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Cell Biology International (2005) 29, 497505 (Printed in Great Britain)
Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer
Roman Paducha*1, Adam Walter‑Croneckb, Barbara Zdzisińskaa, Agnieszka Szuster‑Ciesielskaa and Martyna Kandefer‑Szerszeńa
aDepartment of Virology and Immunology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, ul. Akademicka 19, 20-033 Lublin, Poland
bDepartment of Haematooncology and Bone Marrow Transplantation, Medical University, Jaczewskiego 8, 20-950 Lublin, Poland
Metastasis is a multistep process involving a variety of direct cell–cell, cell–matrix and paracrine interactions. In the present study, we examined some consequences of direct interaction between tumour cells and endothelial cells in vitro. When multicellular spheroids of two human tumour cell lines (HeLa and Hep-2) were transferred onto a human umbilical vein endothelial cell (HUVEC) monolayer, a peri-spheroidal zone of damaged endothelial cells was observed after 24
Keywords: Tumour spheroids, Endothelium, Metastasis, Reactive oxygen species, Metalloproteinase-2, Interleukin-6.
1The Foundation for Polish Science scholarship holder.
*Corresponding author. Tel.: +48 81 537 59 42; fax: +48 81 537 59 59.
Tumour metastasis involves migration in the blood or lymph stream, attachment and migration through the vascular wall, and proliferation in sites distant from the primary tumour. This process depends not only on cell adhesion molecules, which are necessary for direct cell-to-cell interaction, but is also mediated by reactive oxygen species (ROS), angiogenic growth factors and their receptors and proteolytic enzymes (Liekens et al., 2001).
ROS are involved in the pathogenesis of several blood vessel diseases, e.g., sepsis and arteriosclerosis (Lorenz et al., 1998); they cause oxidation of membrane lipids, DNA breakdown and decomposition of proteins in endothelial cells (Lorenz et al., 1998; Yen et al., 2001). Tumour cells may produce ROS that injure endothelial cells and promote metastasis (Offner et al., 1996).
The continued growth and dissemination of solid tumours require proteolysis of the ECM as well as endothelial cell injury. Members of the matrix metalloproteinase (MMP) family are believed to play an important part in this process. MMPs are a group of secreted or transmembrane enzymes that can digest basement membrane and ECM components (Chambers and Matrisian, 1997). They are secreted in a latent form, so they require to be activated (Foda and Zucker, 2001). The MMP family comprises at least 20 enzymes, which can be divided into four subclasses: collagenases, stromelysins, gelatinases and the matrix type-MMPs (MT-MMPs) (Jackson and Nguyen, 1997). MMP-9 (gelatinase B/type IV collagenase) and MMP-2 (gelatinase A/type IV collagenase) are postulated to play crucial roles in tumour invasion (Fridman et al., 1995). The 72
IL-6 is a central proinflammatory cytokine, an important inducer of acute phase proteins and a regulator of the immune response, and it is implicated in cancer development (Lin-Hung et al., 2001). It is multifunctional and is produced by endothelial cells as well as macrophages and T cells. Endothelial-derived IL-6 inhibits tumour cell growth in premalignant and early stages of cancer. However, in intermediate and advanced stages, substantial microenvironmental levels of this cytokine stimulate primary tumour angiogenesis and metastasis (Rak et al., 1996).
A multicellular tumour spheroid (MCTS) is a three-dimensional tumour cell cluster. Its well-defined geometry, or the definite number of cells forming its mass, make the MCTS useful for analysing the mechanisms involved in tumour–endothelium interactions (Hamilton, 1998). It is well known that the adhesion of tumour cells to the luminal surface of the blood vessel wall can modulate the secretive capacity of endothelial cells and, by inducing apoptosis of these cells, alter endothelial integrity (Kebers et al., 1998). Clinical observations as well as in vitro studies indicate that injury to the vascular endothelium can facilitate and promote tumour cell metastasis (Offner et al., 1992). However, the mechanisms involved in endothelial injury are poorly understood.
The present study was conducted to investigate whether direct contact between human tumour cell spheroids and monolayers of endothelial cells induce changes in MMP-2, ROS and IL-6 production in comparison to tumour and endothelial cells cultivated separately.
2 Materials and methods
2.1 Tumour cell lines
Cells of the human cervical carcinoma line (HeLa: ECACC No 85060701) and the human laryngeal carcinoma line (Hep-2: ECACC No 86030501) were used in this study. They were grown as monolayers in 25-cm3 culture flasks (Nunc., Roskilde, Denmark) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Gibco™, Paisley, UK) and antibiotics (100
2.2 Tumour cell spheroid preparation
Tumour cell spheroids were prepared by the liquid overlay method (Offner et al., 1993). An aliquot of tumour cell suspension (200
2.3 Endothelial cells
HUVEC were isolated from human umbilical cords obtained from healthy donors up to 4
2.4 Skin fibroblasts
HSF cells were isolated from freshly excised tissue fragments from healthy donors. The explants were washed twice with RPMI 1640 medium containing 200
2.5 Tumour cell spheroid – HUVEC co-cultures
Tumour spheroids were harvested with glass pipettes from agarose-coated microplates and transferred into a Petri dish filled with warm RPMI 1640 medium. After washing for 5
2.6 Staining of spheroid-HUVEC co-culture
A stock solution containing 0.1
2.7 Confocal microscopy
Co-cultures of tumour spheroids with HUVEC were stained with 3,6-bis(dimethylamino)acridinium chloride (1
2.8 Measurement of superoxide anion production by cytochrome c reduction assay
A HUVEC suspension at 5
MMP activity was measured using SDS-PAGE zymography. Gelatin (300
2.10 MMP-2 and IL-6 assay
The levels of MMP-2 and IL-6 were tested immunoenzymatically (ELISA) using commercially available kits (R&D, Minneapolis, MN and Oncogene, Darmstadt, Germany), according to the manufacturers' instructions. Briefly, samples of supernatants were added to 96-well microplates coated with murine monoclonal antibodies against human MMP-2 or IL-6 and incubated for 2
2.11 Statistical analyses
Significance levels were calculated using student's t-test. P-values lower than 0.05 were considered statistically significant.
Four days after the single-tumour cell suspension was seeded onto 1% agarose-coated microplate wells, tightly packed, rounded spheroids were obtained, approximately 250–300
The structure of a HeLa-cell spheroid. Membrane integrity IP-H assay, analysed under fluorescence inversion microscope (Olympus BX 51). Bar 50
Confocal microscope analysis of co-culture of HeLa spheroids with HUVEC. The co-culture was stained for 2
Migration zone of HeLa cells on HUVECs. Co-culture after 24
In order to identify the factors responsible for cell damage, we tested superoxide generation and metalloproteinase production during direct interactions between tumour cell spheroids and endothelial cell monolayers. After 24
(A) Superoxide anion (O
SDS-PAGE analysis of metalloproteinase (MMP) production. Lane 1 – MMPs in culture medium supplemented with 2% FCS; lane 2 – molecular weight marker; lane 3 – MMPs produced by HUVEC monoculture; lanes 4 and 5 – MMPs produced in co-culture of the HUVEC monolayer with HeLa and Hep-2 cell line spheroids, respectively; lanes 6 and 7 – MMPs produced by HeLa and Hep-2 cell line spheroids monocultures, respectively. Values in the table represent densitometrically quantitated volumes of bands.
Metalloproteinase-2 (MMP-2) production during 24
Immunoenzymatic analysis showed that the endothelium and tumour spheroids alone produced low levels of IL-6 (Fig. 7A). In the co-culture model, the quantities of the cytokine were significantly higher than in the HUVEC monoculture, but they did not exceed the sum of IL-6 production by endothelial cells and tumour spheroids cultured separately. However, after preincubation with LPS, IL-6 production increased significantly in HUVECs and their co-culture with tumour spheroids (Fig. 7B).
(A) Interleukin-6 (IL-6) production during 24
Metastasis is a multistep process involving the escape of cells from the primary tumour, intravasation, dissemination via blood or lymphatic vessels, adhesive interactions with the endothelium, proteolytic degradation of the basement membrane and ECM components, extravasation, and secondary tumour growth in distant sites (Chambers and Matrisian, 1997). The attachment of the tumour cells to the internal blood vessel wall depends crucially on the expression of adhesive molecules on endothelial cell surfaces. Cytokines and growth factors are also implicated in the metastatic process. Moreover, interactions between tumour cell clusters and endothelial cells often occur where inflammatory reactions develop and where proinflammatory cytokines, which play a crucial role in cell–cell adhesions, are released (Takahashi et al., 2001). Cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) are overexpressed in the inflammatory state and play an important role in tumour development, influencing the metastatic potential of the tumour cells (Johnson, 1991; Maruo et al., 2002). ICAM-1 expression is induced by proinflammatory cytokines including interferon-γ (IFN-γ), interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α) (Kirnbauer et al., 1992).
Direct cell–cell interactions and cytokine release are not sufficient for extravasation of tumour cells or their migration into the connective tissue (Voura et al., 1998). The development of an invasive tumour cell phenotype also entails changes in cell motility (Siegel and Malmstein, 1997) and the capacity to evoke local proteolysis. The ability of tumour cells to migrate across the endothelium and basement membrane is considered one of the major characteristics of malignancy (Werner et al., 2002), involving the paracrine production of matrix metalloproteinases, cytokines, and growth factors (Rak et al., 1996). Therefore, in this study, we analysed ROS, MMP-2 and IL-6 production after direct interaction of tumour cell spheroids (HeLa and Hep-2 cell lines) with an endothelial cell (HUVEC) monolayer as an in vitro model of the direct and paracrine interactions between malignant cells and the blood vessel endothelium.
Therefore, the question arises: what are the inducers of ROS production in tumour cells adhering to the endothelium? ROS are generated (inter alia) during cellular respiration, and their most important sources are mitochondria (Jackson and Loeb, 2001). ROS release is stimulated and controlled by cytokines and growth factors, e.g. IL-1β or TNF-α, and these in turn influence the regulatory transcription factors that mediate apoptosis (Zapolska-Downar et al., 2002; Haddad, 2002). Therefore, some cytokines have been shown in several cell models to induce oxidative stress. Also, it has been reported that mitochondrial-derived ROS contribute to tumour necrosis factor-α (TNF-α)-induced cytotoxicity and nuclear factor-κB (NF-κB) activation. NF-κB activation is necessary for the expression of TNF-α-induced cellular adhesion molecules (E-selectin, VCAM-1) (Zapolska-Downar et al., 2002; Haddad, 2002), and these molecules facilitate direct cell–cell interactions. There are also several interdependencies between ROS and IL-6 production. ROS induce IL-6 production that is highly responsive to inflammation. IL-6, in turn, increases superoxide release by neutrophils, hepatocytes or fibroblast-like synoviocytes (Koren et al., 2000; Sung et al., 2000). Therefore, we suspect that co-culture of tumour spheroids with endothelial cells involves the following sequence of events: initial attachment of tumour spheroids to the HUVEC cell monolayer may signal ROS release, then ROS stimulates the expression of endothelial adhesive molecules that facilitate tumour implantation and IL-6 production. IL-6 is one of the cytokines that influence the capacity of tumour cells to migrate.
Oxidative stress is stimulated by LPS as well as by cytokines. Moreover, oxidative stress increases the production of proinflammatory cytokines from endothelial cells, e.g. IL-1β, TNF-α and IL-6, and these are potent oxidative stress mediators (Haddad, 2002; Legrand-Poels et al., 1997). IL-6 is a multifunctional, central proinflammatory cytokine crucial in the acute phase response. It is produced mainly not only by monocytes, macrophages and fibroblasts but is also produced by endothelial cells (Saba et al., 1996). In this study, we observed that IL-6 was produced in HUVEC monocultures and in co-cultures with tumour spheroids, but its level was very low. LPS is one of the most potent stimulants of endothelial IL-6 expression. Von Asmuth et al. (1991) showed that the stimulation of endothelial cells with LPS significantly increases IL-6 release. We found that pretreatment of endothelial cells with LPS increased both superoxide anion release and IL-6 production. The positive correlation between proinflammatory IL-6 production and superoxide anion release strongly suggests that IL-6 is involved in the generation of oxidative stress; this, in turn, enhances proinflammatory cytokine production. Considering the causes of enhanced IL-6 production, we can also speculate that cell-to-cell contact or soluble factors are responsible for the phenomenon. It is well known that sIL-6R strongly sensitises endothelial cells. Endothelial cells do not express membrane-bound IL-6R and therefore cannot be stimulated by IL-6 alone (Rose-John, 2003; Dowdall et al., 2002). In contrast to endothelial cells, cancer cells can express IL-6R and secrete the soluble IL-6R form (Matsuo et al., 2003). Therefore, the increase in IL-6 in our co-culture model may be at least partially a result of co-operation between IL-6 and tumour-derived sIL-6R, which may directly or indirectly enhance IL-6 production in the endothelial and tumour cells.
Apart from ROS, an essential role in tumour metastasis and invasion is also played by MMP expression. Metastasis requires proteolytic degradation of ECM components by MMPs to facilitate the invasion of malignant cells through the basement membrane and, subsequently, the connective tissue. In our experiments, we did not cover the cell-culture surfaces with any extracellular matrix element. Nevertheless, a significant increase in MMP production was observed after interaction between the tumour cell spheroids and the endothelial cell monolayer. Therefore, we suggest that endothelial cell-derived ECM elements and direct cell–cell contact are enough to stimulate an increased MMPs production by tumour cells. It is well known that the expression of metalloproteinases is regulated by various growth factors, cytokines and oncogene products (FGF-2, EGF, TNF-α or Ras) (Hah and Lee, 2003). It has also been shown that enhanced expression of IL-6 during inflammation may augment the expression of MMP-2 (Yano et al., 2003). There is also a significant positive correlation between the activated form of MMP-2 and the expression of vascular endothelial growth factor (VEGF). In consequence, enhanced VEGF expression stimulates tumour vascularization and motility of tumour cells (Kurizaki et al., 1998; Garzetti et al., 1999). Experiments addressing the role of cytokines in tumour cell–endothelium interactions are in progress.
In conclusion, the present study reveals that tumour cell spheroids induce local damage after direct contact with the endothelial monolayer, and the damage is mediated by overproduction of reactive oxygen species (ROS) and increased MMP-2 activity. In addition, the activation of endothelial cells with LPS, as an in vitro model of inflammation, significantly increases superoxide anion and IL-6 production in co-cultures of tumour cell spheroids and HUVEC.
This research was supported by grant No 6 PO4C 048 18 from State Committee for Scientific Research.
Chang CH, Werb, Z. . Colowick SP, Kaplan NO. Methods in enzymology. In: Di Sabato G, Everse J, editors. Phagocytosis and cell-mediated cytotoxicity, 132, Part J; 1986. p. 410–17.
Dowdall JF, Winter, DC, Andrews, E, Laug, WE, Wang, JH, Redmond, HP. Soluble interleukin-6 receptor (sIL-6R) mediates colonic tumor adherence to the vascular endothelium: a mechanism for metastatic initiation? J Surg Res 2002:107:1-6
Garzetti GG, Ciavattini, A, Lucarini, G, Pugnaloni, A, De nictolis, M, Amati, S. Expression of vascular endothelial growth factor related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors. Cancer 1999:85:2219-25
Hah N, Lee, S-T. An absolute role of the PCK-dependent NF-κB activation for induction of MMP-9 in hepatocellular carcinoma cells. Biochem Biophys Res Commun 2003:305:428-33
Jackson ChJ, Nguyen, M. Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases. Int J Biochem Cell Biol 1997:29:1167-77
Johnson JP. Cell adhesion molecules of the immunoglobulin supergene family and their role in malignant transformation and progression to metastatic disease. Cancer Metastasis Rev 1991:10:11-22
Kirnbauer R, Charvat, B, Schauer, E, Kock, A, Urbanski, A, Forster, E. Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF-β, and UVB light. J Invest Dermatol 1992:98:320-6
Koren R, Rocker, D, Kotestiano, O, Liberman, UA, Ravid, A. Synergistic anticancer activity of 1,25-dihydroxyvitamin D
Kräling BM, Wiederschain, DG, Boehm, T, Rehn, M, Mulliken, JB, Moses, MA. The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro. J Cell Sci 1999:112:1599-609
Legrand-Poels S, Maniglia, S, Boelaert, JR, Piette, J. Activation of the transcription factor NF-κB in lipopolysaccharide-stimulated U937 cells. Biochem Pharmacol 1997:53:339-46
Lin-Hung W, Min-Liang, K, Chi-An, Ch, Wen-Fang, Ch, Shao-Pei, Ch, Fon-Jou, H. Interleukin-6 in cervical cancer: the relationship with vascular endothelial growth factor. Gynecol Oncol 2001:82:49-56
Lorenz B, Schlüter, T, Bohnensack, R, Pergande, G, Müller, WEG. Effect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species. Biochem Pharmacol 1998:56:1615-24
Maruo Y, Gochi, A, Kaihara, A, Shimamura, H, Yamada, T, Tanaka, N. ICAM-1 expression and the soluble ICAM-1 level for evaluating the metastatic potential of gastric cancer. Int J Cancer 2002:100:486-90
Offner FA, Bigalke, I, Schiefer, J, Wirtz, HCh, Klosterhalfen, B, Feichtinger, H. Interaction of human malignant melanoma tumor spheroids with endothelium and reconstituted basement membrane: modulation by RGDS. Int J Cancer 1993:54:506-12
Offner FA, Schiefer, J, Wirtz, HCh, Bigalke, I, Pavelka, M, Hollweg, G. Tumor-cell–endothelial interactions: free radicals are mediators of melanoma-induced endothelial cell damage. Virchows Arch 1996:428:99-106
Offner FA, Wirtz, HCh, Schiefer, J, Bigalke, I, Klosterhalfen, B, Bittinger, F. Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayer. Am J Pathol 1992:141:601-10
Rak J, Filmus, J, Kerbel, RS. Reciprocal paracrine interactions between tumour cells and endothelial cells: the ‘angiogenesis progression’ hypothesis. Eur J Cancer 1996:32A:2438-50
Saba AA, Kaidi, AA, Godziachvili, V, Dombi, GW, Dawe, EJ, Libcke, JH. Effect of interleukin-6 and its neutralizing antibodies on peritoneal adhesion formation and wound healing. Am Surg 1996:62:569-72
Sung J-Y, Hong, J-H, Kang, H-S, Choi, I, Lim, S-D, Lee, J-K. Methotrexate suppresses the interleukin-6 induced generation of reactive oxygen species in the synoviocytes of rheumatoid arthritis. Immunopharmacology 2000:47:35-44
Von Asmuth EJ, Leeuwenberg, JF, Ceska, M, Buurman, WA. LPS and cytokine-induced endothelial cell IL-6 release and ELAM-1 expression; involvement of serum. Cytokine Netw 1991:2:291-7
Wartenberg M, Ling, FC, Schallengerg, M, Bäumer, AT, Petrat, K, Hescheler, J. Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumour spheroids by reactive oxygen species. J Biol Chem 2001:276:17420-8
Yano S, Nokihara, H, Yamamoto, A, Goto, H, Ogawa, H, Kanematsu, T. Multifunctional interleukin-1β promotes metastasis of human lung cancer cells in SCID mice via enhanced expression of adhesion-, invasion- and angiogenesis-related molecules. Cancer Sci 2003:94:244-52
Yen CH, Hsieh, CC, Chou, SY, Lau, YT. 17β-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells. Life Sci 2001:70:403-13
Zapolska-Downar D, Zapolski-Downar, A, Naruszewicz, M, Siennicka, A, Krasnodēbska, B, Kołodziej, B. Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes. Life Sci 2002:71:2897-908
Received 14 September 2004/18 December 2004; accepted 23 January 2005doi:10.1016/j.cellbi.2005.01.007