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reoxygenation down-regulates the expression of E-cadherin in human colon cancer cell lines
Cancer Letters 211 (2004) 79–87 www.elsevier.com/locate/canlet
Anoxia/reoxygenation down-regulates the expression of E-cadherin in human colon cancer cell lines Satoshi Kokuraa,*, Norimasa Yoshidaa, Eiko Imamotob, Miho Uedab, Takeshi Ishikawaa, Kazuhiko Uchiyamab, Masashi Kuchideb, Yuji Naitoa, Takeshi Okanouea, Toshikazu Yoshikawab a
Molecular Gastroenterology and hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan b Inflammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566 Japan Received 1 September 2003; received in revised form 20 January 2004; accepted 28 January 2004
Abstract The E-cadherin-mediated cell – cell adhesiveness is a critical factor for carcinoma cell invasion and metastasis. Anoxia/reoxygenation is known to occur in cancer tissues. In this study, we investigated whether anoxia/reoxygenation induces the down-regulation of E-cadherin expression in the human colon cancer cell lines HT-29, and SW1116. Colon cancer cells were exposed to anoxia (2 h) followed by reoxygenation (4– 46 h). The subsequent expression of E-cadherin on the cell surface was examined by immunocytochemistry and enzyme-linked immunosorbent assays, the total amount of E-cadherin protein was examined by Western blotting, and the E-cadherin mRNA level was examined by a real-time polymerase chain reaction assay. The expression of E-cadherin on the cell surface and the total amount of E-cadherin protein were transiently reduced after anoxia/reoxygenation. On the other hand, the E-cadherin mRNA level was not decreased during reoxygenation. Pretreatment with actinomycin D or reagents that interfere with the activation of NF-kB significantly attenuated the downregulation of E-cadherin, which implicated a role for the de novo protein synthesis. These results indicate that anoxia/reoxygenation induces a transient reduction of E-cadherin expression in human colon cancer cells through NF-kB dependent transcriptional pathway. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Anoxia/reoxygenation; E-cadherin; Colon cancer; Metastasis
1. Introduction Metastases of cancers are assumed to occur in multiple steps. The initial step is the detachment * Corresponding author. Tel.: þ81-75-251-5505; fax: þ 81-75252-3721. E-mail address: [email protected] (S. Kokura).
of cells from primary tumors. The integrity and morphology of cancer cell –cell adhesions are maintained by E-cadherin and its associated intracellular catenin molecules. In vitro experimental evidence has suggested that selective loss of E-cadherin causes disruption of cell –cell adhesion [1] and acquisition of invasiveness [2]. Furthermore, immunohistochemical studies have indicated that decreased E-cadherin
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expression in various human cancers in vivo is correlated with invasive and metastatic potential [3]. These findings indicate that down-regulation of E-cadherin facilitates tumor cell invasion by causing separation of cells from the primary tumor. However, mutational inactivation of E-cadherin appears to be common only in diffuse type gastric carcinomas and infiltrating lobular breast carcinomas [4]. In the majority of cancers with altered E-cadherin expression, such as colorectal cancers and esophageal cancers, gene mutations are rare or absent [5]. Thus, researchers are interested in understanding the mechanisms by which the expression of E-cadherin is reduced or eliminated. Cancer tissues develop a pathophysiological microenvironment during growth, characterized by an irregular microvascular network and regions of chronically and transiently ischemic cells. Ischemic cancer cells are known to be reperfused by neovascularization or by a decrease in tissue pressure. Bush et al. reported that 3 h of ischemia clearly reduced the total amount of E-cadherin in canine kidney cells [6]. Rofstad et al. indicated that tumor hypoxia promotes the development of metastasis [7]. These findings suggest that anoxia/reoxygenation induces the reduction of E-cadherin expression, which may promote metastasis. In this study, we investigated whether anoxia/ reoxygenation reduces the expression of E-cadherin in human colon cancer cell lines.
2. Materials and methods 2.1. Cell line and cell culture The human colon cancer cell lines HT-29, and SW1116 were obtained from the American Type Culture Collection (Rockville, MD). The HT-29 cells were grown in McCoy’s 5a medium (Sigma, St Louis, MO) with 10% fetal calf serum (FCS) and penicillin (100 U/ml)/streptomycin (100 mg/ml). SW1116 cells were grown in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% FCS and penicillin (100 U/ml)/streptomycin (100 mg/ml). Cell cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 8C.
2.2. Anoxia/reoxygenation protocol The in vitro model of anoxia/reoxygenation used in this study is similar to the one previously reported [8]. Briefly, confluent HT-29 colon cancer cells or SW1116 cells were exposed to anoxia by incubation in a Plexiglas chamber that was continuously purged (1 l/min) with an anoxic gas mixture (93% N2 – 5% CO2 – 2% H2). Chamber pO2 was monitored during the entire experiment using an oxygen electrode (model OM-1, Microelectrodes, Londonderry, NH). Reoxygenation was performed by exposing cancer cells to normoxia (21% O2 – 5% CO2 – 74% N2) in the CO2 incubator. Control cells were exposed to normoxia. 2.3. Immunocytochemistry The HT-29 cells cultured on cover slips were incubated with anti-E-cadherin antibody (PROGEN, Heidelberg, Germany) in phosphate-buffered saline (PBS) containing 5% FCS and 2 mM CaCl2 for 2 h at room temperature, followed by 30 min of incubation with fluorescence-labeled secondary antibody (CAPPEL, Aurora, OH). Samples were visualized by epiillumination on a photomicroscope (model IX 70, OLYMPUS, Tokyo, Japan). 2.4. E-cadherin expression assay The HT-29 cells or SW1116 cells were plated on 48-well tissue culture dishes. Primary antibody for E-cadherin in PBS with 5% FCS was added to each well and the dishes were incubated for 30 min at 37 8C. The cells were washed and incubated for 30 min at 37 8C with a secondary antibody, horseradish peroxide-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, PA) diluted 1:2500 in PBS with 5% FCS. The wells were then washed and antibody binding was detected by the addition of 100 ml of 0.1 mg/ml 3,30 ,5,50 -tetramethylbenzidine (Sigma) with 0.003% H2O2. The reaction was stopped by the addition of 75 ml of 8 N sulfuric acid. The samples were transferred to 96-well plates and color development was read on a microplate reader (model MPR-A4i, TOSOH COR, Tokyo, Japan) at an optical density of 450 nm after subtracting the background values in cells stained with 5% FCS in place of
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primary antibody and the second-step antibody. All data points were performed in triplicate. To determine whether transcription is involved in anoxia/reoxygenation-induced decrease of E-cadherin expression, HT-29 cells were exposed to Actinomycin D (ActD, 2 mg/ml) 30 min before cells were treated with anoxia/reoxygenation. The contribution of the nuclear transcription factor, NF-kB to anoxia/reoxygenation-induced decrease of E-cadherin was assessed with HT-29 cells treated with lactacyctin or MG132 (proteosome inhibitor). Both inhibitors were added to HT-29 cells 1 h before treatment of cells with anoxia/reoxygenation. 2.5. Western blotting Whole cell extracts were prepared as follows. The HT-29 cells were lysed at 4 8C in a solution of 50 mmol/l Tris –HCL, pH 7.6, 300 mmol/l NaCl, 0.5% Triton X-100, 10 mg/ml aprotinin, 10 mg/ml leuptin, 1 mmol/l phenylmethylsulfonyl fluoride, 1.8 mg/ml iodoacetamide, 50 mmol/l NaF, and 1 mM DTT. The supernatants were collected and stored at 2 70 8C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with anti-E-cadherin mAb and reprobed with a mouse anti-actin mAb (Calbiochem, San Diego, CA) to normalize to equivalent gel loading. The immune complexes were visualized by Western blotting with a commercial kit (ECL by Amersham, Buckinghamshire, England) according to the manufacturer’s recommendations. 2.6. Real-time amplification Total RNA was extracted from HT-29 cells by the guanidine thiocyanate-cesium chloride centrifugation method. The RNA was reverse-transcribed with an oligo(dT) primer and reverse transcriptase (Takara Biomedicals, Shiga, Japan). To determine the levels of E-cadherin mRNA in HT-29 cells, real-time quantitative polymerase chain reaction amplification was performed with the Light Cycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) using the DNA binding dye SYBER
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Green 1 (Roche Molecular Biochemicals) for the detection of amplification products. 2.7. Statistical analysis Results are presented as the mean ^ SEM. The data were analyzed by one-way analysis of variance and differences were considered significant when P values were less than 0.05 in the Scheffe´ multiple comparison test. The StatView 4.11-J software program (Abacus Concepts Inc., Berkeley, CA) on a Macintosh computer was used to conduct the analyses.
3. Results 3.1. Effects of anoxia or anoxia/reoxygenation on the expression of E-cadherin in SW1116 cells and HT-29 cells We exposed SW1116 cells and HT-29 cells to 6 h of normoxia, 6 h of anoxia or 2 h of anoxia followed by 4 h of reoxygenation, respectively. Two hours of anoxia followed by 4 h of reoxygenation resulted in a significant decrease in E-cadherin expression compared with that induced by anoxia alone (Fig. 1a and b). 3.2. Time course kinetics of E-cadherin expression of HT-29 cells after anoxia/reoxygenation In control cells exposed to normoxia for 48 h, E-cadherin expression on the plasma membrane of HT-29 cells was not altered. Two hours of anoxia followed by 4 h of reoxygenation resulted in a significant decrease in E-cadherin expression, and 22 h of reoxygenation induced a further decrease in E-cadherin expression. However, after 46 h of reoxygenation, E-cadherin expression was recovered (Fig. 2). 3.3. Effect of anoxia/reoxygenation on the distribution of E-cadherin and morphology of HT-29 cells Immunofluorescent staining showed that the control cells exposed to normoxia were attached tightly to each other and their E-cadherin was localized to
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3.4. Effect of anoxia/reoxygenation on the degradation of E-cadherin Western blot analysis of anoxia/reoxygenationexposed HT-29 cell lysates revealed that no significant loss of E-cadherin was detectable after 4 h of reoxygenation. In contrast, a clear and consistent reduction in the total amount of E-cadherin was seen after 22 h of reoxygenation relative to that after 24 h of normoxia (Fig. 4). 3.5. Effect of anoxia/reoxygenation on the transcription of E-cadherin The level of E-cadherin mRNA in HT-29 cells was not altered by 2 h of anoxia followed by 4 –22 h of reoxygenation (Fig. 5). 3.6. Effect of inhibitors of NF-kB on the downregulation of E-cadherin after anoxia/reoxygenation
Fig. 1. Effects of anoxia or anoxia/reoxygenation on the expression of E-cadherin in SW1116 cells and HT-29 cells. SW1116 cells (a) and HT-29 cells (b) were exposed to 6 h of normoxia, 6 h of anoxia or 2 h of anoxia followed by 4 h of reoxygenation, respectively. Each value represents the mean ^ SE of three experiments performed in triplicate. * P , 0:05 compared with normoxia, or anoxia alone.
the lateral aspects of the plasma membranes in a continuous, linear staining pattern. After 4 h of reoxygenation, the staining pattern for E-cadherin was altered. The previously seen linear staining appeared to be weaker and discontinuous. After 22 h of reoxygenation, E-cadherin expression on the plasma membrane had almost disappeared and cellcell adhesion was partially destroyed. However, after 46 h of reoxygenation, the linear staining pattern for E-cadherin was recovered and each cell was attached again (Fig. 3).
Previous studies from our laboratory and others have invoked a role for NF-kB in anoxia/reoxygenation-mediated cell reaction [9,10]. To assess the possibility that anoxia/reoxygenation elicits a transcription-dependent down-regulation of E-cadherin, we treated HT-29 cells with inhibitor of macromolecule synthesis, ActD (2 mg/ml). Treatment with ActD alone had no significant effect on the expression
Fig. 2. Time-course of E-cadherin expression in HT-29 colon cancer cells after anoxia/reoxygenation. The HT-29 cells were exposed to 2 h of anoxia (or normoxia) and then reoxygenated. Each value represents the mean ^ SE of three experiments performed in triplicate. * P , 0:05 compared with normoxia.
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Fig. 3. Anoxia/reoxygenation alters the expression of E-cadherin. E-cadherin staining was positive along the area of cell–cell contact in normoxia-exposed HT-29 cells. E-cadherin staining appeared to be weaker and discontinuous after 2 h of anoxia followed by 4 or 22 h of reoxygenation. However, after 46 h of reoxygenation, the previous E-cadherin staining pattern was recovered.
Fig. 4. Effect of anoxia/reoxygenation on the total amount of E-cadherin. The HT-29 cells were exposed to 2 h of anoxia followed by 4 or 22 h of reoxygenation. The HT-29 cells were lysed and proteins (20 mg) were separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were probed with anti-E-cadherin mAb. One representative of three separate immunoblots is shown (top). Densitometric quantification of the proteins normalized to actin was performed (bottom). Each value represents the mean ^ SE of three separate immunoblots.
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of cancer cells from the primary tumors [11], which is the initial step of cancer metastasis. The present results showing that E-cadherin on the plasma membrane had almost disappeared and the attachments of cancer cells to cancer cells were partially destroyed after 22 h of reoxygenation suggest that anoxia/reoxygenation may induce cancer metastasis. The results of previous studies also showed that hypoxia promotes
Fig. 5. E-cadherin mRNA expression in HT-29 cells. Total RNA was extracted from the normoxia (6 or 24 h)-exposed HT-29 cells and the anoxia (2 h)/reoxygenation (4 or 22 h)-exposed HT-29 cells. RNA was reverse-transcribed and subjected to real-time quantitative amplification to determine the levels of each mRNA in HT-29 cells. Each value represents the mean ^ SE of three experiments.
of E-cadherin (data not shown). The decrease of E-cadherin was significantly attenuated when the cells were pretreated with ActD for 30 min (Fig. 6a). To evaluate the contribution of transcription factor, NF-kB, to anoxia/reoxygenation-induced down-regulation of E-cadherin, HT-29 cells were treated with proteasome inhibitors (lactacyctin, or MG132). Treatment with proteasome inhibitors alone had no significant effect on the expression of E-cadherin (data not shown). The decrease of E-cadherin was significantly attenuated when the cells were pretreated with lactacyctin (10 mM), or MG132 (10 mM) for 1 h (Fig. 6b).
4. Discussion This study provided evidence to support the hypothesis that anoxia/reoxygenation induces the reduction of E-cadherin expression in human colon adenocarcinoma cell lines. The reduction of E-cadherin on cancer cell surfaces causes the release
Fig. 6. Effect of inhibitors of transcription (ActD) or NF-kB activation (proteasome inhibitor) on E-cadherin expression after anoxia/reoxygenation. (a) Effect of ActD (2 mg/ml). (b) Effect of lactacyctin (10 mM) or MG132 (10 mM). Responses are shown for E-cadherin expression at 22 h reoxygenation after anoxia (2 h). Each value indicates mean ^ SE of three experiments. * P , 0:05 compared with normoxia. #p , 0:05 compared with anoxia/reoxygenation.
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the development of metastasis [7]. Anoxia can induce the expression of several adhesion molecules on epithelial cells, endothelial cells, and cancer cells. Many studies have indicated that the expression of adhesion molecules is important for tumor invasion and metastasis in colorectal cancer [12 – 15]. The loss of E-cadherin expression might be related to the invasive capacity as well as metastatic potential of cancer cells [16]. Although, the role of E-cadherin in invasion and metastasis is relatively well known, the present knowledge of the detailed mechanisms by which E-cadherin is down-regulated remains incomplete. Previous studies have demonstrated that downregulation of E-cadherin expression is associated with differentiation grade and metastasis in human colorectal carcinomas [17] and squamous cell carcinomas of the head and neck [18]. Expression of E-cadherin has been studied in tissue sections from a variety of differentiation grades, and lower levels of expression were observed in more poorly differentiated cancers [19]. The down-regulation of E-cadherin is not necessarily a causative factor of metastasis to the liver. For example, differentiated gastric cancers, which express high amounts of E-cadherin, often metastasize to the liver [20]. In addition, welldifferentiated colorectal carcinomas frequently develop liver metastasis. However, at the initial step of metastasis, it is necessary for cancer cells to detach from the primary tumor. Furthermore, the downregulation of E-cadherin expression may be essential for cancer cells to detach from the primary tumor [11]. Thus, the transient down-regulation of E-cadherin, by which cancer cells acquire their invasive property, must be organized by some mechanism at the primary site in the process of liver metastasis. In this study, the expression of E-cadherin was significantly reduced during reoxygenation from 4 to 22 h. Western blots of HT-29 colon carcinoma cells exposed to normoxia or anoxia revealed two different patterns of downregulation, depending on the duration of reoxygenation. Four hours of reoxygenation down-regulated the expression of E-cadherin, demonstrated by immunofluorescent staining and enzyme-linked immunosorbent assays. However, no loss in the total amount of E-cadherin was detectable by Western blotting after 4 h of reoxygenation, which indicates that the reduction in E-cadherin expression is probably the result of the internalization of surface-accessible
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E-cadherin. Conversely, the Western blots revealed a clear reduction in the total amount of E-cadherin after 22 h of reoxygenation, suggesting that the loss of E-cadherin is not only the result of the internalization; however, this additional mechanism is unclear. Our real time amplification study revealed that the levels of E-cadherin mRNA expression did not differ between normoxia-exposed HT-29 cells and anoxia/reoxygenation-exposed HT-29 cells. Therefore, the difference in the total amounts of E-cadherin protein might be due to post-translational modifications. This is supported by the previously reported results of Nakayama et al., which showed that E-cadherin expression was decreased in highly metastatic cell lines [21], whereas the E-cadherin mRNA levels did not differ between highly metastatic cell lines and weakly metastatic cell lines [22]. Taken together with another previous report [6], our immunofluorescent staining and Western blots studies suggest that E-cadherin on the cell surface is internalized 4 h after reoxygenation, and degraded in response to further reoxygenation (22 h). However, the present results do not eliminate other possibilities such as an extracellular cleavage of E-cadherin. Insights into the molecular processes that regulate the transcription-dependent alterations in HT-29 cells following anoxia/reoxygenation may be provided by previous studies of cytokine-, oxidants, and anoxia/ reoxygenation-induced cancer cell or endothelial cell responses [23,10]. These studies have revealed that the oxidant stress associated with anoxia/reoxygenation results in a transcription-dependent expression of E-selection, VCAM-1, ICAM-1, and anti-apoptotic proteins. Furthermore, it appears that the activation of nuclear factor NF-kB is a critical event in the oxidantinduced, transcription-dependent expression of endothelial cell adhesion molecules and anti-apoptotic proteins. These observations raise the possibility that other proteins that are also under the control of this signaling pathway may contribute to the transcriptiondependent down-regulation of E-cadherin. Our data demonstrates that treatment of HT-29 cells with inhibitor of transcription or reagents that interfere with the activation of NF-kB significantly attenuated the anoxia/reoxygenation-induced down-regulation of E-cadherin. The mechanism by which anoxia/reoxygenation modulates NF-kB dependent down regulation of E-cadherin is unclear. One possibility may be that
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nitric oxide (NO) induces the degradation of E-cadherin. The transcription-dependent synthesis of inducible NO synthase (iNOS) is known to be regulated by NF-kB [24]. Further support for a role of NO in the degradation of E-cadherin is provided by a recent report that describes beta-catenin as an important target of NO [25]. But in this study, we do not investigate the involvement of NO in the mechanism by which decrease of E-cadherin occurs. Further research is needed to identify the mechanisms by which anoxia/reoxygenation downregulates E-cadherin. Our results indicated that the anoxia/reoxygenation-induced reduction of E-cadherin expression is transient. Our immunofluorescent staining and enzyme-linked immunosorbent assays revealed that the expression of E-cadherin and the HT-29 cell-cell adhesion were recovered 46 h after reoxygenation. Osada et al. reported that E-cadherin participates in secondary tumor formation in the liver [26]. They speculated that increased cell-cell adhesion causes self-aggregation of cancer cells and promotes tumor formation in the liver. This scenario is supported by a previous report from Mayer et al., which shows by immunohistochemical analysis that all liver metastases of gastric cancers showed a high frequency of strongly E-cadherin-positive cells, regardless of the staining pattern in the primary cancer [27]. To initiate metastasis, cancer cells have to become detached from the primary cancer, and in this step downregulation of E-cadherin is known to play an important role. However, primary colorectal cancers and their liver metastatic nodules often show a welldifferentiated histology and retain their E-cadherin expression. Differentiated human gastric cancer, which retains E-cadherin expression, often metastasizes to the liver, whereas undifferentiated gastric cancers, which show reduced amounts of E-cadherin, often show intraperitoneal dissemination and lymphogenous metastasis but not liver metastasis. These findings suggest that in the process of liver metastasis, the transient down-regulation of the E-cadherin, by which cancer cells acquire their invasive property, is organized by some mechanism at the primary site. In the liver, E-cadherin, which is down-regulated at the primary site, might become recovered and thus play a important role in secondary cancer formation.
In our present experimental model with the human colon adenocarcinoma cell lines, we found that anoxia/reoxygenation induces a transient reduction in E-cadherin expression, which may promote metastasis.
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Anoxia/reoxygenation down-regulates the expression of E-cadherin in human colon cancer cell lines Satoshi Kokuraa,*, Norimasa Yoshidaa, Eiko Imamotob, Miho Uedab, Takeshi Ishikawaa, Kazuhiko Uchiyamab, Masashi Kuchideb, Yuji Naitoa, Takeshi Okanouea, Toshikazu Yoshikawab a
Molecular Gastroenterology and hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan b Inflammation and Immunology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566 Japan Received 1 September 2003; received in revised form 20 January 2004; accepted 28 January 2004
Abstract The E-cadherin-mediated cell – cell adhesiveness is a critical factor for carcinoma cell invasion and metastasis. Anoxia/reoxygenation is known to occur in cancer tissues. In this study, we investigated whether anoxia/reoxygenation induces the down-regulation of E-cadherin expression in the human colon cancer cell lines HT-29, and SW1116. Colon cancer cells were exposed to anoxia (2 h) followed by reoxygenation (4– 46 h). The subsequent expression of E-cadherin on the cell surface was examined by immunocytochemistry and enzyme-linked immunosorbent assays, the total amount of E-cadherin protein was examined by Western blotting, and the E-cadherin mRNA level was examined by a real-time polymerase chain reaction assay. The expression of E-cadherin on the cell surface and the total amount of E-cadherin protein were transiently reduced after anoxia/reoxygenation. On the other hand, the E-cadherin mRNA level was not decreased during reoxygenation. Pretreatment with actinomycin D or reagents that interfere with the activation of NF-kB significantly attenuated the downregulation of E-cadherin, which implicated a role for the de novo protein synthesis. These results indicate that anoxia/reoxygenation induces a transient reduction of E-cadherin expression in human colon cancer cells through NF-kB dependent transcriptional pathway. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Anoxia/reoxygenation; E-cadherin; Colon cancer; Metastasis
1. Introduction Metastases of cancers are assumed to occur in multiple steps. The initial step is the detachment * Corresponding author. Tel.: þ81-75-251-5505; fax: þ 81-75252-3721. E-mail address: [email protected] (S. Kokura).
of cells from primary tumors. The integrity and morphology of cancer cell –cell adhesions are maintained by E-cadherin and its associated intracellular catenin molecules. In vitro experimental evidence has suggested that selective loss of E-cadherin causes disruption of cell –cell adhesion [1] and acquisition of invasiveness [2]. Furthermore, immunohistochemical studies have indicated that decreased E-cadherin
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expression in various human cancers in vivo is correlated with invasive and metastatic potential [3]. These findings indicate that down-regulation of E-cadherin facilitates tumor cell invasion by causing separation of cells from the primary tumor. However, mutational inactivation of E-cadherin appears to be common only in diffuse type gastric carcinomas and infiltrating lobular breast carcinomas [4]. In the majority of cancers with altered E-cadherin expression, such as colorectal cancers and esophageal cancers, gene mutations are rare or absent [5]. Thus, researchers are interested in understanding the mechanisms by which the expression of E-cadherin is reduced or eliminated. Cancer tissues develop a pathophysiological microenvironment during growth, characterized by an irregular microvascular network and regions of chronically and transiently ischemic cells. Ischemic cancer cells are known to be reperfused by neovascularization or by a decrease in tissue pressure. Bush et al. reported that 3 h of ischemia clearly reduced the total amount of E-cadherin in canine kidney cells [6]. Rofstad et al. indicated that tumor hypoxia promotes the development of metastasis [7]. These findings suggest that anoxia/reoxygenation induces the reduction of E-cadherin expression, which may promote metastasis. In this study, we investigated whether anoxia/ reoxygenation reduces the expression of E-cadherin in human colon cancer cell lines.
2. Materials and methods 2.1. Cell line and cell culture The human colon cancer cell lines HT-29, and SW1116 were obtained from the American Type Culture Collection (Rockville, MD). The HT-29 cells were grown in McCoy’s 5a medium (Sigma, St Louis, MO) with 10% fetal calf serum (FCS) and penicillin (100 U/ml)/streptomycin (100 mg/ml). SW1116 cells were grown in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% FCS and penicillin (100 U/ml)/streptomycin (100 mg/ml). Cell cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37 8C.
2.2. Anoxia/reoxygenation protocol The in vitro model of anoxia/reoxygenation used in this study is similar to the one previously reported [8]. Briefly, confluent HT-29 colon cancer cells or SW1116 cells were exposed to anoxia by incubation in a Plexiglas chamber that was continuously purged (1 l/min) with an anoxic gas mixture (93% N2 – 5% CO2 – 2% H2). Chamber pO2 was monitored during the entire experiment using an oxygen electrode (model OM-1, Microelectrodes, Londonderry, NH). Reoxygenation was performed by exposing cancer cells to normoxia (21% O2 – 5% CO2 – 74% N2) in the CO2 incubator. Control cells were exposed to normoxia. 2.3. Immunocytochemistry The HT-29 cells cultured on cover slips were incubated with anti-E-cadherin antibody (PROGEN, Heidelberg, Germany) in phosphate-buffered saline (PBS) containing 5% FCS and 2 mM CaCl2 for 2 h at room temperature, followed by 30 min of incubation with fluorescence-labeled secondary antibody (CAPPEL, Aurora, OH). Samples were visualized by epiillumination on a photomicroscope (model IX 70, OLYMPUS, Tokyo, Japan). 2.4. E-cadherin expression assay The HT-29 cells or SW1116 cells were plated on 48-well tissue culture dishes. Primary antibody for E-cadherin in PBS with 5% FCS was added to each well and the dishes were incubated for 30 min at 37 8C. The cells were washed and incubated for 30 min at 37 8C with a secondary antibody, horseradish peroxide-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, PA) diluted 1:2500 in PBS with 5% FCS. The wells were then washed and antibody binding was detected by the addition of 100 ml of 0.1 mg/ml 3,30 ,5,50 -tetramethylbenzidine (Sigma) with 0.003% H2O2. The reaction was stopped by the addition of 75 ml of 8 N sulfuric acid. The samples were transferred to 96-well plates and color development was read on a microplate reader (model MPR-A4i, TOSOH COR, Tokyo, Japan) at an optical density of 450 nm after subtracting the background values in cells stained with 5% FCS in place of
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primary antibody and the second-step antibody. All data points were performed in triplicate. To determine whether transcription is involved in anoxia/reoxygenation-induced decrease of E-cadherin expression, HT-29 cells were exposed to Actinomycin D (ActD, 2 mg/ml) 30 min before cells were treated with anoxia/reoxygenation. The contribution of the nuclear transcription factor, NF-kB to anoxia/reoxygenation-induced decrease of E-cadherin was assessed with HT-29 cells treated with lactacyctin or MG132 (proteosome inhibitor). Both inhibitors were added to HT-29 cells 1 h before treatment of cells with anoxia/reoxygenation. 2.5. Western blotting Whole cell extracts were prepared as follows. The HT-29 cells were lysed at 4 8C in a solution of 50 mmol/l Tris –HCL, pH 7.6, 300 mmol/l NaCl, 0.5% Triton X-100, 10 mg/ml aprotinin, 10 mg/ml leuptin, 1 mmol/l phenylmethylsulfonyl fluoride, 1.8 mg/ml iodoacetamide, 50 mmol/l NaF, and 1 mM DTT. The supernatants were collected and stored at 2 70 8C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with anti-E-cadherin mAb and reprobed with a mouse anti-actin mAb (Calbiochem, San Diego, CA) to normalize to equivalent gel loading. The immune complexes were visualized by Western blotting with a commercial kit (ECL by Amersham, Buckinghamshire, England) according to the manufacturer’s recommendations. 2.6. Real-time amplification Total RNA was extracted from HT-29 cells by the guanidine thiocyanate-cesium chloride centrifugation method. The RNA was reverse-transcribed with an oligo(dT) primer and reverse transcriptase (Takara Biomedicals, Shiga, Japan). To determine the levels of E-cadherin mRNA in HT-29 cells, real-time quantitative polymerase chain reaction amplification was performed with the Light Cycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) using the DNA binding dye SYBER
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Green 1 (Roche Molecular Biochemicals) for the detection of amplification products. 2.7. Statistical analysis Results are presented as the mean ^ SEM. The data were analyzed by one-way analysis of variance and differences were considered significant when P values were less than 0.05 in the Scheffe´ multiple comparison test. The StatView 4.11-J software program (Abacus Concepts Inc., Berkeley, CA) on a Macintosh computer was used to conduct the analyses.
3. Results 3.1. Effects of anoxia or anoxia/reoxygenation on the expression of E-cadherin in SW1116 cells and HT-29 cells We exposed SW1116 cells and HT-29 cells to 6 h of normoxia, 6 h of anoxia or 2 h of anoxia followed by 4 h of reoxygenation, respectively. Two hours of anoxia followed by 4 h of reoxygenation resulted in a significant decrease in E-cadherin expression compared with that induced by anoxia alone (Fig. 1a and b). 3.2. Time course kinetics of E-cadherin expression of HT-29 cells after anoxia/reoxygenation In control cells exposed to normoxia for 48 h, E-cadherin expression on the plasma membrane of HT-29 cells was not altered. Two hours of anoxia followed by 4 h of reoxygenation resulted in a significant decrease in E-cadherin expression, and 22 h of reoxygenation induced a further decrease in E-cadherin expression. However, after 46 h of reoxygenation, E-cadherin expression was recovered (Fig. 2). 3.3. Effect of anoxia/reoxygenation on the distribution of E-cadherin and morphology of HT-29 cells Immunofluorescent staining showed that the control cells exposed to normoxia were attached tightly to each other and their E-cadherin was localized to
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3.4. Effect of anoxia/reoxygenation on the degradation of E-cadherin Western blot analysis of anoxia/reoxygenationexposed HT-29 cell lysates revealed that no significant loss of E-cadherin was detectable after 4 h of reoxygenation. In contrast, a clear and consistent reduction in the total amount of E-cadherin was seen after 22 h of reoxygenation relative to that after 24 h of normoxia (Fig. 4). 3.5. Effect of anoxia/reoxygenation on the transcription of E-cadherin The level of E-cadherin mRNA in HT-29 cells was not altered by 2 h of anoxia followed by 4 –22 h of reoxygenation (Fig. 5). 3.6. Effect of inhibitors of NF-kB on the downregulation of E-cadherin after anoxia/reoxygenation
Fig. 1. Effects of anoxia or anoxia/reoxygenation on the expression of E-cadherin in SW1116 cells and HT-29 cells. SW1116 cells (a) and HT-29 cells (b) were exposed to 6 h of normoxia, 6 h of anoxia or 2 h of anoxia followed by 4 h of reoxygenation, respectively. Each value represents the mean ^ SE of three experiments performed in triplicate. * P , 0:05 compared with normoxia, or anoxia alone.
the lateral aspects of the plasma membranes in a continuous, linear staining pattern. After 4 h of reoxygenation, the staining pattern for E-cadherin was altered. The previously seen linear staining appeared to be weaker and discontinuous. After 22 h of reoxygenation, E-cadherin expression on the plasma membrane had almost disappeared and cellcell adhesion was partially destroyed. However, after 46 h of reoxygenation, the linear staining pattern for E-cadherin was recovered and each cell was attached again (Fig. 3).
Previous studies from our laboratory and others have invoked a role for NF-kB in anoxia/reoxygenation-mediated cell reaction [9,10]. To assess the possibility that anoxia/reoxygenation elicits a transcription-dependent down-regulation of E-cadherin, we treated HT-29 cells with inhibitor of macromolecule synthesis, ActD (2 mg/ml). Treatment with ActD alone had no significant effect on the expression
Fig. 2. Time-course of E-cadherin expression in HT-29 colon cancer cells after anoxia/reoxygenation. The HT-29 cells were exposed to 2 h of anoxia (or normoxia) and then reoxygenated. Each value represents the mean ^ SE of three experiments performed in triplicate. * P , 0:05 compared with normoxia.
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Fig. 3. Anoxia/reoxygenation alters the expression of E-cadherin. E-cadherin staining was positive along the area of cell–cell contact in normoxia-exposed HT-29 cells. E-cadherin staining appeared to be weaker and discontinuous after 2 h of anoxia followed by 4 or 22 h of reoxygenation. However, after 46 h of reoxygenation, the previous E-cadherin staining pattern was recovered.
Fig. 4. Effect of anoxia/reoxygenation on the total amount of E-cadherin. The HT-29 cells were exposed to 2 h of anoxia followed by 4 or 22 h of reoxygenation. The HT-29 cells were lysed and proteins (20 mg) were separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were probed with anti-E-cadherin mAb. One representative of three separate immunoblots is shown (top). Densitometric quantification of the proteins normalized to actin was performed (bottom). Each value represents the mean ^ SE of three separate immunoblots.
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of cancer cells from the primary tumors [11], which is the initial step of cancer metastasis. The present results showing that E-cadherin on the plasma membrane had almost disappeared and the attachments of cancer cells to cancer cells were partially destroyed after 22 h of reoxygenation suggest that anoxia/reoxygenation may induce cancer metastasis. The results of previous studies also showed that hypoxia promotes
Fig. 5. E-cadherin mRNA expression in HT-29 cells. Total RNA was extracted from the normoxia (6 or 24 h)-exposed HT-29 cells and the anoxia (2 h)/reoxygenation (4 or 22 h)-exposed HT-29 cells. RNA was reverse-transcribed and subjected to real-time quantitative amplification to determine the levels of each mRNA in HT-29 cells. Each value represents the mean ^ SE of three experiments.
of E-cadherin (data not shown). The decrease of E-cadherin was significantly attenuated when the cells were pretreated with ActD for 30 min (Fig. 6a). To evaluate the contribution of transcription factor, NF-kB, to anoxia/reoxygenation-induced down-regulation of E-cadherin, HT-29 cells were treated with proteasome inhibitors (lactacyctin, or MG132). Treatment with proteasome inhibitors alone had no significant effect on the expression of E-cadherin (data not shown). The decrease of E-cadherin was significantly attenuated when the cells were pretreated with lactacyctin (10 mM), or MG132 (10 mM) for 1 h (Fig. 6b).
4. Discussion This study provided evidence to support the hypothesis that anoxia/reoxygenation induces the reduction of E-cadherin expression in human colon adenocarcinoma cell lines. The reduction of E-cadherin on cancer cell surfaces causes the release
Fig. 6. Effect of inhibitors of transcription (ActD) or NF-kB activation (proteasome inhibitor) on E-cadherin expression after anoxia/reoxygenation. (a) Effect of ActD (2 mg/ml). (b) Effect of lactacyctin (10 mM) or MG132 (10 mM). Responses are shown for E-cadherin expression at 22 h reoxygenation after anoxia (2 h). Each value indicates mean ^ SE of three experiments. * P , 0:05 compared with normoxia. #p , 0:05 compared with anoxia/reoxygenation.
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the development of metastasis [7]. Anoxia can induce the expression of several adhesion molecules on epithelial cells, endothelial cells, and cancer cells. Many studies have indicated that the expression of adhesion molecules is important for tumor invasion and metastasis in colorectal cancer [12 – 15]. The loss of E-cadherin expression might be related to the invasive capacity as well as metastatic potential of cancer cells [16]. Although, the role of E-cadherin in invasion and metastasis is relatively well known, the present knowledge of the detailed mechanisms by which E-cadherin is down-regulated remains incomplete. Previous studies have demonstrated that downregulation of E-cadherin expression is associated with differentiation grade and metastasis in human colorectal carcinomas [17] and squamous cell carcinomas of the head and neck [18]. Expression of E-cadherin has been studied in tissue sections from a variety of differentiation grades, and lower levels of expression were observed in more poorly differentiated cancers [19]. The down-regulation of E-cadherin is not necessarily a causative factor of metastasis to the liver. For example, differentiated gastric cancers, which express high amounts of E-cadherin, often metastasize to the liver [20]. In addition, welldifferentiated colorectal carcinomas frequently develop liver metastasis. However, at the initial step of metastasis, it is necessary for cancer cells to detach from the primary tumor. Furthermore, the downregulation of E-cadherin expression may be essential for cancer cells to detach from the primary tumor [11]. Thus, the transient down-regulation of E-cadherin, by which cancer cells acquire their invasive property, must be organized by some mechanism at the primary site in the process of liver metastasis. In this study, the expression of E-cadherin was significantly reduced during reoxygenation from 4 to 22 h. Western blots of HT-29 colon carcinoma cells exposed to normoxia or anoxia revealed two different patterns of downregulation, depending on the duration of reoxygenation. Four hours of reoxygenation down-regulated the expression of E-cadherin, demonstrated by immunofluorescent staining and enzyme-linked immunosorbent assays. However, no loss in the total amount of E-cadherin was detectable by Western blotting after 4 h of reoxygenation, which indicates that the reduction in E-cadherin expression is probably the result of the internalization of surface-accessible
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E-cadherin. Conversely, the Western blots revealed a clear reduction in the total amount of E-cadherin after 22 h of reoxygenation, suggesting that the loss of E-cadherin is not only the result of the internalization; however, this additional mechanism is unclear. Our real time amplification study revealed that the levels of E-cadherin mRNA expression did not differ between normoxia-exposed HT-29 cells and anoxia/reoxygenation-exposed HT-29 cells. Therefore, the difference in the total amounts of E-cadherin protein might be due to post-translational modifications. This is supported by the previously reported results of Nakayama et al., which showed that E-cadherin expression was decreased in highly metastatic cell lines [21], whereas the E-cadherin mRNA levels did not differ between highly metastatic cell lines and weakly metastatic cell lines [22]. Taken together with another previous report [6], our immunofluorescent staining and Western blots studies suggest that E-cadherin on the cell surface is internalized 4 h after reoxygenation, and degraded in response to further reoxygenation (22 h). However, the present results do not eliminate other possibilities such as an extracellular cleavage of E-cadherin. Insights into the molecular processes that regulate the transcription-dependent alterations in HT-29 cells following anoxia/reoxygenation may be provided by previous studies of cytokine-, oxidants, and anoxia/ reoxygenation-induced cancer cell or endothelial cell responses [23,10]. These studies have revealed that the oxidant stress associated with anoxia/reoxygenation results in a transcription-dependent expression of E-selection, VCAM-1, ICAM-1, and anti-apoptotic proteins. Furthermore, it appears that the activation of nuclear factor NF-kB is a critical event in the oxidantinduced, transcription-dependent expression of endothelial cell adhesion molecules and anti-apoptotic proteins. These observations raise the possibility that other proteins that are also under the control of this signaling pathway may contribute to the transcriptiondependent down-regulation of E-cadherin. Our data demonstrates that treatment of HT-29 cells with inhibitor of transcription or reagents that interfere with the activation of NF-kB significantly attenuated the anoxia/reoxygenation-induced down-regulation of E-cadherin. The mechanism by which anoxia/reoxygenation modulates NF-kB dependent down regulation of E-cadherin is unclear. One possibility may be that
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nitric oxide (NO) induces the degradation of E-cadherin. The transcription-dependent synthesis of inducible NO synthase (iNOS) is known to be regulated by NF-kB [24]. Further support for a role of NO in the degradation of E-cadherin is provided by a recent report that describes beta-catenin as an important target of NO [25]. But in this study, we do not investigate the involvement of NO in the mechanism by which decrease of E-cadherin occurs. Further research is needed to identify the mechanisms by which anoxia/reoxygenation downregulates E-cadherin. Our results indicated that the anoxia/reoxygenation-induced reduction of E-cadherin expression is transient. Our immunofluorescent staining and enzyme-linked immunosorbent assays revealed that the expression of E-cadherin and the HT-29 cell-cell adhesion were recovered 46 h after reoxygenation. Osada et al. reported that E-cadherin participates in secondary tumor formation in the liver [26]. They speculated that increased cell-cell adhesion causes self-aggregation of cancer cells and promotes tumor formation in the liver. This scenario is supported by a previous report from Mayer et al., which shows by immunohistochemical analysis that all liver metastases of gastric cancers showed a high frequency of strongly E-cadherin-positive cells, regardless of the staining pattern in the primary cancer [27]. To initiate metastasis, cancer cells have to become detached from the primary cancer, and in this step downregulation of E-cadherin is known to play an important role. However, primary colorectal cancers and their liver metastatic nodules often show a welldifferentiated histology and retain their E-cadherin expression. Differentiated human gastric cancer, which retains E-cadherin expression, often metastasizes to the liver, whereas undifferentiated gastric cancers, which show reduced amounts of E-cadherin, often show intraperitoneal dissemination and lymphogenous metastasis but not liver metastasis. These findings suggest that in the process of liver metastasis, the transient down-regulation of the E-cadherin, by which cancer cells acquire their invasive property, is organized by some mechanism at the primary site. In the liver, E-cadherin, which is down-regulated at the primary site, might become recovered and thus play a important role in secondary cancer formation.
In our present experimental model with the human colon adenocarcinoma cell lines, we found that anoxia/reoxygenation induces a transient reduction in E-cadherin expression, which may promote metastasis.
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