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The Role of Nrf2 in Migration and Invasion of Human Glioma Cell U251
PEER-REVIEW REPORTS COCHLEA RADIATION AND HEARING LOSS IN VESTIBULAR SCHWANNOMA RADIOSURGERY
17. Poen JC, Golby AJ, Forster KM, Martin DP, Chinn DM, Hancock SL, Adler JR Jr: Fractionated stereotactic radiosurgery and preservation of hearing in patients with vestibular schwannoma: a preliminary report. Neurosurgery 45:1299-1305; discussion 1305–1297, 1999. 18. Tamura M, Carron R, Yomo S, Arkha Y, Muraciolle X, Porcheron D, Thomassin JM, Roche PH, Regis J: Hearing preservation after gamma knife radiosurgery for vestibular schwannomas presenting with high-level hearing. Neurosurgery 64:289-296; discussion 296, 2009. 19. Thomas C, Di Maio S, Ma R, Vollans E, Chu C, Clark B, Lee R, McKenzie M, Martin M, Toyota B: Hearing preservation following fractionated stereotactic
radiotherapy for vestibular schwannomas: prognostic implications of cochlear dose. J Neurosurg 107: 917-926, 2007. 20. Timmer FC, Hanssens PE, van Haren AE, Mulder JJ, Cremers CW, Beynon AJ, van Overbeeke JJ, Graamans K: Gamma knife radiosurgery for vestibular schwannomas: results of hearing preservation in relation to the cochlear radiation dose. Laryngoscope 119:1076-1081, 2009. Conflict of interest statement: Dr. John Adler is Vice President of Varian Medical, Inc.; Dr. Iris Gibbs is a member of the Clinical Advisory Board and the Speakers’ Bureau of Accuray, Inc., manufacturer of the CyberKnife radiosurgical system. This work is in part supported by
gifts from Robert C. and Jeannette Powell, Alan Wong and Sylvia Tang, and Paula and William Zappettini to Steven D. Chang, MD. M. G. Hayden Gephart is supported by a postdoctoral fellowship from the California Institute of Regenerative Medicine. Received 20 January 2012; accepted 03 April 2012; published online 05 April 2012 Citation: World Neurosurg. (2013) 80, 3/4:359-363. http://dx.doi.org/10.1016/j.wneu.2012.04.001 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter © 2013 Elsevier Inc. All rights reserved.
The Role of Nrf2 in Migration and Invasion of Human Glioma Cell U251 Hao Pan, Handong Wang, Lin Zhu, Lei Mao, Liang Qiao, Xingfen Su
Key words 䡲 Glioma 䡲 Invasion 䡲 Matrix metalloproteinase 9 䡲 Migration 䡲 NF-E2-related factor 2 Abbreviations and Acronyms ARE: antioxidant response element DMEM: Dulbecco’s modified Eagle medium MMP9: Matrix metalloproteinase 9 Nrf2: NF-E2-related factor 2 RT-PCR: Reverse transcriptase–polymerase chain reaction Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People’s Republic of China To whom correspondence should be addressed: Handong Wang, M.D., Ph.D. [E-mail: [email protected]] Citation: World Neurosurg. (2013) 80, 3/4:363-370. http://dx.doi.org/10.1016/j.wneu.2011.06.063 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter © 2013 Elsevier Inc. All rights reserved.
INTRODUCTION The transcription factor NF-E2-related factor 2 (Nrf2) is considered as a critical regulator of intracellular antioxidants and phase II detoxification enzymes by transcriptional upregulation of many antioxidant response element (ARE)-containing genes. It belongs to Keap1Nrf2-ARE signaling pathway. In response to oxidative stress, Nrf2 is activated and dissociated from Keap1. After translocated from cy-
䡲 OBJECTIVE: NF-E2-related factor 2 (Nrf2) is a transcription factor that is related to tumor cell multidrug resistance and proliferation. Here we studied the involvement of Nrf2 in the migration and invasion of human U251 glioma cells. 䡲 METHODS: Two kinds of plasmid, that is, pEGFP-Nrf2 and Si-Nrf2, were constructed and transfected to upregulate or downregulate the expression of Nrf2 in U251 glioma cell line. Blank vectors or random siRNA plasmid were used as negative control. Cells treated with lipofectamine only were set up as blank control. Protein and mRNA level of Nrf2 and matrix metalloproteinase 9 (MMP9) were investigated by reverse transcriptase–polymerase chain reaction and western blot after transfection. Wound healing assay and transwell assay were used to study migration and invasion of U251 after transfection. Gelatin zymography was performed to reveal the change of MMP9 activity after transfection. 䡲 RESULTS: The mRNA and protein level of Nrf2 was upregulated in U251pEGFP-Nrf2 while downregulated in U251-Si-Nrf2 48 hours after transfection. In the wound healing assay, there were more cells in group pEGFP-Nrf2 crossing the scratch line than in group Si-Nrf2. Furthermore, in transwell migration and invasion assay, there were more cells in group pEGFP-Nrf2 penetrating the membranes than in group Si-Nrf2. Then we investigated the change of MMP9 activity, mRNA, and protein levels after transfection. The results suggested that upregulation of Nrf2 led to an increase in MMP9 expression and activity whereas downregulation of Nrf2 led to a decrease in MMP9 expression and activity. 䡲 CONCLUSION: Nrf2 is involved in migration and invasion of U251 cells, which may be related to MMP9.
toplasm to nucleus, Nrf2 forms a heterodimer with Maf, which binds to the ARE sequence to upregulate many kinds of genes expression (21). The Nrf2 downstream genes include 1) intracellular redox-balancing proteins: glutamate cysteine ligase, glutathione peroxidase,
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and heme oxygenase-1; 2) phase II detoxifying ⬎enzymes: glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase–1; and 3) transporters: multidrug resistance–associated protein (21). Recent studies have found more Nrf2 downstream genes that are
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Table 1. Primers and Parameters Used in Reverse Transcriptase–Polymerase Chain Reaction Primer Name
TUMOR
Nrf2
Forward 5=-TCAGCATGCTACGTGATGAAG-3=
Size Reverse 5=-TTTGCTGCAGGGAGTATTCA-3=
Tm (°C)
(bp)
Cycle
58
68
38
MMP9
5=-CGGTGATTGACGACGCCTTTG-3=
5=-TTGGAACCACGACGCCCTTGC-3=
63
255
35
GAPDH
5=-GAAGGTGAAGGTCGGAGTC-3=
5=-GAAGATGGTGATGGGATTTC-3=
58
226
35
Tm, melting temperature; bp, base pair; Nrf2, NF-E2-related factor 2; MMP9: matrix metalloproteinase 9; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
involved in stress response, xenobiotics metabolism, the ubiquitin-mediated proteasomal degradation system, cell proliferation and so on (5, 20, 22, 36, 41). In normal tissue, Nrf2 is considered as an important transcript factor for cells to survive from many kinds of negative stimulus such as injury, ischemia, inflammation, hemorrhage, cancer, pulmonary fibrosis, acute pulmonary injury, and degeneration disease (6, 8, 18, 19, 36, 38, 42, 44). Interestingly, Nrf2 may play a dark role in tumor tissue. It has been verified that Nrf2 is upregulated in lung, head, and neck squamous cell carcinoma tissues (39, 40). Further investigation shows that overexpression of Nrf2 enhances tumor resistance to chemotherapeutic agents in some lung carcinoma, breast adenocarcinoma, and neuroblastoma cell lines (43). Matrix metalloproteinase 9 (MMP9), which belongs to the ECM-degrading enzyme family, is a kind of gelatinase. MMP9 is involved in migration and invasion of tumor cells. It has been found that the transcription and activity levels of MMP9 are higher in glioma than in normal brain and such elevation is strongly correlated with the tumor grade (9). Our previous study (25) proved that MMP9 was upregulated in Nrf2 knockout mice than wild-type mice after spinal cord injury. This result suggests the possible relationship between Nrf2 and MMP9. In this study, we investigated the change of MMP9 level, migration, and invasion ability of human U251 glioma cells after up- or downregulating the expression of Nrf2.
MATERIALS AND METHODS Cell Culture and Transient Transfection Human U251 glioma cells were obtained from ATCC and cultured in Dulbecco’s mod-
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ified Eagle medium (DMEM; HyClone, Illinois, USA) with 10% fetal bovine serum (HyClone) at 37°C and 5% CO2 incubator. The primers for human Nrf2 cDNA were as follows: forward 5=-CCGCTCGAGATGATGGACTTGGAGCTGCC-3=, reverse 5=-GGGGTACCGTGTTTTTCTTAACATCTGGC-3=. Human Nrf2 cDNA was cloned into the cloning site of the vector pEGFP-N1 (GeneChem, Shanghai, China) using the standard re-
combinant DNA technique. The new plasmid was named as pEGFP-Nrf2. And a blank vector (pEGFP) was used as negative control. The vector pGPH1/GPF/Neo used for cloning Nrf2 short hairpin RNA (shRNA) was purchased from GenePharma (Shanghai, China). The target sequence was GCAGTTCAATGAAGCTCAACT. The new plasmid was named as Si-Nrf2. Random sequence was used as
Figure 1. mRNA --⬎level of Nrf2 and MMP9 analyzed by reverse transcriptase–polymerase chain reaction 48 hours after transfection. (A) Group pEGFP-Nrf2 showed higher expression whereas group Si-Nrf2 showed lower expression of Nrf2 and MMP9 mRNA level. There was no obvious difference among group pEGFP, Si-control, and Lipo. (B) Relative mRNA level of Nrf2/GAPDH in group pEGFP-Nrf2 was 1.00 ⫾ 0.01 with 0.55 ⫾ 0.01 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among three control groups. (C) Relative mRNA level of MMP9/GAPDH in group pEGFP-Nrf2 was 0.84 ⫾ 0.04 with 0.64 ⫾ 0.02 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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Reverse Transcription-PCR Total RNA was isolated with Trizol (Invitrogen) and single-stranded cDNA was synthesized from 2 g of total RNA with BU-Script RT-Kit (Biouniquer, Jiangsu, China) according to the manufacturer’s protocol. The cDNA was stored in –20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, Wisconsin, USA) according to the manufacturer’s protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by using agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of GAPDH was used as an internal reference gene.
Figure 2. Protein level of Nrf2 and MMP9 analyzed by Western blot 48 hours after transfection. (A) Group pEGFP-Nrf2 showed higher expression and group Si-Nrf2 showed lower expression of Nrf2 and MMP9 protein level. There was no obvious difference among group pEGFP, Si-control and Lipo. (B) Relative protein level of Nrf2/-actin in group pEGFP-Nrf2 was 1.02 ⫾ 0.06 with 0.24 ⫾ 0.01 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among the three control groups. (C) Relative protein level of MMP9/-actin in group pEGFP-Nrf2 was 1.26 ⫾ 0.01 with 0.54 ⫾ 0.01 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
negative control, which was named as Sicontrol. Cells were seeded in six-well plates at 1 ⫻ 106/well and allowed to attach for 24 hours before transfection. Then pEGFPNrf2, pEGFP, Si-Nrf2, and Si-control were transfected by lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s protocol. Cells treated
with lipofectamine 2000 alone were set up as blank control, which was named as group Lipo. After incubation at 37°C and 5% CO2 for 24, 48, and 72 hours, cells were collected to testify the expression of Nrf2 by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.
Western Blot Analysis To obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DL-dithiothreitol, 1 mM phenylmethane-sulfonyl fluoride, and protease inhibitor cocktail in phosphate-buffered saline pH 7.4) and centrifuged at 12,000g for 15 minutes at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, Illinois, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in the saline buffer overnight at 4°C, and the following antibodies were used: anti-Nrf2 (sc-722 [57 kDa]; Santa Cruz Biotechnology, California,
Table 2. The Results of Wound Healing Assay, Migration and Invasion Assay pEGFP-Nrf2
pEGFP
Si-Nrf2
Si-control
Lipo
116.83 ⫾ 5.27 *
82.17 ⫾ 5.12
31.00 ⫾ 4.69†
82.50 ⫾ 4.04
77.00 ⫾ 4.38
Migration assay
21.50 ⫾ 3.83*
11.67 ⫾ 1.63
4.17 ⫾ 1.47†
11.00 ⫾ 2.28
11.67 ⫾ 1.75
Invasion assay
33.33 ⫾ 4.50*
14.83 ⫾ 2.23
6.00 ⫾ 2.10†
15.17 ⫾ 1.72
14.83 ⫾ 3.06
Wound healing assay
The data indicate the number of cells crossing the scratch line or penetrating the membranes. Cells in group pEGFP-Nrf2 shows enhanced migratory and invasive ability than cells in group Si-Nrf2. There was no statistical difference among three control groups. Nrf2, NF-E2-related factor 2; Lipo, lipofectamine. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. †P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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USA), anti--actin (sc-130301 [42 kDa]; Santa Cruz Biotechnology), and anti-MMP9 (sc-21733 [92 kDa]; Santa Cruz Biotechnology). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 hour at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase–linked secondary antibody at a 1:2000 dilution for 1 hour. Blots were incubated with enhanced chemiluminescence detection system (Amersham Biosciences, Bucks, United Kingdom) and exposed radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of -actin was used as an internal reference gene. Migration and Invasion Assay Forty-eight hours after transfection, cells were treated with trypsin and resuspended to single-cell solutions. A total of 1 ⫻ 105 cells in 200 L DMEM were seeded on the upper chamber of 6.5 mm Transwell with 8.0-m-pore polycarbonate membrane insert (Corning Inc, New York, USA). DMEM (600 L) with 10% fetal bovine serum was added into the lower chamber as a chemoattractant. After the cells were incubated for 8 hours, the insert was washed with phosphate-buffered saline and cells on the top surface of the insert were removed gently by a cotton swab. Cells adhering to the lower surface were fixed by methanol, stained by crystal violet, and counted under microscope in six predetermined fields (⫻200). The matrigel invasion assay was similar to the cell migration assay, except that the transwell membrane was precoated with ECM gel (Sigma-Aldrich, Missouri, USA) and the cells were incubated for 24 hours. All assays were independently repeated at least three times.
Wound Healing Assay Twenty-four hours after transfection, cells were seeded at the density of 1 ⫻ 105/well in a 24-well plate. Incubated for 24 hours (48 hours after transfection), cells were scratched using the back side of a standard 100-L pipette tip. After being washed three times with phosphate-buffered saline, scratches including the flanking front lines of cells were photographed (40-fold magnification). The edges of the wound were marked after scratching (time point 0
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Figure 3. Wound healing assay. (A) There were more cells crossing the edges in group pEGFP-Nrf2 and lesser cells in group Si-Nrf2 as compared with three control groups 48 hours after scratch (96 hours posttransfection) (⫻40). (B) Counting cell numbers migrating over the edges 48 hours after scratch (96 hours posttransfection) with ImageJ, we found that there were 116.83 ⫾ 5.27 cells crossing the scratch line in group pEGFP-Nrf2 with 31.00 ⫾ 4.69 cells in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among the three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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Figure 4. Transwell migration assay. (A–E) Cells of five groups penetrated the membranes 8 hours after seeding in the Transwell upper chamber (⫻100). (F) There were 21.50 ⫾ 3.83 cells through the membranes in group pEGFP-Nrf2 with only 4.17 ⫾ 1.47 cells in group Si-Nrf2 (P ⬍ 0.001).
No statistical difference showed in three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
Figure 5. Transwell invasion assay. (A–E) Cells of five groups penetrated the membranes 24 hours after seeding in the Transwell upper chamber, which was precoated with ECM gel (⫻100). (F) There were 33.33 ⫾ 4.50 cells through the membranes in group pEGFP-Nrf2 compared with 6.00 ⫾
2.10 cells in group Si-Nrf2 (P ⬍ 0.001). No statistical difference showed in the three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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hours). Cells were incubated under standard conditions. The scratched area was photographed 24 and 48 hours after wounding (corresponding to 72 and 96 hours posttransfection). Cells migrating over the edges were counted 48 hours after the scratch. Experiments were performed in triplicate, evaluating four scratches in each experiment.
Gelatin Zymography Cells of five groups were homogenized in the lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, 0.01% Triton, and then mixed with electrophoresis loading buffer. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer’s instruction (Genmed Scientifics Inc, Massachusetts, USA). Briefly, 40 g cytosolic protein extracts were separated by electrophoresis. The proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in the substrate buffer for 40 hours at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 hour and destained in 50% methanol. Proteolytic activities were shown by clear bands in blue gel, indicating the lysis of the substrate. Quantification of MMP9 band density was performed with the image analysis program ImageJ and was described in arbitrary units. Statistical Analysis All experiments were done at least three times. Data were expressed as mean ⫾ SD. Data were evaluated by analysis of variance and least significant difference multiple comparison test. P value ⬍0.05 were considered to be significant. All analyses were performed by SPSS 18.0. RESULTS Transient Transfection Effect on Nrf2 mRNA and Protein Level To validate the transient transfection effect of four plasmids mentioned above, we tested the mRNA and protein level of five cell groups 24, 48, 72 hours after transfection, respectively. RT-PCR and western blot results indicated that transfection efficiency was most predominant at 48 hours after
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transfection (data not shown). Transfection of pEGFP-Nrf2 enhanced the Nrf2 mRNA and protein level ⬎38% and ⬎130% as compared with transfection of pEGFP, respectively (P ⬍ 0.05). Si-Nrf2 transfection reduced the Nrf2 mRNA and protein level ⬎30% and ⬎40% as compared with transfection of Si-control, respectively (P ⬍ 0.05). Group pEGFP, Si-control, and Lipo showed no difference in Nrf2 mRNA and protein level (Figures 1 and 2). So we chose 48 hours after transfection as the time point for further study.
Nrf2 Is Involved in U251 Cell Migration and Invasion To examine the role of Nrf2 in U251 cell migration, we performed wound healing assay and transwell migration assay. As showed in Table 2, cells in group pEGFPNrf2 showed incremental migratory ability than cells in group pEGFP and Lipo, whereas cells in group Si-Nrf2 showed decreased migration ability than cells in group Si-control and Lipo in the wound healing assay (P ⬍ 0.001) (Figure 3). Transwell migration assay showed a similar result, with more cells penetrating the membranes in group pEGFP-Nrf2 than in group pEGFP and Lipo (P ⬍ 0.001). There were fewer cells penetrating the membranes in group Si-Nrf2 than in group Si-control and Lipo (P ⬍ 0.001) (Figure 4). There was no statistical difference among the three control groups. In the transwell invasion assay, ECM gel was used to simulate the extracellular matrix around glioma cells, which would reproduce tumor microenvironment in vitro. As showed in Table 2, there were more cells in group pEGFP-Nrf2 that crossed the membranes than in group pEGFP and Lipo 24 hours after seeding (P ⬍ 0.001) (Figure 5). There were fewer cells that crossed the membranes in group Si-Nrf2 than in group Si-control and Lipo 24 hours after seeding (P ⬍ 0.001) (Figure 5). No statistical difference was found among the three control groups.
Change of MMP9 Activity, mRNA, and Protein Level After Transfection As mentioned above, upregulation of Nrf2 enhanced the invasion and migration of
Figure 6. Gelatin zymography. (A) Gelatin zymography analysis showed brighter band in pEGFP-Nrf2 and bleaker band in Si-Nrf2 than in group pEGFP, Si-control, and Lipo. (B) Quantitative analysis showed that MMP9 activity was higher in pEGFP-Nrf2 than in pEGFP (90.65 ⫾ 5.050 vs. 64.03 ⫾ 2.780, P ⬍ 0.001). The MMP9 activity of Si-Nrf2 was lower than that of Si-control (43.07 ⫾ 2.596 vs. 67.05 ⫾ 2.192, P ⬍ 0.001). Group pEGFP, Si-control, and Lipo showed no difference in MMP9 activity. The data were described in arbitrary units. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
U251 cells whereas downregulation of Nrf2 reduced the invasion and migration of U251 cells. It is believed that MMP9 is a critical enzyme in tumor cell migration and invasion. So here we detected the MMP9 expression after transfection. Forty-eight hours after transfection, cells were collected to check the mRNA and protein level of MMP9 by RT-PCR and Western blot. MMP9 mRNA and protein level of U251pEGFP-Nrf2 were 0.84⫾ 0.04 and 1.26⫾ 0.01, respectively, compared with 0.69⫾ 0.01 and 0.69 ⫾ 0.02 for U251-pEGFP (P ⬍ 0.001) as compared with the internal reference gene. MMP9 mRNA and protein level of U251-Si-Nrf2 were 0.64 ⫾ 0.02 and 0.54 ⫾ 0.01, respectively, compared with 0.69 ⫾ 0.02 and 0.68 ⫾ 0.02 for U251-Si-control (P ⬍ 0.001) as compared with the internal reference gene (Figures 1 and 2). Gelatin zymography revealed higher MMP9 activity in U251-pEGFPNrf2 than in U251-pEGFP (90.65 ⫾ 5.050 vs. 64.03 ⫾ 2.780, P ⬍ 0.001). The MMP9 activity of Si-Nrf2 was lower than that of Sicontrol (43.07 ⫾ 2.596 vs. 67.05 ⫾ 2.192,
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P ⬍ 0.001) (Figure 6). Group pEGFP, Sicontrol, and Lipo showed no difference in MMP9 activity and mRNA and protein level. Summarily, upregulation of Nrf2 led to an increase in MMP9 expression and activity whereas downregulation of Nrf2 led to a decrease in MMP9 expression and activity.
DISCUSSION Nrf2 was first discovered by Moi in 1994 (27). Follow-up study showed that Nrf2 is a critical transcription factor in Keap1-Nrf2ARE signaling pathway to resist oxidative stress. Nrf2 is considered as a “good” protein because many chemopreventive compounds have been found to be its inducer, such as sulforaphane (16), curcumin (1), epigallocatechin-3-gallate (29), resveratrol (4), wasabi (28), cafestol, kahweol (12), lycopene (3), and carnosol (37). All these compounds have been proven to be able to prevent tumorigenesis through activation of Nrf2 and further downstream antioxidant genes. Studies reveal that Nrf2 knockout mice show increased cancer susceptibility to carcinogens and are refractory to the protective actions of some chemopreventive agents, which confirm the role of Nrf2 in chemoprevention on the other side (2, 17, 23). Surprisingly, new emerging results have revealed that Nrf2 not only protects the normal cells from carcinogens but also promotes the tumor cell survival in chemotherapeutics. In 2004, Ikeda (14) found that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells, which started the studies on tumor promotion effect of Nrf2. Most studies focused on lung cancer. It has been revealed that Nrf2 is mutational and overexpressed in many lung tumor tissues and cell lines (11, 31, 39). Similar results were also found in prostate, breast, head, and neck cancer (10, 30, 40). Further research indicates that Nrf2 plays a critical role in tumor cell proliferation and chemoresistance. Nrf2 is highly activated in human lung cancer cell line A549. After knockdown by siRNA technology, cell proliferation of A549 cells is prominently inhibited, accompanied by chemoresistance to cisplatin (13). Cisplatin-resistant human ovarian cancer SK-OV cells show exacerbated cytotoxicity following cisplatin treatment after transfection of Nrf2 siRNA (7).
NRF2 PARTICIPATES IN MIGRATION AND INVASION OF GLIOMA
Similar discovery is also present in different cancer types and chemotherapeutic agents (43). All these results indicate the “dark” side of Nrf2. Meng’s group found that low concentration of arsenic stimulated cell migration and vascular tube formation in human microvascular endothelial cells with overexpression of heme oxygenase-1 mRNA and protein, which is a result of increased Nrf2 binding to the heme oxygenase-1 transcription site (26). This finding suggests that Nrf2 participates not only in cell proliferation and chemoresistance but also possibly in cell migration and invasion. For the first time, our results demonstrate that enhanced expression of Nrf2 promotes glioma cell invasion and migration, whereas reduced expression of Nrf2 attenuates it. Interestingly, Rachakonda et al. (34) found that A549 cells stably expressing Nrf2 shRNA and HepG2 cells stably expressing FLAG/Keap1, which both reduced the expression of Nrf2 and exhibited an increase in migration compared to control. We hypothesize that the differences in migration observed by Rachakonda et al. (34) and the work reported here may be attributed to stable versus transient repression of Nrf2 and different cell lines. To investigate the mechanism of Nrf2’s role in glioma cell invasion and migration, we focused on the enzyme MMP9. MMP9, also named gelatinase B, degrade denatured collagen and type IV collagen. It has been confirmed that MMP9 protein is not only prominently expressed in vascular structures of glioma but also expressed higher in tumor cells than in normal cells. And its expression and activity levels are strongly correlated with the tumor grade (9, 35). Downregulated expression of AKT2 and EGFR by siRNA decreased expression of MMP9 in glioma, accompanied by weakened invasion ability (15, 45). These studies indicate that MMP9 plays an important role in glioma invasion and migration. Recent study shows that arsenic induces malignant transformation of human keratinocytes, which is correlated with elevated expression of Nrf2 and MMP9 (33). In spinal cord injury, Nrf2 knockout mice show higher expression and activity of MMP9 than Nrf2 wild-type mice (25). All these suggest the possible relationship between Nrf2 and MMP9. Our results indicate that upregulated expression of Nrf2 promotes overexpression of MMP9 in mRNA and protein
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levels, while downregulated expression of Nrf2 reduces expression of MMP9. Oxidative stress activates EGFR or PI3K/AKT signaling pathway and then induces translocation of Nrf2 from the cytoplasm to the nucleus. Inhibition of AKT activity markedly attenuates Nrf2 translocation and downstream gene transcription (24, 32). As mentioned above, EGFR and AKT can also regulate the expression of MMP9 (15, 45). So EGFR and AKT may be the link of Nrf2 and MMP9. But the signal pathway between Nrf2 and MMP9 is unknown, which still needs further research.
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Tuder RM, Biswal S: Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 114:1248-1259, 2004. 37. Satoh T, Kosaka K, Itoh K, Kobayashi A, Yamamoto M, Shimojo Y, Kitajima C, Cui J, Kamins J, Okamoto S, Izumi M, Shirasawa T, Lipton SA: Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem 104: 1116-1131, 2008. 38. Shih AY, Li P, Murphy TH: A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo. J Neurosci 25:10321-10335, 2005. 39. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E, Brock MV, Biswal S: Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 3:e420, 2006. 40. Stacy DR, Ely K, Massion PP, Yarbrough WG, Hallahan DE, Sekhar KR, Freeman ML: Increased expression of nuclear factor E2 p45-related factor 2 (NRF2) in head and neck squamous cell carcinomas. Head Neck 28:813-818, 2006. 41. Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamoto M, Biswal S: Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res 62:5196-5203, 2002. 42. Wang J, Fields J, Zhao C, Langer J, Thimmulappa RK, Kensler TW, Yamamoto M, Biswal S, Dore S: Role of Nrf2 in protection against intracerebral hemorrhage injury in mice. Free Radic Biol Med 43:408-414, 2007. 43. Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y, Chen W, Yi X, Zheng W, Wondrak GT, Wong PK, Zhang DD: Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis 29:1235-1243, 2008. 44. Yan W, Wang HD, Feng XM, Ding YS, Jin W, Tang K: The expression of NF-E2-related factor 2 in the rat brain after traumatic brain injury. J Trauma 66:14311435, 2009. 45. Zhang J, Han L, Zhang A, Wang Y, Yue X, You Y, Pu P, Kang C: AKT2 expression is associated with glioma malignant progression and required for cell survival and invasion. Oncol Rep 24:65-72, 2010. Conflict of interest statement: Supported by grants from National Natural Science Foundation of China (No. 81070974), the Jiangsu Provincial Key Subject (X4200722), and Jinling Hospital of Nanjing, China (2010Q017). Received 01 March 2011; accepted 29 June 2011; published online 07 November 2011 Citation: World Neurosurg. (2013) 80, 3/4:363-370. http://dx.doi.org/10.1016/j.wneu.2011.06.063 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter © 2013 Elsevier Inc. All rights reserved.
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17. Poen JC, Golby AJ, Forster KM, Martin DP, Chinn DM, Hancock SL, Adler JR Jr: Fractionated stereotactic radiosurgery and preservation of hearing in patients with vestibular schwannoma: a preliminary report. Neurosurgery 45:1299-1305; discussion 1305–1297, 1999. 18. Tamura M, Carron R, Yomo S, Arkha Y, Muraciolle X, Porcheron D, Thomassin JM, Roche PH, Regis J: Hearing preservation after gamma knife radiosurgery for vestibular schwannomas presenting with high-level hearing. Neurosurgery 64:289-296; discussion 296, 2009. 19. Thomas C, Di Maio S, Ma R, Vollans E, Chu C, Clark B, Lee R, McKenzie M, Martin M, Toyota B: Hearing preservation following fractionated stereotactic
radiotherapy for vestibular schwannomas: prognostic implications of cochlear dose. J Neurosurg 107: 917-926, 2007. 20. Timmer FC, Hanssens PE, van Haren AE, Mulder JJ, Cremers CW, Beynon AJ, van Overbeeke JJ, Graamans K: Gamma knife radiosurgery for vestibular schwannomas: results of hearing preservation in relation to the cochlear radiation dose. Laryngoscope 119:1076-1081, 2009. Conflict of interest statement: Dr. John Adler is Vice President of Varian Medical, Inc.; Dr. Iris Gibbs is a member of the Clinical Advisory Board and the Speakers’ Bureau of Accuray, Inc., manufacturer of the CyberKnife radiosurgical system. This work is in part supported by
gifts from Robert C. and Jeannette Powell, Alan Wong and Sylvia Tang, and Paula and William Zappettini to Steven D. Chang, MD. M. G. Hayden Gephart is supported by a postdoctoral fellowship from the California Institute of Regenerative Medicine. Received 20 January 2012; accepted 03 April 2012; published online 05 April 2012 Citation: World Neurosurg. (2013) 80, 3/4:359-363. http://dx.doi.org/10.1016/j.wneu.2012.04.001 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter © 2013 Elsevier Inc. All rights reserved.
The Role of Nrf2 in Migration and Invasion of Human Glioma Cell U251 Hao Pan, Handong Wang, Lin Zhu, Lei Mao, Liang Qiao, Xingfen Su
Key words 䡲 Glioma 䡲 Invasion 䡲 Matrix metalloproteinase 9 䡲 Migration 䡲 NF-E2-related factor 2 Abbreviations and Acronyms ARE: antioxidant response element DMEM: Dulbecco’s modified Eagle medium MMP9: Matrix metalloproteinase 9 Nrf2: NF-E2-related factor 2 RT-PCR: Reverse transcriptase–polymerase chain reaction Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, People’s Republic of China To whom correspondence should be addressed: Handong Wang, M.D., Ph.D. [E-mail: [email protected]] Citation: World Neurosurg. (2013) 80, 3/4:363-370. http://dx.doi.org/10.1016/j.wneu.2011.06.063 Journal homepage: www.WORLDNEUROSURGERY.org Available online: www.sciencedirect.com 1878-8750/$ - see front matter © 2013 Elsevier Inc. All rights reserved.
INTRODUCTION The transcription factor NF-E2-related factor 2 (Nrf2) is considered as a critical regulator of intracellular antioxidants and phase II detoxification enzymes by transcriptional upregulation of many antioxidant response element (ARE)-containing genes. It belongs to Keap1Nrf2-ARE signaling pathway. In response to oxidative stress, Nrf2 is activated and dissociated from Keap1. After translocated from cy-
䡲 OBJECTIVE: NF-E2-related factor 2 (Nrf2) is a transcription factor that is related to tumor cell multidrug resistance and proliferation. Here we studied the involvement of Nrf2 in the migration and invasion of human U251 glioma cells. 䡲 METHODS: Two kinds of plasmid, that is, pEGFP-Nrf2 and Si-Nrf2, were constructed and transfected to upregulate or downregulate the expression of Nrf2 in U251 glioma cell line. Blank vectors or random siRNA plasmid were used as negative control. Cells treated with lipofectamine only were set up as blank control. Protein and mRNA level of Nrf2 and matrix metalloproteinase 9 (MMP9) were investigated by reverse transcriptase–polymerase chain reaction and western blot after transfection. Wound healing assay and transwell assay were used to study migration and invasion of U251 after transfection. Gelatin zymography was performed to reveal the change of MMP9 activity after transfection. 䡲 RESULTS: The mRNA and protein level of Nrf2 was upregulated in U251pEGFP-Nrf2 while downregulated in U251-Si-Nrf2 48 hours after transfection. In the wound healing assay, there were more cells in group pEGFP-Nrf2 crossing the scratch line than in group Si-Nrf2. Furthermore, in transwell migration and invasion assay, there were more cells in group pEGFP-Nrf2 penetrating the membranes than in group Si-Nrf2. Then we investigated the change of MMP9 activity, mRNA, and protein levels after transfection. The results suggested that upregulation of Nrf2 led to an increase in MMP9 expression and activity whereas downregulation of Nrf2 led to a decrease in MMP9 expression and activity. 䡲 CONCLUSION: Nrf2 is involved in migration and invasion of U251 cells, which may be related to MMP9.
toplasm to nucleus, Nrf2 forms a heterodimer with Maf, which binds to the ARE sequence to upregulate many kinds of genes expression (21). The Nrf2 downstream genes include 1) intracellular redox-balancing proteins: glutamate cysteine ligase, glutathione peroxidase,
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and heme oxygenase-1; 2) phase II detoxifying ⬎enzymes: glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase–1; and 3) transporters: multidrug resistance–associated protein (21). Recent studies have found more Nrf2 downstream genes that are
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Table 1. Primers and Parameters Used in Reverse Transcriptase–Polymerase Chain Reaction Primer Name
TUMOR
Nrf2
Forward 5=-TCAGCATGCTACGTGATGAAG-3=
Size Reverse 5=-TTTGCTGCAGGGAGTATTCA-3=
Tm (°C)
(bp)
Cycle
58
68
38
MMP9
5=-CGGTGATTGACGACGCCTTTG-3=
5=-TTGGAACCACGACGCCCTTGC-3=
63
255
35
GAPDH
5=-GAAGGTGAAGGTCGGAGTC-3=
5=-GAAGATGGTGATGGGATTTC-3=
58
226
35
Tm, melting temperature; bp, base pair; Nrf2, NF-E2-related factor 2; MMP9: matrix metalloproteinase 9; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
involved in stress response, xenobiotics metabolism, the ubiquitin-mediated proteasomal degradation system, cell proliferation and so on (5, 20, 22, 36, 41). In normal tissue, Nrf2 is considered as an important transcript factor for cells to survive from many kinds of negative stimulus such as injury, ischemia, inflammation, hemorrhage, cancer, pulmonary fibrosis, acute pulmonary injury, and degeneration disease (6, 8, 18, 19, 36, 38, 42, 44). Interestingly, Nrf2 may play a dark role in tumor tissue. It has been verified that Nrf2 is upregulated in lung, head, and neck squamous cell carcinoma tissues (39, 40). Further investigation shows that overexpression of Nrf2 enhances tumor resistance to chemotherapeutic agents in some lung carcinoma, breast adenocarcinoma, and neuroblastoma cell lines (43). Matrix metalloproteinase 9 (MMP9), which belongs to the ECM-degrading enzyme family, is a kind of gelatinase. MMP9 is involved in migration and invasion of tumor cells. It has been found that the transcription and activity levels of MMP9 are higher in glioma than in normal brain and such elevation is strongly correlated with the tumor grade (9). Our previous study (25) proved that MMP9 was upregulated in Nrf2 knockout mice than wild-type mice after spinal cord injury. This result suggests the possible relationship between Nrf2 and MMP9. In this study, we investigated the change of MMP9 level, migration, and invasion ability of human U251 glioma cells after up- or downregulating the expression of Nrf2.
MATERIALS AND METHODS Cell Culture and Transient Transfection Human U251 glioma cells were obtained from ATCC and cultured in Dulbecco’s mod-
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ified Eagle medium (DMEM; HyClone, Illinois, USA) with 10% fetal bovine serum (HyClone) at 37°C and 5% CO2 incubator. The primers for human Nrf2 cDNA were as follows: forward 5=-CCGCTCGAGATGATGGACTTGGAGCTGCC-3=, reverse 5=-GGGGTACCGTGTTTTTCTTAACATCTGGC-3=. Human Nrf2 cDNA was cloned into the cloning site of the vector pEGFP-N1 (GeneChem, Shanghai, China) using the standard re-
combinant DNA technique. The new plasmid was named as pEGFP-Nrf2. And a blank vector (pEGFP) was used as negative control. The vector pGPH1/GPF/Neo used for cloning Nrf2 short hairpin RNA (shRNA) was purchased from GenePharma (Shanghai, China). The target sequence was GCAGTTCAATGAAGCTCAACT. The new plasmid was named as Si-Nrf2. Random sequence was used as
Figure 1. mRNA --⬎level of Nrf2 and MMP9 analyzed by reverse transcriptase–polymerase chain reaction 48 hours after transfection. (A) Group pEGFP-Nrf2 showed higher expression whereas group Si-Nrf2 showed lower expression of Nrf2 and MMP9 mRNA level. There was no obvious difference among group pEGFP, Si-control, and Lipo. (B) Relative mRNA level of Nrf2/GAPDH in group pEGFP-Nrf2 was 1.00 ⫾ 0.01 with 0.55 ⫾ 0.01 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among three control groups. (C) Relative mRNA level of MMP9/GAPDH in group pEGFP-Nrf2 was 0.84 ⫾ 0.04 with 0.64 ⫾ 0.02 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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Reverse Transcription-PCR Total RNA was isolated with Trizol (Invitrogen) and single-stranded cDNA was synthesized from 2 g of total RNA with BU-Script RT-Kit (Biouniquer, Jiangsu, China) according to the manufacturer’s protocol. The cDNA was stored in –20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, Wisconsin, USA) according to the manufacturer’s protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by using agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of GAPDH was used as an internal reference gene.
Figure 2. Protein level of Nrf2 and MMP9 analyzed by Western blot 48 hours after transfection. (A) Group pEGFP-Nrf2 showed higher expression and group Si-Nrf2 showed lower expression of Nrf2 and MMP9 protein level. There was no obvious difference among group pEGFP, Si-control and Lipo. (B) Relative protein level of Nrf2/-actin in group pEGFP-Nrf2 was 1.02 ⫾ 0.06 with 0.24 ⫾ 0.01 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among the three control groups. (C) Relative protein level of MMP9/-actin in group pEGFP-Nrf2 was 1.26 ⫾ 0.01 with 0.54 ⫾ 0.01 in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
negative control, which was named as Sicontrol. Cells were seeded in six-well plates at 1 ⫻ 106/well and allowed to attach for 24 hours before transfection. Then pEGFPNrf2, pEGFP, Si-Nrf2, and Si-control were transfected by lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s protocol. Cells treated
with lipofectamine 2000 alone were set up as blank control, which was named as group Lipo. After incubation at 37°C and 5% CO2 for 24, 48, and 72 hours, cells were collected to testify the expression of Nrf2 by reverse transcriptase–polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.
Western Blot Analysis To obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DL-dithiothreitol, 1 mM phenylmethane-sulfonyl fluoride, and protease inhibitor cocktail in phosphate-buffered saline pH 7.4) and centrifuged at 12,000g for 15 minutes at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, Illinois, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in the saline buffer overnight at 4°C, and the following antibodies were used: anti-Nrf2 (sc-722 [57 kDa]; Santa Cruz Biotechnology, California,
Table 2. The Results of Wound Healing Assay, Migration and Invasion Assay pEGFP-Nrf2
pEGFP
Si-Nrf2
Si-control
Lipo
116.83 ⫾ 5.27 *
82.17 ⫾ 5.12
31.00 ⫾ 4.69†
82.50 ⫾ 4.04
77.00 ⫾ 4.38
Migration assay
21.50 ⫾ 3.83*
11.67 ⫾ 1.63
4.17 ⫾ 1.47†
11.00 ⫾ 2.28
11.67 ⫾ 1.75
Invasion assay
33.33 ⫾ 4.50*
14.83 ⫾ 2.23
6.00 ⫾ 2.10†
15.17 ⫾ 1.72
14.83 ⫾ 3.06
Wound healing assay
The data indicate the number of cells crossing the scratch line or penetrating the membranes. Cells in group pEGFP-Nrf2 shows enhanced migratory and invasive ability than cells in group Si-Nrf2. There was no statistical difference among three control groups. Nrf2, NF-E2-related factor 2; Lipo, lipofectamine. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. †P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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USA), anti--actin (sc-130301 [42 kDa]; Santa Cruz Biotechnology), and anti-MMP9 (sc-21733 [92 kDa]; Santa Cruz Biotechnology). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 hour at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase–linked secondary antibody at a 1:2000 dilution for 1 hour. Blots were incubated with enhanced chemiluminescence detection system (Amersham Biosciences, Bucks, United Kingdom) and exposed radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of -actin was used as an internal reference gene. Migration and Invasion Assay Forty-eight hours after transfection, cells were treated with trypsin and resuspended to single-cell solutions. A total of 1 ⫻ 105 cells in 200 L DMEM were seeded on the upper chamber of 6.5 mm Transwell with 8.0-m-pore polycarbonate membrane insert (Corning Inc, New York, USA). DMEM (600 L) with 10% fetal bovine serum was added into the lower chamber as a chemoattractant. After the cells were incubated for 8 hours, the insert was washed with phosphate-buffered saline and cells on the top surface of the insert were removed gently by a cotton swab. Cells adhering to the lower surface were fixed by methanol, stained by crystal violet, and counted under microscope in six predetermined fields (⫻200). The matrigel invasion assay was similar to the cell migration assay, except that the transwell membrane was precoated with ECM gel (Sigma-Aldrich, Missouri, USA) and the cells were incubated for 24 hours. All assays were independently repeated at least three times.
Wound Healing Assay Twenty-four hours after transfection, cells were seeded at the density of 1 ⫻ 105/well in a 24-well plate. Incubated for 24 hours (48 hours after transfection), cells were scratched using the back side of a standard 100-L pipette tip. After being washed three times with phosphate-buffered saline, scratches including the flanking front lines of cells were photographed (40-fold magnification). The edges of the wound were marked after scratching (time point 0
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Figure 3. Wound healing assay. (A) There were more cells crossing the edges in group pEGFP-Nrf2 and lesser cells in group Si-Nrf2 as compared with three control groups 48 hours after scratch (96 hours posttransfection) (⫻40). (B) Counting cell numbers migrating over the edges 48 hours after scratch (96 hours posttransfection) with ImageJ, we found that there were 116.83 ⫾ 5.27 cells crossing the scratch line in group pEGFP-Nrf2 with 31.00 ⫾ 4.69 cells in group Si-Nrf2 (P ⬍ 0.001). There was no statistical difference among the three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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Figure 4. Transwell migration assay. (A–E) Cells of five groups penetrated the membranes 8 hours after seeding in the Transwell upper chamber (⫻100). (F) There were 21.50 ⫾ 3.83 cells through the membranes in group pEGFP-Nrf2 with only 4.17 ⫾ 1.47 cells in group Si-Nrf2 (P ⬍ 0.001).
No statistical difference showed in three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
Figure 5. Transwell invasion assay. (A–E) Cells of five groups penetrated the membranes 24 hours after seeding in the Transwell upper chamber, which was precoated with ECM gel (⫻100). (F) There were 33.33 ⫾ 4.50 cells through the membranes in group pEGFP-Nrf2 compared with 6.00 ⫾
2.10 cells in group Si-Nrf2 (P ⬍ 0.001). No statistical difference showed in the three control groups. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
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hours). Cells were incubated under standard conditions. The scratched area was photographed 24 and 48 hours after wounding (corresponding to 72 and 96 hours posttransfection). Cells migrating over the edges were counted 48 hours after the scratch. Experiments were performed in triplicate, evaluating four scratches in each experiment.
Gelatin Zymography Cells of five groups were homogenized in the lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, 0.01% Triton, and then mixed with electrophoresis loading buffer. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer’s instruction (Genmed Scientifics Inc, Massachusetts, USA). Briefly, 40 g cytosolic protein extracts were separated by electrophoresis. The proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in the substrate buffer for 40 hours at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 hour and destained in 50% methanol. Proteolytic activities were shown by clear bands in blue gel, indicating the lysis of the substrate. Quantification of MMP9 band density was performed with the image analysis program ImageJ and was described in arbitrary units. Statistical Analysis All experiments were done at least three times. Data were expressed as mean ⫾ SD. Data were evaluated by analysis of variance and least significant difference multiple comparison test. P value ⬍0.05 were considered to be significant. All analyses were performed by SPSS 18.0. RESULTS Transient Transfection Effect on Nrf2 mRNA and Protein Level To validate the transient transfection effect of four plasmids mentioned above, we tested the mRNA and protein level of five cell groups 24, 48, 72 hours after transfection, respectively. RT-PCR and western blot results indicated that transfection efficiency was most predominant at 48 hours after
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transfection (data not shown). Transfection of pEGFP-Nrf2 enhanced the Nrf2 mRNA and protein level ⬎38% and ⬎130% as compared with transfection of pEGFP, respectively (P ⬍ 0.05). Si-Nrf2 transfection reduced the Nrf2 mRNA and protein level ⬎30% and ⬎40% as compared with transfection of Si-control, respectively (P ⬍ 0.05). Group pEGFP, Si-control, and Lipo showed no difference in Nrf2 mRNA and protein level (Figures 1 and 2). So we chose 48 hours after transfection as the time point for further study.
Nrf2 Is Involved in U251 Cell Migration and Invasion To examine the role of Nrf2 in U251 cell migration, we performed wound healing assay and transwell migration assay. As showed in Table 2, cells in group pEGFPNrf2 showed incremental migratory ability than cells in group pEGFP and Lipo, whereas cells in group Si-Nrf2 showed decreased migration ability than cells in group Si-control and Lipo in the wound healing assay (P ⬍ 0.001) (Figure 3). Transwell migration assay showed a similar result, with more cells penetrating the membranes in group pEGFP-Nrf2 than in group pEGFP and Lipo (P ⬍ 0.001). There were fewer cells penetrating the membranes in group Si-Nrf2 than in group Si-control and Lipo (P ⬍ 0.001) (Figure 4). There was no statistical difference among the three control groups. In the transwell invasion assay, ECM gel was used to simulate the extracellular matrix around glioma cells, which would reproduce tumor microenvironment in vitro. As showed in Table 2, there were more cells in group pEGFP-Nrf2 that crossed the membranes than in group pEGFP and Lipo 24 hours after seeding (P ⬍ 0.001) (Figure 5). There were fewer cells that crossed the membranes in group Si-Nrf2 than in group Si-control and Lipo 24 hours after seeding (P ⬍ 0.001) (Figure 5). No statistical difference was found among the three control groups.
Change of MMP9 Activity, mRNA, and Protein Level After Transfection As mentioned above, upregulation of Nrf2 enhanced the invasion and migration of
Figure 6. Gelatin zymography. (A) Gelatin zymography analysis showed brighter band in pEGFP-Nrf2 and bleaker band in Si-Nrf2 than in group pEGFP, Si-control, and Lipo. (B) Quantitative analysis showed that MMP9 activity was higher in pEGFP-Nrf2 than in pEGFP (90.65 ⫾ 5.050 vs. 64.03 ⫾ 2.780, P ⬍ 0.001). The MMP9 activity of Si-Nrf2 was lower than that of Si-control (43.07 ⫾ 2.596 vs. 67.05 ⫾ 2.192, P ⬍ 0.001). Group pEGFP, Si-control, and Lipo showed no difference in MMP9 activity. The data were described in arbitrary units. *P ⬍ 0.001, group pEGFP-Nrf2 compared with group pEGFP and Lipo. #P ⬍ 0.001, group Si-Nrf2 compared with group Si-control and Lipo.
U251 cells whereas downregulation of Nrf2 reduced the invasion and migration of U251 cells. It is believed that MMP9 is a critical enzyme in tumor cell migration and invasion. So here we detected the MMP9 expression after transfection. Forty-eight hours after transfection, cells were collected to check the mRNA and protein level of MMP9 by RT-PCR and Western blot. MMP9 mRNA and protein level of U251pEGFP-Nrf2 were 0.84⫾ 0.04 and 1.26⫾ 0.01, respectively, compared with 0.69⫾ 0.01 and 0.69 ⫾ 0.02 for U251-pEGFP (P ⬍ 0.001) as compared with the internal reference gene. MMP9 mRNA and protein level of U251-Si-Nrf2 were 0.64 ⫾ 0.02 and 0.54 ⫾ 0.01, respectively, compared with 0.69 ⫾ 0.02 and 0.68 ⫾ 0.02 for U251-Si-control (P ⬍ 0.001) as compared with the internal reference gene (Figures 1 and 2). Gelatin zymography revealed higher MMP9 activity in U251-pEGFPNrf2 than in U251-pEGFP (90.65 ⫾ 5.050 vs. 64.03 ⫾ 2.780, P ⬍ 0.001). The MMP9 activity of Si-Nrf2 was lower than that of Sicontrol (43.07 ⫾ 2.596 vs. 67.05 ⫾ 2.192,
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P ⬍ 0.001) (Figure 6). Group pEGFP, Sicontrol, and Lipo showed no difference in MMP9 activity and mRNA and protein level. Summarily, upregulation of Nrf2 led to an increase in MMP9 expression and activity whereas downregulation of Nrf2 led to a decrease in MMP9 expression and activity.
DISCUSSION Nrf2 was first discovered by Moi in 1994 (27). Follow-up study showed that Nrf2 is a critical transcription factor in Keap1-Nrf2ARE signaling pathway to resist oxidative stress. Nrf2 is considered as a “good” protein because many chemopreventive compounds have been found to be its inducer, such as sulforaphane (16), curcumin (1), epigallocatechin-3-gallate (29), resveratrol (4), wasabi (28), cafestol, kahweol (12), lycopene (3), and carnosol (37). All these compounds have been proven to be able to prevent tumorigenesis through activation of Nrf2 and further downstream antioxidant genes. Studies reveal that Nrf2 knockout mice show increased cancer susceptibility to carcinogens and are refractory to the protective actions of some chemopreventive agents, which confirm the role of Nrf2 in chemoprevention on the other side (2, 17, 23). Surprisingly, new emerging results have revealed that Nrf2 not only protects the normal cells from carcinogens but also promotes the tumor cell survival in chemotherapeutics. In 2004, Ikeda (14) found that the Nrf2/MafK heterodimer regulates GST-P gene expression during early hepatocarcinogenesis and in hepatoma cells, which started the studies on tumor promotion effect of Nrf2. Most studies focused on lung cancer. It has been revealed that Nrf2 is mutational and overexpressed in many lung tumor tissues and cell lines (11, 31, 39). Similar results were also found in prostate, breast, head, and neck cancer (10, 30, 40). Further research indicates that Nrf2 plays a critical role in tumor cell proliferation and chemoresistance. Nrf2 is highly activated in human lung cancer cell line A549. After knockdown by siRNA technology, cell proliferation of A549 cells is prominently inhibited, accompanied by chemoresistance to cisplatin (13). Cisplatin-resistant human ovarian cancer SK-OV cells show exacerbated cytotoxicity following cisplatin treatment after transfection of Nrf2 siRNA (7).
NRF2 PARTICIPATES IN MIGRATION AND INVASION OF GLIOMA
Similar discovery is also present in different cancer types and chemotherapeutic agents (43). All these results indicate the “dark” side of Nrf2. Meng’s group found that low concentration of arsenic stimulated cell migration and vascular tube formation in human microvascular endothelial cells with overexpression of heme oxygenase-1 mRNA and protein, which is a result of increased Nrf2 binding to the heme oxygenase-1 transcription site (26). This finding suggests that Nrf2 participates not only in cell proliferation and chemoresistance but also possibly in cell migration and invasion. For the first time, our results demonstrate that enhanced expression of Nrf2 promotes glioma cell invasion and migration, whereas reduced expression of Nrf2 attenuates it. Interestingly, Rachakonda et al. (34) found that A549 cells stably expressing Nrf2 shRNA and HepG2 cells stably expressing FLAG/Keap1, which both reduced the expression of Nrf2 and exhibited an increase in migration compared to control. We hypothesize that the differences in migration observed by Rachakonda et al. (34) and the work reported here may be attributed to stable versus transient repression of Nrf2 and different cell lines. To investigate the mechanism of Nrf2’s role in glioma cell invasion and migration, we focused on the enzyme MMP9. MMP9, also named gelatinase B, degrade denatured collagen and type IV collagen. It has been confirmed that MMP9 protein is not only prominently expressed in vascular structures of glioma but also expressed higher in tumor cells than in normal cells. And its expression and activity levels are strongly correlated with the tumor grade (9, 35). Downregulated expression of AKT2 and EGFR by siRNA decreased expression of MMP9 in glioma, accompanied by weakened invasion ability (15, 45). These studies indicate that MMP9 plays an important role in glioma invasion and migration. Recent study shows that arsenic induces malignant transformation of human keratinocytes, which is correlated with elevated expression of Nrf2 and MMP9 (33). In spinal cord injury, Nrf2 knockout mice show higher expression and activity of MMP9 than Nrf2 wild-type mice (25). All these suggest the possible relationship between Nrf2 and MMP9. Our results indicate that upregulated expression of Nrf2 promotes overexpression of MMP9 in mRNA and protein
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levels, while downregulated expression of Nrf2 reduces expression of MMP9. Oxidative stress activates EGFR or PI3K/AKT signaling pathway and then induces translocation of Nrf2 from the cytoplasm to the nucleus. Inhibition of AKT activity markedly attenuates Nrf2 translocation and downstream gene transcription (24, 32). As mentioned above, EGFR and AKT can also regulate the expression of MMP9 (15, 45). So EGFR and AKT may be the link of Nrf2 and MMP9. But the signal pathway between Nrf2 and MMP9 is unknown, which still needs further research.
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