Oncogene (2002) 21, 8347 – 8350 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc
Induction of RECK by nonsteroidal anti-inflammatory drugs in lung cancer cells Li-Teh Liu1, Hui-Chiu Chang2, Lien-Chai Chiang3 and Wen-Chun Hung*,4 1
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan; 2Department of Physiology, Kaohsiung Medical University, Kaohsiung 807, Taiwan; 3Department of Microbiology, Kaohsiung Medical University, Kaohsiung 807, Taiwan; 4School of Technology for Medical Sciences, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to exert anti-angiogenic and anti-metastatic activity both in vitro and in vivo. Block of angiogenesis and metastasis by NSAIDs has been found to be mediated partly via suppression of matrix metalloproteinase (MMP) activity. However, the molecular mechanism of this inhibitory action has not been well defined. Recent works demonstrated that a membraneanchored MMP inhibitor RECK may potently suppress MMP-2 and -9 activity to inhibit angiogenesis and metastasis in vitro and in vivo. In this study, we test the possibility that NSAIDs may up-regulate RECK to inhibit MMP activity. RT – PCR analyses showed that NS398 and aspirin up-regulated RECK mRNA level in CL-1 human lung cancer cells. Additionally, NSAIDs increased RECK protein level as detected by immunoblotting. Since RECK is a membrane-anchored glycoprotein, we also performed immunofluorescent staining to assess the expression of RECK on cell surface. Our results showed that fluorescent intensity of RECK was obviously increased after NSAID treatment. Moreover, induction of RECK by NSAIDs was associated with reduction of MMP-2 activity. We also found that NSAID-activated RECK expression might not be mediated via inhibition of cyclo-oxygenases (COXs) because addition of prostaglandin E2 (PGE2) could not counteract the effect of NSAIDs and overexpression of COX-2 could not down-regulate RECK. Taken together, our results suggest that induction of RECK expression may be one of the mechanisms by which NSAIDs suppress MMP activity to block angiogenesis and metastasis. Oncogene (2002) 21, 8347 – 8350. doi:10.1038/sj.onc. 1206017 Keywords: RECK; NSAIDs; lung cancer Matrix metalloproteinase (MMPs), a family of zincdependent endo-peptidases that selectively degrade
*Correspondence: W-C Hung, School of Technology for Medical Sciences, Kaohsiung Medical University, No 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan; E-mail:
[email protected] Received 15 July 2002; revised 3 September 2002; accepted 3 September 2002
components of the extracellular matrix, are involved not only in normal tissue remodeling but also in tumor angiogenesis and metastasis (Vu and Werb, 2000). The MMP family consists of at least 20 enzymes and may be subgrouped into different types including collagenases, stromelysins, gelatinases, matrilysin, membranetype MMPs, and metalloelastase, based on substrate specificity and sequence characteristic (Sternlicht and Werb, 2001). MMPs are synthesized as inactive precursors and are activated by proteolytic cleavage (Birkedal-Hansen, 1995). In addition, naturally occurring tissue inhibitor of metalloproteinases can bind and suppress MMP function (Ellerbroek and Stack, 1999; Brew et al., 2000). Thus, MMP activity can be regulated at three steps: (1) gene expression; (2) proenzyme processing; and (3) direct inhibition of enzymatic activity. The RECK gene was isolated as a transformation suppressor gene by using an expression cloning strategy designed to identify human cDNA inducing flat reversion in a v-Ki-ras-transformed NIH3T3 cell line (Kitayama et al., 1989; Takahashi et al., 1998). The RECK gene encoding a membrane glycoprotein that negatively regulates MMP-2 and MMP-9 activity and inhibits tumor angiogenesis and metastasis (Takahashi et al., 1998; Oh et al., 2001). While RECK mRNA is expressed in most of human tissues and untransformed cells, it is undetectable in many tumor cell lines or in cells artificially expressed active oncogenes (Takahashi et al., 1998). Clinical investigation also demonstrated that patients with high RECK protein in tumor tissues tended to show better survival and such tumors were less invasive (Furumoto et al., 2001). These data suggest that RECK is a novel suppressor gene for angiogenesis and metastasis. Recent studies showed that nonsteroidal anti-inflammatory drugs (NSAIDs) exert potent anti-angiogenic and anti-metastatic activity both in vitro and in vivo (Rozic et al., 2001; Masferrer et al., 2000). Our previous study showed that human lung cancer cells expressed high levels of MMP-2 and two NSAIDs, NS398 and indomethacin, inhibited MMP-2 expression and enzymatic activity in these cells (Pan et al., 2001). Recently, we demonstrated that these two NSAIDs suppressed MMP-2 via inhibition of gene transcription (Pan and Hung, 2002). Our study provides a possible mechanism by which NSAIDs inhibit angiogenesis and
NSAIDs activate RECK L-T Liu et al
8348
metastasis. However, in addition to direct inhibition of gene expression, NSAIDs may regulate MMP activity by different mechanisms. Therefore, we test the possibility that NSAIDs may enhance RECK expression to suppress MMP activity. CL-1 lung cancer cells were treated without or with various NSAIDs for 48 h and RECK mRNA level was investigated by RT – PCR. The concentrations of NSAIDs suitable for treatment of cells has been determined in our previous work (Pan et al., 2001) and we routinely used 100 mM of NS398 and 500 mM of aspirin to treat cells in this study. As shown in Figure 1a, NS398 and aspirin increased RECK expression in CL-1 cells. We next performed Western blot analysis to investigate the expression of RECK protein. In accordance with the results of RT – PCR analysis, our data demonstrated that RECK protein level was upregulated after NSAID treatment (Figure 1b). Since RECK is a membrane-anchored glycoprotein, we also investigated whether the amount of RECK protein on cell surface of CL-1 cells was increased after NSAID treatment by using immunofluorescent staining. Our data indeed demonstrated that the fluorescent intensity for RECK was obviously increased after NSAID
Figure 1 Up-regulation of RECK by NSAIDs in CL-1 human lung cancer cells. (a) Cells were treated with DMSO (C) or 100 mM of NS398 (NS) or 500 mM of aspirin (ASA) for 48 h. Total RNAs were extracted by RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). One mg of total RNA was subjected to RT – PCR amplifying RECK and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used were: RECK-forward: 5’-CCTCAGTGAGCACAGTTCAGA-3’, RECK-reverse: 5’-GCAGCACACACACTGCTGTA-3’, GAPDH-forward: 5’-CCCATCACCATCTTCCAG-3’, GAPDH-reverse: 5’-CAGTCTTCTGGGTGGCAGT-3’. cDNA was generated by OneStep RT – PCR Kit (QIAGEN, Valencia, CA, USA) and the conditions for PCR were 27 cycles of denaturation (948C/1 min), annealing (608C/1 min), extension (728C/1 min) and one cycle of final extension (728C/10 min). The predicted sizes of PCR products for RECK and GAPDH were 477 and 346 bp, respectively. PCR products were analysed on a 2% 0.56TBE agar gel. (b) Cellular proteins from control or drug-treated cells were extracted and subjected to Western blot analysis to investigate RECK protein level. The blots were then probed with anti-RECK antibody and visualized by secondary antibody conjugated with HRP and ECL chemiluminescence system (NEN Life Sciences, Boston, MA, USA). Beta-actin was also probed as internal control to verify equal loading between samples Oncogene
treatment (Figure 2). These results suggest that NSAIDs can up-regulate RECK expression in CL-1 lung cancer cells. Because NSAIDs could up-regulate RECK expression, we examined whether MMP activity might be suppressed by NSAIDs. We performed gelatin zymography to address this question. Our results indicated that CL-1 cells did not secrete MMP-9. Similar observations had also been reported by the authors who established CL-1 cell line from tumor specimen of a lung cancer patient undergoing surgical resection (Chu et al., 1997). Conversely, we found that CL-1 cells secreted significant amounts of MMP-2. A triplet of bands around 70 kDa corresponding to the molecular weight of the pro-, intermediate, and active forms of MMP-2 were detected in the gelatin zymography (Figure 3). Our results indicated that the amount of active form of MMP-2 was obviously reduced after NSAID treatment. Conversely, the proform MMP-2 was increased in NS398- and aspirintreated cells. It is noteworthy that the total amounts of MMP-2 (including pro-, intermediate, and active form) secreted by NSAID-treated cells were also reduced. Our previous results have shown that NSAIDs may directly inhibit MMP-2 expression (Pan and Hung, 2002). Therefore, NSAIDs may inhibit MMP-2 activity via two mechanisms: direct inhibition of MMP-2 expression by interfering with the ERK/Sp1 signaling pathway and suppression of processing of pro-MMP-2 to its active form by inducing RECK expression. The signaling pathways by which NSAIDs act to stimulate RECK expression are currently unknown. Although the promoter region of mouse RECK gene has been cloned and a number of transcription factor binding sites have been identified (Sasahara et al., 1999), the promoter sequence of human RECK gene has not been published yet. It will be an important issue to clone the human RECK promoter in order to understand the regulation of RECK expression in normal and cancer cells and to clarify the molecular action by which NSAIDs control RECK expression. However, a rational hypothesis is that NSAIDs may enhance
Figure 2 Detection of RECK expression on cell surface by immunofluorescent staining. Cells were seeded in slide chambers and treated with DMSO (C) or NS398 (NS) for 48 h. Cells were fixed with 4% formaldehyde at room temperature for 15 min and blocked by 5% non-fat milk for 30 min. Slides were washed twice with TBST (Tris-buffered saline containing 0.05% Tween 20) and incubated with anti-RECK antibody for 1 h. After being washed extensively with TBST, slides were incubated with FITC-conjugated secondary antibody for another 1 h. Slides were mounted and observed under a fluorescent microscope
NSAIDs activate RECK L-T Liu et al
8349
Figure 3 Analysis of MMP-2 activity by gelatin zymography. CL-1 cells were treated with DMSO (C) or NS398 (NS) or aspiring (ASA) in serum-free medium for 48 h. After incubation, conditioned medium was collected and concentrated by Centricon YM-50 columns (Amicon, Bedford, MA, USA). Attached cells were trypsinized and cell numbers were counted by using a hemocytometer. Conditioned medium from equal number of cells was separated by 10% polyacrylamide gels containing 0.1% gelatin (Invitrogen, Carlsbad, CA, USA). The gels were incubated in 2.5% Triton X-100 solution at room temperature with gentle agitation to remove SDS and soaked in reaction buffer (50 mM TrisHCl pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.5 mM ZnCl2) at 378C overnight. The gels were stained for 1 h with staining solution (0.1% Coomassie brilliant blue, 30% methanol and 10% acetic acid) and destained in the same solution without Coomassie brilliant blue. A triplet of bands around 70 kDa corresponding to the molecular weight of the pro-, intermediate, and active forms of MMP-2 were detected in the zymography
RECK expression via inhibition of the ras/ERK/Sp1 signaling pathway. Previous study has indicated that ras activation may inhibit RECK via the Sp1 binding site in the promoter region (Sasahara et al., 1999). Results of this work showed that CL-1 cells expressed very low levels of RECK. It may be due to overexpression of growth factor receptors (such as Neu), which lead to activation of the ras/ERK signaling pathway, in these cancer cells as reported previously (Chen et al., 2001). Beside CL-1 cells, induction of RECK expression by NSAIDs can also be observed in a number of human lung cancer cell lines harboring ras mutations (data not shown). Our results support the notion that NSAIDs may counteract oncogene-induced down-regulation of RECK and may be useful for the prevention of lung tumorigenesis and metastasis. Indeed, epidemiological studies have shown that aspirin use is associated with reduced risk of lung cancer (Schreinemachers and Everson, 1994; Nelson, 1995; Akhmedkhanov et al., 2002). Previous studies have indicated that overexpression of COX-2 may enhance angiogenic and metastatic potential (Tsujii and DuBois, 1995). So, we also addressed whether NSAIDs acted via inhibition of COX to modulate RECK expression. Figure 4a showed that addition of PGE2 alone could not downregulate RECK expression. Additionally, PGE2 could not counteract NSAID-induced up-regulation of RECK. To further confirm these results, we transfected COX-2 expression vector, pSG5-COX-2, into CL-1 cells and tested the effect of COX-2 overexpression on RECK expression. Our results indicated that high level of COX-2 expression was detected in cells transfected with pSG5-COX-2, but not in cells transfected with control vector or mock transfected cells (Figure 4b). We found that overexpression of COX-2 could not modulate RECK expression. Our results suggest that
Figure 4 NSAIDs may regulate RECK via a COX-2-independent pathway. (a) Cells were treated DMSO (C) or 2 mM of PGE2 or 100 mM of NS398 (NS) or both (NS+PG) for 48 h. Total RNAs were extracted and subjected to analysis of RECK expression by RT – PCR. GAPDH was used as an internal control to check the efficiency of cDNA synthesis and PCR amplification. (b) Cells were mock transfected or transfected with control or pSG5-COX-2 expression vectors and cultured in 10% FCS medium for 48 h. Total RNAs were extracted and expression of COX-2 and RECK was investigated by RT – PCR. (c) Cells were transfected with various expression vectors as described above and Western blot analysis was performed to investigate the protein level of RECK in cells
NSAIDs induce RECK expression in a COX-independent manner. Lines of evidence have suggested that RECK is a key regulator of angiogenesis and metastasis during tumor invasion. First, the RECK gene was ubiquitously expressed in various normal tissues and was downregulated by a number of oncogenes (Takahashi et al., 1998). In addition, enforced expression of RECK potently suppressed oncogene-induced metastasis (Takahashi et al., 1998; Oh et al., 2001). Second, tumor transplantation experiments in mice clearly indicated that angiogenesis and metastasis is dramatically suppressed in tumors derived from RECKexpressing cancer cells (Oh et al., 2001). Third, investigation of RECK expression in primary tumor specimens showed that RECK expression is inversely correlated with the invasion-related clinico-pathological features (Furumoto et al., 2001). In the present study, we have demonstrated that NSAIDs are effective drugs in the induction of RECK in human lung cancers. The therapeutic concentration for aspirin is about 100 mM (Frantz and O’Neill, 1995). However, it has been Oncogene
NSAIDs activate RECK L-T Liu et al
8350
shown that aspirin concentrations of 1 to 2 mM in serum are required to achieve an anti-inflammatory effect (Meredith and Vale, 1992). The therapeutic concentration for NS398 is currently unclear. However, administration of 100 – 300 mg/kg of NS398 into mice did not cause significant side-effect (Futaki et al., 1993). Although the concentrations of NSAIDs used in this study is a little higher than the therapeutic concentrations, these doses are tolerable and show no significant side effect. Taken together, our results provide the first evidence that RECK expression can
be up-regulated or restored by chemical agents and suggest that identification of new compounds that may increase RECK expression may provide a new strategy for cancer treatment.
Acknowledgements We thank Dr ML Kuo for providing the experimental materials. This work was supported by the grant NSC 912320-B-037-015.
References Akhmedkhanov A, Toniolo P, Zeleniuch-Jacquotte A, Koenig KL and Shore RE. (2002). Br. J. Cancer, 87, 49 – 53. Birkedal-Hansen H. (1995). Curr. Opin. Cell. Biol., 7, 728 – 735. Brew K, Dinakarpandian D and Nagase H. (2000). Biochim. Biophys. Acta, 1477, 267 – 283. Chen JW, Peck K, Hong TM, Yang SC, Sher YP, Shih JY, Wu R, Cheng JL, Roffler SR, Wu CW and Yang PC. (2001). Cancer Res., 61, 5223 – 5230. Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJC, Wu R and Wu CW. (1997). Am. J. Resir. Cell Mol. Biol., 17, 353 – 360. Ellerbroek SM and Stack MS. (1999). Bioassays, 21, 940 – 949. Frantz B and O’Neill EA. (1995). Science, 270, 2017 – 2019. Furumoto K, Arii S, Mori A, Furuyama H, Gorrin Rivas MJ, Nakao T, Isobe N, Murata T, Takahashi C, Noda M and Imamura M. (2001). Hepatology, 33, 189 – 195. Futaki N, Arai I, Hamasaka Y, Takahashi S, Highchi S and Otomo S. (1993). J. Pharm. Pharmacol., 45, 753 – 755. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y and Noda M. (1989). Cell, 56, 77 – 84. Masferrer JL, Leahy KM, Koki AT, Zweifel BS, Settle SL, Mark Woerner SB, Edwards DA, Flickinger AG, Moore RJ and Seilbert K. (2000). Cancer Res., 60, 1306 – 1311. Meredith TJ and Vale JA. (1992). Therapeutic Applications of NSAIDs, Subpopulations and New Formulations. Famaey JP and Paulus HE (eds). New York: Dekker, pp 67 – 96.
Oncogene
Nelson C. (1995). J. Natl. Cancer Inst., 87, 567 – 569. Oh J, Takahashi R, Kondo S, Mizoguchi A, Adachi E, Sasahara RM, Nishimura S, Imamura Y, Kitayama H, Alexander DB, Ide C, Horan TP, Arakawa T, Yoshida H, Nishikawa S, Itoh Y, Seiki M, Itohara S, Takahashi C and Noda M. (2001). Cell, 107, 789 – 800. Pan MR, Chuang LY and Hung WC. (2001). FEBS Lett., 508, 365 – 368. Pan MR and Hung WC. (2002). J. Biol. Chem., 277, 32775 – 32780. Rozic JG, Chakraborty C and Lala PK. (2001). Int. J. Cancer, 93, 497 – 506. Sasahara RM, Takahashi C and Noda M. (1999). Biochem. Biophys. Res. Commun., 264, 668 – 675. Schreinemachers DM and Everson RB. (1994). Epidermiology, 5, 138 – 146. Sternlicht MD and Werb Z. (2001). Annu. Rev. Cell Dev. Biol., 17, 463 – 516. Takahashi C, Sheng Z, Horan TP, Kitayama H, Maki M, Hitomi K, Kitaura Y, Takai S, Sasahara RM, Horimoto A, Ikawa Y, Ratzkin BJ, Arakawa T and Noda M. (1998). Proc. Natl. Acad. Sci. USA, 95, 13221 – 13226. Tsujii M and DuBois RN. (1995). Cell, 83, 493 – 501. Vu TH and Werb Z. (2000). Genes Dev., 14, 2123 – 2133.
