pegylated catalase prevents metastatic tumor growth aggravated by tumor removal

Free Radical Biology & Medicine 41 (2006) 1449–1458www.elsevier.com/locate/freeradbiomed

Original Contribution

PEGylated catalase prevents metastatic tumor growth aggravatedby tumor removal

Kenji Hyoudou a, Makiya Nishikawa b, Yuki Kobayashi a, Yukari Umeyama a,Fumiyoshi Yamashita a, Mitsuru Hashida a,⁎

a Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japanb Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Received 20 December 2005; revised 14 July 2006; accepted 7 August 2006Available online 11 August 2006

Abstract

Although surgical removal is a primary option for treating tumors, it can lead to the increased growth of metastatic tumors. Because surgicalprocedures may generate reactive oxygen species (ROS), known promoters of tumor metastasis and growth, we investigated whether PEGylatedcatalase (PEG-catalase, plasma half-life of 13.6 h) was able to prevent this after surgical removal of a footpad tumor in mice. Murine melanomacells labeled with the firefly luciferase gene were used to monitor the distribution of tumor cells. After inoculation into the footpad, tumor cellswere found in the lung, and the number increased with time. The surgical removal of the footpad tumor significantly (p < 0.05) increased thenumber of metastatic tumor cells and the level of plasma lipoperoxides. An intravenous injection of PEG-catalase significantly (p < 0.05)suppressed the metastatic tumor growth as well as the peroxidation. Quantitative RT-PCR and Western blot analyses indicated that PEG-catalasemarkedly reduced the increase in the expression of epidermal growth factor receptor. These findings indicate that the removal of tumor producesROS, which then aggravate metastatic tumor growth by activating several growth factors. PEG-catalase can effectively prevent this metastatictumor growth by detoxifying the ROS.© 2006 Elsevier Inc. All rights reserved.

Keywords: Reactive oxygen species; Catalase; Surgical removal; Quantitative analysis; Tumor dormancy; Metastatic recurrence; Free radicals

Tumor metastasis is the major cause of death in cancerpatients, and it can occur many years after treatment of theprimary tumor. However, the mechanisms of metastaticrecurrence are not fully understood and, therefore, effectivetherapeutic approaches have not been established. Tumormetastasis can be roughly divided into the following steps:tumor cell dissociation, invasion, intravasation, distribution todistant organs, arrest in small vessels, adhesion to endothelialcells, extravasation, invasion of the target organ, and prolifera-tion [1]. It is important to note that metastatic tumor cells

Abbreviations: PEG-catalase, polyethylene glycol-conjugated catalase;ROS, reactive oxygen species; EGF, epidermal growth factor; EGFR, epidermalgrowth factor receptor; EGR-1, early growth response gene-1; HBSS, Hanks'balanced salt solution; TBARS, thiobarbituric acid-reactive substances;GAPDH, glyceraldehyde-3-phosphate dehydrogenase.⁎ Corresponding author. Fax: +81 75 753 4575.E-mail address: [email protected] (M. Hashida).

0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.freeradbiomed.2006.08.004

sometimes remain dormant in tissues to which they havemigrated, but they can then start to proliferate after a longinterval after dissociation from primary tumors. These clinicaland experimental observations [2–4] confirm that migratingtumor cells do not indiscriminately produce secondary tumorswherever they become located. A number of factors andprocesses are involved in regulating the dormancy and growthof tumor cells. They include angiogenesis, cell cycle arrest,immune regulation, and interactions of tumor cells with theirmicroenvironment [5–8]. In previous studies, tumor dormancywas thought to involve preangiogenic micrometastases in whichproliferation and apoptosis are balanced [9,10]. These dormantmicrometastases may start to proliferate when factors releasedfrom the host or tumor cells promote neovascularization andprogressive growth of tumor cells [11,12]. However, recentstudies have suggested another possibility of tumor dormancy;solitary tumor cells exhibit very little proliferation or apoptosis[13–15]. If these quiescent cells remain viable in sufficiently

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1450 K. Hyoudou et al. / Free Radical Biology & Medicine 41 (2006) 1449–1458

large numbers, they might contribute to metastatic recurrenceafter a period of clinical dormancy.

A correlation between surgical trauma and locoregionaltumor recurrence was previously demonstrated [16,17]. It wasalso reported that surgical trauma results in the production ofROS from inflammatory cells entering damaged tissues via arespiratory burst [18]. The ROS generated destroy invadingorganisms as well as damaged tissues, but the overwhelmingoxidative stress can result in additional tissue destruction.Moreover, the expression of some growth factors andangiogenic factors secreted after surgery for tissue repair ismediated by ROS. These pieces of evidence suggest that arobust production of ROS is involved in the mechanisms oftumor recurrence after the surgical removal of a primarytumor.

In previous reports, we have shown that an experimentalpulmonary metastasis in mice can be effectively inhibited bypolyethylene glycol-conjugated catalase (PEG-catalase) [19].The number of the metastatic colonies on the lung surface wassignificantly smaller in mice treated with PEG-catalase than inuntreated (saline-injected) mice. This inhibitory effect of PEG-catalase was due to the inhibition of multiple steps of tumormetastasis, including cell adhesion, invasion, and proliferation[20]. Similar results were obtained by targeted delivery ofcatalase to the liver parenchymal cells for inhibition of ex-perimental hepatic metastasis [21] or by retention of catalase inthe peritoneal cavity for inhibition of peritoneal dissemination(unpublished data). As shown in these reports, scavenginghydrogen peroxide is thought to be very effective in inhibitingtumor metastasis.

In the present study, we first evaluated the tissue dispositionof tumor cells by inoculating firefly luciferase-expressingmouse melanoma cells into the footpad of mice, in which thefootpad tumor was considered a model of primary tumor.Then, we examined the effects of catalase and PEG-catalase onthe robust proliferation of metastatic tumors after removalof the footpad tumor and found that PEG-catalase largelyprevented both the production of ROS and metastatic tumorgrowth. Based on these results, we have tried to elucidatethe inhibitory mechanisms of PEG-catalase by quantitativelyevaluating mRNA expression in metastatic tumor-bearingmouse lung.

Materials and methods

Animals

Male C57BL/6 (6-week-old) mice were purchased from theShizuoka Agricultural Cooperative Association for LaboratoryAnimals (Shizuoka, Japan). Animals were maintained underconventional housing conditions. All animal experiments wereconducted in accordance with the principles and proceduresoutlined in the National Institutes of Health Guide for the Careand Use of Laboratory Animals. The protocols for animalexperiments were approved by the Animal ExperimentationCommittee of the Graduate School of Pharmaceutical Sciencesof Kyoto University.

Chemicals

Dulbecco's modified Eagle's minimum essential mediumand Hanks' balanced salt solution (HBSS) were obtained fromNissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum wasobtained from Biowhittaker (Walkersville, MD, USA). Bovineliver catalase (C-100; 40,000 units/mg) and bovine serumalbumin (BSA) were purchased from Sigma Chemical (St.Louis, MO, USA). A product of PEG (2,4-bis (O-methoxypoly-ethylene glycol)-6-chloro-s-triazin) was obtained from Seika-gaku Corp. (Japan), and PEG-catalase was synthesized and itsenzymatic activity measured as reported previously [22]. PEG-BSAwas also synthesized in a similar manner to that for PEG-catalase. Inactivated PEG-catalase, devoid of catalase activity,was prepared by heating the PEG-catalase at 95°C for 3 min. Allother chemicals were of the highest grade available.

Tumor cells

Murine melanoma B16-BL6 tumor cells [23] were obtainedfrom the Cancer Chemotherapy Center of the JapaneseFoundation for Cancer Research (Tokyo, Japan). B16-BL6cells expressing firefly luciferase (B16-BL6/Luc) were estab-lished as previously described [20].

Pulmonary metastasis after intrafootpad inoculation in mice

The footpads of mice were inoculated with 2 × 105 B16-BL6/Luc cells in 0.02 ml HBSS. At 1, 7, 14, 21, or 28 days aftertumor inoculation, mice were killed and the footpad and thelung were excised and weighed and the luciferase activity in thetissues was measured.

Growth inhibition of metastatic tumor cells by PEG-catalaseafter footpad tumor removal

Spontaneous pulmonary metastasis was induced by inoculat-ing 2 × 105 B16-BL6/Luc cells as described above. The footpadtumor was surgically removed at 20 or 21 days, when the tumorhad grown to 267 ± 23 mm3. Saline (vehicle), catalase, PEG-catalase, inactivated PEG-catalase, or PEG-BSA (12.5 μg/mouse, an amount equivalent to that of PEG-catalase) wasintravenously injected at a dose of 500 catalase units/mouse justbefore removal of the footpad tumor. At 7 days after the removal,mice were euthanized and the lung was excised and weighed andthe luciferase activity in the tissue was measured.

Separately, the effects of PEG-catalase on the survival ofmice with spontaneous lung metastases were examined.Spontaneous pulmonary metastasis was induced by injecting2 × 105 B16-BL6 cells as described above. Then, saline or PEG-catalase (1000 catalase units) was intravenously injected justbefore removal of the tumor.

Measurement of luciferase activity

The cells or tissues were homogenized with a lysis buffer(0.05% Triton X-100, 2 mM EDTA, 0.1 M Tris, pH 7.8) and

1451K. Hyoudou et al. / Free Radical Biology & Medicine 41 (2006) 1449–1458

subjected to three cycles of freezing (liquid N2 for 3 min) andthawing (37°C for 3 min), followed by centrifugation at10,000 g for 10 min. Ten microliters of the supernatant wasmixed with 100 μl luciferase assay buffer (Picagene; Toyo Ink,Tokyo, Japan) and the light produced was immediatelymeasured using a luminometer (Lumat LB 9507; EG&GBerthold, Bad Wild-bad, Germany). The luciferase activitywas converted to the number of tumor cells using a regressionline (1 cell/tissue = 0.83 RLU/s/μl).

Measurement of lipid peroxides in plasma after footpad tumorremoval

At 1 h after the removal of the footpad tumor, blood wascollected from the vena cava under ether anesthesia. Then,plasmawas obtained by centrifugation of the blood collected andthe concentration of lipoperoxides in the plasma was measuredby reaction with thiobarbituric acid. Briefly, 0.2 ml of plasmawas mixed with 0.025 ml of 2,6-di-tert-butyl-4-methylphenol(5 mM in ethanol) and 0.2 ml of orthophosphoric acid (0.2 M).Then 0.025 ml of TBA reagent (0.11 M 2-thiobarbituric acid in0.1 M NaOH) was added, and the reaction mixture wasincubated at 90°C for 45 min. Thiobarbituric acid-reactivesubstances (TBARS) were extracted with 0.5 ml of n-butanol.Then the TBARS-derived fluorescence was measured by amultilabel counter (excitation at 530 nm, emission at 560 nm;Wallac 1420 ARVO MX-2; Perkin–Elmer Life Sciences,Boston, MA, USA) as previously reported [24].

Separately, the antioxidant capacity of naïve mice wasexamined. Experimental acute oxidative stress was induced by abolus injection of 200 or 2000 nmol hydrogen peroxide into thetail vein of the mice. Assuming that the total amount of blood is8% of body weight, i.e., about 2 ml per 25 g mouse, the initialconcentrations of hydrogen peroxide in the blood wereestimated to be about 100 and 1000 μM, respectively. PEG-catalase (100 or 1000 catalase units/mouse) was intravenouslyinjected just before the hydrogen peroxide injection. At 15 minafter hydrogen peroxide injection, the concentration of lipo-peroxides in the plasma was measured as described above.

RT-PCR and real-time quantitative PCR

At 2 h after the removal of the footpad tumor, mice wereeuthanized and the lung was harvested. Total RNAwas isolatedfrom the lung using a nucleic acid purification system,MagExtractor MFX-2100 (Toyobo Co., Ltd., Osaka, Japan),according to the manufacturer's instructions. Reverse transcrip-tion of RNAwas performed using a reverse transcription reagent(Takara, Otsu, Japan). The expression of epidermal growthfactor (EGF), EGF receptor (EGFR), transforming growthfactor-α (TGF-α), c-jun, c-fos, c-myc, and early growthresponse gene-1 (EGR-1) in the lung was determined by real-time quantitative PCR in a LightCycler system (Roche Diag-nostics, Mannheim, Germany) using SYBR Premix Ex Taq(Takara). The primers for the PCR were designed and producedby Nihon Gene Research Laboratories (Sendai, Japan) asfollows: EGF, forward 5′-GTCCTAGAGAAACACCAAGAC-

C-3′, reverse 5′-CACGTAGACTGAAGTACCTCT-3′; EGFR,forward 5′-ATCATGGGAGAGAACAACAC-3′, reverse 5′-CAATCCCAGTGGCAATAGA-3′; TGF-α, forward 5′-GGTA-TCCTGGTAGCTGTGTGTC-3′, reverse 5′-GCTTCTCTTCCTG-CACCAA-3′; c-jun, forward 5′-GTGCCAACTCATGCTAACG-3′,reverse5′-GCAACCAGTCAAGTTCTCAAG-3′; c-fos, forward5′-GCTGACAGATACACTCCAA-3′, reverse 5′-GACCTC-CAGTCAAATCCA-3′; c-myc, forward 5′-ACCACCAGCA-GCGACTCT-3′, reverse 5′-AGACGTGGCACCTCTTGA-3′;EGR-1, forward 5′-AGCGAACAACCCTATGAGC-3′, reverse5′-GGGTTCAGGCCACAAAGT-3′; and GAPDH, forward 5′-TCTCCTGCGACTTCAACA-3′, reverse 5′-GCTGTAGCCGT-ATTCATTGT-3′. Thermocycling for each reaction was done ina final volume of 20 μl containing 2 μl of cDNA sample (orstandard), 0.2 μmol/L each primer, and 10 μl SYBR Premix ExTaq. After 10 s of initial denaturation at 95°C, the cyclingprotocol consisted of 60 cycles of denaturation at 95°C for 5 s,annealing at 60°C for 10 s, and elongation at 72°C for 10 s. TheLightCycler apparatus measured the fluorescence of eachsample in every cycle at the end of the annealing step. AfterPCR was completed, the LightCycler software (RocheDiagnostics) converted the raw data into copies of targetmolecules. The amount of target mRNAwas normalized to thatof GAPDH mRNA from the same cDNA sample.

Western blot analysis

Cells or tissues were harvested 6 h after treatment (e.g.,removal of the footpad tumor or addition of hydrogen peroxide)and homogenized with RIPA lysis buffer (Santa Cruz Biotech-nology, Inc., Santa Cruz, CA, USA), followed by centrifugationat 10,000 g for 10 min. The samples were diluted in a samplebuffer (0.25 mM Tris–HCl (pH 6.8), 10% 2-mercaptoethanol,4% SDS, 10% sucrose, and 0.004% bromphenol blue) andboiled at 95°C for 3 min. Proteins were resolved on 7.5 or 12.5%SDS–PAGE for EGFR and EGF, respectively, and transferredonto PVDF membranes (Hybond-P; Amersham Biosciences,Buckinghamshire, UK), which then were blocked with 1% BSAin Tween–PBS for 1 h. The membrane was probed with anti-EGF (Upstate, Charlottesville, VA, USA) or anti-EGFR(Calbiochem, La Jolla, CA, USA) antibody, incubated overnightin Tween–PBS containing 1% BSA, washed three times withTween–PBS, and incubated for 1 h at room temperature withhorseradish peroxidase-conjugated sheep anti-rabbit immuno-globulin (Amersham Biosciences) in Tween–PBS. Proteinbands were visualized by the ECL system (Amersham Bio-sciences) and the luminescence was detected by a cooled CCDcamera (Light Capture AE-6962; Atto Corp., Tokyo, Japan)according to the manufacturer's instructions. Quantitative densi-tometry analysis was performed by CS analyzer (Atto Corp.).

Effect of hydrogen peroxide on the expression of EGF, EGFR,and immediate early response genes in tumor cells

B16-BL6 cells were seeded on six-well plates at a density of1 × 105 cells per well in culture medium and cultured for 24 hbefore any treatment (e.g., hydrogen peroxide or catalase). After


replacement of culture medium with serum-free medium,300 nmol hydrogen peroxide (100 μM in the final concentra-tion) was added to the B16-BL6 tumor cells followed by theaddition of catalase (333 units/ml). At 1 h after treatment, cellswere harvested and total RNAwas isolated using a nucleic acidpurification system, then RT-PCR and real-time quantitativePCR were performed as described above.

Statistical analysis

Differences were statistically evaluated by one-way ANOVAfollowed by the Student–Newman–Keuls multiple comparisontest and Kaplan–Meier analysis with a log-rank test to dete-rmine survival; the level of statistical significance was p < 0.05.

Results

Disposition and proliferation of B16-BL6/Luc cells afterinoculation into the footpad

The characteristics of B16-BL6/Luc cells have been reportedpreviously [20]. Neither the lung nor the footpad excised fromnaïve mice showed any detectable luciferase activity. Theaddition of B16-BL6/Luc cells to the lung tissue proportionallyincreased the luciferase activity [20], indicating that theluciferase activity of the lung and footpad can be used forcalculating the number of tumor cells. Based on theseproperties, the luciferase activity measured was converted intothe number of B16-BL6/Luc cells in the lung or footpad by theequation given under Materials and methods.

Fig. 1 shows the number of B16-BL6/Luc cells in the lungand footpad of mice after inoculation of cells into the footpad.As early as 1 day after tumor inoculation, metastatic tumor cellscould be detected in the lung. Little luciferase activity wasdetected in other organs, such as the liver and spleen (data notshown). The number and proliferation rate of the metastatictumor cells in the lung increased with time, whereas the growthrate of the tumor cells in the footpad gradually decreased.

Fig. 1. Number of tumor cells in mouse lung and footpad after inoculation of2 × 105 B16-BL6/Luc cells into the footpad. Results are expressed as the means ±SD of at least three mice.

Colonies on the lung surface were visible only at 4 weeks afterinoculation into the footpad.

Growth inhibition of metastatic tumor cells by catalasederivatives after footpad tumor removal

The footpad tumor was surgically removed at 21 days afterinoculation into the footpad. Fig. 2A shows the mouse lungswith metastatic tumors at 7 days after footpad tumor removal.The number and diameter of the tumor colonies dramaticallyincreased when the tumor tissue in the footpad was surgicallyremoved (Fig. 2A, image b). The number and diameter of thetumor colonies in the removed, PEG-catalase-treated groupwere clearly smaller than those in the removed, saline-treatedgroup (Fig. 2A, image d). Fig. 2B shows the number ofmetastatic tumor cells in the lung, which increased about 1000-fold in the 7-day period after removal, and the number of tumorcells in the removed, saline-treated group (3.3 ± 2.8 × 107 cells/lung) was significantly (p < 0.05) greater than that in the no-removal, saline-treated group (3.0 ± 1.4 × 106 cells/lung). Asingle intravenous administration of catalase just before theremoval suppressed the rapid growth of the tumor cells in thelung. Moreover, PEG-catalase greatly inhibited the proliferationof the metastatic tumor cells; only 0.91% of tumor cells in thesaline group were detected (p < 0.05). Neither heat-inactivatedPEG-catalase nor PEG-BSA had significant effects on thegrowth of metastatic tumor cells after tumor removal, suggest-ing that the catalase activity within the blood circulation iscritical for the inhibition by PEG-catalase. A treatment withPEG-catalase of the no-removal group (no-removal, PEG-catalase-treated group) somewhat reduced the growth of tumorcells in the lung, but the effect was not significant. We alsoexamined sham surgery to evaluate whether the increase in thegrowth is due to the removal of the footpad tumor or to surgicaltrauma. The number of tumor cells in the lung also increased inthe removed (non-tumor-bearing footpad), saline-treated group,although the number was smaller than that in the tumor-removed, saline-treated group.

Reduced lipid peroxidation in plasma by PEG-catalase aftertumor removal

Before assessing the effects of PEG-catalase on the plasmalipoperoxide concentration after the removal of footpad tumor,we examined the endogenous antioxidant capacity in naïvemice receiving an intravenous injection of hydrogen peroxide toinduce acute oxidative stress. The TBARS level was measuredas an indicator of lipid peroxidation. As shown in Fig. 3A, theplasma lipoperoxide concentration increased with theincrease in the dose of hydrogen peroxide. Then, we examinedthe effects of exogenously administered catalase on thisexperimental lipid peroxidation. An injection of PEG-catalasebefore the injection of hydrogen peroxide significantly inhibitedthe increase in the amount of the lipid peroxidation in plasma.

Next, we examined whether the lipid peroxide concentrationwas altered by surgical removal of the footpad tumor. TheTBARS level hardly changed with the growth of the tumor cells

Fig. 3. Concentration of lipid peroxide in plasma. (A) Antioxidant capacity ofnaïve mice and the effect of PEG-catalase injection on ROS scavenging ability.Hydrogen peroxide was injected into the tail vein of mice at the indicated dose.PEG-catalase was intravenously injected just before hydrogen peroxideinjection. Fifteen minutes after injection, the concentration of plasmalipoperoxides was measured. Results are expressed as the means + SD of fivemice. *p < 0.05 compared with control (hydrogen peroxide 0 nmol; PEG-catalase 0 unit) group; †p < 0.05 compared only with hydrogen peroxide-treated(hydrogen peroxide 2000 nmol; PEG-catalase 0 unit) group. (B) Plasma lipidperoxide concentration at 1 h after removal of the footpad tumor. Saline(vehicle) or catalase derivatives (500 units/body) were intravenously injectedjust before removal of the tumor. Results are expressed as the means + SD of atleast five mice. *Statistically significant difference compared with the no-removal, saline-treated group (p < 0.05); †statistically significant differencecompared with the removed, saline-treated group (p < 0.05).

Fig. 2. Effects of catalase derivatives on the growth of metastatic tumor cells afterremoval of the footpad tumor. Saline (vehicle) or catalase derivatives (500 units/body) were intravenously injected just before removal of the tumor. (A) Typicalexamples of pulmonary metastases at 7 days after tumor removal. (Images a)Footpad tumor not removed; (b) removed, saline (vehicle)-treated; (c) removed,native catalase-treated; (d) removed, PEG-catalase-treated. (B) Mice were killedat 21 (at the time of removal) or 28 days after tumor inoculation and the luciferaseactivity in the lung was assayed. Results are expressed as the means + SD of atleast five mice. *Statistically significant difference compared with the no-removal, saline-treated group (p < 0.05); †statistically significant differencecompared with the removed, saline-treated group (p < 0.05).


in the footpad up to 4 weeks (Fig. 3B). However, as previouslyreported [18], the surgery significantly increased the lipoper-oxides in the plasma. This increase was significantly prevented

by an intravenous administration of PEG-catalase just beforethe removal of the tumor-bearing footpad (p < 0.05 against thesaline group). The removal of non-tumor-bearing footpad also


significantly increased the plasma lipoperoxide level, whichwas again almost completely prevented by PEG-catalase. Theseresults indicate that surgery itself generates ROS, irrespective ofthe removal of the footpad tumor, and PEG-catalase is effectivein preventing the lipid peroxidation induced by the surgery.

Effects of catalase on expression of EGF, EGFR, andimmediate early response genes in B16-BL6 cells

To address whether catalase derivatives can inhibit theexpression of molecules involved in the growth of tumor cells,the mRNA levels in B16-BL6 cells were evaluated byquantitative PCR. The expression levels of major EGFRligands, EGF and TGF-α, were hardly changed by the additionof hydrogen peroxide or catalase (Fig. 4A). However, theexpression of EGFR increased dramatically after the addition ofhydrogen peroxide, which was efficiently prevented by catalase.To examine the activation downstream of the EGFR signaltransduction pathway after the activation of EGFR, wemeasured the expression of immediate early response genes.These genes have been previously reported as transcriptionfactors upregulated by the activation of EGFR [25–27]. As aresult, c-jun expression was significantly increased by theaddition of hydrogen peroxide, and other gene expression, suchas that of c-fos, c-myc, and EGR-1, showed a tendency toincrease after the addition of hydrogen peroxide (Fig. 4B).Moreover, Western blot analysis revealed an increase in EGFand EGFR expression after hydrogen peroxide treatment (Fig.4C). Quantitative densitometry evaluations showed that theexpression was significantly (p < 0.05) increased by addition ofhydrogen peroxide at concentrations as low as 10 μM (Fig. 4D).The increase in these genes' expression was completelyinhibited by the presence of catalase.

Effects of PEG-catalase on EGF and EGFR expression in thelung after tumor removal

In the above experiments, we demonstrated that ROS weregenerated after removal of the footpad tumor. We also showed

Fig. 4. Effects of hydrogen peroxide and catalase on the expression of cellgrowth-related genes after treatment with hydrogen peroxide and catalase invitro. (A, B) Real-time quantitative PCR analysis of (A) EGFR ligands (EGFand TGF-α) and EGFR and (B) immediate early response genes. Cells weretreated with hydrogen peroxide (100 μM) and/or catalase (333 units/ml) for 1 h.The amount of target mRNAwas normalized to that of GAPDHmRNA from thesame cDNA sample. Data are represented as an X-fold increase relative to thecontrol (hydrogen peroxide (−), catalase (−)) group. Results are expressed as themean + SD of three wells. *Statistically significant difference compared with thecontrol (hydrogen peroxide (−), catalase (−)) group (p < 0.05); †statisticallysignificant difference compared only with hydrogen peroxide-treated (hydrogenperoxide (+), catalase (−)) group (p < 0.05). (C) Western blot analysis of EGFand EGFR expression. Cells were treated with hydrogen peroxide and catalasefor 6 h. (D) Quantitative densitometry evaluations of Western blot analysis. Dataare represented as an X-fold increase relative to the control (hydrogen peroxide0 μM, catalase 0 units/ml) group. Results are expressed as the mean + SD ofthree wells. *Statistically significant difference compared with the control(hydrogen peroxide 0 μM, catalase 0 units/ml) group (p < 0.05); †statisticallysignificant difference compared only with hydrogen peroxide-treated (hydrogenperoxide 100 μM, catalase 0 unit/ml) group (p < 0.05).

that hydrogen peroxide aggravated the malignancy of tumorcells by increasing the expression of EGFR and activation of theEGFR signal transduction pathway. Next, we tried to measurethe alterations in the expression of molecules involved in the

Fig. 5. Expression levels of EGF and EGFR after removal of the footpad tumor.Saline (vehicle) or PEG-catalase (500 units/body) was intravenously injectedjust before removal of the tumor. (A) Real-time quantitative PCR analysis ofEGF and EGFR. The amounts of EGF and EGFR mRNA were normalized tothat of GAPDHmRNA from the same cDNA sample. Data are represented as anX-fold increase relative to naïve mice. Results are expressed as the means + SDof at least four mice. *Statistically significant difference compared with thenaïve or no-removal group (p < 0.05); †statistically significant differencecompared with the removed, saline-treated group (p < 0.05). (B) Western blotanalysis of EGF and EGFR 6 h after tumor removal. (C) Quantitativedensitometry evaluations of Western blot analysis. Data are represented as an X-fold increase relative to the no-removal group. Results are expressed as themeans + SD of three mice. *Statistically significant difference compared withthe no-removal group (p < 0.05); †statistically significant difference comparedwith removed, saline-treated group (p < 0.05).


growth of tumor cells in the lung after removal of the tumor. ThemRNA levels in the tumor-bearing lung were evaluated byquantitative PCR. EGF expression in the lung was hardlychanged by the inoculation of tumor cells or by the removal oftumor, but was reduced (although not significantly) by theadministration of PEG-catalase to mice undergoing removal ofthe tumor (Fig. 5A). EGFR expression was significantlyincreased by removal of the tumor and this increase wascompletely inhibited by the administration of PEG-catalase. Wealso measured the expression of immediate early genes in thelung after the removal, but no marked changes were observed(data not shown). Because such genes are ubiquitous transcrip-tion factors expressed in various cells, the alteration of theirexpression in tumor cells may be masked even when it has takenplace. The amounts of EGF and EGFR proteins in the lung werealso evaluated by Western blot analysis. A marked increase inthe expression of both EGF and EGFR was observed in thetumor-removed, saline-treated group (Figs. 5B and 5C). Again,PEG-catalase significantly (p < 0.05) inhibited this increase.

PEG-catalase extended the survival period of mice after tumorremoval

Fig. 6 shows the survival of mice with spontaneouspulmonary metastases of B16-BL6 cells. Intravenous adminis-tration of PEG-catalase just before the removal significantlyprolonged the survival time of mice compared with the saline-treated group (p < 0.0001).

Discussion

Although metastasis is a major target of cancer therapy, itsometimes occurs many years after treatment of the primarytumors, clearly demonstrating the difficulty of achievingcomplete prevention. One of the major reasons for this difficultyis the fact that the tissue disposition of tumor cells in vivo ispoorly understood even in animal models. In a previous study,we introduced the firefly luciferase gene into B16-BL6 cells asa marker and succeeded in establishing a highly sensitive andquantitative method to analyze the tissue disposition of thetumor cells [20]. In the present study, we first examined thetissue disposition of B16-BL6/Luc cells after inoculation intothe footpad and identified the characteristic features of the invivo fate of the cells, focusing on the metastasis andproliferation in the lung.

Because B16-BL6/Luc melanoma cells were inoculated intothe footpad of syngeneic C57BL/6 mice, the first step involvedin pulmonary metastasis would be proliferation of the tumorcells in the footpad followed by their entrance into the bloodcirculation and growth at secondary sites. As shown in Fig. 1,tumor cells were detected in the lung as early as 1 day afterinoculation. Thereafter, the number of tumor cells in the lungincreased with time, probably due to their proliferation in thelung and the arrival of additional tumor cells from the primarysite. Although there were more than 10,000 B16-BL6/Luccells in the lung at 3 weeks after tumor inoculation, nometastatic colonies could be seen on the lung surface under a

Fig. 6. Survival of mice bearing spontaneous pulmonary metastases. Thefootpad tumor foci were removed at 20 days after tumor inoculation into thefootpad. Saline (vehicle) or PEG-catalase (1000 units/mouse) was injected justbefore removal of the tumor. The survival of the removed, PEG-catalase-treatedgroup was significantly longer than that of the no-removal (p < 0.0001) orremoved, saline-treated (p < 0.0001) group (n = 10).


dissecting microscope, indicating the very high sensitivity ofthe assay method used in this study. As previously reported inboth animal models and patients [28–31], a robust prolifera-tion of metastatic tumor cells was observed under ourexperimental conditions after removal of the tumor (Figs. 2Aand 2B).

Even under normal conditions, ROS are continuouslyproduced as by-products of metabolism by enzymes, such assuperoxide dismutase, xanthine oxidase, and NADPH oxidase[32]. ROS scavenging enzymes also naturally exist in livingorganisms; however, there are many reports showing that lowactivity of ROS scavenging enzymes can be the cause of anumber of diseases. To evaluate the antioxidant capacity withinthe blood circulation, we injected hydrogen peroxide into thetail vein and measured lipid peroxidation in plasma. Althougherythrocytes contain a large amount of catalase activity, there islittle catalase activity in the plasma. As a result, theconcentration of plasma lipoperoxides significantly increasedas the dose of intravenously delivered hydrogen peroxideincreased. Moreover, exogenously delivered PEG-catalaseeffectively inhibited the increase in this lipid peroxidation.These results indicate that endogenous catalase activity is notenough to prevent oxidative damage induced by large amountsof ROS, perhaps because of the need for some time to allowhydrogen peroxide to cross the erythrocyte membrane. Asshown in Fig. 3B, the concentration of plasma lipoperoxideswas significantly increased by surgical removal of a footpad,irrespective of the presence of tumor on it, clearly indicatingthat the surgery generates ROS at a level that cannot beefficiently scavenged by the endogenous antioxidant system.ROS are reported to be involved in various processes of tumormetastasis, such as adhesion, invasion, and proliferation [33–38]. Therefore, the scavenging of ROS by antioxidant enzymescan be an effective approach for inhibiting tumor metastasis.

We have reported that experimental pulmonary or hepaticmetastasis of tumor cells can be effectively inhibited by a singleinjection of PEG-catalase or other catalase derivatives [19–21].In these studies, tumor cells were injected into the bloodcirculation through the tail vein or the portal vein, so that tumor

cells will immediately arrive at downstream organs. On theother hand, we found that tumor cells inoculated into thefootpad gradually migrated to the lung with time. A single,bolus injection of PEG-catalase had little effect on the numberof tumor cells in the lung when the footpad tumor was notsurgically removed (Fig. 2B). However, the same treatment wassignificantly effective in inhibiting the growth of tumor cellsafter tumor removal even compared to the no-removal, saline-treated group. Removal of the non-tumor-bearing footpadincreased the plasma lipoperoxides to a level as high as thatobserved in the tumor removal, saline-treated group, but itseffect on the number of tumor cells in the lung was not soevident. It is reported that primary tumors could releasemolecules, such as thrombospondin, that inhibit the prolifera-tion of tumor cells in micrometastases [39]. Therefore, we thinkthat the metastatic tumor growth in the non-tumor-bearingfootpad removed, saline-treated mice would be influenced byboth ROS generated by the surgery and inhibitory moleculesreleased from the footpad tumor. Further studies are needed toelucidate the effects of molecules from primary tumors on theproliferation of tumor cells in micrometastases. The findings ofthe present study suggest that (i) the footpad tumor continuouslyprovides metastatic tumor cells that eventually migrate to andproliferate in the lung, (ii) it may release molecules thatsomewhat inhibit or retard the proliferation of tumor cells inmicrometastases, and (iii) surgical trauma generates a largeamount of ROS, which would aggravate the growth of tumorcells in the lung. In the present study, we clearly demonstratedthat a single injection of PEG-catalase is highly effective ininhibiting the robust growth of tumor cells after removal of thefootpad tumor, but not in inhibiting the continuous migration oftumor cells from the tumor to the lung.

It has already been reported that ROS have effects on theexpression of molecules involved in cell proliferation andangiogenesis [40–42]. EGF is a potent mitogen for severaltypes of epithelial cells and it has been implicated in thedevelopment and progression of various malignancies throughautocrine and paracrine pathways [43]. In actual fact, EGFR ishighly expressed in a variety of tumors, such as breast, colon,gastric, pancreatic, ovarian, and prostate cancers; gliomas; andmelanomas; and it plays an important role in the processes oftumor growth, invasion, and metastasis [43]. Many cancercells depend on EGFR activity for proliferation, and blockingthe EGFR-associated signal transduction pathway has dramaticeffects on the growth and tumorigenicity of several tumor celllines [44]. Therefore, suppression of the expression of EGFand EGFR by PEG-catalase after removal of the tumor wouldbe a critical factor in the antimetastatic effects of PEG-catalase. Previously, it has been reported that exogenoushydrogen peroxide can induce tyrosine phosphorylation ofEGFR directly and exert effects on the induction of earlygrowth response genes, disruption of gap junction commu-nication, triggering of calcium inflow, and promotion oftransformation [26]. Moreover, EGR-1, a by-product of EGFRactivation, can activate the basal transcription activity of theEGFR promoter and also enhance endogenous EGFR expres-sion [45]. Taken together, generation of hydrogen peroxide


after removal of the tumor is thought to trigger theproliferation of dormant tumor cells.

It has previously been reported that primary tumor-derivedendogenous antiangiogenic factors, such as angiostatin andendostatin, contribute to the dormancy of metastatic tumors[46,47]. However, the size of metastatic tumors in a dormantstate is less than 1 mm in diameter, to which sufficient nutrientand oxygen can be supplied by diffusion, therefore angiogenesisis not necessary for tumor growth [48]. Consequently, changesin the levels of those primary tumor-derived antiangiogenicfactors may not be involved in the rapid growth of metastatictumor cells after removal of the tumor. A reduction in anti-angiogenic factors may accelerate tumor growth only after thetumor tissue has become relatively large.

In conclusion, PEG-catalase markedly suppressed the robustgrowth of metastatic tumor cells after removal of the tumor.Detoxification of hydrogen peroxide in the plasma by PEG-catalase reduced the expression of EGF and EGFR in the lungcontaining metastatic tumor cells, and this may be a criticalfactor for the inhibitory effects of PEG-catalase on the rapidgrowth of metastatic tumor cells after removal of the tumor.These findings indicate that sustained catalase activity in theblood circulation after removal of the tumor is a promisingapproach to preventing metastatic recurrence of a variety oftumors.

Acknowledgments

This work was supported in part by Grants-in-Aid forScientific Research from the Ministry of Education, Culture,Sports, Science, and Technology of Japan; by Health and LaborSciences Research Grants for Research on Hepatitis and BSEfrom the Ministry of Health, Labor, and Welfare of Japan; andby the 21st Century COE Program “Knowledge InformationInfrastructure for Genome Science."

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pegylated catalase prevents metastatic tumor growth aggravated by tumor removal

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