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Signaling and regulatory mechanisms of integrin ανβ6 on the apoptosis of colon cancer cells
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Cancer Letters 266 (2008) 209–215 www.elsevier.com/locate/canlet
Signaling and regulatory mechanisms of integrin amb6 on the apoptosis of colon cancer cells Zhang Zhao-yang a,1, Xu Ke-sen a,1, He Qing-si a,1, Niu Wei-bo a, Wang Jia-yong a, Mi Yue-tang a, Wang Jin-shen b, Wang Guo-qiang c, Yang Guang-yun a, Niu Jun a,* a
Department of General Surgery, QiLu Hospital of Shandong University, Jinan 250012, Shandong, China b Department of General Surgery of Shandong Provincial Hospital, Jinan 250001, Shandong, China c Shandong Medicinal Biotechnology Center, Jinan 250062, Shandong, China Received 22 December 2007; received in revised form 21 February 2008; accepted 22 February 2008
Abstract Considerable researches have been done about integrin amb6 and carcinomas, but little information has been shown about the relationship between integrin amb6 and apoptosis. In this study, we investigated the apoptosis and its related signal pathways to integrin avb6 in colon cancer cells. After we blocked the function of integrin avb6 in HT29 cells used the monoclonal antibody, the apoptotic cells increased markedly. Meanwhile, cytochrome C released from mitochondria into cytosol, Bcl-2 decreased while Bax increased significantly, and Fas and Fas-ligand had no change. The activities of caspase-3 and caspase-9 increased, while caspase-8 remained no change. Moreover, the expression of phosphorylated extracellular signal-related kinase (P-ERK) decreased. We confirmed that integrin avb6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involved the mitochondrial pathway. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Integrin amb6; Apoptosis; Colon cancer
1. Introduction Adhesion to extracellular matrix (ECM) is necessary for the survival of epithelial cells. Inadequate or inappropriate cell–ECM interactions result in apoptosis, a phenomenon known as anoikis [1,2]. These interactions are dependent on the cell surface receptors. Integrins are a group of cell adhesion *
Corresponding author. Tel: +86 531 82169203; fax: +86 531 82169203. E-mail address: [email protected] (N. Jun). 1 The first three authors contributed equally to this manuscript.
molecules consisting of two non-covalently bound transmembrane subunits (a and b) [3], and integrin-mediated adhesion to ECM triggers intracellular signaling pathways to modulate cell proliferation, morphology, migration, invasion, and survival [4]. Although several different integrins can promote cell survival, specific integrin–ligand interactions may be required to inhibit anoikis in a distinct cell type [5]. Among the various families of cell adhesion molecules, integrin expression patterns appear to be directly implicated in the progression of malignant disease [6]. The avb6 integrin does not express itself
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within the normal epithelial tissues [7], but only in those that have undergone malignant transformation [8]. Research has confirmed that heterologous expression of avb6 in colon cancer cells promotes tumor cell growth in vitro and in vivo [9], moreover, avb6 expression in colon cancer cells leads to increased gelatinase B secretion in a protein kinase C (PKC)-dependent manner [10,11], and that integrin avb6 upregulates its own expression via PKCmediated signaling as tumor cells become crowded through a system of integrin autoregulation [12]. However, little information has been shown about the relationship between integrin avb6 and apoptosis. Inhibition of av integrin activity by monoclonal antibodies (mAbs), cyclic RGD peptide antagonists, and peptidomimetics has been shown to induce endothelial apoptosis, inhibit angiogenesis, and increase endothelial monolayer permeability [13,14]. Silencing av integrin with siRNA resulted in apoptosis in breast cancer cells [15]. But either silencing or inhibiting of av integrin will probably reduce the expression level or block the function of integrin avb1, avb3, avb5, avb6, and avb8, so they have a more broad-spectrum anti-integrin effect. In this study, we used the monoclonal antibody against integrin avb6 to block the function of avb6 integrin so as to investigate the role of integrin avb6 in apoptosis of colon cancer cells and the related mechanisms in a more specific pathway.
4.5 g/l of glucose) (Sigma, USA) containing 10% heatinactivated foetal calf serum (FCS) (Sigma, USA) and supplemented with 20 mM HEPES, 100 IU/ml penicillin and 100 lg/ml streptomycin (Merck, Germany). The cells were incubated in 37 °C, 5% CO2, and saturated humidity. 2.3. Effects of antibody For treatment of integrin avb6 antibody, cells were harvested, rinsed with PBS and re-suspended in serumfree DMEM, then added mAb 10D5 into the culture solution at the final concentration of 0.1 mg/ml and incubated for 30 min on ice for the sufficient combination of mAb and antigen. Negative controls were performed with the identical concentration of mouse immunoglobulins IgG2a (Dako). Then the cells were plated on fibronectin-coated chamber slides with standard medium in the density of 40,000/cm2. After 6 h incubation, the cells were rinsed with PBS to stop the reaction. 2.4. Cell proliferation assay Cell viability was determined by measurement of the cellular metabolism of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. In brief, cells were incubated with MTT at 5 mg/ml for 4 h at 37 °C. The medium was then aspirated and dimethyl sulfoxide (DMSO) (Sigma, USA) was used to dissolve the crystals. Absorbance was measured at 570 nm in a Microplate Reader (Bio-Tech Instruments, USA). 2.5. Detection of apoptosis by Hoechst 33258
2. Materials and methods 2.1. Antibodies and reagents The mouse-anti-human monoclonal antibody 10D5(IgG2a) against integrin avb6 were obtained from Chemicon (Temecula, CA, USA). Antibodies against Bcl-2, Bax, Fas, Fas-ligand, and P-ERK were purchased from Biosource (California, USA), mouse immunoglobulins IgG2a from DAKO (Copenhagen, Denmark), and reagents for SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular weight markers from BioRad Laboratories (Hercules, CA, USA). Cytochrome C Apoptosis Assay Kit was purchased from Biovision (California, USA), and Hoechst 33258 Cell Apoptosis Assay Kit from Keygentec (Nanjing, China). 2.2. Cell line and culture conditions The human colon cancer cell lines HT29 was obtained from American Type Culture Collection (ATCC), and maintained as monolayers in standard medium comprising Dulbecco’s modified Eagle’s medium (DMEM:
After the cells had been treated with 10D5 for 6 h, the slides were washed twice with iced PBS, and then be fixed with 4% (w/v) paraformaldehyde for 10 min at 4 °C following twice washing with PBS. Then Hoechst 33258 fluorescent dye was added to the slides and incubated for 10 min at room temperature. Slides were then washed twice with PBS and examined under a fluorescence microscope (Nikon, Japan). Apoptotic feature was assessed by observing chromatin condensation and fragments staining by Hoechst 33258. In each case 10 random fields and more than 500 cells were counted. 2.6. Caspase-3, 8, 9 activity assay Caspase activity assay was carried out using a fluorometric protease assay kit (Biovision, USA) following the instructions provided by the manufacturer. In brief, cells were homogenized on ice with kit-provided lysis buffer. An aliquot of 50 ll of supernatants was incubated with an equal volume of the reaction buffer containing fluorogenic peptide substrate at 37 °C for 1–2 h. Enzymatic release of free fluorogenic moiety was measured by a fluorometer.
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2.7. Preparation of whole cell protein lysate The cells were lysed in 500 ll of extraction buffer (100 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100, 0.1% SDS, 0.1% NP-40, 1 mM vanadate, 1 lg/ml pepstatin, 1 mM PMSF, 5 lg/ml aprotinin, and 1 lg/ml of leupeptin) for 30 min on ice. The extracts were cleared by centrifugation for 20 min at 10,000g. 2.8. Extraction of cytosol and mitochondria lysate Extraction of cytosol and mitochondria lysate was practiced using the cytochrome C Apoptosis Assay Kit. According to the directions of the manufacturer, cells were suspended in kit-provided buffer for 30 min on ice, and then the cells were sonicated five times during this period. After centrifugation 10 min at 3500g, the supernatants were collected and were further centrifuged for 15 min at 1400g. The supernatant was the cytosol lysate, and the pellet was suspended in protein extraction buffer and centrifuged to obtain the mitochondrial lysate. 2.9. Western blotting Proteins of the cells were normalized to 30 lg/lane and were electrophoresed using SDS–PAGE, after which the proteins were transferred to nitrocellulose membranes (Amersham–Pharmacia Biotech, UK). The membranes were probed with the first antibody (1:1000) overnight at 4 °C followed by peroxidase-labelled secondary antibodies. Immunoreactive bands were visualized using DAB method, and the optical density was analysis with the Scion image software. 2.10. Statistical analysis Data were reported as the means ± SD and were the representative of an average of at least three independent experiments. Statistical comparisons were made by t test and One-Way ANOVA, and P<0.01 was considered statistically significant. 3. Results 3.1. Antibody against integrin avb6 induced apoptosis in the cells The MTT assay showed (Fig. 1A) that after incubation for 6 h together with mAb 10D5, the survival rate of HT29 cells decreased to (73.83 ± 2.65)% compared to the control cells, while in the cells treated with IgG2a, it was similar to the control cells. Hoechst 33258 staining showed that there were significant morphological changes in the nuclear chromatin (Fig. 1B). In the control and IgG2a treated groups, the nuclei were stained a less bright blue and the color was homogeneous, while in the 10D5 treated group, the
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blue emission light in apoptotic cells was much bright than the control cells. Condensed chromatin could also be found in part of the treated cells and some of them formed the structure of apoptotic bodies, which is one of the classic characteristics of apoptotic cells. The percentage that apoptotic cells shared in the counted fields in control, IgG2a and mAb 10D5 treated cells of HT29 were (1.39 ± 0.74)%, (1.22 ± 0.81)%, and (23.60 ± 1.92)%, respectively. By comparison, we can see that the apoptotic rate in mAb 10D5 treated cells are different from those in the control and IgG2a treated cells (P < 0.01). 3.2. Antibody against integrin avb6 upregulated the activity of caspase-3 and caspase-9, but had no effect on caspase-8 The results of caspase activity assay showed that after blocking the function of integrin amb6, the activities of caspase-3 and caspase-9 increased markedly (P < 0.0001 vs the control), while the activity of caspase-8 remained no change (P > 0.05 vs the control). In the IgG2a treated cells, there was no obvious change (P > 0.05 vs the control) (Fig. 1C). The results of Western blotting indicated that the changes of the expressions of caspase-3, 8, 9 were coincident to the changes of their activity (Fig. 1D). 3.3. Antibody against integrin avb6 induced cytochrome C release from mitochondria The results of Western blotting indicated that, comparing to the control cells, the treatment of mAb 10D5 resulted in a significant shift of cytochrome C from the mitochondrial fraction to the cytosolic fraction of cell extracts (P < 0.01 vs the control), while in the IgG2a treated cells, there was no change (P > 0.05 vs the control) (Fig. 2). 3.4. Antibody against integrin avb6 upregulated Bax and downregulated Bcl-2 in HT29 cells, but had no change on Fas and Fas-ligand Fig. 3 indicated that after the treatment of mAb 10D5, the expression of Bax increased, but the expression of Bcl2 decreased, comparing to those in the control cells, the difference is significant (P<0.01 vs the control). While the expressions of Fas and Fas-ligand had no obvious change (P > 0.05). In the IgG2a treated cells, there was no change in the expression of Bax, Bcl-2, Fas, and Fasligand (P > 0.05 vs the control). 3.5. Antibody against integrin avb6 downregulated P-ERK expression Fig. 3 indicated that after the treatment of mAb 10D5, the expression of P-ERK1/2 decreased, comparing to those in the control and IgG2a treated cells, the difference is significant (P < 0.01).
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Fig. 1. Monoclonal antibody 10D5 induced apoptosis in HT29 cells. *P < 0.01 vs control and IgG2a treated cells. (A) Cell survival was determined by MTT assay. Values are expressed as a percentage of each control. (B) Detection of apoptosis by Hoechst 33258. Values are expressed as a percentage of apoptotic cells in the counted fields. (C) Caspase activity assay. Values are expressed as a fold of each control. (D) Caspase expressions were assayed by Western blotting. Values are expressed as a fold of b-actin.
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Fig. 2. Cytochrome C distribution was assayed by Western blotting. Values are expressed as a fold of the loading control, respectively. * P < 0.01 vs control and IgG2a treated cells.
Fig. 3. Results of Western blotting for the expression of Bcl-2, Bax, Fas, Fas-ligand, and P-ERK. Values are expressed as a fold of b-actin. * P < 0.01 vs control and IgG2a treated cells.
4. Discussion The aim of this study was to investigate the role of integrin amb6 in apoptosis of colon cancer cells.
We found that after blocking the function of integrin amb6, the percentage of the apoptotic cells increased obviously. In a reverse way, we confirmed that integrin avb6 acted as an important role in
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inhibiting apoptosis in colon cancer cells. Moreover, we found that after blocking the function of integrin amb6, the expression of Bax raised while Bcl-2 reduced, and that more cytochrome C released into the cytoplasm. Meanwhile the Fas and Fas-ligand expressions did not change. As to the caspase activities, caspase-8 remained no change, while those of caspase-3 and caspase-9 increased markedly. The results suggested that the mitochondrial pathway may play a major role for the apoptosis of HT29 cells induced by blocking the function of integrin amb6. Integrin has been reported to have either pro-survival or pro-apoptotic roles in the regulation of cell survival [16]. It has been reported that integrin a3b1 is necessary for the prevention of apoptosis in the culture rat potocytes [17], and promotes apoptosis in T cells and mammal epithelial cells [18,19]. Integrin a5b1 delays apoptosis of Ntera2 neuronal cells [20], and integrin amb3 promotes apoptosis in endothelial and non-small-cell lung cancer cells [21]. But the relationship between integrin amb6 and apoptosis has not been clear. In the present study we confirmed that integrin amb6 had an important role of inhibiting apoptosis in colon cancer cells. Apoptosis signals can be activated by various stimuli and converge toward a common death pathway [17]. Two major pathways are included in regulating apoptosis: the death receptor pathway and the mitochondrial pathway. The death receptor pathway is usually found to cause apoptosis in some types of epithelial and endothelial cells [22,23], and the mitochondrial pathway has also been demonstrated to involve apoptosis of epithelial cells [24]. Several components of both pathways have been shown to be regulated by integrins [16]. In this study, we confirmed that mitochondrial pathway may play a major role for the apoptosis of HT29 cells induced by blocking the function of integrin amb6. Activation of ERK pathway plays an important role in the response of many cell types to extracellular stimuli. Activated ERK can phosphorylate several transcription factor targets and thus alter the pattern of gene transcription. Several reports showed that activation of ERK signaling promotes adhesion-dependent cell survival in several cell types [17]. In our previous study [25] we have confirmed that there is a direct integrin amb6-ERK binding, and that amb6 serves to direct growth factor-activated ERK to downstream cytoplasmic targets involved in regulating cell growth and/or cytoskeletal reorganization. Activation of ERK by serum-
derived growth factors is greatly amplified in cancer cells expressing amb6. Whether non-phosphorylated ERK, when bound to b6, is more efficiently phosphorylated at that location because of conformational changes at the phosphorylation lip of ERK remains unknown. It is also possible that activated ERK, when bound to b6, is protected from deactivation by cellular phosphatases. In the present study, we found that after blocking integrin amb6 with mAb 10D5, the expression of P-ERK1/2 decreased significantly, which is a positive support for our previous study above. We supposed that when integrin amb6 was blocked by the monoclonal antibody, the effects related to it as described above were also be blocked, and the cells took on apoptosis because of losing the grow-activation from the integrin amb6-ERK binding. Meanwhile, this study indicates that ERK activation has anti-apoptosis effect in colon cancer cells, which is agreed with the study of Bijian [26] that demonstrated ERK activation-mediated podocytes survival; but disagreed with the study of Tang [27] who demonstrated that ERK activation contributed to either cell cycle arrest or apoptosis. We think that the reason of the difference is that the cells and the stimulus to induce apoptosis are different. These indicate that activation of ERK may have either anti-apoptotic or pro-apoptotic effects, depending on the kinds of insult and the cell type. In our later study, we will do more work to investigate the relationship between integrin amb6, ERK and apoptosis. In conclusion, we confirmed in this study that integrin amb6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involves the mitochondrial pathways. In addition, ERK activation has anti-apoptotic effect in colon cancer cells. Moreover, an important potential consequence of our work is that amb6 must be an attractive therapeutic candidate for colon cancer. Acknowledgements This study was supported by Research Grants (No. 30570833) from National Natural Sciences Foundation of China, and Research Grants (No. Y2005C42) from Natural Sciences Foundation of Shandong Province. References [1] S.M. Frisch, E. Ruoslahti, Integrins and anoikis, Curr. Opin. Cell Biol. 9 (1997) 701–706.
Z. Zhao-yang et al. / Cancer Letters 266 (2008) 209–215 [2] S.M. Frisch, R.A. Screaton, Anoikis mechanisms, Curr. Opin. Cell Biol. 13 (2001) 555–562. [3] R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, Cell 110 (2002) 673–687. [4] J.D. Hood, D.A. Cheresh, Role of integrins in cell invasion and migration, Nat. Rev. Cancer 2 (2002) 91–100. [5] Chien-An, Jer-Chia Tsai, Pin-Wen Su, Yung-Hsiung Lai, Hung-Chun Chen, Signaling and regulatory mechanisms of integrin a3b1 on the apoptosis of cultured rat podocytes, J. Lab. Clin. Med. 147 (2006) 274–280. [6] G. Mizejiwski, Role of integrins in cancer: survey of expression patterns, Proc. Soc. Biol. Med. 222 (1999) 124– 138. [7] N. Dalvi, G.J. Thornas, J.F. Marshall, M. Morgan, R. Bass, V. Ellis, P.M. Speight, S.A. Whawell, Modulation of the urokinase-type plasminogen activator receptor by the beta 6 integrin subunit, Biochem. Biophys. Res. Commun. 317 (2004) 92–99. [8] K. Arihero, M. Kaneko, S. Fujii, K. Inai, Y. Yokosaki, Significance of alpha 9 beta 1 and alpha m beta 6 integrin expression in breast carcinoma, Breast Cancer 7 (2000) 19– 26. [9] M.V. Agrez, A. Chen, R.I. Cone, R. Pytela, D. Sheppard, The avb6 integrin promotes proliferation of colon carcinoma cells through a unique region of the b6 cytoplasmic domain, J. Cell Biol. 127 (1994) 547–556. [10] J. Niu, X. Gu, J. Turton, C. Meldrum, E.W.W. Howard, M.V. Agrez, Integrin-mediated signaling of gelatinase B secretion in colon cancer cells, Biochem. Biophys. Res. Commun. 249 (1998) 287–291. [11] M.V. Agrez, X. Gu, J. Turton, C. Meldrum, J. Niu, T. Antalis, W.E. Howard, The avb6 integrin induces gelatinase B secretion in colon cancer cells, Int. J. Cancer 81 (1999) 90–97. [12] J. Niu, X. Gu, N. Ahmed, S. Andrews, J. Turton, R. Bates, M. Agrez, The avb6 integrin regulating its own expression with cell crowding: implications for tumor progression, Int. J. Cancer 92 (2001) 40–48. [13] C.C. Kumar, Integrin avb3 as a therapeutic target for blocking tumor induced angiogenesis, Curr. Drug Targets 4 (2003) 123–131. [14] R. Qiao, W. Yan, H. Lum, A.B. Malik, Arg-Gly-Asp peptide increases endothelial hydraulic conductivity: comparison with thrombin response, Am. J. Physiol. 269 (1995) C110– C117. [15] Qizhen Cao, Weibo Cai, Tianfang Li, Yong Yang, Kai Chen, Lei Xing, Xiaoyuan Chen, Combination of integrin siRNA and irradiation for breast cancer therapy, Biochem. Biophys. Res. Commun. 351 (2006) 726–732.
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[16] D.G. Stupack, D.A. Cheresh, Get a ligand, get a life: integrins, signaling and cell survival, J. Cell Sci. 115 (2002) 3729–3738. [17] Chien-an Chen, Jer-chia Tsai, Pin-wen Su, Yung-hsiung Lai, Hung-chun Chen, Signaling and regulatory mechanisms of integrin a3b1 on the apoptosis of cultured rat podocytes, J. Lab. Clin. Med. 147 (2005) 274–280. [18] K. Sato, K. Katagiri, S. Hattori, T. Tsuji, T. Irimura, S. Irie, Laminin 5 promotes activation and apoptosis of the T cells expressing alpha3beta1 integrin, Exp. Cell Res. 247 (1999) 451–460. [19] V.L. Seewaldt, K. Mrozek, R. Sigle, E.C. Dietze, K. Heine, D.M. Hockenbery, Suppression of p53 function in normal human mammary epithelial cells increases sensitivity to extracellular matrix-induced apoptosis, J. Cell Biol. 155 (2001) 471–486. [20] Rosemary M. Gibson, Susan E. Craig, Laura Heenan, Cathy Tournier, Martin J. Humphries, Activation of integrin a5b1 delays apoptosis of Ntera2 neuronal cells, Mol. Cell. Neurosci. 28 (2005) 588–598. [21] M. Jeffrey, B.S. Albert, Carolyn Cao, Ling Geng, Lauren Leavitt, Dennis E. Hallahan, Bo Lu, Integrin avb3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models, Int. J. Radiat. Oncol. 65 (2006) 1536–1543. [22] M. Rytomaa, L.M. Martins, J. Downward, Involvement of FADD and caspase-8 signaling in detachment-induced apoptosis, Curr. Biol. 9 (1999) 1043–1046. [23] S.M. Frish, Evidence for a function of death-receptorrelated, death-domain-containing proteins in anoikis, Curr. Biol. 9 (1999) 1047–1049. [24] A.P. Gilmore, A.D. Metcalfe, L.H. Romer, C.H. Streuli, Integrin mediated survival signals regulate the apoptotic function of Bax through its conformation and subcellular localization, J. Cell Biol. 149 (2000) 431–445. [25] Nuzhat Ahmed, Jun Niu, Douglas J. Dorahy, Xinhua Gu, Sarah Andrews, Cliff J. Meldrum, Rodney J. Scott, Mark S. Baker, Ian G. Macreadie, Michael V. Agrez, Direct integrin amb6-ERK binding: implication for tumor growth, Oncogene 21 (2002) 1370–1380. [26] K. Bijian, T. Takano, J. Papillon, A. Khadir, A.V. Cybulsky, Extracellular matrix regulates glomerular epithelial cell survival and proliferation, Am. J. Physiol. Renal Physiol. 286 (2004) F255–F266. [27] D. Tang, D. Wu, A. Hirao, J.M. Lahti, L. Liu, B. Mazza, ERK activation mediate cell cycle arrest and apoptosis after DNA damage independently of p53, J. Biol. Chem. 277 (2002) 12710–12717.
Cancer Letters 266 (2008) 209–215 www.elsevier.com/locate/canlet
Signaling and regulatory mechanisms of integrin amb6 on the apoptosis of colon cancer cells Zhang Zhao-yang a,1, Xu Ke-sen a,1, He Qing-si a,1, Niu Wei-bo a, Wang Jia-yong a, Mi Yue-tang a, Wang Jin-shen b, Wang Guo-qiang c, Yang Guang-yun a, Niu Jun a,* a
Department of General Surgery, QiLu Hospital of Shandong University, Jinan 250012, Shandong, China b Department of General Surgery of Shandong Provincial Hospital, Jinan 250001, Shandong, China c Shandong Medicinal Biotechnology Center, Jinan 250062, Shandong, China Received 22 December 2007; received in revised form 21 February 2008; accepted 22 February 2008
Abstract Considerable researches have been done about integrin amb6 and carcinomas, but little information has been shown about the relationship between integrin amb6 and apoptosis. In this study, we investigated the apoptosis and its related signal pathways to integrin avb6 in colon cancer cells. After we blocked the function of integrin avb6 in HT29 cells used the monoclonal antibody, the apoptotic cells increased markedly. Meanwhile, cytochrome C released from mitochondria into cytosol, Bcl-2 decreased while Bax increased significantly, and Fas and Fas-ligand had no change. The activities of caspase-3 and caspase-9 increased, while caspase-8 remained no change. Moreover, the expression of phosphorylated extracellular signal-related kinase (P-ERK) decreased. We confirmed that integrin avb6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involved the mitochondrial pathway. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Integrin amb6; Apoptosis; Colon cancer
1. Introduction Adhesion to extracellular matrix (ECM) is necessary for the survival of epithelial cells. Inadequate or inappropriate cell–ECM interactions result in apoptosis, a phenomenon known as anoikis [1,2]. These interactions are dependent on the cell surface receptors. Integrins are a group of cell adhesion *
Corresponding author. Tel: +86 531 82169203; fax: +86 531 82169203. E-mail address: [email protected] (N. Jun). 1 The first three authors contributed equally to this manuscript.
molecules consisting of two non-covalently bound transmembrane subunits (a and b) [3], and integrin-mediated adhesion to ECM triggers intracellular signaling pathways to modulate cell proliferation, morphology, migration, invasion, and survival [4]. Although several different integrins can promote cell survival, specific integrin–ligand interactions may be required to inhibit anoikis in a distinct cell type [5]. Among the various families of cell adhesion molecules, integrin expression patterns appear to be directly implicated in the progression of malignant disease [6]. The avb6 integrin does not express itself
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within the normal epithelial tissues [7], but only in those that have undergone malignant transformation [8]. Research has confirmed that heterologous expression of avb6 in colon cancer cells promotes tumor cell growth in vitro and in vivo [9], moreover, avb6 expression in colon cancer cells leads to increased gelatinase B secretion in a protein kinase C (PKC)-dependent manner [10,11], and that integrin avb6 upregulates its own expression via PKCmediated signaling as tumor cells become crowded through a system of integrin autoregulation [12]. However, little information has been shown about the relationship between integrin avb6 and apoptosis. Inhibition of av integrin activity by monoclonal antibodies (mAbs), cyclic RGD peptide antagonists, and peptidomimetics has been shown to induce endothelial apoptosis, inhibit angiogenesis, and increase endothelial monolayer permeability [13,14]. Silencing av integrin with siRNA resulted in apoptosis in breast cancer cells [15]. But either silencing or inhibiting of av integrin will probably reduce the expression level or block the function of integrin avb1, avb3, avb5, avb6, and avb8, so they have a more broad-spectrum anti-integrin effect. In this study, we used the monoclonal antibody against integrin avb6 to block the function of avb6 integrin so as to investigate the role of integrin avb6 in apoptosis of colon cancer cells and the related mechanisms in a more specific pathway.
4.5 g/l of glucose) (Sigma, USA) containing 10% heatinactivated foetal calf serum (FCS) (Sigma, USA) and supplemented with 20 mM HEPES, 100 IU/ml penicillin and 100 lg/ml streptomycin (Merck, Germany). The cells were incubated in 37 °C, 5% CO2, and saturated humidity. 2.3. Effects of antibody For treatment of integrin avb6 antibody, cells were harvested, rinsed with PBS and re-suspended in serumfree DMEM, then added mAb 10D5 into the culture solution at the final concentration of 0.1 mg/ml and incubated for 30 min on ice for the sufficient combination of mAb and antigen. Negative controls were performed with the identical concentration of mouse immunoglobulins IgG2a (Dako). Then the cells were plated on fibronectin-coated chamber slides with standard medium in the density of 40,000/cm2. After 6 h incubation, the cells were rinsed with PBS to stop the reaction. 2.4. Cell proliferation assay Cell viability was determined by measurement of the cellular metabolism of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. In brief, cells were incubated with MTT at 5 mg/ml for 4 h at 37 °C. The medium was then aspirated and dimethyl sulfoxide (DMSO) (Sigma, USA) was used to dissolve the crystals. Absorbance was measured at 570 nm in a Microplate Reader (Bio-Tech Instruments, USA). 2.5. Detection of apoptosis by Hoechst 33258
2. Materials and methods 2.1. Antibodies and reagents The mouse-anti-human monoclonal antibody 10D5(IgG2a) against integrin avb6 were obtained from Chemicon (Temecula, CA, USA). Antibodies against Bcl-2, Bax, Fas, Fas-ligand, and P-ERK were purchased from Biosource (California, USA), mouse immunoglobulins IgG2a from DAKO (Copenhagen, Denmark), and reagents for SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular weight markers from BioRad Laboratories (Hercules, CA, USA). Cytochrome C Apoptosis Assay Kit was purchased from Biovision (California, USA), and Hoechst 33258 Cell Apoptosis Assay Kit from Keygentec (Nanjing, China). 2.2. Cell line and culture conditions The human colon cancer cell lines HT29 was obtained from American Type Culture Collection (ATCC), and maintained as monolayers in standard medium comprising Dulbecco’s modified Eagle’s medium (DMEM:
After the cells had been treated with 10D5 for 6 h, the slides were washed twice with iced PBS, and then be fixed with 4% (w/v) paraformaldehyde for 10 min at 4 °C following twice washing with PBS. Then Hoechst 33258 fluorescent dye was added to the slides and incubated for 10 min at room temperature. Slides were then washed twice with PBS and examined under a fluorescence microscope (Nikon, Japan). Apoptotic feature was assessed by observing chromatin condensation and fragments staining by Hoechst 33258. In each case 10 random fields and more than 500 cells were counted. 2.6. Caspase-3, 8, 9 activity assay Caspase activity assay was carried out using a fluorometric protease assay kit (Biovision, USA) following the instructions provided by the manufacturer. In brief, cells were homogenized on ice with kit-provided lysis buffer. An aliquot of 50 ll of supernatants was incubated with an equal volume of the reaction buffer containing fluorogenic peptide substrate at 37 °C for 1–2 h. Enzymatic release of free fluorogenic moiety was measured by a fluorometer.
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2.7. Preparation of whole cell protein lysate The cells were lysed in 500 ll of extraction buffer (100 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100, 0.1% SDS, 0.1% NP-40, 1 mM vanadate, 1 lg/ml pepstatin, 1 mM PMSF, 5 lg/ml aprotinin, and 1 lg/ml of leupeptin) for 30 min on ice. The extracts were cleared by centrifugation for 20 min at 10,000g. 2.8. Extraction of cytosol and mitochondria lysate Extraction of cytosol and mitochondria lysate was practiced using the cytochrome C Apoptosis Assay Kit. According to the directions of the manufacturer, cells were suspended in kit-provided buffer for 30 min on ice, and then the cells were sonicated five times during this period. After centrifugation 10 min at 3500g, the supernatants were collected and were further centrifuged for 15 min at 1400g. The supernatant was the cytosol lysate, and the pellet was suspended in protein extraction buffer and centrifuged to obtain the mitochondrial lysate. 2.9. Western blotting Proteins of the cells were normalized to 30 lg/lane and were electrophoresed using SDS–PAGE, after which the proteins were transferred to nitrocellulose membranes (Amersham–Pharmacia Biotech, UK). The membranes were probed with the first antibody (1:1000) overnight at 4 °C followed by peroxidase-labelled secondary antibodies. Immunoreactive bands were visualized using DAB method, and the optical density was analysis with the Scion image software. 2.10. Statistical analysis Data were reported as the means ± SD and were the representative of an average of at least three independent experiments. Statistical comparisons were made by t test and One-Way ANOVA, and P<0.01 was considered statistically significant. 3. Results 3.1. Antibody against integrin avb6 induced apoptosis in the cells The MTT assay showed (Fig. 1A) that after incubation for 6 h together with mAb 10D5, the survival rate of HT29 cells decreased to (73.83 ± 2.65)% compared to the control cells, while in the cells treated with IgG2a, it was similar to the control cells. Hoechst 33258 staining showed that there were significant morphological changes in the nuclear chromatin (Fig. 1B). In the control and IgG2a treated groups, the nuclei were stained a less bright blue and the color was homogeneous, while in the 10D5 treated group, the
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blue emission light in apoptotic cells was much bright than the control cells. Condensed chromatin could also be found in part of the treated cells and some of them formed the structure of apoptotic bodies, which is one of the classic characteristics of apoptotic cells. The percentage that apoptotic cells shared in the counted fields in control, IgG2a and mAb 10D5 treated cells of HT29 were (1.39 ± 0.74)%, (1.22 ± 0.81)%, and (23.60 ± 1.92)%, respectively. By comparison, we can see that the apoptotic rate in mAb 10D5 treated cells are different from those in the control and IgG2a treated cells (P < 0.01). 3.2. Antibody against integrin avb6 upregulated the activity of caspase-3 and caspase-9, but had no effect on caspase-8 The results of caspase activity assay showed that after blocking the function of integrin amb6, the activities of caspase-3 and caspase-9 increased markedly (P < 0.0001 vs the control), while the activity of caspase-8 remained no change (P > 0.05 vs the control). In the IgG2a treated cells, there was no obvious change (P > 0.05 vs the control) (Fig. 1C). The results of Western blotting indicated that the changes of the expressions of caspase-3, 8, 9 were coincident to the changes of their activity (Fig. 1D). 3.3. Antibody against integrin avb6 induced cytochrome C release from mitochondria The results of Western blotting indicated that, comparing to the control cells, the treatment of mAb 10D5 resulted in a significant shift of cytochrome C from the mitochondrial fraction to the cytosolic fraction of cell extracts (P < 0.01 vs the control), while in the IgG2a treated cells, there was no change (P > 0.05 vs the control) (Fig. 2). 3.4. Antibody against integrin avb6 upregulated Bax and downregulated Bcl-2 in HT29 cells, but had no change on Fas and Fas-ligand Fig. 3 indicated that after the treatment of mAb 10D5, the expression of Bax increased, but the expression of Bcl2 decreased, comparing to those in the control cells, the difference is significant (P<0.01 vs the control). While the expressions of Fas and Fas-ligand had no obvious change (P > 0.05). In the IgG2a treated cells, there was no change in the expression of Bax, Bcl-2, Fas, and Fasligand (P > 0.05 vs the control). 3.5. Antibody against integrin avb6 downregulated P-ERK expression Fig. 3 indicated that after the treatment of mAb 10D5, the expression of P-ERK1/2 decreased, comparing to those in the control and IgG2a treated cells, the difference is significant (P < 0.01).
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Fig. 1. Monoclonal antibody 10D5 induced apoptosis in HT29 cells. *P < 0.01 vs control and IgG2a treated cells. (A) Cell survival was determined by MTT assay. Values are expressed as a percentage of each control. (B) Detection of apoptosis by Hoechst 33258. Values are expressed as a percentage of apoptotic cells in the counted fields. (C) Caspase activity assay. Values are expressed as a fold of each control. (D) Caspase expressions were assayed by Western blotting. Values are expressed as a fold of b-actin.
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Fig. 2. Cytochrome C distribution was assayed by Western blotting. Values are expressed as a fold of the loading control, respectively. * P < 0.01 vs control and IgG2a treated cells.
Fig. 3. Results of Western blotting for the expression of Bcl-2, Bax, Fas, Fas-ligand, and P-ERK. Values are expressed as a fold of b-actin. * P < 0.01 vs control and IgG2a treated cells.
4. Discussion The aim of this study was to investigate the role of integrin amb6 in apoptosis of colon cancer cells.
We found that after blocking the function of integrin amb6, the percentage of the apoptotic cells increased obviously. In a reverse way, we confirmed that integrin avb6 acted as an important role in
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inhibiting apoptosis in colon cancer cells. Moreover, we found that after blocking the function of integrin amb6, the expression of Bax raised while Bcl-2 reduced, and that more cytochrome C released into the cytoplasm. Meanwhile the Fas and Fas-ligand expressions did not change. As to the caspase activities, caspase-8 remained no change, while those of caspase-3 and caspase-9 increased markedly. The results suggested that the mitochondrial pathway may play a major role for the apoptosis of HT29 cells induced by blocking the function of integrin amb6. Integrin has been reported to have either pro-survival or pro-apoptotic roles in the regulation of cell survival [16]. It has been reported that integrin a3b1 is necessary for the prevention of apoptosis in the culture rat potocytes [17], and promotes apoptosis in T cells and mammal epithelial cells [18,19]. Integrin a5b1 delays apoptosis of Ntera2 neuronal cells [20], and integrin amb3 promotes apoptosis in endothelial and non-small-cell lung cancer cells [21]. But the relationship between integrin amb6 and apoptosis has not been clear. In the present study we confirmed that integrin amb6 had an important role of inhibiting apoptosis in colon cancer cells. Apoptosis signals can be activated by various stimuli and converge toward a common death pathway [17]. Two major pathways are included in regulating apoptosis: the death receptor pathway and the mitochondrial pathway. The death receptor pathway is usually found to cause apoptosis in some types of epithelial and endothelial cells [22,23], and the mitochondrial pathway has also been demonstrated to involve apoptosis of epithelial cells [24]. Several components of both pathways have been shown to be regulated by integrins [16]. In this study, we confirmed that mitochondrial pathway may play a major role for the apoptosis of HT29 cells induced by blocking the function of integrin amb6. Activation of ERK pathway plays an important role in the response of many cell types to extracellular stimuli. Activated ERK can phosphorylate several transcription factor targets and thus alter the pattern of gene transcription. Several reports showed that activation of ERK signaling promotes adhesion-dependent cell survival in several cell types [17]. In our previous study [25] we have confirmed that there is a direct integrin amb6-ERK binding, and that amb6 serves to direct growth factor-activated ERK to downstream cytoplasmic targets involved in regulating cell growth and/or cytoskeletal reorganization. Activation of ERK by serum-
derived growth factors is greatly amplified in cancer cells expressing amb6. Whether non-phosphorylated ERK, when bound to b6, is more efficiently phosphorylated at that location because of conformational changes at the phosphorylation lip of ERK remains unknown. It is also possible that activated ERK, when bound to b6, is protected from deactivation by cellular phosphatases. In the present study, we found that after blocking integrin amb6 with mAb 10D5, the expression of P-ERK1/2 decreased significantly, which is a positive support for our previous study above. We supposed that when integrin amb6 was blocked by the monoclonal antibody, the effects related to it as described above were also be blocked, and the cells took on apoptosis because of losing the grow-activation from the integrin amb6-ERK binding. Meanwhile, this study indicates that ERK activation has anti-apoptosis effect in colon cancer cells, which is agreed with the study of Bijian [26] that demonstrated ERK activation-mediated podocytes survival; but disagreed with the study of Tang [27] who demonstrated that ERK activation contributed to either cell cycle arrest or apoptosis. We think that the reason of the difference is that the cells and the stimulus to induce apoptosis are different. These indicate that activation of ERK may have either anti-apoptotic or pro-apoptotic effects, depending on the kinds of insult and the cell type. In our later study, we will do more work to investigate the relationship between integrin amb6, ERK and apoptosis. In conclusion, we confirmed in this study that integrin amb6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involves the mitochondrial pathways. In addition, ERK activation has anti-apoptotic effect in colon cancer cells. Moreover, an important potential consequence of our work is that amb6 must be an attractive therapeutic candidate for colon cancer. Acknowledgements This study was supported by Research Grants (No. 30570833) from National Natural Sciences Foundation of China, and Research Grants (No. Y2005C42) from Natural Sciences Foundation of Shandong Province. References [1] S.M. Frisch, E. Ruoslahti, Integrins and anoikis, Curr. Opin. Cell Biol. 9 (1997) 701–706.
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