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• W2017209306 abstract "To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38α+/- mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38α+/- mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38α+/- mice. Platelets of p38α+/- mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38α+/- mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38α+/- mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38α+/- mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38α+/- mice was similar to that of WT mice. These results suggest that p38α plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis. To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38α+/- mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38α+/- mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38α+/- mice. Platelets of p38α+/- mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38α+/- mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38α+/- mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38α+/- mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38α+/- mice was similar to that of WT mice. These results suggest that p38α plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis. Mitogen-activated protein kinases (MAPKs) 3The abbreviations used are: MAPK, mitogen-activated protein kinase; PECAM-1, platelet endothelial cell adhesion molecule-1; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; WT, wild type; B16, mouse B16 melanoma F10; LLC, mouse Lewis lung carcinoma; B16-β-gal, B16 cells stably expressing β-galactosidase; PBS, phosphate-buffered saline. 3The abbreviations used are: MAPK, mitogen-activated protein kinase; PECAM-1, platelet endothelial cell adhesion molecule-1; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; WT, wild type; B16, mouse B16 melanoma F10; LLC, mouse Lewis lung carcinoma; B16-β-gal, B16 cells stably expressing β-galactosidase; PBS, phosphate-buffered saline. transduce a variety of extracellular signals to the transcriptional machinery via cascades of protein phosphorylation. There are three genetically distinct MAPKs in mammals, consisting of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK (p38). All three members are activated by dual phosphorylation of the conserved TXY motif and then phosphorylate their respective substrates on serine or threonine residues (1Minden A. Lin A. McMahon M. Lange-Carter C. Dérijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar, 3Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (684) Google Scholar). There are four mammalian isoforms of p38 (α, β, γ, and δ). Among them, p38α and -β are expressed relatively ubiquitously, as shown by Northern blot analysis of adult tissues (4Jiang Y. Chen C. Li Z. Guo W. Gegner J.A. Lin S. Han J. J. Biol. Chem. 1994; 266: 17920-17926Google Scholar). Although targeted disruption of the p38α gene results in homozygous embryonic lethality because of defects in erythropoiesis and placental organogenesis (5Adams R.H. Porras A. Alonso G. Jones M. Vintersten K. Panelli S. Valladares A. Perez L. Klein R. Nebreda A.R. Mol. Cell. 2000; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 6Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), the p38α+/- mouse is a useful tool for analyzing the in vivo role of p38 in disease models (7Otsu K. Yamashita N. Nishida K. Hirotani S. Yamaguchi O. Watanabe T. Hikoso S. Higuchi Y. Matsumura Y. Maruyama M. Sudo T. Osada H. Hori M. Biochem. Biophys. Res. Commun. 2003; 302: 56-60Crossref PubMed Scopus (66) Google Scholar, 9Sakurai K. Matsuo Y. Sudo T. Takuwa Y. Kimura S. Kasuya Y. J. Recept. Signal Transduct. Res. 2004; 24: 283-296Crossref PubMed Scopus (30) Google Scholar). Tumor metastasis is a significant process resulting in unexpected death in cancer patients. Recent advances in molecular cancer research have clarified a variety of therapeutic targets, some of which are being tested in clinical trials (10Tímár J. Ladányi A. Peták I. Jeney A. Kopper L. Pathol. Oncol. Res. 2003; 9: 49-72Crossref PubMed Scopus (16) Google Scholar). Very recently, the relationship between tumor metastasis and MAPKs has been investigated (11Steeg P.S. Nat. Rev. Cancer. 2003; 3: 55-63Crossref PubMed Scopus (435) Google Scholar, 12Aguirre-Ghiso J.A. Ossowski L. Rosenbaum S.K. Cancer Res. 2004; 64: 7336-7345Crossref PubMed Scopus (138) Google Scholar). It is reported that p38α is important for the maintenance of breast cancer with an invasive phenotype by promoting the stability of urokinase plasminogen activator and its receptor mRNA (13Huang S. New L. Pan Z. Han J. Nemerow G.R. J. Biol. Chem. 2000; 275: 12266-12272Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). It has also been demonstrated that p38 is involved in various metastatic processes (14Käkönen S.M. Selander K.S. Chirgwin J.M. Yin J.J. Burns S. Rankin W.A. Grubbs B.G. Dallas M. Cui Y. Guise T.A. J. Biol. Chem. 2002; 277: 24571-24578Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 17Huang X. Chen S. Xu L. Liu Y. Deb D.K. Platanias L.C. Bergan R.C. Cancer Res. 2005; 65: 3470-3478Crossref PubMed Scopus (236) Google Scholar). On the other hand, MKK4, a common upstream prerequisite for c-Jun N-terminal kinase and p38 activation as an MAPK kinase, is characterized as a metastasis suppressor gene in human ovarian carcinoma (18Yamada S.D. Hickson J.A. Hrobowski Y. Vander Griend D.J. Benson D. Montag A. Karrison T. Huo D. Rutgers J. Adams S. Rinker-Schaeffer C.W. Cancer Res. 2002; 62: 6717-6723PubMed Google Scholar, 19Hickson J.A. Huo D. Vander Griend D.J. Lin A. Rinker-Schaeffer C.W. Yamada S.D. Cancer Res. 2006; 66: 2264-2270Crossref PubMed Scopus (101) Google Scholar). Likewise, loss of p38 activation leads to an increase in tumorigenesis because of a cell cycle defect (20Brancho D. Tanaka N. Jaeschke A. Ventura J.J. Kelkar N. Tanaka Y. Kyuuma M. Takeshita T. Flavell R.A. Davis R.J. Genes Dev. 2003; 17: 1969-1978Crossref PubMed Scopus (404) Google Scholar). These findings clearly suggest that p38 activity in tumor cells regulates tumor progression and metastasis. However, there is no in vivo confirmation of a pathophysiological role of p38 in hosts during tumor metastasis. To elucidate this point, we used p38α+/- mice to examine the in vivo role of p38α during tumor metastasis. Here, we showed that tumor metastasis is suppressed in p38α+/- mice. Experimental Animals—The use of animals in all of our experiments was in accordance with the guidelines for animal care of Chiba University and RIKEN. Female mice heterozygous for targeted disruption of the p38α gene (6Tamura K. Sudo T. Senftleben U. Dadak A.M. Johnson R. Karin M. Cell. 2000; 102: 221-231Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) were crossed with C57BL/6J male mice (Saitama Experimental Animal Supply Co.) to generate p38α+/- and p38α+/+ (wild-type; WT) mice. Offspring (>8 generations) were genotyped by PCR analysis of tail-derived DNA. Multiplex PCR with three primers per reaction was used. The primers were as follows: A, 5′-CCCTATACTCCCTCTCTGTGTAACTTTTG-3′; B, 5′-CCCAAACCCCAGAAAGAAATGATG-3′; C, 5′-TTCAGTGACAACGTCGAGCACAGCTG-3′. Using these primers for one cycle at 94 °C for 5 min followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, with an extension step of 7 min at 72 °C at the end of the last cycle, produced 800- and 450-bp fragments from the mutant and WT alleles, respectively. 8-12-week-old WT and p38α+/- littermates were used for each experiment. Cells—Mouse B16 melanoma F10 (B16) cells, mouse Lewis lung carcinoma (LLC) cells, and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10% (v/v) fetal calf serum under a humidified atmosphere of 95% air and 5% CO2 at 37 °C. B16 cells stably expressing β-galactosidase (B16-β-gal) were generated by cotransfection of pCMVβ (BD Biosciences) and pBLAST (InvivoGen), using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Stable transfectants were selected in the medium containing 50 μg/ml blasticidin S (Funakoshi). After 2-3 weeks, clones were isolated by limiting dilution, and β-galactosidase-expressing clones were identified by X-gal staining after fixation with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 m sodium phosphate buffer (pH 7.4). Experimental Lung Metastasis Model—B16 and LLC cells were trypsinized and recovered from the medium by brief centrifugation. They were suspended with physiological saline to a concentration of 107 cells/ml. Mice (WT and p38α+/-) were anesthetized with an intraperitoneal injection of avertin, and 106 cells (in a volume of 100 μl) were systemically injected into the tail veins. In all experiments, injection of tumor cells was performed at a speed of 10 μl/min using a syringe pump (model CFV-3200; NIHON KOHDEN). 3 and 4 weeks after injection of LLC and B16 cells, respectively, the lungs were dissected out and rinsed in phosphate-buffered saline (PBS). Then they were separated into individual lobes, and the number of surface metastatic foci was counted. In the case of using B16-β-gal, the lungs were dissected out from WT and p38α+/- mice 1 week after injection. Their lungs were immersed in 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 m sodium phosphate buffer (pH 7.4), followed by X-gal buffer (1 mg/ml 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside, 10 mm K4Fe(CN)6, 10 mm K3Fe(CN)6, and 2 mm MgCl2 in PBS), and then embedded in paraffin and cut into sections 4 μm in thickness. The sections were placed on poly-l-lysine-coated slides, and blue-dyed colonies were counted under a light microscope (Axioplan; Zeiss). Tumor Growth Experiment—B16 and LLC cells were suspended with physiological saline to a concentration of 107 cells/ml. Each cell type (106 cells in a volume of 100 μl) was inoculated subcutaneously into the left flank of mice (WT and p38α+/-) under anesthesia with an intraperitoneal injection of avertin. Tumors were measured along the plane of the body cavity using a hand caliper, and tumor volume was calculated as (length × width)2/2. In Vitro Binding of Platelets to Tumor Cells—Platelets were prepared from WT and p38α+/- mice by the method previously described (9Sakurai K. Matsuo Y. Sudo T. Takuwa Y. Kimura S. Kasuya Y. J. Recept. Signal Transduct. Res. 2004; 24: 283-296Crossref PubMed Scopus (30) Google Scholar). Then platelets were fluorescently labeled with calcein by incubation with 1 μm calcein-acetoxymethyl ester (Molecular Probes) for 30 min at 37 °C and washed twice with Hepes-Tyrode's buffer (10 mm Hepes, 137 mm NaCl, 2.68 mm KCl, 0.42 mm NaH2PO4, 1.7 mm MgCl2, 11.9 mm NaHCO3, 5 mm glucose, pH 7.2). B16, LLC, and HEK293T cells were seeded on 8-well chamber slides and incubated with fluorescently labeled platelets at a density of 107/ml for 1 h at 37 °C. In the case of activating platelets, 1 unit/ml thrombin and 20 μg/ml collagen were added to each well just after the application of platelets. For elucidating the effect of heparin, 20 units/ml heparin (Wako Chemicals) was added to each well just after the application of platelets. After washing the chamber slides with Hepes-Tyrode's buffer twice, binding of platelets to tumor cells was examined by a fluorescence microscope (Axioplan; Zeiss). In Vivo Binding of Platelets to Tumor Cells—B16 cells labeled with calcein were resuspended and used for a systemic injection into WT and p38α+/- mice. After 30 min, the lungs were dissected out, fixed with 4% paraformaldehyde in 0.1 m sodium phosphate buffer (pH 7.4), and frozen in Tissue Tek OCT compound (Miles Inc.). Freshly cut lung sections (10 μm in thickness) were placed on poly-l-lysine-coated slides. The sections were stained with a monoclonal anti-mouse CD41 (integrin αIIb) antibody (Pharmingen) in combination with a secondary antibody (Alexa Fluor 594-labeled goat anti-rat IgG (Molecular Probes, Inc., Eugene, OR)) and observed under a fluorescence microscope (Axioplan; Zeiss). The resulting fluorescence profile was analyzed by an Intelligent Quantifier (Bio Image) to quantify the fluorescence intensity of platelets adherent to calcein-labeled tumor cells. Reverse Transcription-PCR Detection of P- and E-selectin mRNAs—B16 cells were injected into WT and p38α+/- mice. As a negative control, B16-free PBS was injected into WT and p38α+/- mice. The lungs were taken from these mice 4 h after injection. Total RNA was prepared from the lungs using ISOGEN (Wako Chemicals) according to the manufacturer's instructions. Single strand cDNA was synthesized from prepared RNA (3 μg), with Moloney murine leukemia virus reverse transcriptase (Invitrogen) using an oligo(dT) primer (Invitrogen) in a total volume of 20 μl. The resultant cDNA sample (1 μl) was subjected to PCR for amplification of mouse P- or E-selectin cDNA using specific primers (for P-selectin, sense primer, 5′-TGCAGCTTTTCCTGTGATGAAGGC-3′; antisense primer, 5′-ATAGAGCCAACACCAAACTCTCCG-3′; for E-selectin, sense primer, 5′-GACCTTTCCAAAAATGGGTCCAG-3′; antisense primer, 5′-AGAGCAATGAGGACGATGTCAGGAG-3′). As an internal control, mouse glyceraldehyde-3-phosphate dehydrogenase cDNA was amplified using specific primers (sense primer, 5′-GACCACAGTCCATGACATCACT-3′; antisense primer, 5′-TCCACCACCCTGTTGCTGTAG-3′). The settings of the thermal cycler were 30 cycles of 30 s at 94 °C, 30 s at 59 °C, and 1 min at 72 °C for mouse E- and P-selectin and 25 cycles of 40 s at 94 °C, 1 min at 60 °C, and 1 min at 72 °C for mouse glyceraldehyde-3-phosphate dehydrogenase. For detecting P-selectin mRNA in platelets, total RNA sample (0.25 μg) was subjected to reverse transcription reaction with Moloney murine leukemia virus in a total volume of 20 μl, and then the resultant sample (1 μl) was subjected to PCR with 35 cycles. The amplified products were separated in 1.2% agarose gel and visualized with ethidium bromide staining under UV radiation. Specific amplification of the expected size (mouse E-selectin, 570 bp; P-selectin, 371 bp; and mouse glyceraldehyde-3-phosphate dehydrogenase, 453 bp) was observed. Isolation of Lung Endothelial Cells—The lungs were dissected out from WT and p38α+/- mice and washed with ice-cold PBS three times. The tissues were cut into small pieces and incubated in Dulbecco's modified Eagle's medium supplemented with 2 mg/ml collagenase (Worthington), 2 mg/ml hyaluronidase (Sigma), and 1 mg/ml dispase (Invitrogen) at 37 °C for 30 min with shaking. Then the tissues were suspended well by a pipette, and the resultant suspension was passed through a 70-μm nylon mesh filter (Falcon). The cells were collected by centrifugation at 400 × g for 10 min. After washing twice with PBS, the cells suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 50 units/ml heparin were put on the top of Percoll solution (50% lower and 30% upper) layers and centrifuged at 800 × g for 30 min. The resultant interface fraction was collected and washed with PBS twice. The cells were suspended in 0.1% bovine serum albumin/PBS (Type V; Sigma) and subjected to MACS separation with rat monoclonal anti-PECAM-1 (BD Biosciences) and anti-rat IgG microbeads (Miltenyi Biotec.). The isolation of PECAM-1-positive endothelial cells was performed according to the manufacturer's protocol (Miltenyi Biotec.). The cells were seeded on a collagen-coated 6-well plate and maintained for 2 days in CS-C medium (Sigma) supplemented with 10% fetal bovine serum and EC growth supplement (Sigma). After confirming over 90% purity by the uptake of DiI-acetylated low density lipoprotein, the cells were used for experiments. Western Blot Analysis—The cell lysates of lung endothelial cells (7.5 μg) and platelets (15 μg) were subjected to Western blot analysis with anti-α-catenin, anti-PECAM-1, and anti-CD41 mouse monoclonal antibodies (BD Biosciences, Sigma, and BD Biosciences), anti-p38, anti-phospho-p38, and anti-phospho-ATF2 rabbit polyclonal antibodies (Cell Signaling), anti-E-selectin rat monoclonal antibody (R & D Systems), and anti-P-selectin goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Flow Cytometry—Liver endothelial cells were isolated from WT and p38α+/- mice by the method previously described (21Morin O. Patry P. Lafleur L. J. Cell. Physiol. 1984; 119: 327-334Crossref PubMed Scopus (29) Google Scholar) with modification. Cells were cultured for 2 days, and over 90% purity was confirmed by the uptake of DiI-acetylated low density lipoprotein and immunostaining with antibodies to VE-cadherin and vascular endothelial growth factor receptor-2. Then cells were stained for P-selectin, E-selectin, or PECAM-1 (CD31), followed by fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin (BD Biosciences). The labeled cells were analyzed on a FACScan flow cytometer (BD Biosciences), and rates of positive cells were quantified using CELLQest software 2.1.1 (BD Biosciences). Assay for Attachment and Transmigration of Tumor Cells— Primary cultured lung endothelial cells (105 cells) were seeded on 24-well cell culture inserts (apical chamber; Falcon) with pore size of 8 μm and maintained in CS-C medium overnight. After replacing the medium with Opti-MEM I (Invitrogen), DiI-labeled B16 cells (3 × 104 cells) were added to the apical chambers. At the same time, platelets (3 × 106) freshly isolated from WT and p38α+/- mice were added to the apical chambers for determination of the effect of platelets. As a negative control, DiI-labeled B16 cells with or without platelets were added to the endothelial cell-free apical chambers. All apical chambers were washed with Opti-MEM I twice for removing nonattaching cells 30 min after applying B16 cells to the apical chambers. Some apical chambers were fixed with 4% paraformaldehyde in 0.1 m sodium phosphate buffer (pH 7.4) for counting the number of B16 cells attached on endothelial cells by a fluorescence microscope (Axioplan; Zeiss). Some other apical chambers were further incubated to determine the transendothelial migration of B16 cells. After incubation for 8 h, nonmigrating cells were removed by scraping the apical side of the apical chambers with a cotton swab. Then the apical chambers were fixed and subjected to the examination of transmigrating B16 cells by a fluorescence microscope (Axioplan; Zeiss). Resistance of p38α+/- Mice to Experimental Lung Metastasis of Tumor Cells but Not to Tumor Growth—Two transplantable tumor cell lines, B16 and LLC, were used in the present study, because both are highly metastatic to the lung (22Zhu D. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (123) Google Scholar, 23Palumbo J.S. Kombrinck K.W. Drew A.F. Grimes T.S. Kiser J.H. Degen J.L. Bugge T.H. Blood. 2000; 96: 3302-3309Crossref PubMed Google Scholar). As shown in Fig. 1, A and B, there was a significant difference in surface pulmonary metastatic foci between WT and p38α+/- mice following intravenous injection of B16 cells (WT, 91.8 ± 21.0 cells/mouse; p38α+/-, 9.3 ± 3.2 cells/mouse). Also in the case of LLC cell injection, the formation of pulmonary metastatic foci was significantly decreased in p38α+/- mice compared with WT mice (WT, 31.2 ± 5.4 cells/mouse; p38α+/-, 11.2 ± 4.3 cells/mouse). To explore in greater detail whether the significant reduction in surface pulmonary metastases in p38α+/- mice resulted from tumor growth, we established B16 cells stably expressing β-galactosidase (B16-β-gal) in order to visualize a small focus of B16 cells. By using B16-β-gal, we investigated the formation of pulmonary metastatic foci at an earlier time after injection of tumor cells. Also in this case, a significant reduction in pulmonary metastasis was observed in p38α+/- mice (Fig. 1C). The size of metastatic foci dyed blue was comparable between WT and p38α+/- mice, indicating that tumor growth is not impaired in p38α+/- mice (data not shown). To further clarify this notion, we investigated the time-dependent increase in tumor volume after subcutaneous tumor cell transplantation. Both B16 and LLC cells when injected subcutaneously formed tumors with 100% penetrance, in which the steady growth of tumors showed no difference between WT and p38α+/- mice (Fig. 2). These results suggest that host p38α affects the metastatic potential of tumor cells but neither tumor cell growth nor tumor rejection.FIGURE 2Time-dependent tumor growth of subcutaneously transplanted tumor cells. A, tumor volume after subcutaneous injection of B16 cells in WT and p38α+/- littermates (open circles, WT; closed circles, p38α+/- littermates). B, tumor volume after subcutaneous injection of LLC cells in WT and p38α+/- littermates (open circles, WT; closed circles, p38α+/- littermates). Data are mean (n = 6). No significant difference in tumor growth of both B16 and LLC cells in WT and p38α+/- littermates was found by Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Angiogenesis is well known to be an important process in tumor cell growth (24Nicholson B. Theodorescu D. J. Cell. Biochem. 2004; 91: 125-150Crossref PubMed Scopus (104) Google Scholar, 25Tonini T. Rossi F. Claudio P.P. Oncogene. 2003; 22: 6549-6556Crossref PubMed Scopus (265) Google Scholar). The microenvironment of the local host tissue appears to be an active participant in exchanging cytokines and enzymes with tumor cells that modify the local extracellular matrix, stimulate migration, and promote tumor angiogenesis, proliferation, and survival. It has been demonstrated that p38 plays a role in angiogenesis via regulating the production of inflammatory mediators (26Jackson J.R. Bolognese B. Hillegass L. Kassis S. Adams J. Griswold D.E. Winkler J.D. J. Pharmacol. Exp. Ther. 1998; 284: 687-692PubMed Google Scholar). Likewise, targeted disruption of the p38α gene results in homozygous embryonic lethality because of defects in placental organogenesis, in which angiogenesis plays a crucial role (5Adams R.H. Porras A. Alonso G. Jones M. Vintersten K. Panelli S. Valladares A. Perez L. Klein R. Nebreda A.R. Mol. Cell. 2000; 6: 109-116Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Thus, the reduction of p38 intrinsic activity in the local host tissue by a single copy disruption of the p38α gene is thought to cause some impairment of angiogenesis. However, the comparable tumor growth between WT and p38α+/- mice suggests that angiogenesis during tumor growth may not show impairment in p38α+/- mice in the present experimental model. Reduction of Tumor Cell-Platelet Interaction in p38α+/-Mice—Tumor cells are frequently observed in the vasculature in complexes with platelets, and this association, together with the hypercoagulable state of malignant disease, appears to be essential for successful metastasis. The ability of tumor cells to induce platelet aggregation is widespread among cancers, including colon adenocarcinoma (27Kato Y. Fujita N. Yano H. Tsuruo T. Cancer Res. 1997; 57: 3040-3045PubMed Google Scholar), lung carcinoma (28Janowska-Wieczorek A. Wysoczynski M. Kijowski J. Marquez-Curtis L. Machalinski B. Ratajczak J. Ratajczak M.Z. Int. J. Cancer. 2005; 113: 752-760Crossref PubMed Scopus (601) Google Scholar), melanomas (29Im J.H. Fu W. Wang H. Bhatia S.K. Hammer D.A. Kowalska M.A. Muschel R.J. Cancer Res. 2004; 64: 8613-8619Crossref PubMed Scopus (224) Google Scholar), and others. The ability of tumor cells to induce platelet aggregation is of benefit for the survival of tumor cells for the following reasons: tumor cells coated with platelets acquire the ability to evade the body's immune system and blood flow's high shear forces; large tumor-platelet aggregate tends to embolize in the microvasculature; and tumor cells efficiently receive a number of growth factors from platelets (30Jurasz P. Alonso-Escolano D. Radomski M.W. Br. J. Pharmacol. 2004; 143: 819-826Crossref PubMed Scopus (266) Google Scholar). Thus, platelets possibly act to facilitate all of the intermediate steps of transvascular metastasis, including tumor cell retention and arrest, subendothelial interaction, and extravasation from the microvasculature. We recently demonstrated that the binding activity of activated platelets from p38α+/- mice to fibrinogen, possibly mediated through integrin αIIbβ3, is suppressed compared with the case of platelets from WT mice (9Sakurai K. Matsuo Y. Sudo T. Takuwa Y. Kimura S. Kasuya Y. J. Recept. Signal Transduct. Res. 2004; 24: 283-296Crossref PubMed Scopus (30) Google Scholar). This clearly indicates that the function of platelets is attenuated in p38α+/- mice compared with WT mice. Then we investigated whether p38α mediates the interaction of platelets with tumor cells in vitro and in vivo. We labeled platelets freshly isolated from WT and p38α+/- mice with calcein and applied them to B16 and LLC cells in vitro. We used HEK293T cells as a negative control and confirmed that resting and activated platelets from WT mice did not show the activity of rosetting on the cells. As shown in Fig. 3A, resting and activated platelets from WT mice rosetted on B16 and LLC cells, and this interaction was clearly reduced in the case of p38α+/- mice. Heparin markedly inhibited the interaction of platelets from WT and p38α+/- mice with tumor cells. Quantitative analysis showed that resting and activated platelets from p38α+/- mice had significantly decreased activity of rosetting on tumor cells compared with those from WT mice. The stimulation of platelets from WT mice with thrombin and collagen increased the number of platelets rosetted on tumor cells. This increase was moderate in the case of p38α+/- mice (WT, 1.8 times; p38α+/-, 1.5 times), indicating that agonist-induced activation is reduced in platelets from p38α+/- mice. The tumor cell-platelet interaction was significantly heparin-sensitive (Fig. 3B). To examine the tumor cell-platelet interaction in vivo, we injected calcein-labeled B16 cells, and those that lodged in the capillaries of the lung were further stained with anti-CD41, a defined platelet marker. As shown in Fig. 4A, platelets almost covered B16 cells in the lung of WT mice. In contrast, platelets weakly and partly covered B16 cells in the lung of p38α+/- mice. Quantitative analysis of the ratio of red fluorescence (CD41-like immunoreactivity) versus green fluorescence (B16) showed that the interaction of B16 cells with platelets in the lung was significantly decreased in p38α+/- mice compared with WT mice (Fig. 4B). These results suggest that suppression of the interaction of platelets with tumor cells can affect the metastatic potential of tumor cells in p38α+/- mice.FIGURE 4In vivo interaction of platelets with tumor cells. A, typical examples of platelet clumps attached on tumor cells in the lung. WT (A-C) and p38α+/- littermates (D-F) were injected with calcein-labeled B16 cells. Lung sections were stained with monoclonal anti-mouse CD41 antibody in combination with Alexa Fluor 594-labeled secondary antibody, and B16 (A and D) and attached platelets (B and E) were determined as green fluorescence and red fluorescence, respectively, under a fluorescence microscope. Merged profiles (C and F) show the decrease of platelets attached on tumor cells in p38α+/- mice. Bar, 20 μm. B, quantitative analysis of platelet/tumor cell adhesion in the lung of WT and p38α+/- littermates. The interaction of platelets with B16 in the lung was expressed as the ratio of fluorescent intensity (red/green). Data are mean ± S.D. (n = 5). *, significantly different from WT mice (Student's t test).View Large Image Figure ViewerDownload Hi-res image" @default.
• W2017209306 created "2016-06-24" @default.
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• W2017209306 date "2006-12-01" @default.
• W2017209306 modified "2023-10-14" @default.
• W2017209306 title "Involvement of p38α Mitogen-activated Protein Kinase in Lung Metastasis of Tumor Cells" @default.
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