal Dunn cells (3). These findings suggested that therapyagainst NF-nB activity is a prime target and has the potential toimprove the prognosis of osteosarcoma.

NF-nB is an inducible dimeric transcription factor thatbelongs to the Rel/NF-nB family of transcription factors.NF-nB consists of two major polypeptides, p50 and p65 (4).In resting cells, NF-nB is sequestered in the cytoplasm by InBproteins. Stimulus-mediated phosphorylation and subsequentproteolytic degradation of InB allows the release and nucleartranslocation of NF-nB, where it transactivates several targetgenes (5).

NF-nB plays a key role in the malignant behavior of tumors.It has been shown to regulate a whole cadre of genes importantfor angiogenesis, invasion, and metastasis (6–8). Studies of celllines in vitro have shown that NF-nB is constitutively activatedin many malignancies (9, 10). In addition, some investigatorshave shown that inhibition of NF-nB by insertion of mutatedInB into cancer cell lines causes decreased tumor growth andmetastasis in vivo (11–14).

Human Cancer Biology

Authors’ Affiliations: 1Departments of Kampo Medicine and 2Orthopaedics,Osaka University Graduate School of Medicine; and 3Medical Center forTranslational Research, Osaka University Hospital, Osaka, JapanReceived 6/28/06; revised10/3/06; accepted10/13/06.Grant support: Ministry of Education, Culture, Sports, Science and Technology,Japan grants 17390417 and 18591631 (H. Yoshikawa and A. Myoui) and theMinistry of Health, Labor, and Welfare, Japan (Cancer Research grant 14-17;A. Myoui).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Akira Myoui, Department of Orthopaedics, OsakaUniversity Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871,Japan. Phone: 81-6-6879-3552; Fax: 81-6-6879-3559; E-mail: [email protected].

F2007 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-06-1559

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Parthenolide is one of the main sesquiterpene lactonesresponsible for the bioactivities of feverfew, a traditional folkremedy that has been used for various inflammatory con-ditions, such as rheumatoid arthritis, fever, and migraine(15, 16). Several studies have proposed that the effect of par-thenolide is due to its ability to inhibit NF-nB activation. Inaddition, parthenolide was also reported to have microtubule-interfering properties (17) that induce apoptotic cell death bymultiple pathways, including oxidative stress, endoplasmicreticulum stress, intracellular thiol depletion, caspase activation,and mitochondrial dysfunction (18, 19); inhibit 5-lipoxygenaseand cyclooxygenase (20); and sensitize cancer cells to chemo-therapeutic drugs, such as paclitaxel and CPT11 (21). Despite itspotential as an anticancer drug, few studies have clarified theeffect of parthenolide on metastasis (22) or the role of antitumoractivities in the course of metastasis in vivo. We showed thatparthenolide suppressed lung metastasis in LM8, a highly metas-tatic murine osteosarcoma cell line, by interference with tumorcell integration in lung tissue.

Materials and Methods

Cell culture. The cloned murine osteosarcoma cell line LM8, whichshows a high metastatic incidence to the lung after s.c. inoculation intothe back of mice, was cultured in DMEM containing 10% fetal bovineserum (Life Technologies, Gaithersburg, MD) in an air incubator with5% CO2 at 37jC. In some cultures, parthenolide (Sigma Chemical, St.Louis, MO) was added at final concentrations of 0.2 to 200 Ag/mL.

Animals. C3H male mice, ages 5 weeks, were purchased from JapanOriental Yeast Co., Ltd. (Tokyo, Japan). All mice were housed underspecific pathogen-free conditions with a 12-h light and dark cycle. Thehousing care rules and experimental protocol were approved by theAnimal Care and Use Committee of Osaka University.

Electrophoretic mobility shift assay. Nuclear extracts of cellswere prepared with the NE-PER nuclear extraction reagent (PierceBiotechnology, Rockford, IL). Biotin end-labeled double-strandedoligonucleotides 5¶-AGTTGAGGGGACTTTCCCAGGC-biotin-3¶ and5¶-TCAACTCCCCTGAAAGGGTCCG-biotin-3¶ were purchased fromInvitrogen (Carlsbad, CA). The binding reactions contained 7.5 Agnuclear extract protein, buffer [10 mmol/L Tris (pH 7.5), 50 mmol/LKCl, 5 mmol/L MgCl2, 1 mmol/L DTT, 0.05% NP40, 2.5% glycerol],1 Ag poly(deoxyinosinic-deoxycytidylic acid), and 2 nmol/L biotin-labeled DNA. The reactions were incubated at room temperature for20 min. The reactions were electrophoresed on a 10% precast Tris-borate-EDTA gel at 100 V for 1 h in 100 mmol/L Tris-borate-EDTAbuffer and transferred to a nylon membrane. The biotin-labeled DNAwas detected with a LightShift chemiluminescent electrophoreticmobility shift assay (EMSA) kit (Pierce Biotechnology). We repeatedthe assay for four times.

Luciferase assays. LM8 cells seeded in 24-well plates were trans-fected with pNF-nB-Luc (firefly luciferase driven by a TATA box with fiveNF-nB sites in the enhancer element; Stratagene, La Jolla, CA) by jetPEI(Polyplus Transfection, Biopark, France). Normalization of transfectionefficiency was done by cotransfection with pRL-TK (Promega, Madison,WI) that contained cDNA encoding Renilla luciferase. Luciferaseactivities were quantified at 24 h using the Dual-Luciferase ReporterAssay System (Promega) following the manufacturer’s instructions. Wedid the assay for four times.

Western blotting. LM8 cells in a 10-cm dish were starved withserum-free DMEM and incubated with various concentrations (0, 0.2, 2,20, and 200 Ag/mL) of parthenolide for 48 h. Ten-fold concentratedconditioned media were subjected to electrophoresis on a 12% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose filters.

The membrane was incubated with rabbit polyclonal anti-mouse VEGFantibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed byhorseradish peroxidase–conjugated secondary antibody (Santa CruzBiotechnology). Visualization was done using Amersham enhancedchemiluminescence detection system (Amersham Biosciences, Piscat-away, NJ) according to the manufacturer’s instructions. We repeatedthis assay thrice.

Invasion assay. Invasiveness into the reconstituted basementmembrane Matrigel (Becton Dickinson, Franklin Lakes, NJ) wasassayed. Cells were added into the upper compartment of a Transwellcell culture chamber and incubated with or without different concen-trations of parthenolide. Fetal bovine serum at a final concentration of10% (v/v) in culture medium was used as chemoattractant in the lowerchamber. After 22 h of incubation, cells remaining on the upper surfaceof the membrane were removed with a cotton swab. Cells that invadedthrough the Matrigel-precoated membrane filter were fixed, stained byGiemsa, and then counted under a microscope. We repeated the assayfor four times.

Flow cytometric analysis of apoptosis and the cell cycle. The effect ofparthenolide on the cell cycle of LM8 cells was investigated by flowcytometric analysis of cells stained with propidium iodide (SigmaChemical). Briefly, cells were trypsinized, washed in PBS, and fixed in70% ethanol overnight at 4jC until used. Fixed cells were incubatedwith 1 mg/mL RNase (Sigma Chemical) for 60 min at 37jC. Then, 0.25mg/mL propidium iodide was added for 15 min at 4jC in the dark. Weacquired 10,000 cells using FACSCalibur (Becton Dickinson). Datawere analyzed using ModFitLT 3.0 software (Becton Dickinson). Theseexperiments were repeated for four times.

Spontaneous metastasis model. C3H male mice, ages 5 weeks, were

used to estimate the in vivo metastatic potential to the lung. Tumor cells

(1 � 105) were suspended in 200 AL DMEM and inoculated s.c. into the

back of mice. To investigate whether parthenolide prevents tumorigen-

esis and lung metastasis in vivo , we developed two experimental

protocols. In the prophylactic treatment model, in which parthenolide

administration was initiated from the time of tumor cell inoculation,

six groups of mice were used in the experiment. The first group (n = 8)

was injected i.p. with a vehicle (PBS) every day. The other experimental

groups (n = 4-14) received injections of parthenolide dissolved in

distilled water at a dosage of 0.01, 0.1, 1, or 100 Ag/kg or 1 mg/kg daily.

In the therapeutic treatment model, treatment was started from day 17

after the onset of microscopic lung metastasis, confirmed in a

preliminary experiment. The control group (n = 5) was injected i.p.

with a vehicle (PBS), and the experimental groups received partheno-

lide at a dosage of 100 Ag/kg (n = 4) or 1 mg/kg (n = 4) daily. All mice

were killed on day 25, and primary tumors and lungs were collected for

biochemical and histologic studies. We evaluated the volume of the

primary tumor by measuring the three dimensions of the excised

tumor.Tail vein injection model. A total of 2 � 105 cells of LM8 was then

resuspended in 200 AL PBS and injected into the tail vein of C3H malemice. Mice were divided into three groups and the first group (n = 7)was injected with a vehicle (PBS) every day. The second group (n = 7)received parthenolide at a dosage of 1 mg/kg daily, starting immediatelyafter LM8 cell injection. The third (n = 7) group received the same doseof parthenolide every day starting from 48 h after cell injection.Seventeen days after cell injection, the mice were killed and the lungswere removed.

Histologic and immunohistochemical analyses. The tumors derivedfrom implanted LM8 and the lungs were removed, fixed in 10%formalin, embedded in paraffin, and then cut into 4-Am sections. Theywere stained with H&E and the lungs were evaluated microscopically toconfirm the presence of pulmonary metastasis. The proportional area oflung metastasis (the sum of the areas of lung metastasis/area of entirelung tissue) was calculated in lung sections with the largest tissue area.Immunohistochemistry was done using the streptavidin-peroxidasemethod with Histofine SAB-PO kits and an aminoethylcarbazolesubstrate kit (Nichirei, Tokyo, Japan) according to the manufacturer’s


www.aacrjournals.orgClin Cancer Res 2007;13(1) January1, 2007 60



protocol. Tissue sections were deparaffinized, hydrated in PBS, andincubated in 3% H2O2 in methanol for 20 min at room temperature toblock endogenous peroxidase activity. After being washed in PBS, thesections were preincubated with 10% normal serum from the samespecies as the secondary antibody (to minimize background staining)for 20 min at room temperature and then incubated with an anti-p65polyclonal antibody (Santa Cruz Biotechnology) or an anti-VEGFpolyclonal antibody (Santa Cruz Biotechnology) overnight at 4jC. Afterwashing in PBS, the sections were incubated with secondary antibodyfor 20 min at room temperature, then incubated with peroxidase-conjugated streptavidin for 20 min at room temperature, and washed inPBS. Finally, visualization was done using aminoethylcarbazole as asubstrate. As a control for immunostaining, normal serum from thesame species as the primary antibodies was used instead of the primaryantibodies.

Tumor cell homing in lung tissue. LM8 cells were additionallylabeled by the membrane dye PKH using the fluorescence cell linkerkit PKH26 (Sigma Chemical). Then, a total of 2 � 105 cells wassuspended in 200 AL PBS and injected into the tail vein of C3H mice(n = 15). Parthenolide treatment at a dosage of 1 mg/kg daily wasinitiated concomitantly with injection of LM8. At 4 (n = 5), 24(n = 5), and 48 h (n = 5) after cell injection, the mice were killed andthe lungs were removed. The lungs were then homogenized in PBS.The fluorescence of the released PKH26 was measured at 551 nm.Data were expressed as units of PKH26 released (1 unit = 1 unit offluorescence at 551 nm) per milligram of protein in each culture. Theassay was done in triplicate.

Stable transfection of LM8 cells with IkBaM and control vector. LM8cells were transfected with pCMV-InBaM expression vector or controlpCMV-InBa vector (Clontech, Mountain View, CA) using jetPEI. TheIjBaM gene differed from wild-type InBa by serine-to-alaninemutations at residues 32 and 36. These mutations protected InBa from

ubiquitination and proteasome-mediated proteolysis. Thus, the productof pCMV-InBaM blocked NF-nB activation in a dominant-negativefashion. Cells were selected with standard medium containing G418(Life Technologies). Two clones (M7 and M9) that overexpress InBaMand a control clone (CTL), which was transfected with control pCMV-InBa vector, were selected.

Statistical analysis. Data are presented as mean F SD. Groupswere compared by one-way ANOVA and individual groups werecompared using the Mann-Whitney U test for unpaired analysis.Differences between treatment groups were considered significant atP < 0.05.

Results

NF-kB DNA binding and transcription activity of NF-kB wasinhibited by parthenolide. To assess the influence of partheno-lide on NF-nB signaling in LM8 cells, EMSAs for NF-nB weredone. We added parthenolide or vehicle to subconfluent LM8cell cultures for 1 h and nuclear protein was extracted. Asshown in Fig. 1A, NF-nB DNA binding was inhibited byparthenolide dose dependently. Additionally, we determinedthe effect of parthenolide on the transactivational activity ofNF-nB in LM8 cells by promoter reporter assay. As shown in Fig.1B, NF-nB transcriptional activity was inhibited by parthenolidein a dose-dependent manner. The NF-nB activity in the cellsincubated with high-dose parthenolide (200 Ag/mL) was 10-fold lower than the control.

VEGF expression was inhibited by parthenolide. When cul-tured in serum-free medium, LM8 cells released VEGF in culturemedium as determined by Western blotting. Parthenolide

Fig. 1. In vitro effect of parthenolide onmurine osteosarcoma cellline LM8. A, EMSA for NF-nB. LM8 cells were pretreated withparthenolide for 1h. Nuclear extracts were subjected to EMSA.Parthenolide inhibits the DNA binding of NF-nB. B, transcriptionalactivity of NF-nB. A Luciferase reporter assay for NF-nBtranscriptional activity was done as described in Materials andMethods. NF-nB transcriptional activity was inhibited byparthenolide dose dependently. Representative data from fourdifferent experiments. C,VEGF secretion was decreased byparthenolide. Conditioned medium of LM8 cell cultures treated byparthenolide was analyzed forVEGF expression byWestern blotting.D, parthenolide reduced the invasiveness of LM8. Invasiveness wasdetermined using a Matrigel-precoated cell culture insert (n = 4).Representative data from three different experiments. Columns,mean; bars, SD. *, P < 0.05 to controls without parthenolide(ANOVA).

Parthenolide Decreases LungMetastasis




treatment inhibited the secretion of VEGF in a dose-dependentmanner (Fig. 1C).

Invasive potential was inhibited by parthenolide. Invasivepotential of LM8 cells after incubating with parthenolide wasdetermined using the Matrigel invasion assay. Figure 1D showsthat tumor cell invasion was decreased by parthenolide in adose-dependent manner.

Parthenolide inhibited cell growth and induced apoptosis inLM8 cell culture. We determined the effect of parthenolide onthe cell cycle of LM8 cells using FACS analysis. Parthenolidetreatment reduced the growth fraction (S + G2-M) and increasedthe apoptosis fraction (sub-G1) at 4, 12, and 24 h. At 48 h,parthenolide treatment abolished the G1 peak and no cellsseemed to be viable under the microscope (Fig. 2).

Parthenolide had a distinct effect on lung metastasis but noeffect on tumorigenesis in the prophylactic treatment model. Weevaluated the effect of parthenolide on tumors in vivo using amurine LM8 model of metastasis. After s.c. inoculation oftumor cells, all mice developed large primary tumors at theinjection site. In the prophylactic treatment model, the primary

tumors treated with parthenolide of >1 Ag/kg/d tended to besmaller than the vehicle-treated control tumors but the differ-ences were not statistically significant (Fig. 3A). However, weobserved that pulmonary metastatic nodules were dramaticallysuppressed by parthenolide. There were significant differencesin the proportional area of lung metastasis between vehicle-treated and parthenolide-treated mice (Fig. 3B-F). Especially,treatment with parthenolide of >1 Ag/kg/d effectively sup-pressed lung metastasis. Immunohistchemical analysis showedstrong p65 expression in the lung metastatic nodules of theuntreated mice. However, metastatic tumors treated withparthenolide of 1 mg/kg/d were negative for p65 immunoloc-alization. Immunohistochemistry also showed that VEGFexpression of metastatic lung tumors and surrounding lungtissue was clearly suppressed by parthenolide treatmentcompared with untreated metastatic tumors of similar size(Fig. 3G-N).

Parthenolide had no effect on lung metastasis or tumorigenesisin the therapeutic treatment model. Next, we did the experimentto determine whether parthenolide can decrease pulmonary

Fig. 2. Cell cycle analysis in LM8 cellstreatedwith or without parthenolide at 4,12,24, and 48 h. Representative data from fourdifferent experiments. Results are expressedas a percentage of the cells in each fraction.*, P < 0.05 from untreated control at eachtime point. ND, not determined.





Fig. 3. The effect of parthenolide on tumor metastasis in the prophylactic model. LM8 cells (1 �105) were injected s.c. into the back of mice.The mice were treated withi.p. injection of parthenolide or vehicle (PBS) starting from the day of tumor cell inoculation. A, the primary tumors treated by parthenolide tended to be smaller than thecontrols but no statistically significant difference was found. B, pulmonary metastatic nodules were dramatically suppressed by parthenolide. n = 4-14 from three differentexperiments. Columns, mean; bars, SD. *, P < 0.05 from PBS control.C to F, microphotographs of the lungs treated by PBS (C) and by parthenolide at a dosage of1 Ag/kg/d(D),100 Ag/kg/d (E), and1mg/kg/d (F). Bar,1mm.Magnification,�40. Immunohistochemical analyses of the tumor nodules in lung tissue treatedwith PBS (G-J) andwithparthenolide at a dosage of1mg/kg/d (K-N).The expression of p65 was suppressed in parthenolide-treated mice (L) compared with control mice (H).The expression ofVEGF was also suppressed in the parthenolide-treated mice (M) compared with control mice (I). An intrinsic positive control ofVEGF was vascular endothelial wall.G and K,H&E staining. Arrowheads, blood vessels; arrows, tumor nodules. J and N, negative controls without primary antibodies. Bar, 100 Am. Magnification, �100.





lung nodules after the development of lung metastasis. Theprimary tumor volumes and the proportional area of lungmetastasis showed no significant difference between vehicle-treated and parthenolide-treated mice (Fig. 4).

Parthenolide interferes with early events of tumor cellcolonization in lung. To clarify where parthenolide worksduring the multistep metastatic process, we tested its effect in alung metastasis model by tail vein injection. The lungmetastasis of mice that received parthenolide concomitantlywith tumor cell inoculation was significantly reduced comparedwith other groups (Fig. 5A-D). In the mice treated withparthenolide starting from 48 h after the injection of tumorcells, the lung metastasis was not suppressed (Fig. 5B and D),suggesting that parthenolide inhibited metastasis not byworking on the steps before extravasation but by working onearly events of lung colonization that occur within 48 h aftercell intravasation. Then, we examined the effect of parthenolideon the homing of tumor cells to lung tissue. After 4 or 24 h,there was no significant difference in the fluorescence of labeledLM8 cells in the lung between the control group and theparthenolide-treated group. However, the fluorescence in themice treated with parthenolide gradually decreased with timeand, at 48 h, it was significantly lower than that in mice thatwere treated with PBS of 48 h (Fig. 5E).

Blocking NF-kB activation inhibited VEGF expression and lungmetastasis in vivo. We established LM8 cell clones that werestably transfected with an InBa mutant expression vectorpCMV-InBaM. Two clones (M7 and M9) were selected. Nuclearproteins from transfectants and wild-type cells were analyzedby EMSA for NF-nB binding activity. DNA binding of NF-nBwas inhibited in both mutant clones (Fig. 6A). Consistent withthese results, the NF-nB-luciferase reporter assay (n = 4) showed

that the transcription activity of NF-nB was significantlyreduced in those clones compared with control clone, CTL(Fig. 6B).

Next, we tested the metastatic potential of those clones in vivousing a spontaneous metastasis model to determine whetherselective inhibition of NF-nB suppresses lung metastasis. Asshown in Fig. 6C, the primary tumor volumes were notstatistically different between the mice implanted with InBaMclones and the mice implanted with CTL (n = 6). However, wefound that pulmonary metastatic nodules in the miceimplanted with the clones overexpressing InBa mutant weredramatically suppressed, especially in the M7 clone with thelowest NF-nB activity. There were significant differences in theproportional area of metastasis between the mice with CTL andthe mice with M7 or M9 (Fig. 6D-G).

Discussion

Suppression of various genes that are involved in tumor cellinvasion and angiogenesis on blockade of NF-nB activity hasbeen reported in cancer cells (12, 23–27). Although previousstudies claimed the involvement of several cytokines under theregulation of NF-nB, such as interleukin-8, tumor necrosisfactor-a, and VEGF, in the regulatory mechanisms (12, 22–25,28, 29), little has been known when and how NF-nB isimplicated during the multistep cascade of events in thedevelopment of metastasis. In addition, although partheno-lide, an inhibitor of NF-nB, has also shown antimetastaticactivity in vitro (21, 30), there have been few reports aboutits in vivo antimetastatic activity or its mode of action (22).Our in vivo experiments showed the following: (a) lungmetastasis was suppressed when parthenolide was given

Fig. 4. Parthenolide did not suppresstumor metastasis in the therapeutictreatmentmodel.Themicewere treatedwithparthenolide or vehicle (PBS) initiating fromday17 after the onset of lung metastasis.No statistically significant difference wasobserved in the volume of the primarytumors (A) or the proportional ratio of lungmetastasis (B) between vehicle-treated andparthenolide-treated mice. Control group,n = 5; treatment groups, n = 4. Columns,mean; bars, SD. H&E stainings of the lungwith PBS (C), 100 Ag/kg/d parthenolide(D), and1mg/kg/d parthenolide (E).Magnification, �40.





starting concurrently with s.c. inoculation of tumor cells intothe back of mice or i.v. administration of tumor cells; (b) lungmetastasis was not reduced when parthenolide was given afterhoming of the tumor cells; (c) parthenolide had little effect onthe size of the primary tumor; and (d) inhibiting NF-nB activityby overexpressing InBaM suppressed lung metastasis but nottumorigenesis. These data suggest that parthenolide, perhapsby inhibiting NF-nB activity for the most part, prevented lungmetastasis mainly by working on early events in lungcolonization involving interactions between tumor cellsarrested in the capillary bed and the microenvironment at thetarget site not just by suppressing tumor cell proliferation.

It is now recognized that resistance to cell death, particularlyapoptotic cell death, is an important aspect of metastaticprogression (31). During the multistep metastatic cascade, it isbelieved that tumor cells withstand severe proapoptoticpressures from host cell cytokines and growth factors (32–34).Constitutively activated NF-nB-mediated transcription hasbeen well documented in association with the induction ofantiapoptotic genes and the ability of cancer cells to evadeapoptosis induced by various stimuli (35). Parthenolide hasbeen reported to induce apoptosis effectively (19, 36, 37). Ourdata also showed that inhibition of NF-nB activity byparthenolide resulted in strong induction of LM8 cell apoptosisin vitro. Recent in vivo videomicroscopic studies showed thatmost tumor cells arrested in microvasculature at the metastaticsite successfully extravasated and survived as solitary cells for3 days but only a few percentage of the cells started to replicateto form micrometastases (38). In our study, an experimentalmetastasis model by i.v. inoculation of fluorescence-labeledtumor cells showed that parthenolide did not affect the tumorcell arrest in lung vasculature at 4 h. However, fluorescence

derived from tumor cells in lung tissue gradually decreased overtime with parthenolide treatment with a significant differenceat 48 h, suggesting that parthenolide, at least in part, inducedtumor cell death before the cells can form multicellularmicrometastatic foci.

NF-nB is one of the most important upstream regulators ofVEGF, a major angiogenic factor that induces endothelial cellproliferation. In our data, parthenolide clearly decreased VEGFsecretion by LM8 cells in vitro. Moreover, in the spontaneousmetastasis model, it suppressed the expression of immunor-eacting VEGF at lung metastatic foci where intracellularaccumulation of NF-nB was suppressed as well. Theseobservations suggest that one of the mechanisms by whichparthenolide inhibits lung metastasis is the suppression ofVEGF expression via NF-nB inactivation. A recent study showedthat disrupting VEGF signaling leading to vascular leakage bythe pretreatment with pharmacologic inhibitors of VEGF couldalleviate lung metastasis. It showed that the inhibitor could getthe beneficial effect within the first days after tumor cellinoculation, when solitary tumor cells are beginning toextravasate and proliferate (39). These observations wereconsistent with our results in the i.v. tumor cell inoculationmodel, which revealed failure in the blockade of metastasiswith parthenolide treatment starting from 48 h after cellinoculation. This supports our notion that the metastasis-preventing effect of parthenolide is mediated at least in part byinhibition of VEGF secretion.

Although the inhibition of NF-nB activity by expressingInBaM has been shown to suppress metastasis in a variety oftumors, the reports differ about the effect on the growth ofprimary tumors (11, 12, 14, 25). Our study showed thatparthenolide effectively blocked the development of lung

Fig. 5. The effect of parthenolide on lungmetastasis in a tail vein injection model.TheH&E staining of lung tissue of the mice thatreceived PBS (A), parthenolide startingfrom 48 h after cell injection (B), andparthenolide concomitantly with theinjection of LM8 (C).The lung metastasisof mice that received parthenolideconcomitantly with the injection of tumorcells was significantly reduced comparedwith other groups (n = 7; D). LM8 cellswere labeled by themembrane dye PKHandinjected into the tail vein. Parthenolidetreatment at a dosage of1mg/kg daily wasinitiated concomitantly injection of cells.At 4, 24, and 48 h after cell injection, thefluorescence of the released PKH in lungtissue was measured. At 4 and 24 h,there was no significant difference in thefluorescence of labeled cells in the lungbetween the control group and theparthenolide-treated group. However, thefluorescence treated with parthenolidegradually decreased with time and wassignificantly lower than that treated withPBS at 48 h (n = 5; E). Points, mean; bars,SD. *, P < 0.05 to PBS control.





metastasis of osteosarcoma but did not block the proliferationof the primary site tumor or established lung meta-stases. Despite its suppressive effect on the cell cycle in vitro,its poor effect on established tumors in vivo might spoil theimportance of proliferation suppression among the putativemechanisms of metastasis inhibition by parthenolide. Howev-er, the pharmacodynamic properties of parthenolide are stillunder investigation (40) and there remains the possibility thatparthenolide is not to be delivered in high enough concen-trations to established tumors when given systemically.

Several antitumor studies on the inhibition of NF-nB activityin vivo used a gene therapy, including an InB mutant.Parthenolide is currently used commonly as a food supplementfor the treatment of migraines and reportedly has no severeside effects compared with the placebo group; therefore, it maybe easier and more efficient than gene therapy in vivo (15).Mutation of p53 is a common genetic alteration in cancers

that is closely associated with resistance to systemic therapy.A recent study revealed that one possible pathway throughwhich mutants of p53 may induce loss of drug sensitivity is viathe NF-nB pathway (41). Parthenolide directly blocks NF-nBactivity regardless of p53 status and also induces apoptosis viathe c-Jun NH2-terminal kinase pathway independent of NF-nBand p53. Thus, parthenolide could be expected to have an effecton tumors with the p53 mutation that poorly respond toanticancer drugs (30).

In conclusion, we showed that parthenolide, a sesquiterpenelactone derived from natural herbs, inhibited lung metastasis ina spontaneous metastasis model and an i.v. metastasis modelusing the highly metastatic murine osteosarcoma cell line LM8.Although extensive clinical studies are needed to clarify whetherthis is also the case in human patients with cancers, our resultssuggest that NF-nB is a potential molecular target for designingspecific prophylactic interventions against distant metastasis.

Fig. 6. Suppression of NF-nB activityby the overexpressing mutant InBareducedVEGF secretion and blocked lungmetastasis. An InBaM construct wastransfected to LM8 cells and two clones(M7 and M9) were selected according tothe activity of NF-nB for in vitro and in vivostudies. EMSA of NF-nBDNA binding inM7 and M9 and in CTL, a clone thatwas transfected with control vector (A),and transcriptional activity of NF-nB byluciferase reporter assay (n = 4; B).Columns, mean; bars, SD. *, P < 0.05 to CTLwithout tumor necrosis factor-a (TNF-a).#, P < 0.01to CTL with tumor necrosisfactor-a. These clones were subjected tothe spontaneous metastasis model fortheir metastatic potential. No statisticallysignificant difference was observed in thevolume of the primary tumors (C); however,the proportional area of lung metastasis inthe mice inoculated with InBaM cloneswas significantly smaller than that with thecontrol clone (n = 6; D). Columns, mean;bars, SD. *, P < 0.05 to CTL. Representativemicrophotographs of the lungs of the miceinjected with M7 (E), M9 (F), and CTL (G)cells. H&E staining. Magnification, �40.





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2007;13:59-67. Clin Cancer Res Yuki Kishida, Hideki Yoshikawa and Akira Myoui Inhibits Lung Colonization of Murine Osteosarcoma Cells

B,κParthenolide, a Natural Inhibitor of Nuclear Factor-

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