Senescent Peritoneal Mesothelial Cells Promote Ovarian Cancer Cell Adhesion
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080613
ISSN1525-2191
AutoresKrzysztof Książek, Justyna Mikuła‐Pietrasik, Katarzyna Korybalska, Grzegorz Dworacki, Achim Jörres, Janusz Witowski,
Tópico(s)Cancer Research and Treatments
ResumoAdhesion of ovarian cancer cells to the peritoneal mesothelium is a key step in the malignant progression of the disease. In an in vitro study, we showed that the adherence of ovarian cancer cells (of the OVCAR-3, SKOV-3, and A2780 cell lines) to senescent human omentum-derived peritoneal mesothelial cells (HOMCs) was greater than to early passage cells. The process was mediated primarily by the increased interaction of the α5β1 integrin on cancer cells with HOMC-associated fibronectin (FN). In comparison with early passage HOMCs, senescent cells exhibited increased FN mRNA expression levels and produced significantly more FN. To assess the effect of senescence-associated oxidative stress on FN release, HOMCs were rendered senescent by exposure to an oxidant, tert-butyl hydroperoxide. Treatment with tert-butyl hydroperoxide resulted in a significant increase in HOMC FN mRNA and protein expression levels. The effect of oxidative stress on FN synthesis was found to be mediated by transforming growth factor-β1, whose signaling pathway was controlled at upstream and downstream levels by p38 MAPK. The activity of p38 MAPK increased markedly in senescent HOMCs. Treatment of HOMCs with antioxidants significantly attenuated senescence-associated increases in p38 MAPK activity, production of both transforming growth factor-β1 and FN, and ovarian cancer cell adhesion. These data indicate that oxidative stress that accompanies senescence may increase FN production by HOMCs and thus facilitate binding and dissemination of ovarian cancer cells. Adhesion of ovarian cancer cells to the peritoneal mesothelium is a key step in the malignant progression of the disease. In an in vitro study, we showed that the adherence of ovarian cancer cells (of the OVCAR-3, SKOV-3, and A2780 cell lines) to senescent human omentum-derived peritoneal mesothelial cells (HOMCs) was greater than to early passage cells. The process was mediated primarily by the increased interaction of the α5β1 integrin on cancer cells with HOMC-associated fibronectin (FN). In comparison with early passage HOMCs, senescent cells exhibited increased FN mRNA expression levels and produced significantly more FN. To assess the effect of senescence-associated oxidative stress on FN release, HOMCs were rendered senescent by exposure to an oxidant, tert-butyl hydroperoxide. Treatment with tert-butyl hydroperoxide resulted in a significant increase in HOMC FN mRNA and protein expression levels. The effect of oxidative stress on FN synthesis was found to be mediated by transforming growth factor-β1, whose signaling pathway was controlled at upstream and downstream levels by p38 MAPK. The activity of p38 MAPK increased markedly in senescent HOMCs. Treatment of HOMCs with antioxidants significantly attenuated senescence-associated increases in p38 MAPK activity, production of both transforming growth factor-β1 and FN, and ovarian cancer cell adhesion. These data indicate that oxidative stress that accompanies senescence may increase FN production by HOMCs and thus facilitate binding and dissemination of ovarian cancer cells. Ovarian cancer is still associated with high mortality. The poor outcome is related to a large extent to metastatic spreading of cancer cells within the peritoneal cavity.1Amadori D Sansoni E Amadori A Ovarian cancer: natural history and metastatic pattern.Front Biosci. 1997; 2: g8-g10PubMed Google Scholar Attachment of malignant cells to the peritoneal mesothelium is thought to be a critical step in peritoneal dissemination of the disease. Available data indicate that the process is mediated by interactions between extracellular matrix components produced by mesothelial cells and the corresponding adhesion molecules on ovarian cancer cells. A key role is thought to be played by CD44 and β1 integrin receptors and their ligands—hyaluronan, fibronectin (FN), and vitronectin.2Ahmed N Riley C Rice G Quinn M Role of integrin receptors for fibronectin, collagen and laminin in the regulation of ovarian carcinoma functions in response to a matrix microenvironment.Clin Exp Metastasis. 2005; 22: 391-402Crossref PubMed Scopus (120) Google Scholar, 3Gardner MJ Catterall JB Jones LM Turner GA Human ovarian tumour cells can bind hyaluronic acid via membrane CD44: a possible step in peritoneal metastasis.Clin Exp Metastasis. 1996; 14: 325-334Crossref PubMed Scopus (88) Google Scholar, 4Lessan K Aguiar DJ Oegema T Siebenson L Skubitz AP CD44 and beta1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells.Am J Pathol. 1999; 154: 1525-1537Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 5Strobel T Cannistra SA Beta1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.Gynecol Oncol. 1999; 73: 362-367Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 6Kenny HA Kaur S Coussens LM Lengyel E The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin.J Clin Invest. 2008; 118: 1367-1379Crossref PubMed Scopus (291) Google Scholar FN is a ubiquitous constituent of extracellular matrix. Secreted by cells as a soluble dimer, it is then processed and assembled into insoluble fibrils at the cell surface.7Wierzbicka-Patynowski I Schwarzbauer JE The ins and outs of fibronectin matrix assembly.J Cell Sci. 2003; 116: 3269-3276Crossref PubMed Scopus (393) Google Scholar FN is involved in many cellular functions including the maintenance of cell shape, tissue repair, cell differentiation, adhesion, and migration. These processes are primarily mediated by binding of the Arg-Gly-Asp (RGD) sequence of the FN molecule to integrin receptors (especially to α5β1 and αVβ3).8Pankov R Yamada KM Fibronectin at a glance.J Cell Sci. 2002; 115: 3861-3863Crossref PubMed Scopus (1507) Google Scholar It has been demonstrated that FN released by peritoneal mesothelial cells stimulates ovarian cancer cell motility in vitro.9Rieppi M Vergani V Gatto C Zanetta G Allavena P Taraboletti G Giavazzi R Mesothelial cells induce the motility of human ovarian carcinoma cells.Int J Cancer. 1999; 80: 303-307Crossref PubMed Scopus (45) Google Scholar Moreover, competitive inhibition of FN by RGD-containing peptides has been reported to decrease peritoneal spreading of ovarian cancer cells in mice.10Yamamoto K Murae M Yasuda M RGD-containing peptides inhibit experimental peritoneal seeding of human ovarian cancer cells.Nippon Sanka Fujinka Gakkai Zasshi. 1991; 43: 1687-1692PubMed Google Scholar More recently it has been demonstrated that the attachment of ovarian cancer cells to the peritoneum results in up-regulation of their matrix metalloproteinase-2, which cleaves mesothelial cell-derived FN into small fragments that further augment cancer cell binding.6Kenny HA Kaur S Coussens LM Lengyel E The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin.J Clin Invest. 2008; 118: 1367-1379Crossref PubMed Scopus (291) Google Scholar Interestingly, increased production of FN appears to be one of the most typical features of senescent cells.11Fodil-Bourahla I Drubaix I Robert L Labat-Robert J Effect of in vitro aging on the modulation of protein and fibronectin biosynthesis by the elastin-laminin receptor in human skin fibroblasts.Gerontology. 1999; 45: 23-30Crossref PubMed Scopus (15) Google Scholar, 12Grillari J Hohenwarter O Grabherr RM Katinger H Subtractive hybridization of mRNA from early passage and senescent endothelial cells.Exp Gerontol. 2000; 35: 187-197Crossref PubMed Scopus (83) Google Scholar Increased levels of FN have also been detected in plasma and cells from aged individuals.13Kumazaki T Kobayashi M Mitsui Y Enhanced expression of fibronectin during in vivo cellular aging of human vascular endothelial cells and skin fibroblasts.Exp Cell Res. 1993; 205: 396-402Crossref PubMed Scopus (65) Google Scholar, 14Labat-Robert J Fibronectin in malignancy.Semin Cancer Biol. 2002; 12: 187-195Crossref PubMed Scopus (99) Google Scholar Although a link between cancer progression and the age-dependent FN production and processing has been postulated to occur,14Labat-Robert J Fibronectin in malignancy.Semin Cancer Biol. 2002; 12: 187-195Crossref PubMed Scopus (99) Google Scholar the exact nature of such a relationship remains unclear. Cellular senescence occurs in response to irreparable DNA damage with potentially oncogenic potential and such DNA injuries are caused primarily by oxidative stress.15Kregel KC Zhang HJ An integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations.Am J Physiol. 2007; 292: R18-R36Crossref PubMed Scopus (715) Google Scholar, 16Passos JF von Zglinicki T Oxygen free radicals in cell senescence: are they signal transducers?.Free Radic Res. 2006; 40: 1277-1283Crossref PubMed Scopus (100) Google Scholar By inhibiting cell proliferation, cellular senescence may thus act as a powerful tumor-suppressing mechanism.17Campisi J d'Adda di Fagagna F Cellular senescence: when bad things happen to good cells.Nat Rev Mol Cell Biol. 2007; 8: 729-740Crossref PubMed Scopus (3098) Google Scholar It is believed to protect effectively young organisms against neoplasia, however, it may produce some unfavorable effects later in life. Paradoxically, they may include accelerated cancer progression. It appears that the accumulation of senescent cells with age may alter the tissue architecture to the extent that it provides a permissive environment for uncontrolled growth of adjacent premalignant cells.18Krtolica A Campisi J Cancer and aging: a model for the cancer promoting effects of the aging stroma.Int J Biochem Cell Biol. 2002; 34: 1401-1414Crossref PubMed Scopus (256) Google Scholar Indeed, it has been demonstrated that proliferation of premalignant breast epithelial cells in vitro and tumor growth in vivo increased in the presence of senescent rather than early-passage fibroblasts.19Krtolica A Parrinello S Lockett S Desprez PY Campisi J Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.Proc Natl Acad Sci USA. 2001; 98: 12072-12077Crossref PubMed Scopus (1227) Google Scholar, 20Liu D Hornsby PJ Senescent human fibroblasts increase the early growth of xenograft tumors via matrix metalloproteinase secretion.Cancer Res. 2007; 67: 3117-3126Crossref PubMed Scopus (335) Google Scholar Moreover, fibroblasts adjacent to the malignant tissue in ovarian cancer specimens, in contrast to those neighboring the normal epithelium, have been found to be senescent.21Yang G Rosen DG Zhang Z Bast Jr, RC Mills GB Colacino JA Mercado-Uribe I Liu J The chemokine growth-regulated oncogene 1 (Gro-1) links RAS signaling to the senescence of stromal fibroblasts and ovarian tumorigenesis.Proc Natl Acad Sci USA. 2006; 103: 16472-16477Crossref PubMed Scopus (265) Google Scholar It has also been demonstrated that senescent fibroblasts promoted tumor-associated angiogenesis,22Coppé JP Kauser K Campisi J Beausejour CM Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence.J Biol Chem. 2006; 281: 29568-29574Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar which could facilitate malignant progression. We have recently analyzed mechanisms of senescence in human peritoneal mesothelial cells in vitro23Ksiazek K Piwocka K Brzezinska A Sikora E Zabel M Breborowicz A Jörres A Witowski J Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence.J Appl Physiol. 2006; 100: 988-995Crossref PubMed Scopus (61) Google Scholar, 24Ksiazek K Passos JF Olijslagers S Saretzki G Martin-Ruiz C von Zglinicki T Premature senescence of mesothelial cells is associated with non-telomeric DNA damage.Biochem Biophys Res Commun. 2007; 362: 707-711Crossref PubMed Scopus (44) Google Scholar and detected senescent mesothelial cells in freshly explanted specimens of omentum.25Ksiazek K Mikula-Pietrasik J Jörres A Witowski J Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells.Free Radic Biol Med. 2008; 45: 460-467Crossref PubMed Scopus (32) Google Scholar Because the omentum is the commonest location of ovarian cancer metastases,26Doig T Monaghan H Sampling the omentum in ovarian neoplasia: when one block is enough.Int J Gynecol Cancer. 2006; 16: 36-40Crossref PubMed Scopus (32) Google Scholar in the present study we have set out to examine whether and how senescence of human omentum-derived mesothelial cells (HOMCs) modulates the adhesion of ovarian cancer. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Corp. (St. Louis, MO). All tissue culture plastics were from Nunc (Roskilde, Denmark). Monoclonal antibodies against α5β1 integrin (clones HA5 and JBS5) and against αVβ3 integrin (clone LM609), as well as isotype control antibodies and fluorescein-conjugated anti-mouse IgG were obtained from Chemicon/Millipore (Bedford, MA). Monoclonal mouse anti-β1 integrin (clone P5D2), affinity-purified polyclonal anti-transforming growth factor (TGF)-β (AF-101-NA), appropriate control antibodies, and horseradish peroxidase-labeled polyclonal goat anti-rabbit IgG were purchased from R&D Systems (Wiesbaden, Germany). Polyclonal rabbit anti-FN antibody was obtained from DAKO (Hamburg, Germany). GRGDSP, a FN binding blocking peptide and GRADSP, an inactive control peptide, as well as SB202190 were obtained from Calbiochem/Merck (Warsaw, Poland). HOMCs were isolated from specimens of omentum obtained from consenting patients undergoing elective abdominal surgery. Cells were isolated, propagated, and characterized as described in detail elsewhere.27Yung S Li FK Chan TM Peritoneal mesothelial cell culture and biology.Perit Dial Int. 2006; 26: 162-173Crossref PubMed Scopus (103) Google Scholar The donors had no evidence of peritoneal inflammation and/or malignancy. The study was approved by the institutional ethics committee. Cells were cultured in medium M199 supplemented with l-glutamine (2 mmol/L), penicillin (100 U/ml), streptomycin (100 μg/ml), hydrocortisone (0.4 μg/ml), and 10% (v/v) fetal bovine serum. The ovarian cancer cell line OVCAR-3 was purchased from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium supplemented with l-glutamine (2 mmol/L), HEPES (10 mmol/L), sodium pyruvate (1 mmol/L), glucose (4500 mg/L), and 10% fetal bovine serum. The ovarian cancer cell lines SKOV-3 and A2780 were obtained from the European Collection of Cell Cultures (Porton Down, UK) and propagated in McCoy's 5a and RPMI 1640 media, respectively, both supplemented with l-glutamine (2 mmol/L), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum. HOMCs were grown to senescence by serial passages performed at 4- to 5-day intervals with a fixed seeding density of 3 × 104 cells/cm2. Cultures were considered senescent when cells failed to increase in number during 4 weeks, showed enlarged morphology, and when >70% of cells stained positively for senescence-associated β-galactosidase (SA-β-Gal).28Parrinello S Coppe JP Krtolica A Campisi J Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation.J Cell Sci. 2005; 118: 485-496Crossref PubMed Scopus (461) Google Scholar SA-β-Gal was detected essentially as described by Dimri and colleagues.29Dimri GP Lee X Basile G Acosta M Scott G Roskelley C Medrano EE Linskens M Rubelj I Pereira-Smith O Peacocke M Campisi J A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc Natl Acad Sci USA. 1995; 92: 9363-9367Crossref PubMed Scopus (5834) Google Scholar To prepare co-cultures of early-passage and senescent cells, an aliquot of early-passage cells was frozen in liquid nitrogen and stored until the remaining cells became senescent.25Ksiazek K Mikula-Pietrasik J Jörres A Witowski J Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells.Free Radic Biol Med. 2008; 45: 460-467Crossref PubMed Scopus (32) Google Scholar Then, the early-passage cells were defrosted and mixed with senescent cells to produce co-cultures containing required fraction of senescent cells. In selected experiments HOMCs were cultured to senescence in the presence of reactive oxygen species (ROS) scavenger α-phenyl-N-tert-butyl-nitrone (PBN) at a dose of 800 μmol/L.30von Zglinicki T Pilger R Sitte N Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts.Free Radic Biol Med. 2000; 28: 64-74Crossref PubMed Scopus (442) Google Scholar In some tests HOMC senescence was induced by oxidative stress using tert-butyl hydroperoxide (t-BHP).31Dumont P Burton M Chen QM Gonos ES Frippiat C Mazarati JB Eliaers F Remacle J Toussaint O Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.Free Radic Biol Med. 2000; 28: 361-373Crossref PubMed Scopus (311) Google Scholar Briefly, early-passage cells at 70% confluence were treated with 30 μmol/L t-BHP for 1 hour daily for 7 consecutive days. Between the exposures cells were maintained in standard medium supplemented with or without PBN (800 μmol/L). After 7 days of such treatment cells were allowed to recover in standard growth medium for 5 days, during which they developed the senescence phenotype, as evidenced by hypertrophic morphology and extensive positive staining for SA-β-Gal. When required, the incubation was performed in the presence of specific inhibitors or antibodies, as detailed in legends to figures. Adhesion of ovarian cancer cells to HOMCs was determined as described by Alkhamesi and colleagues32Alkhamesi NA Ziprin P Pfistermuller K Peck DH Darzi AW ICAM-1 mediated peritoneal carcinomatosis, a target for therapeutic intervention.Clin Exp Metastasis. 2005; 22: 449-459Crossref PubMed Scopus (58) Google Scholar with minor modifications. Briefly, HOMCs were plated in flat-bottom 96-well plates (8 × 104 cells/well) and left to settle overnight. Ovarian cancer cells were detached by trypsinization, washed with phosphate-buffered saline (PBS), and probed with 5 μmol/L calcein-AM (Molecular Probes, Invitrogen, Eugene, OR) for 30 minutes at 37°C. Calcein-labeled cells were washed with M199 medium with 0.1% fetal bovine serum to remove the free dye and added (3 × 104 cells/well) on top of mesothelial cells. After 45 minutes of incubation at 37°C, total fluorescence in each well was recorded in a spectrofluorimeter (Perkin-Elmer, Turku, Finland) using 485-nm and 535-nm wavelengths for excitation and emission, respectively. Then, the nonadherent cells were removed by gentle washing and the measurement of fluorescence was repeated. To calculate the percentage of bound cells, the values recorded were compared with those representing total fluorescence. In the inhibition studies, HOMCs and/or ovarian cancer cells were treated with appropriate antibodies or inhibitors for 30 minutes before and during the adhesion assay. To confirm the role of FN in ovarian cancer cell adhesion, a 96-well plate was coated overnight with increasing doses of human plasma FN in PBS. After the blockade of nonspecific binding with 1% bovine serum albumin for 2 hours, the adhesion assay was performed as described above. Cell-bound FN was measured with an immunoassay. Briefly, HOMCs in 96-well plates were incubated with 10% Roti-Block (Carl Roth, Karlsruhe, Germany) in PBS for 2 hours to block unspecific binding sites, then washed three times with PBS and incubated with anti-FN antibody (2.5 μg/ml) overnight. After extensive washing, bound antibodies were detected with secondary horseradish peroxidase-labeled anti-IgG antibody (1:2000 for 2 hours) and subsequent exposure to horseradish peroxidase substrate reagent (BD Pharmingen, Franklin Lakes, NJ). The color reaction was stopped with 2 N H2SO4 and OD was read at 450 nm. The results were normalized per 105Strobel T Cannistra SA Beta1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.Gynecol Oncol. 1999; 73: 362-367Abstract Full Text PDF PubMed Scopus (149) Google Scholar cells. Expression of α5β1 integrin on ovarian cancer cells was assessed by flow cytometry, as described.33Said N Najwer I Motamed K Secreted protein acidic and rich in cysteine (SPARC) inhibits integrin-mediated adhesion and growth factor-dependent survival signaling in ovarian cancer.Am J Pathol. 2007; 170: 1054-1063Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Briefly, harvested cells were washed twice in cold PBS, divided into 105Strobel T Cannistra SA Beta1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.Gynecol Oncol. 1999; 73: 362-367Abstract Full Text PDF PubMed Scopus (149) Google Scholar cell aliquots, and incubated for 45 minutes on ice with either the anti-human integrin α5β1 monoclonal antibody or the isotype control antibody. After washing with cold PBS, cells were incubated for 45 minutes on ice with the anti-mouse fluorescein-conjugated secondary antibody. Cells were then washed and analyzed with the FACSCanto flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The data obtained were analyzed and graphically presented using PC-Lysis software (Becton Dickinson). Total RNA was extracted from HOMCs with the TRIzol reagent (Invitrogen, Carlsbad, CA), purified, and reverse-transcribed into cDNA with random hexamer primers, as described.34Jörres A Dinter H Topley N Gahl GM Frei U Scholz P Inhibition of tumour necrosis factor production in endotoxin-stimulated human mononuclear leukocytes by the prostacyclin analogue iloprost: cellular mechanisms.Cytokine. 1997; 9: 119-125Crossref PubMed Scopus (49) Google Scholar PCR was performed with Platinum TaqDNA polymerase (Invitrogen) using oligonucleotide primers synthesized by TIB MolBiol SyntheseLabor (Berlin, Germany). Because alternative RNA splicing may produce several transcript variants of the FN gene, the assessment of FN mRNA expression was performed with a primer pair detecting the most typical variants 1 to 5. Detailed evaluation of FN transcript variants was performed using primer specific for each variant, as shown in Table 1.35O'Bryan JP Frye RA Cogswell PC Neubauer A Kitch B Prokop C Espinosa III, R Le Beau MM Earp HS Liu ET axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.Mol Cell Biol. 1991; 11: 5016-5031Crossref PubMed Scopus (644) Google Scholar, 36Phillips AO Steadman R Morrisey K Martin J Eynstone L Williams JD Exposure of human renal proximal tubular cells to glucose leads to accumulation of type IV collagen and fibronectin by decreased degradation.Kidney Int. 1997; 52: 973-984Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 37Parkar MH Bakalios P Newman HN Olsen I Expression and splicing of the fibronectin gene in healthy and diseased periodontal tissue.Eur J Oral Sci. 1997; 105: 264-270Crossref PubMed Scopus (6) Google Scholar, 38Phillips AO Steadman R Topley N Williams JD Elevated D-glucose concentrations modulate TGF-beta 1 synthesis by human cultured renal proximal tubular cells. The permissive role of platelet-derived growth factor.Am J Pathol. 1995; 147: 362-374PubMed Google Scholar, 39Cheuk BL Cheng SW Differential expression of integrin alpha5beta1 in human abdominal aortic aneurysm and healthy aortic tissues and its significance in pathogenesis.J Surg Res. 2004; 118: 176-182Abstract Full Text Full Text PDF PubMed Scopus (23) Google ScholarTable 1PCR Primer SequencesSequenceProduct size (bp)Referenceα-Actin20435 F: 5′-GGAGCAATGATCTTGATCTT-3′ R: 5′-TGCTCACAGGCAAGGTGTAG-3′FN43936 F: 5′-AGCCGCCACGTGCCAGGATTAC-3′ R: 5′-CTTATGGGGGTGGCCGTTGTGG-3′FN ED-A151, 42037 F: 5′-GACTATTGAAGGCTTGCAGCC-3′ R: 5′-CTTGTGGGTGTGCCTGAGTG-3′FN ED-B185, 45837 F: 5′-GGTCCATGCTGAATCAGAGCTC-3′ R: 5′-CAGGTGACACGCATGGTGTCTG-3′FN IIICS124, 316, 391, 409, 48437 F: 5′-GGCTACTATTACTGGCCTGG-3′ R: 5′-CTGAGAGAGAGCTTCTTGTCC-3′TGF-β151038 F: 5′-GGCAGTGGTTGAGCCGTGGA-3′ R: 5′-TGTTGGACAGCTGCTCCACCT-3′Integrin α531339 F: 5′-ACCTGCAGCTGGAAGTGTTT-3′ R: 5′-TTAGTGTCCCGGAGATGAGG-3′Integrin-β152639 F: 5′-AGACCTGCCTTGGTGTGTCT-3′ R: 5′-CAGTGTTGTGGGATTTGCAC-3′F, forward; R, reverse; bp, base pairs Open table in a new tab F, forward; R, reverse; bp, base pairs TGF-β1 concentration in cell culture supernatants was measured with an immunoassay using the DuoSet ELISA development system (R&D Systems). The samples to be measured were activated with 1 N HCI for 15 minutes and subsequently neutralized with 1.2 N NaOH/0.5 mol/L HEPES. The assay was performed as per the manufacturer's instructions. Its sensitivity was 15 pg/ml. Activation by phosphorylation of p38 MAPK was quantified by in-cell Western technique using Fast Activated Cell-Based ELISA (FACE) p38 kit (Active Motif, Rixensart, Belgium) according to the manufacturer's instructions. The results were expressed as a ratio of phosphorylated to total p38 activity. Statistical analysis was performed using GraphPad Prism 4.00 software (GraphPad Software, San Diego, CA). The data were analyzed with the t-test or repeated measures analysis of variance, as appropriate. Results were expressed as means ± SD. Differences with a P value <0.05 were considered significant. The number of experiments indicated (n) always refers to the number of independent experiments performed with HOMCs from different donors. Adhesion of OVCAR-3 cells to freshly isolated HOMCs increased in a time-dependent manner (Figure 1A). The plateau was reached after 45 minutes. Therefore, this time point was chosen for all subsequent experiments. Because the attachment of ovarian cancer cells to the mesothelium is thought to be partly mediated by β1 integrins, and these are expressed on both OVCAR-3 cells2Ahmed N Riley C Rice G Quinn M Role of integrin receptors for fibronectin, collagen and laminin in the regulation of ovarian carcinoma functions in response to a matrix microenvironment.Clin Exp Metastasis. 2005; 22: 391-402Crossref PubMed Scopus (120) Google Scholar, 40Cannistra SA DeFranzo B Niloff J Ottensmeir C Functional heterogeneity of CD44 molecules in ovarian cancer cell lines.Clin Cancer Res. 1995; 1: 333-342PubMed Google Scholar and HOMCs,41Gardner MJ Jones LM Catterall JB Turner GA Expression of cell adhesion molecules on ovarian tumour cell lines and mesothelial cells, in relation to ovarian cancer metastasis.Cancer Lett. 1995; 91: 229-234Crossref PubMed Scopus (105) Google Scholar we began by performing the adhesion assay with cells treated separately with blocking antibodies. The pretreatment of OVCAR-3 cells with an anti-β1 integrin antibody, but not with control IgG, led to a significant dose-dependent inhibition of OVCAR-3 cell adhesion to early-passage HOMCs. The maximal inhibition observed was 55.4 ± 6.4% (n = 3, P < 0.05). In contrast, when the same antibody was applied to HOMCs, the inhibition of OVCAR-3 cell adhesion was of considerably lower magnitude and reached only 11.5 ± 10.6% (n = 3, P < 0.05). Simultaneous blockade of β1 integrins on OVCAR-3 cells and HOMCs did not produce any greater inhibition compared with neutralization of OVCAR-3 cell β1 integrins only. The above data suggested that OVCAR-3 cell binding was mediated primarily through their β1 integrins. Because β1 integrin is one of the subunits of the main FN receptor, integrin α5β1, to characterize the involvement of the receptor more precisely, the adhesion assay was performed with OVCAR-3 cells treated with a specific integrin α5β1-neutralizing antibody (Figure 1B). These experiments showed that the α5β1 integrin blockade on OVCAR-3 cells reduced their binding to HOMCs by up to 46.4 ± 2.9%. Because FN may also bind to an additional receptor (integrin αVβ3), the adhesion of OVCAR-3 cells was assessed after its neutralization (Figure 1C). This led to a 18.5 ± 2.3% inhibition of OVCAR-3 adherence. To determine the role of HOMC-derived FN as a ligand for FN receptors on OVCAR-3 cell adhesion, HOMCs were incubated with GRGDSP, a peptide with attachment sequence of FN that acts as a competitive inhibitor of integrin binding.33Said N Najwer I Motamed K Secreted protein acidic and rich in cysteine (SPARC) inhibits integrin-mediated adhesion and growth factor-dependent survival signaling in ovarian cancer.Am J Pathol. 2007; 170: 1054-1063Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar The presence of GRGDSP, but not of a control peptide GRADSP, resulted in a significant inhibition of OVCAR-3 cell adhesion to HOMCs (Figure 1D). The most effective inhibition (by 26 ± 11%) was achieved with GRGDSP at 10 μmol/L. The assessment of adhesion of OVCAR-3 cells to FN-coated plates showed a significant dose-dependent effect of FN (Figure 1E). The adhesion-promoting effect of FN became significant at a dose of 0.5 μg/ml and at the highest dose tested (10 μg/ml) the OVCAR-3 adhesion was increased by 81 ± 15%. The attachment of OVCAR-3 cells to senescent HOMCs was greater by up to 50% than to their young counterparts (Figure 2, A and B). To confirm the effect of senescent HOMCs, the adhesion assay was performed with HOMC cultures prepared by mixing young and senescent cells at different ratios.25Ksiazek K Mikula-Pietrasik J Jörres A Witowski J Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells.Free Radic Biol Med. 2008; 45: 460-467Crossref PubMed Scopus (32) Google Scholar The adhesion of OVCAR-3 cells was found to increase in proportion to the fraction of senescent cells (Figure 2C). The ef
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