The Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Derived Reactive Oxygen Species in the Acquisition of Metastatic Ability of Tumor Cells
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.060073
ISSN1525-2191
AutoresFutoshi Okada, Masanobu Kobayashi, Hiroki Tanaka, Tokushige Kobayashi, Hiroshi Tazawa, Yoshihito Iuchi, Kunishige Onuma, Masuo Hosokawa, Mary C. Dinauer, Nicholas H. Hunt,
Tópico(s)bioluminescence and chemiluminescence research
ResumoWe examined the role of phagocyte-derived oxygen radicals in tumor cell acquisition of metastatic phenotype by comparing gp91phox−/− mice and C57BL/6J wild-type (WT) mice. The gp91phox−/− mouse is deficient in the gp91phox gene, an essential subunit of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase that generates superoxide anion. QR-32 fibrosarcoma cells are nonmetastatic but are converted into metastatic tumors once in contact with foreign body (gelatin sponge)-induced phagocytes in vivo. Compared to QR-32 cells co-implanted with the foreign body in WT mice, those in gp91phox−/− mice exhibited reduced metastasis. There was no difference in the incidence of primary tumors after injection of B16BL6 melanoma cells in WT and gp91phox−/− mice. However, after resection of the primary tumors, metastases were reduced in gp91phox−/− mice. Thymosin β4 gene expression and cell motility/invasion were seen in the tumors from WT mice but not in those from gp91phox−/− mice. Adoptive transfer of phagocytes from WT mice, but not those from gp91phox−/− mice, restored the metastatic ability of tumors grown in gp91phox−/− mice. These findings show that tumor metastatic behavior can primarily be endowed by phagocyte-derived superoxide anion and its oxidative metabolites, which are generated through activation of nicotinamide adenine dinucleotide phosphate oxidase. We examined the role of phagocyte-derived oxygen radicals in tumor cell acquisition of metastatic phenotype by comparing gp91phox−/− mice and C57BL/6J wild-type (WT) mice. The gp91phox−/− mouse is deficient in the gp91phox gene, an essential subunit of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase that generates superoxide anion. QR-32 fibrosarcoma cells are nonmetastatic but are converted into metastatic tumors once in contact with foreign body (gelatin sponge)-induced phagocytes in vivo. Compared to QR-32 cells co-implanted with the foreign body in WT mice, those in gp91phox−/− mice exhibited reduced metastasis. There was no difference in the incidence of primary tumors after injection of B16BL6 melanoma cells in WT and gp91phox−/− mice. However, after resection of the primary tumors, metastases were reduced in gp91phox−/− mice. Thymosin β4 gene expression and cell motility/invasion were seen in the tumors from WT mice but not in those from gp91phox−/− mice. Adoptive transfer of phagocytes from WT mice, but not those from gp91phox−/− mice, restored the metastatic ability of tumors grown in gp91phox−/− mice. These findings show that tumor metastatic behavior can primarily be endowed by phagocyte-derived superoxide anion and its oxidative metabolites, which are generated through activation of nicotinamide adenine dinucleotide phosphate oxidase. Metastasis has a significant impact on the mortality in cancer patients, as shown by the overall 5-year survival rate being less than 20% because of metastases.1Landis SH Murray T Bolden S Wingo PA Cancer statistics.CA Cancer J Clin. 1998; 48: 6-29Crossref PubMed Scopus (2453) Google Scholar Metastatic disease is generally difficult to treat with currently available therapies, and thus an improved understanding of the underlying molecular and cellular mechanisms of tumor metastasis is needed. A tumor undergoes a complex series of processes to achieve metastasis,2Fidler IJ Critical determinants of cancer metastasis: rationale for therapy.Cancer Chemother Pharmacol. 1999; 43: S3-S10Crossref PubMed Scopus (155) Google Scholar, 3Hart IR Saini A Biology of tumour metastasis.Lancet. 1992; 339: 1453-1457Abstract PubMed Scopus (322) Google Scholar which is termed “multistep carcinogenesis.” Tumor metastasis is promoted by genetic alterations in each step,4Welch DR Steeg PS Rinker-Schaeffer CW Molecular biology of breast cancer metastasis. Genetic regulation of human breast carcinoma metastasis.Breast Cancer Res. 2000; 2: 408-416Crossref PubMed Scopus (115) Google Scholar and epigenetic changes of crucial genes related to organ-specific colonization have been elucidated to some extent.5Nicolson GL Cancer metastasis: tumor cell and host organ properties important in metastasis to secondary sites.Biochem Biophys Acta. 1988; 948: 175-224PubMed Google Scholar, 6Zetter BR Cellular basis of site-specific tumor metastasis.N Engl J Med. 1990; 322: 605-612Crossref PubMed Scopus (321) Google Scholar, 7Radinsky R Modulation of tumor cell gene expression and phenotype by organ specific metastatic environment.Cancer Metastasis Rev. 1995; 14: 323-338Crossref PubMed Scopus (98) Google ScholarAlthough little is known about the causative factors that convert benign tumors into malignant ones, it has been proposed that metastatic tumor cells originate in a pre-existing or dynamic heterogeneity of tumor cells with metastatic potential that reside in the primary tumor8Fidler IJ Kripke ML Metastasis results from preexisting variant cells within a malignant tumor.Science. 1977; 197: 893-895Crossref PubMed Scopus (1149) Google Scholar, 9Weigelt B Peterse JL van't Veer LJ Breast cancer metastasis: markers and models.Nat Rev Cancer. 2005; 5: 591-602Crossref PubMed Scopus (1652) Google Scholar and that expand to clinically distinguishable metastasis in the environment of the host (selection). In the current study, we aimed to identify the endogenous factors that can promote tumor metastasis.Among endogenous factors potentially related to tumor development and progression, oxygen radicals are thought to have a major influence. Epidemiological studies have indicated that genotoxic reactive oxygen species (ROS) are implicated in one-fourth to one-third of all cancers.10Doll R Peto RJ The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today.Natl Cancer Inst. 1981; 66: 1191-1308Google Scholar, 11Parkin DM Global cancer statistics in the year 2000.Lancet Oncol. 2001; 2: 533-543Abstract Full Text Full Text PDF PubMed Scopus (2144) Google Scholar In vitro studies have revealed that oxygen radicals may be associated with tumor development.12Yoon SO Park SJ Yoon SY Yun CH Chung AS Sustained production of H2O2 activates pro-matrix metalloproteinase-2 through receptor tyrosine kinases/phosphatidylinositol 3-kinase/NF-kappaB pathway.J Biol Chem. 2002; 277: 30271-30282Crossref PubMed Scopus (141) Google Scholar However, in general, research approaches have been indirect, ie, they are performed using in vitro systems or by using antioxidative enzymes,13Yoshizaki N Mogi Y Muramatsu H Koike K Kogawa K Niitsu Y Suppressive effect of recombinant human Cu, Zn-superoxide dismutase on lung metastasis of murine tumor cells.Int J Cancer. 1994; 57: 287-292Crossref PubMed Scopus (52) Google Scholar, 14Tanaka M Kogawa K Nishihori Y Kuribayashi K Nakamura K Muramatsu H Koike K Sakamaki S Niitsu Y Suppression of intracellular Cu-Zn SOD results in enhanced motility and metastasis of Meth A sarcoma cells.Int J Cancer. 1997; 73: 187-192Crossref PubMed Scopus (41) Google Scholar and thus interpretation of the results remains ambiguous. To verify the involvement of ROS in tumor development and progression, we used mice that have targeted disruption of a gene related to ROS production in inflammatory phagocytes.The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a membrane-bound cytochrome b558 composed of two subunits (gp91phox and p22 phox) that coordinate flavin adenine dinucleotide and two heme moieties.15Yu L Zhen L Dinauer MC Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits.J Biol Chem. 1997; 272: 27288-27294Crossref PubMed Scopus (126) Google Scholar With inflammatory stimulation, the cytosolic subunits p40phox, p47phox, p67phox, and the small GTP-binding protein Rac1/Rac2 translocate to the membrane and interact with cytochrome b558.16DeLeo FR Burritt JB Yu L Jesaitis AJ Dinauer MC Nauseef WM Processing and maturation of flavocytochrome b588 include incorporation of heme as a prerequisite for heterodimer assembly.J Biol Chem. 2000; 275: 13986-13993Crossref PubMed Scopus (137) Google Scholar, 17Abo A Pick E Hall A Totty N Teahan CG Segal AW Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1.Nature. 1991; 353: 668-670Crossref PubMed Scopus (758) Google Scholar, 18Rotrosen D Yeung CL Leto TL Malech HL Kwong CH Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase.Science. 1992; 256: 1459-1462Crossref PubMed Scopus (313) Google Scholar The assembled complex mediates electron transfer to extracellular molecular oxygen (O2), resulting in the generation of superoxide anion (O2−) during the phagocyte respiratory burst.19Babior BM NADPH oxidase: an update.Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 20Yu L Quinn MT Cross AR Dinauer CM Gp91phox is the heme binding subunit of the superoxide-generating NADPH oxidase.Proc Natl Acad Sci USA. 1998; 95: 7993-7998Crossref PubMed Scopus (177) Google Scholar The physiological significance of the phagocyte NADPH oxidase in host defense is indicated by the severe, recurrent bacterial or fungal infections that occur in patients with chronic granulomatous disease. Phagocytes in those patients are unable to generate O2− because of various mutations in four of the oxidase proteins (p22phox, p47phox, p67phox, and gp91phox),21Dinauer M The respiratory burst oxidase and the molecular genetics of chronic granulomatous disease.Crit Rev Clin Lab Sci. 1993; 30: 329-369Crossref PubMed Scopus (110) Google Scholar especially a glycosylated 91-kd glycoprotein (gp91phox).19Babior BM NADPH oxidase: an update.Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar gp91phox−/− mice generated by disruption of the X-linked murine cytochrome b558 lack a functional NADPH oxidase, and their phagocytes are unable to generate superoxide anion, thus providing a murine model of chronic granulomatous disease.22Pollock JD Williams DA Gifford MA Li LL Du X Fishman J Orkin SH Doerschuk CM Dinauer MC Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.Nat Genet. 1995; 9: 202-209Crossref PubMed Scopus (749) Google ScholarIn this study, we demonstrated the contribution of NADPH oxidase-derived superoxide and its oxidative metabolites to acquisition of the metastatic phenotype, using two different models in which we were able to observe the natural transition in vivo of nonmetastatic (or metastatic) tumor cells to metastatic (or highly metastatic) ones. We also present evidence that inflammatory phagocytes can serve as an endogenous factor to irreversibly convert tumor cells into metastatic ones (ie, genetic alteration) by generating ROS in vivo.Materials and MethodsTumor Cell Lines and Culture ConditionsThe origin and characteristics of the tumor cell lines used in this study have been described previously.23Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N Kobayashi H Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (59) Google Scholar Briefly, the QR-32 tumor is a clone obtained from 3-methyl-cholanthrene-induced fibrosarcoma in a C57BL/6J mouse that spontaneously regressed when injected into normal syngeneic mice.24Ishikawa M Okada F Hamada J Hosokawa M Kobayashi H Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine.Int J Cancer. 1987; 39: 338-342Crossref PubMed Scopus (53) Google Scholar QR-32 and its derivative metastatic cell line (QRsP-11) were maintained in Eagle's minimum essential medium (MEM; Nissui Pharm., Tokyo, Japan) supplemented with 8% (v/v) fetal bovine serum (Filtron Pty., Ltd., Brookyln, Australia). B16BL6 melanoma cells were maintained in Dulbecco's modified Eagle's medium (Sigma, Tokyo, Japan) with 10% fetal bovine serum.Micegp91phox−/− mice, established as previously described,22Pollock JD Williams DA Gifford MA Li LL Du X Fishman J Orkin SH Doerschuk CM Dinauer MC Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.Nat Genet. 1995; 9: 202-209Crossref PubMed Scopus (749) Google Scholar lack the membrane gp91phox subunit of the NADPH oxidase multicomponent system. The knockout mice were backcrossed with C57BL6J mice for more than 20 generations. C57BL/6J mice (introduced from the Jackson Laboratory, Bar Harbor, ME) were obtained from Nippon SLC (Hamamatsu, Japan) and used as WT mice. Age- and sex-matched mice were used for each set of experiments. All experiments were approved by the Committee of Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine (no. 02048 and no. 03085).Model of Inflammation-Based Tumor ProgressionInflammation was induced by insertion of a foreign body, gelatin sponge, as described previously.23Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N Kobayashi H Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (59) Google Scholar Sterile gelatin sponge (Yamanouchi Pharm., Tokyo, Japan) was cut into 10 × 5 × 3-mm pieces and inserted into a subcutaneous pocket in the right flank of the pelvic region of a mouse. Then QR-32 tumor cells (1 × 105 cells/0.1 ml) were immediately injected into the preinserted gelatin sponge. On day 25, the arising tumors were aseptically removed, and individual culture cell lines were established for evaluation of their metastatic potential in normal mice (1 × 106 cells injected intravenously). On day 25, the mice were sacrificed, and metastatic nodules on the surface of the lungs or other organs were counted macroscopically.Model of Spontaneous Metastasis of B16BL6 Melanoma CellsB16BL6 cells (4 × 105 in 0.05 ml) were injected into the hind footpad. On day 25, the primary tumors were surgically removed under anesthesia. The mice were sacrificed when they were in a moribund state, followed by necropsy.Subcutaneous Tumor Growth and Experimental MetastasisFor evaluation of subcutaneous tumor growth, QRsP-11 tumor cells (2 × 105) or B16BL6 cells (1 × 106) were injected, and the tumor diameters were measured twice a week. For experimental metastasis assay, tumor cells (QRsP-11, 1 × 106 cells; B16BL6, 1 × 105 cells) were injected intravenously. The mice were maintained for 3 and 2 weeks, respectively. After euthanasia, the lungs and other organs were removed and weighed, and surface metastatic lesions were counted macroscopically.Adoptive Transfer of Inflammatory PhagocytesIn the recipient gp91phox−/− mice, QR-32 cells with the foreign body had been implanted or B16BL6 cells had been injected into the footpad. Inflammatory phagocytes (2 × 106 cells), prepared as described below, were then transferred adoptively to the growing site of QR-32 cells daily from day 0 (simultaneously) to day 5 and to the growing site of B16BL6 cells three times each week for 4 weeks.The inflammatory phagocytes were collected by lavage of the peritoneal cavity of WT and gp91phox−/− mice in which a sterile gelatin sponge had been inserted 5 days before. The peritoneal exudate cells were then purified for phagocytes by using Mono-Poly resolving medium (Dainippon Pharmaceutical Co. Ltd., Tokyo, Japan). Histological examination revealed that the purified cellular population was composed of ∼70% neutrophils and 30% macrophages/lymphocytes.Determination of the Total Number and the Types of the Cells Infiltrated into Gelatin SpongeThe gelatin sponge pieces subcutaneously injected into WT and gp91phox−/− mice were removed and digested with 0.2% collagenase in serum-free minimum essential medium for a few minutes at 37°C. After collecting all of the infiltrated cells by centrifugation, we counted the total number of the cells per piece of gelatin sponge. We also performed differential counts of more than 200 cells in smear preparations of the collected cells stained with May-Grünwald's and Giemsa solution (Wako Pure Chemical Inc., Osaka, Japan). Mean percentages of differential cells were obtained from the mean values of independent counts by two pathologists.Assay for Determining Scavenging Capacity of Plasma AntioxidantsThe aim of this assay was to determine scavenging capacity of plasma antioxidants by evaluation of Cu+ derived from Cu++ by the combined action of all antioxidants in plasma (TA01; Oxford Biomedical Research, Oxford, MI). Generation of Cu+ was detected after stable complex formation between Cu+ and bathocuproine as monitored at 490 nm wavelength on a microplate reader (model 550; Bio-Rad, Tokyo, Japan). The decrease in absorbance after addition of the plasma was plotted on a calibration curve estimated by application of known concentrations of uric acid as standard. Results were expressed as μmol/L uric acid equivalents.In Vitro Cell Motility Assay (Phagokinetic Track Assay)Uniform carpets of gold particles were prepared on glass coverslips (22 × 22 mm). The coverslips were placed in 35-mm culture dishes (Greiner Labortechnik, Tokyo, Japan) and 2 × 103 tumor cells were placed in each dish. After 48 hours, phagokinetic tracks of 40 cells were traced under a microscope. The area cleared of gold particles by cells was quantified by using a microscope analyzer (Cosmozone R500; Nikon, Japan).In Vitro Cell Invasion AssayA nucleopore filter (8-μm-pore size) was coated with Matrigel matrix (156 μg/cm2; Becton, Dickinson and Company, Tokyo, Japan) and placed between blind-well chemotactic chamber compartments (Costar Transwell, Tokyo, Japan), and then 5 × 103 tumor cells were placed in the upper compartment. Fibronectin (25 μg/ml) was used as chemoattractant. After 6 hours of incubation, the filters were removed, fixed in 5% glutaraldehyde, and stained with May-Grünwald Giemsa stain. The number of cells that had penetrated into the filter was counted under a microscope.Reverse Transcriptase-Polymerase Chain Reaction (RT- PCR) AnalysisThe detail of RT-PCR for gene amplification of thymosin β4 or GAPDH has been described previously.25Kobayashi T Okada F Fujii N Tomita N Ito S Tazawa H Aoyama T Choi SK Shibata T Fujita H Hosokawa M Thymosin-β4 regulates motility and metastasis of malignant mouse fibrosarcoma cells.Am J Pathol. 2002; 160: 869-882Abstract Full Text Full Text PDF PubMed Scopus (116) Google ScholarStatistical AnalysisThe significance of the differences in tumor and metastatic incidences was evaluated by the χ2Fidler IJ Critical determinants of cancer metastasis: rationale for therapy.Cancer Chemother Pharmacol. 1999; 43: S3-S10Crossref PubMed Scopus (155) Google Scholar test, and the differences in organ weight, number of lung colonies, and antioxidative capacity were calculated by Student's t-test.ResultsDecreased Acquisition of Spontaneous Metastatic Phenotype in gp91phox−/− MiceThe QR-32 fibrosarcoma cells did not produce tumors or form metastases after subcutaneous (2 × 105 cells) or intravenous (1 × 106 cells) injection into C57BL/6J mice.24Ishikawa M Okada F Hamada J Hosokawa M Kobayashi H Changes in the tumorigenic and metastatic properties of tumor cells treated with quercetin or 5-azacytidine.Int J Cancer. 1987; 39: 338-342Crossref PubMed Scopus (53) Google Scholar However, they were converted to grow lethally after being co-implanted with a piece of gelatin sponge, which induced inflammation at the site of implantation, and the tumors arising from them acquired a metastatic phenotype.23Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N Kobayashi H Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (59) Google Scholar Previous studies indicate that infiltrated neutrophils are predominantly involved in the progression process, and oxygen radicals derived from neutrophils play a role in acquisition of metastases.26Tazawa H Okada F Kobayashi T Tada M Mori Y Une Y Sendo F Kobayashi M Hosokawa M Infiltration of neutrophils is required for acquisition of metastatic phenotype of benign murine fibrosarcoma cells. Implication of inflammation-associated carcinogenesis and tumor progression.Am J Pathol. 2003; 163: 2221-2232Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 27Okada F Nakai K Kobayashi T Shibata T Tagami S Kawakami Y Kitazawa T Kominami R Yoshimura S Suzuki K Taniguchi N Inanami O Kuwabara M Kishida H Nakae D Konishi Y Moriuchi T Hosokawa M Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells.Br J Cancer. 1999; 79: 377-385Crossref PubMed Scopus (27) Google ScholarFigure 1A shows that lethal growth of QR-32 cells co-implanted with gelatin sponge was observed in 30 of 39 wild-type (WT) mice in contrast to 8 of 34 gp91phox gene knockout mice (gp91phox−/−) that lack a functional NADPH oxidase. In contrast to the tumor cells grown in WT mice, tumors arising in gp91phox−/− mice acquired a significantly weaker metastatic phenotype, both in the metastases of the lung (Figure 1, B and C; P < 0.001) and other organs (data not shown).The subcutaneously injected gelatin sponge pieces were removed from WT and gp91phox−/− mice, and the exact number of infiltrated cells was counted per gelatin sponge. Table 1 shows that a greater number of cells infiltrated into sponge pieces in gp91phox−/− mice. When we stained the infiltrated cells and determined their cell types by histological examination, there were no differences in the types of cells infiltrated into gelatin sponge between WT and gp91phox−/− mice.Table 1Differential Leukocyte Counts and the Numbers of Cells Infiltrated into Gelatin Sponge in gp91phox−/− and WT MiceMice*A piece of gelatin sponge was implanted into the subcutaneous space of gp91phox−/− and WT mice.Number of mice examinedTotal number of gelatin sponge-infiltrated cells (×106)Percentage of differential leukocytes per gelatin sponge-infiltrated cellsMϕ/MO†Mϕ/MO, macrophages/monocytes.PMN‡PMN, polymorphonuclear neutrophils.LC§LC, lymphocytes.EOS¶EOS, eosinophils.OthersWT616.4 ± 1.5‖p < 0.001.12.5 ± 7.157.8 ± 7.726.9 ± 3.51.0 ± 1.12.0 ± 1.2gp91phox−/−622.6 ± 2.7‖p < 0.001.9.8 ± 4.361.5 ± 5.126.9 ± 2.30.8 ± 1.01.4 ± 0.5* A piece of gelatin sponge was implanted into the subcutaneous space of gp91phox−/− and WT mice.† Mϕ/MO, macrophages/monocytes.‡ PMN, polymorphonuclear neutrophils.§ LC, lymphocytes.¶ EOS, eosinophils.‖ p < 0.001. Open table in a new tab We next examined spontaneous metastasis of B16BL6 melanoma, another in vivo murine tumor model. B16BL6 cells (4 × 105 cells) were injected into the right footpad of mice, and the arising tumors were surgically resected on day 25. No significant difference was observed between the mice in the incidence of primary tumor growth (Figure 1D), which was also evaluated by tumor thickness in proportion to the left footpad (Figure 2C) and by the weight of amputated legs (data not shown). However, local tumor invasion in vivo was evident in the WT mice compared to gp91phox−/− mice when the primary footpad tumors were removed. Grossly, in 12 of 20 WT mice, tumor invaded the underlying calf muscle and could not be removed completely; in contrast, tumors invaded calf muscle in only 4 of 27 gp91phox−/− mice and were easily removed with minimal damage to the surrounding tissues. Figure 1E shows that the incidence of lung metastasis was significantly higher in WT mice than in gp91phox−/− mice (P < 0.001). Furthermore, the number of metastatic nodules on the lung surface was significantly higher in WT mice than in gp91phox−/− mice (Figure 1F, P < 0.001).Figure 2Growth of primary tumors in gp91phox−/− mice and WT mice after subcutaneous or intrafootpad injections of tumor cells. Growth curves of QR-32 cells (1 × 105 cells) co-implanted with gelatin sponge (A), QRsP-11 tumor cells (1 × 106 cells) injected subcutaneously (B), B16BL6 cells (4 × 105 cells) injected into hind footpad (C), and B16BL6 cells (1 × 106 cells) injected subcutaneously (D) into WT and gp91phox−/− mice, respectively. ○, WT mice; •, gp91phox−/− mice; ▵, gp91phox−/− mice with adoptively transferred WT phagocytes; ▴, gp91phox−/− mice with adoptively transferred gp91phox−/− phagocytes. *P < 0.01; gp91phox−/− versus WT mice, and gp91phox−/− mice with adoptively transferred WT phagocytes versus gp91phox−/− mice with adoptively transferred gp91phox−/− phagocytes. Data are mean diameters of the arising tumors at each injection and are representative results of at least two separate experiments producing similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that ROS derived from NADPH oxidase in phagocytes actually contributed to the acquisition of metastatic properties of tumor cells, we tested whether the reduced metastatic ability of tumor cells in gp91phox−/− mice could be restored by adoptive transfer of inflammatory phagocytes. Since we had found that QR-32 cell progression was promoted by the early-phase inflammation in this model,26Tazawa H Okada F Kobayashi T Tada M Mori Y Une Y Sendo F Kobayashi M Hosokawa M Infiltration of neutrophils is required for acquisition of metastatic phenotype of benign murine fibrosarcoma cells. Implication of inflammation-associated carcinogenesis and tumor progression.Am J Pathol. 2003; 163: 2221-2232Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar we adoptively transferred phagocytes into the gelatin sponge co-implantation site from day 0 to day 5. Figure 3A shows that tumor growth occurred in 7 of 10 WT mice and 2 of 10 gp91phox−/− mice without adoptive transfer, 6 of 10 gp91phox−/− mice with adoptively transferred WT mice-derived phagocytes and 2 of 10 gp91phox−/− mice with gp91phox−/− mouse-derived phagocytes. Figure 2A shows their growth curves. More interestingly, tumors grown in the gp91phox−/− mice with WT mouse-derived phagocytes acquired a malignant phenotype, as evidenced by the development of lung metastasis; in contrast, those in gp91phox−/− mice with gp91phox−/− mouse-derived phagocytes did not (Figure 3, B and C).Figure 3Restored metastatic ability of tumor cells by adoptive transfer of inflammatory phagocytes obtained from WT mice. Adoptively transferred inflammatory phagocytes obtained from WT mice recovered acquisition of metastatic potential of tumor cells of both tumor cell lines. *P < 0.05 and **P < 0.001 versus adjacent group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we confirmed recovery of metastatic potential in the B16BL6 model. Phagocytes isolated from WT or gp91phox−/− mice were injected into the B16BL6 tumor bed in gp91phox−/− mice. As shown in Figure 3D, tumor incidence did not differ between the mice; however, adoptive transfer of phagocytes from WT mice significantly enhanced metastatic ability, whereas transfer of phagocytes of gp91phox−/− mice did not (Figure 3, F and G).We also examined the effects of the wild-type versus gp91phox−/− background on growth of the primary tumor. For subcutaneous injection, we used highly tumorigenic and metastatic QRsP-11 tumor cells derived from QR-32 tumor cells in contact with inflammation.23Okada F Hosokawa M Hamada J Hasegawa J Kato M Mizutani M Ren J Takeichi N Kobayashi H Malignant progression of a mouse fibrosarcoma by host cells reactive to a foreign body (gelatin sponge).Br J Cancer. 1992; 66: 635-639Crossref PubMed Scopus (59) Google Scholar There were no differences in the growth incidence and growth rate between gp91phox−/− mice and WT mice (Figure 2B). In addition, we subcutaneously injected B16BL6 cells, finding no difference either in the growth rate (Figure 2D) or in the growth incidence (Figure 4A) between WT and gp91phox−/− mice. However, we found dramatic differences in the incidence of spontaneous distant metastases. Of the 12 WT mice, 9 mice had prominent lung metastasis at necropsy, in contrast to 2 of the 14 gp91phox−/− mice (Figure 4B, P < 0.01). The number of metastatic nodules on the lung surface was significantly higher in WT mice than in gp91phox−/− mice (Figure 4C, P < 0.05). Furthermore, the majority of B16BL6-bearing WT mice developed extrapulmonary metastases, frequently accompanied by metastases to regional lymph nodes (inguinal and axilla), whereas in gp91phox−/− mice metastases were significantly less frequent (Figure 4D). We then used an experimental metastasis assay to evaluate the primary growth properties in the lungs after intravenous injection of QRsP-11 cells or B16BL6 cells. The number and size of lung metastases of the respective tumor lines did not differ between WT mice and gp91phox−/− mice (Table 2). Oxidative stress is balanced on the match of oxidant stimuli and various antioxidants in vivo. We next confirmed the scavenging capacity of plasma antioxidants in WT and gp91phox−/− mice. There was no significant difference between the mice either at 8 or 16 weeks of age (Table 3).Figure 4Decreased acquisition of spontaneous metastatic ability of B16BL6 melanoma cells after subcutaneous injection into gp91phox−/− mice. B16BL6 melanoma cells (1 × 106) were injected subcutaneously into WT mice and gp91phox−/− mice. The mice were sacrificed for necropsy when they were in a moribund state. *P < 0.05 and **P < 0.01 versus WT mice.View Large
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