| To whom correspondence should be addressed: Kaori Tsutsumi, Department of Biomedical Science and Engineering, Faculty of Health Sciences, Hokkaido University, N12W5, Kita-ku, Sapporo 060-0812, Japan. Tel/Fax: +81–11–706–3421 E-mail: tsutsumi@hs.hokudai.ac.jp |
Radiotherapy is an effective approach for treatment of many types of cancer. Recent developments in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates (Onishi et al., 2004; Palazzi et al., 2009). However, the emergence of tolerant cells during or after radiotherapy remains problematic (Peters et al., 1985; Peters et al., 1982).
Non-small cell lung cancer (NSCLC) is one of the most common causes of mortality in Japan and elsewhere, accounting for circa 80% of all lung cancer cases. Approximately 37% of patients present with stage IIIA or IIIB disease (Jemal et al., 2006). Despite recent developments in radiotherapeutic techniques, the 5-year survival rate for this advanced disease is only 10–20% (Spira and Ettinger, 2004). This low survival rate is partly due to high metastasis rates in advanced NSCLC cases (Spira and Ettinger, 2004).
Tumor metastasis occurs in a series of steps, including cell proliferation at primary sites, angiogenesis, invasion to surrounding matrices, and penetration into lymphatic or blood vessels (Fidler et al., 2007). Matrix metalloproteinases (MMPs), members of the matrixin subfamily of the zinc metalloproteinases, possess proteolytic activity and play a key role in tumor invasion and metastasis (Chambers and Matrisian, 1997; Sauter et al., 2008) through degradation of extracellular matrices (Fidler et al., 2007; Nelson et al., 2000). The members of the MMP family each have preferential substrates, such as type I collagen, type IV collagen, or laminin (Nagase et al., 2006), which constitute tumor stroma; these molecules are therefore considered to be possible therapeutic targets to inhibit cancer metastasis.
Radiotherapy combined with platinum-based chemotherapy results in a higher survival rate for patients with stage III NSCLC than radiation alone (Spira and Ettinger, 2004); however, this combined chemo-radiotherapy regimen only increases survival by approximately 5% (Naito et al., 2008). Molecular-targeted therapy combined with chemo-radiotherapy would be expected to further improve the survival of NSCLC patients. In this study, we have identified molecules responsible for enhanced tumor migration, invasiveness, and adhesion in tumor cells that survive irradiation. These molecules, including MMPs, paxillin, FAK, integrin β1, and vinculin, are promising therapeutic targets to improve tumor control using radiotherapy.
The human non-small cell lung cancer cell line H1299 (CRL-5803) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The establishment of irradiation surviving H1299 cells was described previously (Tsutsumi et al., 2006). Briefly, H1299 cells were irradiated at a dose of 10 Gy, seeded onto dishes, and maintained for 14 days. All colonies formed were harvested together, and the resulting cells were designated as H1299-IR cells. Both IR cells and their respective parental cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Cansera, Canada), 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma) at 37°C in a humidified atmosphere containing 5% CO2.
Total RNA was isolated from cells using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). One microgram of the resulting cDNA was used as a template and amplified by PCR using Ex-taq DNA polymerase (Takara Bio Inc., Tokyo, Japan). Sequences of the oligonucleotide primer sets used were as follows: mmp1 forward, 5'-GCTAACCTTTGATGCTATAACTACGA-3', and mmp1 reverse, 5'-TTTGTGCGCATGTAGAATCTG-3'; mmp2 forward, 5'-ATAACCTGGATGCCGTCGT-3', and mmp2 reverse, 5'-AGGCACCCTTGAAGAAGTAGC-3'; mmp9 forward, 5'-GAACCAATCTCACCGACAGG-3', and mmp9 reverse, 5'-GCCACCCGAGTGTAACCATA. Real-time RT-PCR was performed using a Light Cycler 480 and the Universal Probe Library system (Roche, Basel, Switzerland).
Cells (2.5×105) were allowed to grow to semiconfluence in 5 ml of DMEM containing 10% FBS, and then the culture media was replaced by 1 ml of DMEM without FBS. After 24 h of incubation, the conditioned media were collected and clarified by centrifugation at 1,200 rpm for 15 min. The resulting supernatant was mixed with 6×SDS sample buffer without heating. Equal amounts of protein were separated on 7.5% SDS-PAGE gels containing 50 mg/ml of gelatin. The gels were washed in 2.5% Triton X-100 for 1 h at room temperature, incubated in buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, and 150 mM NaCl for 16 h at 37°C, and then stained with 0.25% (w/v) Coomassie Brilliant Blue (CBB) in 45% (v/v) methanol/1% (v/v) acetic acid for 6 h. Then, the gels were de-stained in 10% (v/v) acetic acid/25% (v/v) methanol.
Membrane filter inserts with 8.0-μm pores were purchased from Nunc (Roskilde, Denmark), and coated with type I collagen (Type I-C; Nitta Gelatin Inc. Osaka, Japan) according to the manufacturer’s protocol. Cells were cultured for 24 h in DMEM containing 10% FBS, trypsinized, and then resuspended in DMEM containing 0.1% FBS. The cells were then transferred to the upper surface of each insert. After incubation for 16 h using 10% FBS as a stimulus in the lower chamber, the cells remaining on the upper side of the membrane were removed by wiping, and the cells that had passed through the insert to the bottom side were fixed with 100% methanol and stained with 0.04% crystal violet. For the wound healing assay, confluent cell monolayers were manually scratched with a pipette tip, washed with PBS, allowed to migrate in DMEM containing 10% FBS for 3 h, and then photographed. Cell migration was calculated as the area covered by migrating cells divided by that of the original scratch and expressed as the fold increase over parental cells on type I collagen-coated dishes.
Cells were grown to semi-confluence, trypsinized, and counted. 4×104 cells were then seeded into each well of a 96-well plate. After incubation for 1 h at 37°C, the medium was completely removed. Cells were stained with 0.04% crystal-violet for 10 min at room temperature, and then lysed in DMSO. Absorbance at 590 nm was measured using a micro-plate reader (model 680, BIO-RAD, Hercules, CA, USA).
Cells were grown to semi-confluence on glass coverslips for 24 h. Cells were then fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked with PBS containing 1% BSA for 30 min, and incubated with appropriately diluted primary antibodies specific for paxillin (1:2,500 dilution; BD Bioscience, Franklin Lakes, NJ, USA), β-catenin (1:250 dilution; BD Bioscience), ZO-1 (1:125 dilution; BD Bioscience), phosphorylated FAK (Tyr397; 1:1,000; Invitrogen), integrin β1 (AIIB2; 350 ng/ml in PBS; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), or vinculin (1:800; Sigma). After overnight incubation at 4°C, the cells were washed in PBS with gentle agitation and then incubated with 1:250 diluted AlexaFluor488-conjugated goat-anti-mouse secondary antibody (Molecular Probes/Invitrogen), with 1:200 diluted AlexaFluor594-conjugated goat-anti-mouse secondary antibody (Molecular Probes/Invitrogen), and with 1:200 diluted AlexaFluor594-conjugated phalloidin (Molecular Probes/Invitrogen) for 60 min at room temperature. The coverslips were again washed with PBS, and then mounted on slides. The cells were observed under a Fluoview confocal microscope (Olympus Corp., Tokyo, Japan).
Cells were lysed in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mM Na3VO4] and complete (EDTA-free) protease inhibitor (Roche, Indianapolis, IN, USA), and clarified by microcentrifugation. The supernatants were subjected to SDS-PAGE, and the separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were incubated with primary antibodies specific for p38 (1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated p38 (Thr180/Tyr182; 1:1,000 dilution; Cell Signaling Technology), FAK (1:200 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phosphorylated FAK (Tyr397; 1:1,000; Invitrogen), integrin β1 (1:500; Cell Signaling Technology), paxillin (1:1,000 dilution; BD Bioscience), or β-actin (1:5,000 dilution; Santa Cruz Biotechnology), followed by further incubation with peroxidase-labeled secondary antibodies. Signals were developed using the ECL Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and detected with an LAS-1000UV mini image analyzer (FUJIFILM, Tokyo, Japan).
Basal levels of gene expression in IR cells and parental cells were compared by microarray analysis (Agilent) (Nishioka et al., 2007; Tsutsumi et al., 2006). Of the 30,000 genes examined, we focused on MMP1, which was upregulated 4.4-fold in IR cells. The altered expression of the MMP gene was confirmed by semiquantitative RT-PCR analysis (Fig. 1A) and real-time RT-PCR (Fig. 1B). In addition to mmp1, the expression of mmp2 and mmp9 mRNA appeared to be increased in H1299-IR cells compared with parental cells. Expression of mmp1, mmp2, and mmp9 was upregulated 1.3-, 1.8-, and 2.8-fold, respectively, in IR cells (Fig. 1B).
 View Details | Fig. 1. Expression levels of matrix metalloproteinase mRNA. Cells were grown to semi-confluence, total RNA was extracted, and semiquantitative reverse transcription PCR (RT-PCR; A) and real time RT-PCR (B) were performed for mmp1, mmp2, and mmp9. For real-time RT-PCR, relative expression levels of mmp were normalized to GAPDH expression, and are expressed as the fold increase over parental cells. |
Given that the expression of matrix metalloproteinase mRNA was upregulated in IR cells, and that degradation of the extracellular matrix by MMP is required for tumor cell invasion into surrounding tissue, we next investigated the gelatinase activity of H1299-IR cells by zymography. Under our experimental conditions, we observed two gelatinolytic bands representing gelatinase/active-MMP9 (MW 88 kDa) and gelatinase/active-MMP2 (MW 66 kDa); both were significantly upregulated in IR cells (Fig. 2A). This result encouraged us to investigate the invasive activity of IR cells. As expected, IR cells were approximately 4.5-fold more invasive than parental cells as assessed by migration through a type I collagen-coated membrane (Fig. 2B). Furthermore, wound healing assays on type I collagen-coated dishes showed that the motility of IR cells was enhanced 1.8-fold (Student’s t-test, P<0.001) (Fig. 2C).
 View Details | Fig. 2. Cell migration and invasion of IR cells. (A) Gelatin zymogram. The conditioned medium from IR cells and parental cells was subjected to gelatin PAGE. After electrophoresis, gelatinase activity was observed as described in Materials and Methods. Protease activity is visible as a clear band against a blue background. (B) Cell invasion. Cells were seeded onto a type I collagen-coated filter at a density of 5×103 cells/well. After 16 h, the total number of cells that had migrated to the lower surface of the filter was counted. (C) Cell migration. Cells were cultured to monolayer confluence on a type I collagen-coated or an uncoated dish. Then, the cells were wounded and incubated for 3 h. Relative wound closure was calculated as described in Materials and Methods, and is shown as the mean of three independent experiments with S.D. ***, P<0.001. |
Since cell migration, which is upregulated in H1299-IR cells as we showed using type I collagen-coated dishes, is intimately associated with interactions with extracellular matrices, we next performed a cell adhesion assay. IR cell adhesion to type I collagen was approximately 1.5-fold higher (Student’s t-test, P<0.001) than that of parental cells, while there was no significant difference in adhesion to the uncoated dish (Fig. 3A). We also assessed the formation of F-actin stress fibers and the localization of paxillin (a major constituent of focal adhesions) by confocal microscopy. Quantitative immunofluorescence assessment revealed more abundant focal adhesions, as visualized by paxillin, which were accompanied by more efficient F-actin fiber formation in IR cells compared with parental cells (Fig. 3B and 3C). In contrast, expression of the cell-cell junction molecules β-catenin and ZO-1 did not differ in parental cells and IR cells (Fig. 3D).
 View Details | Fig. 3. Cell adhesion in IR cells. (A) Adhesion assay. Cells grown to semi-confluence were trypsinized and then seeded in each well of a 96-well plate. Cell adhesion was monitored as described in Materials and Methods. The mean values from three independent experiments are shown with S.D. ***, P<0.001. (B) (D) Focal adhesion and cell-cell adhesion were detected by immunostaining for paxillin (green), β-catenin (green), and ZO-1 (green) (D) and observed by confocal microscopy. F-actin was also visualized using AlexaFluor-594-conjugated phalloidin (B; red). (C) Localization of paxillin at focal adhesions. The focal adhesions per cell configured by paxillin were counted, and mean values from five different randomly selected cells are shown with S.D. *, P<0.05. |
Since it has been reported that transient phosphorylation of p38 mediates enhancement of cell motility immediately after irradiation (Jung et al., 2007), we performed an immunoblotting assay to assess p38 phosphorylation (p-p38) in IR cells; unexpectedly, p38 phosphorylation was not significantly different in IR cells and parental cells (Fig. 4A). Furthermore, expression of integrin β1, paxillin, and FAK, as well as phosphorylation of FAK (p-FAK) showed similar levels in IR cells and parental cells as determined by immunoblotting. Interestingly, however, immunofluorescence analysis revealed that p-FAK and vinculin were more abundantly localized at focal adhesions in IR cells than in parental cells (P<0.05; Fig. 4C and 4D). Integrin β1 also tended to accumulate at focal adhesions in IR cells, but the difference between IR and parental cells was not statistically significant (P=0.069; Fig. 4D). Taken together, these results indicate that the mechanisms responsible for the enhanced cell adhesion in IR cells is distinct from those observed in the cells immediately after irradiation, where levels of FAK phosphorylation, paxillin, and integrin are upregulated (Jung et al., 2007; Wild-Bode et al., 2001).
 View Details | Fig. 4. (A) Phosphorylation of p38 in IR cells. Semi-confluent cells were irradiated with 30 mJ/cm2 of ultraviolet (UV) radiation or left untreated, and were then lysed in lysis buffer. Immunoblotting analysis was carried out on the resulting cell lysate as described in Materials and Methods. (B) Semi-confluent IR and parental cells were lysed in lysis buffer, and subjected to immunoblotting using antibodies against p-FAK, FAK, integrin β1, paxillin, and β-actin. (C and D) Localization of p-FAK, integrin β1, and vinculin at focal adhesions. Cells were fixed with paraformaldehyde, permeabilized (except for the samples incubated with anti-integrin β1), and incubated with the indicated primary antibodies, followed by incubation with fluorescence-labeled secondary antibody. Representative photographs are shown (C). (D) The number of focal adhesions observed for each molecule was quantitated as shown in Fig. 3C. The mean values are shown with S.D. *, P<0.05; N.S., not significant. |
The present study provides evidence that tumor cells that survive 10 Gy irradiation acquire malignant potency through increased motility and invasiveness, as well as an enhanced capacity for adhesion. These are important factors in tumor metastasis (Fidler et al., 2007). In order to reach distant organs away from the primary tumor site, tumor cells have to break off and interact with the extracellular matrix, and then enter the lymphatic capillary system or blood flow (Nelson et al., 2000; Tarin and Matsumura, 1994). It is well established that the MMP family plays a key role in all of these processes (Brinckerhoff et al., 2000; Chambers and Matrisian, 1997). MMP activity is regulated at both the transcriptional and post-transcriptional level through activation by other proteases and inactivation by tissue inhibitors of metalloproteinases (Nagase et al., 2006). Increased MMP activity in cancer cells may largely account for their transcriptional regulation (Egeblad and Werb, 2002). MMP1 is upregulated in many advanced tumors and appears to be associated with tumor invasion and metastasis (Chambers and Matrisian, 1997). Also, a number of studies have revealed the important role of gelatinase/type IV collagenase (MMP2 and MMP9) and collagenase/type-I collagenase (MMP1) in tumor invasion (Nelson et al., 2000; Sauter et al., 2008). In the present study, we showed that mmp1, mmp2, and mmp9 mRNA is upregulated in IR cells, which leads to increased invasion and gelatinase activity in IR cells (Fig. 1 and Fig. 2). Therefore, these results suggest that the tumor cells that survive irradiation become more aggressive, arousing concern about therapeutic strategies for repopulated tumors after radiotherapy.
Tumor cell invasiveness is associated with the formation of focal contacts and alteration of the expression or localization of focal adhesion proteins (Alexandrova et al., 2007), including paxillin and FAK, the phosphorylations of which are critically involved in regulation of cell migration (Petit et al., 2000; Tsubouchi et al., 2002). Therefore, both facilitated cell migration and increased adhesion in IR cells can be accounted for by enhanced translocation of these molecules to focal adhesions (Fig. 3 and Fig. 4).
Recently, Jung et al. reported that irradiation promoted tumor cell motility and focal adhesion formation, and diminished cell-cell junctions (Jung et al., 2007). These events occur within 24 h after irradiation and are mediated by increased phosphorylation of p38 and FAK (Jung et al., 2007). In addition, in glioma cells, enhanced integrin expression was found to account for increased cell migration, an early response to irradiation (Wild-Bode et al., 2001). Conversely, in our experiment, p38 and FAK phosphorylation levels, and integrin expression levels, were comparable in IR cells and in parental cells (Fig. 3B and 3C, Fig. 4A, 4B, and 4C). The difference between these two previous reports and our current work appears to be due to the difference in the elapsed time after irradiation. IR cells in our study were prepared 14 days after 10 Gy irradiation, which was a considerably longer time after irradiation than that used in previous reports, in which cells were examined 1–3 days after irradiation. Therefore, it appears that the mechanisms that regulate cell adhesion and migration later in IR cells are distinct from those seen immediately after irradiation, i.e., promotion of cell adhesion and cell migration in irradiation-surviving cells are regulated primarily by the localization of focal adhesion molecules, and not by their expression levels. Indeed, the localization of paxillin, integrin β1, and p-FAK was more consolidated at the focal adhesions in IR cells (Fig. 4C and 4D).
In conclusion, the present study is the first to demonstrate that the increased motility and invasiveness of human NSCLC cells that survive 10 Gy X-ray irradiation result from upregulation of MMPs as well as enhanced localization of paxillin and phosphorylated FAK at focal adhesions. Although additional in-depth studies are required to identify other factors associated with tumor invasion and metastasis after irradiation, MMPs and cell adhesion-related molecules may become an add-on therapeutic target to improve tumor control in radiotherapy.
The authors would like to thank S. Tanaka for use of the confocal laser-scanning microscope, N. Toyoda for technical assistance, and the members of our laboratories for helpful discussion. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Japan Society for the Promotion of Science.
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