- Email: [email protected]
SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell
Biochemical and Biophysical Research Communications 382 (2009) 571–576
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
CXCR4/SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell Lin Xu a, Ya Zhou b, Qi Liu c, Jun-Min Luo a, Min Qing a, Xian-Yin Tang a, Xin-Shen Yao a, Chun-Hong Wang d, Zhen-Ke Wen c,* a
Department of Immunology, Zunyi Medical College, Guizhou, PR China Department of Medical Physics, Zunyi Medical College, Guizhou, PR China c Department of Central Laboratory, Qingdao Blood Center, 9 Longde Road, Shandong Province 266071, PR China d Department of Chest Medicine, Qingdao Chest Hospital, Shandong, PR China b
a r t i c l e
i n f o
Article history: Received 7 March 2009 Available online 18 March 2009
Keywords: TLR9 CpG oligonucleotide 95D cell Metastasis CXCR4
a b s t r a c t Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Toll-like receptors (TLRs), which recognize a variety of pathogen-associated molecular patterns, are mainly expressed on the immune cells and play an important role in innate and adaptive immunity. However, accumulating data demonstrated that functional TLRs were also widely expressed on a variety of tumor cells [1,2]. Activation of TLR signaling in tumor cells could promote tumor cell proliferation, enhance tumor cell invasion and metastasis, suggesting that TLRs may play important roles in tumor biology [3]. There is increasing evidence to suggest that TLR9 expression is not confined to cells of the immune system. TLR9 expression has been detected in various epithelial cells and tumor cells including breast, brain, lung, and gastric cancer cells [4–6]. Furthermore, accumulating data indicate that TLR9 agonist from pathogens could promote tumor metastasis through inflammation dependent mechanisms [7,8]. The CpG ODN could also promote the invasion of tumor cells in vitro [9]. These results indicated that TLR9 pathway might be related to tumor progress. Our previous study demonstrated that the CpG ODN could significantly enhance the metastatic potential of human lung cancer cells in vitro [10]. How-
* Corresponding author. Fax: +86 532 85717544. E-mail address: [email protected] (Z.-K. Wen). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.072
ever, the underlying mechanism of TLR9 pathway on tumor biology remains undefined. Tumor cell migration and metastasis, as an important part of tumor biology, shared many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors [11]. Accumulating data suggested that CXCR4 (CXC chemokine receptor-4) and SDF-1 (stroma cell-derived factor-1, or CXCL12) could regulate the migration and metastasis of a variety of tumors including lung cancer, breast cancer and prostate cancer [12–14]. SDF-1, secreted by stroma cells, could attract tumor cells via the stimulation of CXCR4 expressed on tumor cells [15,16]. Recent studies demonstrated that CXCR4/SDF-1 signaling could promote the metastasis of tumor cells via the Akt pathway and transcription factor AP-1 [17,18]. Importantly, latest findings showed that the CXCR4 pathway could modulate TLR9 signaling in innate immunity [19]. All these results promoted us to further investigate whether the CXCR4/SDF-1 pathway was involved in the enhanced metastasis of human lung cancer induced by CpG ODN via TLR9 signaling pathway. To address this issue, in this study, we performed Real-time PCR and Western blot to detect the expression of CXCR4 on human lung cancer cell line 95D cells in response to CpG ODN. Furthermore, we down-regulated the expression of CXCR4 using CXCR4 siRNA and investigated its effect on the metastasis of 95D cells enhanced by CpG ODN in vitro and in vivo. Our results suggested that the TLR9 agonist could enhance the metastatic potential of 95D cells
572
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
via CXCR4/SDF-1 pathway, which may provide a novel insight on the TLR9 pathway on tumor metastasis.
Materials and methods Reagents and cell line. The following ODNs were used and purchased from Integrated DNA Technologies (Coralville, IO): CpG Control CpG ODN2216: 50 -GGGGGACGATCGTCGGGGG-30 . 0 ODN1612: 5 -GCTAGAGCTTAGGCT-30 . CpG ODN has a phosphorothioate backbone that provides a high degree of nuclease resistance. All other reagents were purchased from Sigma–Aldrich unless stated otherwise. Human lung cancer cell line 95D cells were cultured at 37 °C under 5% CO2 in completed RPMI 1640 (GIBICO, Grand land, NY, USA) medium containing 10% heat-inactivated fetal bovine serum and supplemented with 2 mM glutamine, 100 IU/ml penicillin, and 100 lg/ml streptomycin sulfate. In vitro invasive assay. The invasive assay was done in 24-well cell culture chambers using inserts with 8-mm pore membranes precoated with Matrigel (28Ag/insert; Sigma, Saint Louis, MO) as previously described [10]. Cell suspensions (1 106/ml) were placed in the upper wells and the lower wells were filled with fibroblast-conditioned medium. After incubation for 24 h, cells on the lower surface of the membrane were stained by the H&E method and counted under a light microscope (400). RT-PCR assay. Total cellular RNA were extracted from 107 cells by using the mRNA Purification Kit (Shanghai Shenergy Biocolor Bioscience & Technology Company, China) according to the manufac-
B
15
*
10
*
Relative expression of CXCR4
Relative expression of CXCR4
A
turer’s guidelines. Then the concentration of mRNA was measured and reverse transcription was performed on 2 lg of mRNA by using reverse transcriptase MMLV (Promega, USA) for first-strand cDNA synthesis with Oligo (dT) primer. The cDNA was amplified by PCR with the following primers: CXCR4 (394 bp), 50 -CAGTCTGGACCGCTACCT-30 (sense) and 50 -GATGCTGATCCCAATGT AG30 (anti-sense); b-actin (115 bp), 50 -CCCACAATGTCCCCATCT-30 (sense) and 50 -TAGCCACGCTCAGTCAGG-30 (anti-sense). PCR was carried out as follows: an initial denaturation of 3 min at 94 °C was followed by 28 cycles of 45 s at 94 °C, 45s at 56 °C, and 30s at 72 °C, followed by 10 min of final elongation at 72 °C. Real-time PCR. Total cellular RNA and cDNA were prepared as previously described. CXCR4 levels were measured by SYBR Green-based Realtime PCR using Light Cycler (Roche, USA). The PCR contained 0.3 mM of each primer, 3 mM MgCl2 and 0.75 U of Platinum Taq polymerase (Invitrogen). The primer sequences were designed to bracket an intron to avoid amplification of genomic DNA. Their sequences were as follows; primers: 50 -CCCTCC TGCTGACTATTCCC-30 and 50 -TAAGGCCAACCATGATGTGC-30 . PCR cycling conditions consisted of 95 °C for 6 min, followed by 45 cycles of 95 °C for 15s, 60 °C for 30s, and 72 °C for 30s. Cycle threshold (CT) values were compared against a standard curve to estimate starting amounts of mRNA, and the relative expression of CXCR4 mRNA between samples was estimated by normalizing these values against 18S rRNA CT values were generated using a preoptimized 18S rRNA primer set (Applied Biosystems, FosterCity, CA). Western blotting. Western blotting was performed on cytosolic cellular extracts. Equal amounts of protein were resolved under reducing conditions on a 12% SDS–polyacrylamide gel. Protein
20
10
0 5
diu me
D
*
No.of invaded cells
C
7.5
m
pG lC tro n co
0
Relative expression of CXCR4
30
N OD
G Cp
N OD
* 120
*
90
60
30
5.0
0 2.5
0.0
N N m OD OD diu e G G M p Cp lC tro n Co
Fig. 1. CpG ODN enhanced the expression of CXCR4 on 95D cells. The 95D cells (3 103 cells/well) were stimulated with 10 lg/ml of CpG ODN or control CpG ODN in 24-well plate for 72 h. RT-PCR (A) and Real-time PCR (B) were performed to determinate the mRNA expression of CXCR4 in 95D cells. (C) Western blot was performed to detect the expression of CXCR4 protein in 95D cells. Three independent assays were performed. (D) 95D cells were assayed in 24-well cell-culture chambers using inserts with 8-mm pore membranes pre-coated with Matrigel for their invasive potentials. *p < 0.05.
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
migration was assessed using protein standards (Biorad, CA). Transfer to a nitrocellulose membrane was performed overnight at 30 V using a wet transfer system. Equal protein loading was confirmed with Ponceau staining. The membrane was washed in 5% skim milk in phosphate buffered saline + 0.03% Tween 20 (PBS-T) for 1 h in order to block nonspecific protein binding sites on the membrane. Immunoblotting was performed using a monoclonal antibody to CXCR4, Akt, I-jBa (Santa Cruz Biotechnology) at a dilution of 1/1000 in a nonfat milk–Tris buffer. The membrane was then washed in PBS-T and subsequently probed with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (Amersham Life Sciences) at a dilution of 1:5000 and developed with chemiluminescence (Pierce, IL). The membrane was then exposed to X-ray film (Kodak, NY) which was subsequently developed. Design of artificial siRNAs and transfection. We designed the CXCR4 targeting sequence (sense, 50 -GGAAGCUGUUGGCUGAAAAdTdT-30 ) as reported [20]. The nonspecific control siRNA duplexes were purchased from Dhamacon with the same GC content as CXCR4 siRNAs (42%, D001206-10). These siRNA gene double-strands were ligated with pSilencer4.1 RNAi Expression Vector (Ambion). Then, the vector with CXCR4–siRNA or control siRNA plasmids were transfected into the 95D cells using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s instruction. Migration assay. Wound healing assays were used to evaluate the migration of 95D cells transfected with siRNA against CXCR4 (termed as 95D-siR-CXCR4 cells) and 95D cells transfected with control siRNA (termed as 95D-ctrl-CXCR4 cells). Cells were seeded in six-well plates at 1 105 per well in growth medium. Confluent monolayers were starved overnight in assay medium and a single scratch wound was created using a micropipette tip. The cells were washed with PBS to remove cell debris, supplemented with assay medium containing 10 lg/ ml CpG ODN and monitored. Images were captured with a microscope using a 5 objective at 0 and 24 h post-wounding. For a quantitative measure, we counted the number of migrating cells to wound area based on the picture at 0 h post-wounding. Animal experiments. 95D cells (5 106) transfected with CXCR4–siRNA (treated group) or control-siRNA (control group) were pretreated with 10 lg/ml CpG ODN. After 3 days, the cell were collected and then injected into each mouse of 16 nude mice (8 mice each group) by the tail vein. At 30 days post-injection of cells, the mice were sacrificed. Lungs were harvested in optimum cutting temperature compound and frozen in liquid nitrogen. The collected lung tissue sections were subjected to H&E histostaining. The metastasis foci also were counted under microscope. Luciferase reporter assay plasmids. pNF-jB Luc and pcMV-lacZ were kindly supplied by Professor Ren Tao (Tongji University, China). 95D-siR-CXCR4 cells and 95D-ctrl-CXCR4 cells were co-transfected with various plasmids, respectively, and treated with 200 ng/ml SDF-1a in the presence of 10 lg/ml CpG ODN. After 24 h, luciferase and b-gal activity in 100 ll of cell lysates were measured by the Luciferase Assay System and the b-Galactosidase Enzyme Assay System (Promega), respectively. Transfection efficiency was normalized using b-gal activity. Statistical analyses. Statistical analyses of the data were performed with the SPSS12.0 software. Data were analyzed using a one-way analysis of variance (ANOVA) or Kruskal–Wallis test with PRISM 4.0 (GraphPad Soft-ware Inc, San Diego, CA, USA). *p < 0.05 was considered statistically significant in all comparisons.
Results TLR9 agonist enhanced the expression of CXCR4 on 95D cells Our previous data showed that TLR9 agonist could enhance the metastatic potential of lung cancer cell line 95D cells in vitro [10].
573
To further investigate the possible role of CXCR4/SDF-1 pathway in the metastatic potential of 95D cells enhanced by CpG ODN, we carefully estimated the expression of CXCR4 on 95D cells in response to CpG ODN. As shown in Fig. 1A and B, RT-PCR and Realtime PCR analysis revealed that the CXCR4 signal intensities was dramatically higher in CpG ODN treated 95D cells than that in control CpG ODN treated 95D cells and the control group (p < 0.05). To confirm the above findings, we further examined the expression of CXCR4 protein using Western blot. As shown in Fig. 1C, we found that the expression of CXCR4 protein was significantly higher in CpG ODN treated 95D cells (p < 0.05). In contrast, only weak expression of CXCR4 protein was detected in the control CpG ODN treated 95D cells and the control group (p < 0.05). Finally, we confirmed the enhanced metastasis of 95D cells induced by CpG ODN (Fig. 1D). These data strongly demonstrated that the TLR9 agonist could enhance the expression of CXCR4 on 95D cells. siRNA against CXCR4 down-regulated the expression of CXCR4 on CpG ODN treated 95D cell To determine the effect of CXCR4 expression level on the enhanced metastasis of 95D cells induced by CpG ODN, the expression of CXCR4 in 95D cells was down-regulated using siRNA against CXCR4. 95D cells were stably transfected with the CXCR4 siRNA (termed as 95D-siR-CXCR4) or control siRNA (termed as 95D-ctrl-CXCR4) and treated with CpG ODN, respectively. Then
Fig. 2. SiRNA against CXCR4 down-regulated the expression of CXCR4 on CpG ODN treated 95D cells. 95D cells were stably transfected with CXCR4 siRNA or control siRNA and then were stimulated with the indicated dose of CpG ODN at 3 103 cells/well in 24-well plate for 72 h, respectively. (A) RT-PCR was performed to determinate the mRNA expression of CXCR4 in indicated cells. (B) Western blot was performed to detect the expression of CXCR4 protein. Three independent assays were performed. *p < 0.05.
574
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
A
*
* *
* Relative Migration (%)
No. of invaded cells
125
B 125
*
*
100 75 50 25
100 75 50 25
CpG ODN
(0ug/ml)
(10ug/ml)
CpG ODN untreated
CXCR4-siRNA
CpG ODN treated
Control siRNA
CpG ODN
CpG ODN
(0ug/ml)
D
CXCR4-siRNA
Control-siRNA
CXCR4-siRNA
Control-siRNA
Control-siRNA
CpG ODN
0
Lung metastasis foci
C
CXCR4-siRNA
Control-siRNA
0
(10ug/ml)
*
*
15
10
5
CpG
ODN
untreated
CpG
CXCR4-siRNA
Control-siRNA
CXCR4-siRNA
CXCR4 siRNA
Control-siRNA
0
ODN
treated
Fig. 3. Down-regulation of CXCR4 expression reduced the metastasis of CpG ODN treated 95D cells in vitro and in vivo. (A) The indicated cells were stimulated with 10 lg/ml of CpG ODN in vitro for 72 h and then were assayed in 24-well cell-culture chambers using inserts with 8-mm pore membranes pre-coated with Matrigel for their invasive potentials. (B) Wound healing assays were used to evaluate the migration of 95D-siR-CXCR4 cells. For a quantitative measure, we counted the number of migrating cells to wound area based on the picture at 0 h post-wounding. (C) Eight nude mice were challenged with CpG ODN pretreated 95D cells for 8 weeks. The collected lung tissue sections were subjected to H&E histostaining. The arrows showed the metastatic cells (original magnification: 200). (D) The lung metastasis foci were calculated under microscope. Three independent assays were performed. *p < 0.05.
the expression of CXCR4 was determined. As shown in Fig. 2A, the mRNA expression of CXCR4 in 95D-siR-CXCR4 cells in response to CpG ODN was dramatically down-regulated compared with that in 95D-ctrl-CXCR4 cells (p < 0.05). To further confirm this phenomenon, Western blot was performed to detect the expression of CXCR4 protein in the CpG ODN treated 95-siR-CXCR4 cells and 95-ctrl-CXCR4 cells. As shown in Fig. 2B, compared with that in 95D-ctrl-siRNA group, the expression of CXCR4 protein was significantly reduced in 95D-siRNA group (p < 0.05), which was even lower than that of the untreated 95D-ctrl-CXCR4 cells. These data suggested that siRNA against CXCR4 could effectively down-regulated the expression of CXCR4 on CpG ODN treated 95D cells. Down-regulation of CXCR4 expression reduced the metastasis of CpG ODN treated 95D cells in vitro and in vivo To determine whether down-regulation of CXCR4 could affect the metastasis of 95D cells induced by CpG ODN, 95D-siR-CXCR4 cells and 95D-ctrl-CXCR4 cells were stimulated with CpG ODN,
respectively. Then their invasive potentials and migration in response to SDF-1a was determined in vitro. We investigated that down-regulation of CXCR4 with siRNA potently reduced the invasion and migration of 95D cells induced by CpG ODN (Fig. 3A and B, p < 0.05). To further confirm the effect of CXCR4 expression on the enhanced metastasis of 95D cells induced by CpG ODN in vivo, the nude mice were challenged with 95D-siR-CXCR4 cells pretreated with CpG ODN as previous described [10] and then the lung metastasis were evaluated. We found that the metastatic potential of 95D cells was dramatically abrogated by the CXCR4 siRNA in vivo compared with the control group (Fig. 3C and D, p < 0.05). These results strongly suggested that CXCR4 pathway was crucial for CpG ODN to enhance the metastasis of 95D cells. Down-regulation of CXCR4 expression reduced the Akt phosphorylation in CpG ODN treated 95D cells Previous findings showed that CXCR4/SDF-1 signaling could promote the metastasis of tumor cells via the Akt pathway [21].
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
To further verify whether CpG ODN enhanced the metastasis of 95D cells through CXCR4/SDF-1/Akt pathway, the expression of phosphorylated Akt in the 95D-siR-CXCR4 cells in response to CpG ODN was determined. 95D-ctrl-CXCR4 cells were used as the control. As shown in Fig. 4A, Western blot analysis revealed that SDF-1a induced the phosphorylation of Akt in 95D-ctrlCXCR4 cells at 15 min. Conversely, phosphorylation of Akt in 95D-siR-CXCR4 cells could not be induced after SDF-1a addition (p < 0.05). In addition, there were no changes in the expression level and phosphorylation of I-jBa in both the two groups. To further confirm these results, luciferase reporter assay was performed to detect the activity of NF-jB promoter. As shown in Fig. 4B, SDF-1a could not alter the activity of NF-jB promoter in CpG ODN treated 95D-ctrl-CXCR4 cells and 95D-siR-CXCR4 cells. All of these data indicated that CXCR4/SDF-1/Akt pathway might be important for CpG ODN enhanced metastasis of 95D cells. Discussion Both TLR9 and CXCR4 are recognized individually as tumor enhancer involved in tumor progress [1,22]. Although CXCR4 has been demonstrated to play roles in metastasis of tumors [23,24], the possible role of CXCR4 pathway in TLR9 signaling pathway on metastasis of tumor is under investigation. In this study, we showed that CpG ODN enhanced the expression of CXCR4 on 95D cells, which was consistent with our previous findings [10]. Fur-
575
thermore, we extended our previous work by demonstrating that down-regulation of CXCR4 expression could significantly inhibit the metastasis of 95D cells enhanced by CpG ODN in vitro and in vivo. In addition, we also showed that down-regulation of CXCR4 expression could significantly reduce the phosphorylation of Akt but not the phosphorylation of I-jB and activation of NFjB in 95D cells in response to CpG ODN. These results were consistent with the previous findings which showed that CXCL12/ CXCR4 signaling could activate Akt-1 expression in prostate cancer cells [18]. Our results suggested that the CXCR4/SDF-1/Akt pathway was essential for TLR9 pathway to enhance the metastasis of 95D cells. In fact, recent studies showed that CXCR4 pathway could modulate TLR9 signaling in macrophage [19]. Therefore, it is worthy and interesting to study the exact mechanism underlying the cross-talk between CXCR4/SDF-1 pathway and TLR9 pathway on the metastasis of tumor cells in the following experiments. In summary, here we demonstrated that CXCR4/SDF-1 pathway was critical for TLR9 agonist to enhance the metastasis of human lung cancer cells. These finding may provide a novel insight on the understanding of underlying mechanism of TLR9 pathway on tumor metastasis. Acknowledgments This work was supported by Zunyi Medical College start-up Fund (F-329), Fund of Qingdao Municipal Science and Technology Commission (07-2-1-13-nsh), Zunyi Medical College start-up Fund (F-297), and The National Prophase Project on Basic Research of China (2008CB517310). References
Fig. 4. Down-regulation of CXCR4 expression reduced the Akt phosphorylation in CpG ODN treated 95D cells. (A) 95D-siR-CXCR4 cells were treated with 200 ng/ml SDF-1a in the presence of CpG ODNs. The phosphorylation of Akt and I-jBa were detected by Western blot. 95D-ctrl-CXCR4 cells were used as control. (B) The activity of NF-jB promoter also was determined by luciferase reporter assay. Three independent assays were performed. *p < 0.05.
[1] B. Huang, J. Zhao, J.C. Unkeless, Z.H. Feng, H. Xiong, TLR signaling by tumor and immune cells: a double-edged sword, Oncogene 27 (2008) 218–224. [2] R. Chen, A.B. Alvero, D.A. Silasi, K.D. Steffensen, G. Mor, Cancers take their Toll— the function and regulation of Toll-like receptors in cancer cells, Oncogene 27 (2008) 225–233. [3] B. Huang, J. Zhao, H. Li, Toll-like receptors on tumor cells facilitate evasion of immune surveillance, Cancer Res. 65 (2005) 5009–5014. [4] C. Song, L.Z. Chen, R.H. Zhang, X.J. Yu, Y.X. Zeng, Functional variant in the 30 untranslated region of Toll-like receptor 4 is associated with nasopharyngeal carcinoma risk, Cancer Biol. Ther. 5 (2006) 1285–1291. [5] B. Schmausser, M. Andrulis, S. Endrich, H.K. Muller-Hermelink, M. Eck, Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori, Int. J. Med. Microbiol. 295 (2005) 179–185. [6] M. Zaks-Zilberman, T.Z. Zaks, S.N. Vogel, Induction of proinflammatory and chemokine genes by lipopolysaccharide and paclitaxel (Taxol) in murine and human breast cancer cell lines, Cytokine 15 (2001) 156–165. [7] D. Droemann, D. Albrecht, J. Gerdes, A.J. Ulmer, D. Branscheid, E. Vollmer, K. Dalhoff, P. Zabel, T. Goldmann, Human lung cancer cells express functionally active Toll-like receptor 9, Respir. Res. 6 (2005) 1. [8] F. Tsushima, K. Tanaka, N. Otsuki, P. Youngnak, H. Iwai, K. Omura, M. Azuma, Predominant expression of B7–H1 and its immunoregulatory roles in oral squamous cell carcinoma, Oral Oncol. 42 (2006) 268–274. [9] J.M. Ilvesaro, M.A. Merrell, T.M. Swain, J. Davidson, M. Zayzafoon, K.W. Harris, K.S. Selander, Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro, Prostate 67 (2007) 774–781. [10] T. Ren, Z.K. Wen, Z.M. Liu, Y.J. Liang, Z.L. Guo, L. Xu, Functional expression of TLR9 is associated to the metastatic potential of human lung cancer cell: functional active role of TLR9 on tumor metastasis, Cancer Biol. Ther. 6 (2007) 1704–1709. [11] R.M. Strieter, Chemokines: not just leukocyte chemo-attractants in the promotion of cancer, Nat. Immunol. 2 (2001) 285–286. [12] R.J. Phillips, M.D. Burdick, M. Lutz, J.A. Belperio, M.P. Keane, R.M. Strieter, The stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis innon-small cell lung cancer metastases, Am. J. Respir. Cell Mol. Biol. 167 (2003) 1676–1686. [13] K. Oonakahara, W. Matsuyama, I. Higashimoto, M. Kawabata, K. Arimura, M. Osame, Stromal-derived factor-1a/CXCL12-CXCR4 axis is involved in the dissemination of NSCLC cells into pleural space, Am. J. Respir. Cell Mol. Biol. 30 (2004) 671–677. [14] H.H. Oliveira-Neto, E.T. Silva, C.R. Leles, E.F. Mendonça, C. Alencar Rde, T.A. Silva, A.C. Batista, Involvement of CXCL12 and CXCR4 in lymph node metastases and development of oral squamous cell carcinomas, Tumour Biol. 29 (2008) 262–271.
576
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
[15] J. Wang, R. Loberg, R.S. Taichman, The pivotal role of CXCL12 (SDF-1)/CXCR4 axis in bone metastasis, Cancer Metastasis Rev. 25 (2006) 573–587. [16] M.K. Wendt, A.N. Cooper, M.B. Dwinell, Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells, Oncogene 27 (2008) 1461–1471. [17] C.T. Tan, C.Y. Chu, Y.C. Lu, C.C. Chang, B.R. Lin, H.H. Wu, H.L. Liu, S.T. Cha, E. Prakash, J.Y. Ko, M.L. Kuo, CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway, Carcinogenesis 29 (2008) 1519–1527. [18] S.R. Chinni, S. Sivalogan, Z. Dong, J.C. Filho, X. Deng, R.D. Bonfil, M.L. Cher, CXCL12/CXCR4 signaling activates Akt-1 and MMP-9 expression in prostate cancer cells: the role of bone microenvironment-associated CXCL12, Prostate 66 (2006) 32–48. [19] M. Ishii, C.M. Hogaboam, A. Joshi, T. Ito, D.J. Fong, S.L. Kunkel, CC chemokine receptor 4 modulates Toll-like receptor 9-mediated innate immunity and signaling, Eur. J. Immunol. 38 (2008) 2290–2302.
[20] Z. Liang, Y. Yoon, J. Votaw, M.M. Goodman, L. Williams, H. Shim, Silencing of CXCR4 blocks breast cancer metastasis, Cancer Res. 65 (2005) 967–971. [21] Z. Liang, J. Brooks, M. Willard, K. Liang, Y. Yoon, S. Kang, H. Shim, CXCR4/CXCL12 axis promotes VEGF-mediated tumor angi-ogenesis through Akt signaling pathway, Biochem. Biophys. Res. Commun. 359 (2007) 716–722. [22] S. Gelmini, M. Mangoni, M. Serio, P. Romagnani, E.J. Lazzeri, The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis, Endocrinol. Invest. 31 (2008) 809–819. [23] M.Z. Ratajczak, E. Zuba-Surma, M. Kucia, R. Reca, W. Wojakowski, J. Ratajczak, The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis, Leukemia 20 (2006) 1915–1924. [24] M.Z. Dewan, S. Ahmed, Y. Iwasaki, K. Ohba, M. Toi, N. Yamamoto, Stromal cell-derived factor-1 and CXCR4 receptor interaction in tumor growth and metastasis of breast cancer, Biomed. Pharmacother. 60 (2006) 273–276.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
CXCR4/SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell Lin Xu a, Ya Zhou b, Qi Liu c, Jun-Min Luo a, Min Qing a, Xian-Yin Tang a, Xin-Shen Yao a, Chun-Hong Wang d, Zhen-Ke Wen c,* a
Department of Immunology, Zunyi Medical College, Guizhou, PR China Department of Medical Physics, Zunyi Medical College, Guizhou, PR China c Department of Central Laboratory, Qingdao Blood Center, 9 Longde Road, Shandong Province 266071, PR China d Department of Chest Medicine, Qingdao Chest Hospital, Shandong, PR China b
a r t i c l e
i n f o
Article history: Received 7 March 2009 Available online 18 March 2009
Keywords: TLR9 CpG oligonucleotide 95D cell Metastasis CXCR4
a b s t r a c t Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Toll-like receptors (TLRs), which recognize a variety of pathogen-associated molecular patterns, are mainly expressed on the immune cells and play an important role in innate and adaptive immunity. However, accumulating data demonstrated that functional TLRs were also widely expressed on a variety of tumor cells [1,2]. Activation of TLR signaling in tumor cells could promote tumor cell proliferation, enhance tumor cell invasion and metastasis, suggesting that TLRs may play important roles in tumor biology [3]. There is increasing evidence to suggest that TLR9 expression is not confined to cells of the immune system. TLR9 expression has been detected in various epithelial cells and tumor cells including breast, brain, lung, and gastric cancer cells [4–6]. Furthermore, accumulating data indicate that TLR9 agonist from pathogens could promote tumor metastasis through inflammation dependent mechanisms [7,8]. The CpG ODN could also promote the invasion of tumor cells in vitro [9]. These results indicated that TLR9 pathway might be related to tumor progress. Our previous study demonstrated that the CpG ODN could significantly enhance the metastatic potential of human lung cancer cells in vitro [10]. How-
* Corresponding author. Fax: +86 532 85717544. E-mail address: [email protected] (Z.-K. Wen). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.072
ever, the underlying mechanism of TLR9 pathway on tumor biology remains undefined. Tumor cell migration and metastasis, as an important part of tumor biology, shared many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors [11]. Accumulating data suggested that CXCR4 (CXC chemokine receptor-4) and SDF-1 (stroma cell-derived factor-1, or CXCL12) could regulate the migration and metastasis of a variety of tumors including lung cancer, breast cancer and prostate cancer [12–14]. SDF-1, secreted by stroma cells, could attract tumor cells via the stimulation of CXCR4 expressed on tumor cells [15,16]. Recent studies demonstrated that CXCR4/SDF-1 signaling could promote the metastasis of tumor cells via the Akt pathway and transcription factor AP-1 [17,18]. Importantly, latest findings showed that the CXCR4 pathway could modulate TLR9 signaling in innate immunity [19]. All these results promoted us to further investigate whether the CXCR4/SDF-1 pathway was involved in the enhanced metastasis of human lung cancer induced by CpG ODN via TLR9 signaling pathway. To address this issue, in this study, we performed Real-time PCR and Western blot to detect the expression of CXCR4 on human lung cancer cell line 95D cells in response to CpG ODN. Furthermore, we down-regulated the expression of CXCR4 using CXCR4 siRNA and investigated its effect on the metastasis of 95D cells enhanced by CpG ODN in vitro and in vivo. Our results suggested that the TLR9 agonist could enhance the metastatic potential of 95D cells
572
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
via CXCR4/SDF-1 pathway, which may provide a novel insight on the TLR9 pathway on tumor metastasis.
Materials and methods Reagents and cell line. The following ODNs were used and purchased from Integrated DNA Technologies (Coralville, IO): CpG Control CpG ODN2216: 50 -GGGGGACGATCGTCGGGGG-30 . 0 ODN1612: 5 -GCTAGAGCTTAGGCT-30 . CpG ODN has a phosphorothioate backbone that provides a high degree of nuclease resistance. All other reagents were purchased from Sigma–Aldrich unless stated otherwise. Human lung cancer cell line 95D cells were cultured at 37 °C under 5% CO2 in completed RPMI 1640 (GIBICO, Grand land, NY, USA) medium containing 10% heat-inactivated fetal bovine serum and supplemented with 2 mM glutamine, 100 IU/ml penicillin, and 100 lg/ml streptomycin sulfate. In vitro invasive assay. The invasive assay was done in 24-well cell culture chambers using inserts with 8-mm pore membranes precoated with Matrigel (28Ag/insert; Sigma, Saint Louis, MO) as previously described [10]. Cell suspensions (1 106/ml) were placed in the upper wells and the lower wells were filled with fibroblast-conditioned medium. After incubation for 24 h, cells on the lower surface of the membrane were stained by the H&E method and counted under a light microscope (400). RT-PCR assay. Total cellular RNA were extracted from 107 cells by using the mRNA Purification Kit (Shanghai Shenergy Biocolor Bioscience & Technology Company, China) according to the manufac-
B
15
*
10
*
Relative expression of CXCR4
Relative expression of CXCR4
A
turer’s guidelines. Then the concentration of mRNA was measured and reverse transcription was performed on 2 lg of mRNA by using reverse transcriptase MMLV (Promega, USA) for first-strand cDNA synthesis with Oligo (dT) primer. The cDNA was amplified by PCR with the following primers: CXCR4 (394 bp), 50 -CAGTCTGGACCGCTACCT-30 (sense) and 50 -GATGCTGATCCCAATGT AG30 (anti-sense); b-actin (115 bp), 50 -CCCACAATGTCCCCATCT-30 (sense) and 50 -TAGCCACGCTCAGTCAGG-30 (anti-sense). PCR was carried out as follows: an initial denaturation of 3 min at 94 °C was followed by 28 cycles of 45 s at 94 °C, 45s at 56 °C, and 30s at 72 °C, followed by 10 min of final elongation at 72 °C. Real-time PCR. Total cellular RNA and cDNA were prepared as previously described. CXCR4 levels were measured by SYBR Green-based Realtime PCR using Light Cycler (Roche, USA). The PCR contained 0.3 mM of each primer, 3 mM MgCl2 and 0.75 U of Platinum Taq polymerase (Invitrogen). The primer sequences were designed to bracket an intron to avoid amplification of genomic DNA. Their sequences were as follows; primers: 50 -CCCTCC TGCTGACTATTCCC-30 and 50 -TAAGGCCAACCATGATGTGC-30 . PCR cycling conditions consisted of 95 °C for 6 min, followed by 45 cycles of 95 °C for 15s, 60 °C for 30s, and 72 °C for 30s. Cycle threshold (CT) values were compared against a standard curve to estimate starting amounts of mRNA, and the relative expression of CXCR4 mRNA between samples was estimated by normalizing these values against 18S rRNA CT values were generated using a preoptimized 18S rRNA primer set (Applied Biosystems, FosterCity, CA). Western blotting. Western blotting was performed on cytosolic cellular extracts. Equal amounts of protein were resolved under reducing conditions on a 12% SDS–polyacrylamide gel. Protein
20
10
0 5
diu me
D
*
No.of invaded cells
C
7.5
m
pG lC tro n co
0
Relative expression of CXCR4
30
N OD
G Cp
N OD
* 120
*
90
60
30
5.0
0 2.5
0.0
N N m OD OD diu e G G M p Cp lC tro n Co
Fig. 1. CpG ODN enhanced the expression of CXCR4 on 95D cells. The 95D cells (3 103 cells/well) were stimulated with 10 lg/ml of CpG ODN or control CpG ODN in 24-well plate for 72 h. RT-PCR (A) and Real-time PCR (B) were performed to determinate the mRNA expression of CXCR4 in 95D cells. (C) Western blot was performed to detect the expression of CXCR4 protein in 95D cells. Three independent assays were performed. (D) 95D cells were assayed in 24-well cell-culture chambers using inserts with 8-mm pore membranes pre-coated with Matrigel for their invasive potentials. *p < 0.05.
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
migration was assessed using protein standards (Biorad, CA). Transfer to a nitrocellulose membrane was performed overnight at 30 V using a wet transfer system. Equal protein loading was confirmed with Ponceau staining. The membrane was washed in 5% skim milk in phosphate buffered saline + 0.03% Tween 20 (PBS-T) for 1 h in order to block nonspecific protein binding sites on the membrane. Immunoblotting was performed using a monoclonal antibody to CXCR4, Akt, I-jBa (Santa Cruz Biotechnology) at a dilution of 1/1000 in a nonfat milk–Tris buffer. The membrane was then washed in PBS-T and subsequently probed with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (Amersham Life Sciences) at a dilution of 1:5000 and developed with chemiluminescence (Pierce, IL). The membrane was then exposed to X-ray film (Kodak, NY) which was subsequently developed. Design of artificial siRNAs and transfection. We designed the CXCR4 targeting sequence (sense, 50 -GGAAGCUGUUGGCUGAAAAdTdT-30 ) as reported [20]. The nonspecific control siRNA duplexes were purchased from Dhamacon with the same GC content as CXCR4 siRNAs (42%, D001206-10). These siRNA gene double-strands were ligated with pSilencer4.1 RNAi Expression Vector (Ambion). Then, the vector with CXCR4–siRNA or control siRNA plasmids were transfected into the 95D cells using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s instruction. Migration assay. Wound healing assays were used to evaluate the migration of 95D cells transfected with siRNA against CXCR4 (termed as 95D-siR-CXCR4 cells) and 95D cells transfected with control siRNA (termed as 95D-ctrl-CXCR4 cells). Cells were seeded in six-well plates at 1 105 per well in growth medium. Confluent monolayers were starved overnight in assay medium and a single scratch wound was created using a micropipette tip. The cells were washed with PBS to remove cell debris, supplemented with assay medium containing 10 lg/ ml CpG ODN and monitored. Images were captured with a microscope using a 5 objective at 0 and 24 h post-wounding. For a quantitative measure, we counted the number of migrating cells to wound area based on the picture at 0 h post-wounding. Animal experiments. 95D cells (5 106) transfected with CXCR4–siRNA (treated group) or control-siRNA (control group) were pretreated with 10 lg/ml CpG ODN. After 3 days, the cell were collected and then injected into each mouse of 16 nude mice (8 mice each group) by the tail vein. At 30 days post-injection of cells, the mice were sacrificed. Lungs were harvested in optimum cutting temperature compound and frozen in liquid nitrogen. The collected lung tissue sections were subjected to H&E histostaining. The metastasis foci also were counted under microscope. Luciferase reporter assay plasmids. pNF-jB Luc and pcMV-lacZ were kindly supplied by Professor Ren Tao (Tongji University, China). 95D-siR-CXCR4 cells and 95D-ctrl-CXCR4 cells were co-transfected with various plasmids, respectively, and treated with 200 ng/ml SDF-1a in the presence of 10 lg/ml CpG ODN. After 24 h, luciferase and b-gal activity in 100 ll of cell lysates were measured by the Luciferase Assay System and the b-Galactosidase Enzyme Assay System (Promega), respectively. Transfection efficiency was normalized using b-gal activity. Statistical analyses. Statistical analyses of the data were performed with the SPSS12.0 software. Data were analyzed using a one-way analysis of variance (ANOVA) or Kruskal–Wallis test with PRISM 4.0 (GraphPad Soft-ware Inc, San Diego, CA, USA). *p < 0.05 was considered statistically significant in all comparisons.
Results TLR9 agonist enhanced the expression of CXCR4 on 95D cells Our previous data showed that TLR9 agonist could enhance the metastatic potential of lung cancer cell line 95D cells in vitro [10].
573
To further investigate the possible role of CXCR4/SDF-1 pathway in the metastatic potential of 95D cells enhanced by CpG ODN, we carefully estimated the expression of CXCR4 on 95D cells in response to CpG ODN. As shown in Fig. 1A and B, RT-PCR and Realtime PCR analysis revealed that the CXCR4 signal intensities was dramatically higher in CpG ODN treated 95D cells than that in control CpG ODN treated 95D cells and the control group (p < 0.05). To confirm the above findings, we further examined the expression of CXCR4 protein using Western blot. As shown in Fig. 1C, we found that the expression of CXCR4 protein was significantly higher in CpG ODN treated 95D cells (p < 0.05). In contrast, only weak expression of CXCR4 protein was detected in the control CpG ODN treated 95D cells and the control group (p < 0.05). Finally, we confirmed the enhanced metastasis of 95D cells induced by CpG ODN (Fig. 1D). These data strongly demonstrated that the TLR9 agonist could enhance the expression of CXCR4 on 95D cells. siRNA against CXCR4 down-regulated the expression of CXCR4 on CpG ODN treated 95D cell To determine the effect of CXCR4 expression level on the enhanced metastasis of 95D cells induced by CpG ODN, the expression of CXCR4 in 95D cells was down-regulated using siRNA against CXCR4. 95D cells were stably transfected with the CXCR4 siRNA (termed as 95D-siR-CXCR4) or control siRNA (termed as 95D-ctrl-CXCR4) and treated with CpG ODN, respectively. Then
Fig. 2. SiRNA against CXCR4 down-regulated the expression of CXCR4 on CpG ODN treated 95D cells. 95D cells were stably transfected with CXCR4 siRNA or control siRNA and then were stimulated with the indicated dose of CpG ODN at 3 103 cells/well in 24-well plate for 72 h, respectively. (A) RT-PCR was performed to determinate the mRNA expression of CXCR4 in indicated cells. (B) Western blot was performed to detect the expression of CXCR4 protein. Three independent assays were performed. *p < 0.05.
574
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
A
*
* *
* Relative Migration (%)
No. of invaded cells
125
B 125
*
*
100 75 50 25
100 75 50 25
CpG ODN
(0ug/ml)
(10ug/ml)
CpG ODN untreated
CXCR4-siRNA
CpG ODN treated
Control siRNA
CpG ODN
CpG ODN
(0ug/ml)
D
CXCR4-siRNA
Control-siRNA
CXCR4-siRNA
Control-siRNA
Control-siRNA
CpG ODN
0
Lung metastasis foci
C
CXCR4-siRNA
Control-siRNA
0
(10ug/ml)
*
*
15
10
5
CpG
ODN
untreated
CpG
CXCR4-siRNA
Control-siRNA
CXCR4-siRNA
CXCR4 siRNA
Control-siRNA
0
ODN
treated
Fig. 3. Down-regulation of CXCR4 expression reduced the metastasis of CpG ODN treated 95D cells in vitro and in vivo. (A) The indicated cells were stimulated with 10 lg/ml of CpG ODN in vitro for 72 h and then were assayed in 24-well cell-culture chambers using inserts with 8-mm pore membranes pre-coated with Matrigel for their invasive potentials. (B) Wound healing assays were used to evaluate the migration of 95D-siR-CXCR4 cells. For a quantitative measure, we counted the number of migrating cells to wound area based on the picture at 0 h post-wounding. (C) Eight nude mice were challenged with CpG ODN pretreated 95D cells for 8 weeks. The collected lung tissue sections were subjected to H&E histostaining. The arrows showed the metastatic cells (original magnification: 200). (D) The lung metastasis foci were calculated under microscope. Three independent assays were performed. *p < 0.05.
the expression of CXCR4 was determined. As shown in Fig. 2A, the mRNA expression of CXCR4 in 95D-siR-CXCR4 cells in response to CpG ODN was dramatically down-regulated compared with that in 95D-ctrl-CXCR4 cells (p < 0.05). To further confirm this phenomenon, Western blot was performed to detect the expression of CXCR4 protein in the CpG ODN treated 95-siR-CXCR4 cells and 95-ctrl-CXCR4 cells. As shown in Fig. 2B, compared with that in 95D-ctrl-siRNA group, the expression of CXCR4 protein was significantly reduced in 95D-siRNA group (p < 0.05), which was even lower than that of the untreated 95D-ctrl-CXCR4 cells. These data suggested that siRNA against CXCR4 could effectively down-regulated the expression of CXCR4 on CpG ODN treated 95D cells. Down-regulation of CXCR4 expression reduced the metastasis of CpG ODN treated 95D cells in vitro and in vivo To determine whether down-regulation of CXCR4 could affect the metastasis of 95D cells induced by CpG ODN, 95D-siR-CXCR4 cells and 95D-ctrl-CXCR4 cells were stimulated with CpG ODN,
respectively. Then their invasive potentials and migration in response to SDF-1a was determined in vitro. We investigated that down-regulation of CXCR4 with siRNA potently reduced the invasion and migration of 95D cells induced by CpG ODN (Fig. 3A and B, p < 0.05). To further confirm the effect of CXCR4 expression on the enhanced metastasis of 95D cells induced by CpG ODN in vivo, the nude mice were challenged with 95D-siR-CXCR4 cells pretreated with CpG ODN as previous described [10] and then the lung metastasis were evaluated. We found that the metastatic potential of 95D cells was dramatically abrogated by the CXCR4 siRNA in vivo compared with the control group (Fig. 3C and D, p < 0.05). These results strongly suggested that CXCR4 pathway was crucial for CpG ODN to enhance the metastasis of 95D cells. Down-regulation of CXCR4 expression reduced the Akt phosphorylation in CpG ODN treated 95D cells Previous findings showed that CXCR4/SDF-1 signaling could promote the metastasis of tumor cells via the Akt pathway [21].
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
To further verify whether CpG ODN enhanced the metastasis of 95D cells through CXCR4/SDF-1/Akt pathway, the expression of phosphorylated Akt in the 95D-siR-CXCR4 cells in response to CpG ODN was determined. 95D-ctrl-CXCR4 cells were used as the control. As shown in Fig. 4A, Western blot analysis revealed that SDF-1a induced the phosphorylation of Akt in 95D-ctrlCXCR4 cells at 15 min. Conversely, phosphorylation of Akt in 95D-siR-CXCR4 cells could not be induced after SDF-1a addition (p < 0.05). In addition, there were no changes in the expression level and phosphorylation of I-jBa in both the two groups. To further confirm these results, luciferase reporter assay was performed to detect the activity of NF-jB promoter. As shown in Fig. 4B, SDF-1a could not alter the activity of NF-jB promoter in CpG ODN treated 95D-ctrl-CXCR4 cells and 95D-siR-CXCR4 cells. All of these data indicated that CXCR4/SDF-1/Akt pathway might be important for CpG ODN enhanced metastasis of 95D cells. Discussion Both TLR9 and CXCR4 are recognized individually as tumor enhancer involved in tumor progress [1,22]. Although CXCR4 has been demonstrated to play roles in metastasis of tumors [23,24], the possible role of CXCR4 pathway in TLR9 signaling pathway on metastasis of tumor is under investigation. In this study, we showed that CpG ODN enhanced the expression of CXCR4 on 95D cells, which was consistent with our previous findings [10]. Fur-
575
thermore, we extended our previous work by demonstrating that down-regulation of CXCR4 expression could significantly inhibit the metastasis of 95D cells enhanced by CpG ODN in vitro and in vivo. In addition, we also showed that down-regulation of CXCR4 expression could significantly reduce the phosphorylation of Akt but not the phosphorylation of I-jB and activation of NFjB in 95D cells in response to CpG ODN. These results were consistent with the previous findings which showed that CXCL12/ CXCR4 signaling could activate Akt-1 expression in prostate cancer cells [18]. Our results suggested that the CXCR4/SDF-1/Akt pathway was essential for TLR9 pathway to enhance the metastasis of 95D cells. In fact, recent studies showed that CXCR4 pathway could modulate TLR9 signaling in macrophage [19]. Therefore, it is worthy and interesting to study the exact mechanism underlying the cross-talk between CXCR4/SDF-1 pathway and TLR9 pathway on the metastasis of tumor cells in the following experiments. In summary, here we demonstrated that CXCR4/SDF-1 pathway was critical for TLR9 agonist to enhance the metastasis of human lung cancer cells. These finding may provide a novel insight on the understanding of underlying mechanism of TLR9 pathway on tumor metastasis. Acknowledgments This work was supported by Zunyi Medical College start-up Fund (F-329), Fund of Qingdao Municipal Science and Technology Commission (07-2-1-13-nsh), Zunyi Medical College start-up Fund (F-297), and The National Prophase Project on Basic Research of China (2008CB517310). References
Fig. 4. Down-regulation of CXCR4 expression reduced the Akt phosphorylation in CpG ODN treated 95D cells. (A) 95D-siR-CXCR4 cells were treated with 200 ng/ml SDF-1a in the presence of CpG ODNs. The phosphorylation of Akt and I-jBa were detected by Western blot. 95D-ctrl-CXCR4 cells were used as control. (B) The activity of NF-jB promoter also was determined by luciferase reporter assay. Three independent assays were performed. *p < 0.05.
[1] B. Huang, J. Zhao, J.C. Unkeless, Z.H. Feng, H. Xiong, TLR signaling by tumor and immune cells: a double-edged sword, Oncogene 27 (2008) 218–224. [2] R. Chen, A.B. Alvero, D.A. Silasi, K.D. Steffensen, G. Mor, Cancers take their Toll— the function and regulation of Toll-like receptors in cancer cells, Oncogene 27 (2008) 225–233. [3] B. Huang, J. Zhao, H. Li, Toll-like receptors on tumor cells facilitate evasion of immune surveillance, Cancer Res. 65 (2005) 5009–5014. [4] C. Song, L.Z. Chen, R.H. Zhang, X.J. Yu, Y.X. Zeng, Functional variant in the 30 untranslated region of Toll-like receptor 4 is associated with nasopharyngeal carcinoma risk, Cancer Biol. Ther. 5 (2006) 1285–1291. [5] B. Schmausser, M. Andrulis, S. Endrich, H.K. Muller-Hermelink, M. Eck, Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori, Int. J. Med. Microbiol. 295 (2005) 179–185. [6] M. Zaks-Zilberman, T.Z. Zaks, S.N. Vogel, Induction of proinflammatory and chemokine genes by lipopolysaccharide and paclitaxel (Taxol) in murine and human breast cancer cell lines, Cytokine 15 (2001) 156–165. [7] D. Droemann, D. Albrecht, J. Gerdes, A.J. Ulmer, D. Branscheid, E. Vollmer, K. Dalhoff, P. Zabel, T. Goldmann, Human lung cancer cells express functionally active Toll-like receptor 9, Respir. Res. 6 (2005) 1. [8] F. Tsushima, K. Tanaka, N. Otsuki, P. Youngnak, H. Iwai, K. Omura, M. Azuma, Predominant expression of B7–H1 and its immunoregulatory roles in oral squamous cell carcinoma, Oral Oncol. 42 (2006) 268–274. [9] J.M. Ilvesaro, M.A. Merrell, T.M. Swain, J. Davidson, M. Zayzafoon, K.W. Harris, K.S. Selander, Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro, Prostate 67 (2007) 774–781. [10] T. Ren, Z.K. Wen, Z.M. Liu, Y.J. Liang, Z.L. Guo, L. Xu, Functional expression of TLR9 is associated to the metastatic potential of human lung cancer cell: functional active role of TLR9 on tumor metastasis, Cancer Biol. Ther. 6 (2007) 1704–1709. [11] R.M. Strieter, Chemokines: not just leukocyte chemo-attractants in the promotion of cancer, Nat. Immunol. 2 (2001) 285–286. [12] R.J. Phillips, M.D. Burdick, M. Lutz, J.A. Belperio, M.P. Keane, R.M. Strieter, The stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis innon-small cell lung cancer metastases, Am. J. Respir. Cell Mol. Biol. 167 (2003) 1676–1686. [13] K. Oonakahara, W. Matsuyama, I. Higashimoto, M. Kawabata, K. Arimura, M. Osame, Stromal-derived factor-1a/CXCL12-CXCR4 axis is involved in the dissemination of NSCLC cells into pleural space, Am. J. Respir. Cell Mol. Biol. 30 (2004) 671–677. [14] H.H. Oliveira-Neto, E.T. Silva, C.R. Leles, E.F. Mendonça, C. Alencar Rde, T.A. Silva, A.C. Batista, Involvement of CXCL12 and CXCR4 in lymph node metastases and development of oral squamous cell carcinomas, Tumour Biol. 29 (2008) 262–271.
576
L. Xu et al. / Biochemical and Biophysical Research Communications 382 (2009) 571–576
[15] J. Wang, R. Loberg, R.S. Taichman, The pivotal role of CXCL12 (SDF-1)/CXCR4 axis in bone metastasis, Cancer Metastasis Rev. 25 (2006) 573–587. [16] M.K. Wendt, A.N. Cooper, M.B. Dwinell, Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells, Oncogene 27 (2008) 1461–1471. [17] C.T. Tan, C.Y. Chu, Y.C. Lu, C.C. Chang, B.R. Lin, H.H. Wu, H.L. Liu, S.T. Cha, E. Prakash, J.Y. Ko, M.L. Kuo, CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway, Carcinogenesis 29 (2008) 1519–1527. [18] S.R. Chinni, S. Sivalogan, Z. Dong, J.C. Filho, X. Deng, R.D. Bonfil, M.L. Cher, CXCL12/CXCR4 signaling activates Akt-1 and MMP-9 expression in prostate cancer cells: the role of bone microenvironment-associated CXCL12, Prostate 66 (2006) 32–48. [19] M. Ishii, C.M. Hogaboam, A. Joshi, T. Ito, D.J. Fong, S.L. Kunkel, CC chemokine receptor 4 modulates Toll-like receptor 9-mediated innate immunity and signaling, Eur. J. Immunol. 38 (2008) 2290–2302.
[20] Z. Liang, Y. Yoon, J. Votaw, M.M. Goodman, L. Williams, H. Shim, Silencing of CXCR4 blocks breast cancer metastasis, Cancer Res. 65 (2005) 967–971. [21] Z. Liang, J. Brooks, M. Willard, K. Liang, Y. Yoon, S. Kang, H. Shim, CXCR4/CXCL12 axis promotes VEGF-mediated tumor angi-ogenesis through Akt signaling pathway, Biochem. Biophys. Res. Commun. 359 (2007) 716–722. [22] S. Gelmini, M. Mangoni, M. Serio, P. Romagnani, E.J. Lazzeri, The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis, Endocrinol. Invest. 31 (2008) 809–819. [23] M.Z. Ratajczak, E. Zuba-Surma, M. Kucia, R. Reca, W. Wojakowski, J. Ratajczak, The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis, Leukemia 20 (2006) 1915–1924. [24] M.Z. Dewan, S. Ahmed, Y. Iwasaki, K. Ohba, M. Toi, N. Yamamoto, Stromal cell-derived factor-1 and CXCR4 receptor interaction in tumor growth and metastasis of breast cancer, Biomed. Pharmacother. 60 (2006) 273–276.