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Department of Pathology, Wakayama Medical University (Y.N., H.Y., H.Z., M.N., K.K.), 641-8509 Wakayama City, Japan; and Department of Surgery (Y.T., A.M.), Kuma Hospital, 650-0011 Kobe, Japan
Address all correspondence and requests for reprints to: Dr. Yasushi Nakamura, Department of Pathology, Wakayama Medical University, 811-1 Kimiidera, 641-8509 Wakayama City, Japan. E-mail: ynakamur{at}wakayama-med.ac.jp.
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Abstract |
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Experimental Design: Nitrite/nitrate levels and VEGF-D production were assessed in K1 papillary thyroid carcinoma cells after induction and/or inhibition of nitric oxide (NO) synthesis. Formation of nitrotyrosine, a biomarker for peroxynitrate formation from NO in vivo, was analyzed in primary human PTC.
Results: The production of nitrite/nitrate and VEGF-D in K1 cells was increased by treatment with the NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The NO synthase inhibitor NG-nitro-L-arginine methyl ester inhibited the increase in nitrate/nitrite and eliminated the increase in VEGF-D. High-grade nitrotyrosine staining was observed in 51.8% (29 of 56) of PTCs. Nitrotyrosine levels were significantly correlated with VEGF-D immunoreactivity and lymph node metastasis.
Conclusions: Our data showed a role for NO in stimulating VEGF-D expression in vitro. The formation of its biomarker, nitrotyrosine, was also correlated with VEGF-D expression in human PTC. NO may induce lymph node metastasis via VEGF-D stimulation in PTC.
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Introduction |
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Nitric oxide (NO) is a pleiotropic regulator that is critical to numerous biological processes, including vasodilatation, neurotransmission, and macrophage-mediated immunity (5). It also has genotoxic and angiogenic properties. Increased NO generation in cancer cells may contribute to tumor hemangiogenesis by up-regulating VEGF-A, and VEGF-A-induced neovascularization may increase the tumor’s metastatic ability (6). The effects of NO are mediated in part by its metabolites, such as peroxynitrite. Peroxynitrite can oxidize and nitrate DNA and can also nitrate tyrosine in proteins to produce nitrotyrosine (7). Thus, the presence of nitrotyrosine in tissues has been used as a biomarker for peroxynitrite formation in vivo from NO.
In this study, incubation of K1 papillary thyroid carcinoma cells with an NO donor resulted in the induction of VEGF-D expression. This induction was significantly inhibited by the addition of the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In addition, we investigated how nitrotyrosine formation relates to lymph node metastasis and VEGF-D expression in human PTC.
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Materials and Methods |
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The K1 papillary thyroid carcinoma cell line was purchased from the European Collection of Cell Cultures (Wiltshire, UK). Cells were maintained at 37 C in 5% CO2 as monolayers in tissue culture dishes containing DMEM/Ham’s F-12 medium (Invitrogen Life Technologies, Inc., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (HyClone, Logan, UT). For the experiments, 6-cm tissue culture plates (Corning, Corning, NY) were seeded with 3 x 105 cells in 3 ml medium. Medium was changed on d 3; when the cells were subconfluent (d 5), 10 mM L-NAME (Sigma-Aldrich Corp., Tokyo, Japan), if administered, was added 2 h before 1 mM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) (Cayman Chemical Co., Ann Arbor, MI). These concentrations of L-NAME or DETA NONOate had no effect on cell viability, as measured by the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI; data not shown).
Measurement of nitrate/nitrite production
After DETA NONOate administration, cells were incubated for 1, 2, 3, 4, 6, or 8 h. The supernatants were collected and centrifuged to remove cell debris. The amount of nitrate/nitrite was determined using nitrate/nitrite fluorometric assay kit (Cayman Chemical Co.) (8). The amount of nitrate/nitrite in the supernatants collected from control cells, to which neither DETA NONOate nor L-NAME was administered, was below detectable levels.
Determination of VEGF-D mRNA production
After DETA NONOate administration, cells were incubated for 1, 2, 3, 4, 6, or 8 h. For total RNA extraction, the cells were washed with PBS, scraped from the plates, and stored at –70 C if not processed immediately. Total RNA was extracted using the TRIzol method (Invitrogen Life Technologies, Inc.). After deoxyribonuclease treatment using DNA-free (Ambion, Inc., Austin, TX), mRNA was reverse transcribed for single-strand cDNA using oligo-(dT)20 primer and Thermoscript (Invitrogen Life Technologies, Inc.) according to the manufacturer’s protocol. VEGF-D transcription was measured by quantitative real-time PCR of the resulting cDNA (100 ng), using Universal TaqMan PCR reagents and an ABI PRISM 7000 sequence detector equipped with a 96-well thermal cycler (PerkinElmer Applied Biosystems, Foster City, CA). The primer and probe mixture for VEGF-D and GAPDH was purchased from PerkinElmer Applied Biosystems, and PCR was carried out according to the manufacturer’s protocol. VEGF-D mRNA expression was quantitated relative to control cells (treated with neither DETA NONOate or L-NAME) based on a real-time PCR standard curve constructed with templates consisting of serially diluted solutions of a VEGF-D cDNA-containing plasmid. Mean values were used for statistical testing.
Determination of VEGF-D protein production
For the determination of VEGF-D protein production, cells were incubated for 12 h after DETA NONOate administration and harvested as described above. Cell lysates were prepared using T-PER Tissue Protein Extraction Kit (Pierce Chemical Co., Rockford, IL) containing Halt Protease Inhibitor Cocktail (Pierce Chemical Co.). Total conditioned media from cells were concentrated 100-fold, using Amicon Ultra-4 columns (Millipore Corp., Bilerica, MA). Protein concentration was measured using Coomassie Plus Protein Assay Reagent Kit (Pierce Chemical Co.). For Western blot analysis of VEGF-D, 40-µg samples of whole-cell lysate or concentrated culture media were separated by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (SureBlot F1 System, Astellas Pharma, Inc., Tokyo, Japan) by electroblotting. The membrane was blocked with 5% skim milk in PBS for 1 h at room temperature, incubated overnight with antihuman VEGF-D mouse antibody (R&D Systems, Inc., Minneapolis, MN), rinsed with PBS, and labeled with peroxidase-conjugated antimouse secondary antibody (Amersham Biosciences, Arlington Heights, IL) for 1 h at room temperature. The signals were visualized using the LumiGLO Reserve chemiluminescence substrate kit (Kirkgaard & Perry Laboratories, Inc., Gaithersburg, MD) and recorded by luminocapture (ATTO, Tokyo, Japan). Anti-ß2-microglobulin antibody (DakoCytomation, Glostrup, Denmark) was used for the internal control. To compare levels of proteins, the density of each band was measured by densitometry.
Patients and tumor samples
The study included 56 patients with PTC, diagnosed and treated in Kuma Hospital, Japan, in 2003. None of these cases had a family history of thyroid cancer or malignancy. The patients had received total or subtotal thyroidectomy with regional lymphadenectomy (central neck dissection, lateral neck dissection, superior mediastinal dissection, or a combination of the above). All sections of the excised tumors were histologically evaluated by three pathologists (Y.N., H.Y., and K.K.). Of the 56 patients selected, 49 had classical PTC, three had a poorly differentiated form, and four had a follicular variant as assessed by histopathology. Patients with multifocality were excluded. Patients and tumor characteristics are shown in Table 1
. The median age at surgery for the selected patients was 49.5 yr (range, 16–76 yr). All patients have been followed up, and none has had a recurrence.
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Immunohistochemistry for nitrotyrosine was performed as described previously (4), and 1:100-diluted antihuman nitrotyrosine mouse antibody (Hyclut Biotechnology, Uden, The Netherlands) was used. The immunohistochemical scoring was performed blindly. The intensity of nitrotyrosine immunostainings was evaluated by dividing the nuclear and cytoplasmic staining reaction into four groups: 1, weak; 2, moderate; 3, strong; and 4, very strong. The quantity of immunostained cells was evaluated as follows: 1, less than 25%; 2, 25–50%; 3, 50–75%; and 4, more than 75% of tumor cells showing positivity. A combined score for nitrotyrosine immunostainings was generated by adding the qualitative and quantitative scores. These summed scores were then divided into two groups as low-grade (2–4) and high-grade (5–8) for statistical testing. Immunohistochemistry and scoring for VEGF-D were performed as described previously (4).
Statistics
The effects of drug treatment were analyzed by ANOVA, followed by Student’s t test. Fisher’s exact test was used to examine the association between nitrotyrosine levels and other clinicopathological factors. P < 0.05 was considered significant. A software package (JMP IN 5.1.1, SAS Institute, Cary, NC) was used for all statistical testing and management of the database.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the effect of NO on VEGF-D induction, K1 cells were treated with the NO donor DETA NONOate. This was observed to increase nitrate/nitrite production in the supernatants (Fig. 1A). Pretreatment of the cells with the NOS inhibitor L-NAME substantially inhibited this increase. As shown in Fig. 1, B and C, DETA NONOate also increased VEGF-D mRNA expression, and VEGF-D protein expression in both cell lysates and supernatants. Again, L-NAME inhibited these effects of DETA NONOate. All these effects of DETA NONOate and L-NAME were statistically significant.
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Nitrotyrosine was detected by immunohistochemistry in all PTCs. It was observed mainly in the cytoplasm and focally in the nucleus (data not shown). High-grade nitrotyrosine staining was observed in 51.8% (29 of 56) of the PTC. We have previously reported that VEGF-D protein was expressed as diffuse cytoplasmic staining in PTC cells. As in our previous report (4), grading of intensity and extent of staining of the malignant epithelium was performed as follows: 0, negative; 1, weak/very limited moderate staining; 2, moderate widespread/strong localized staining; and 3, strong widespread. Then VEGF-D antibody-stained sections were grouped as low-grade (0–2) and high-grade (3) for statistical analysis. According to this criterion, high-grade VEGF-D expression was observed in 35.7% (20 of 56) of the 56 papillary thyroid carcinomas. VEGF-D protein expression was significantly correlated with lymph node metastasis (P = 0.0100). As shown in Table 1
, high-grade nitrotyrosine staining was correlated with VEGF-D immunoreactivity (P = 0.0002) and lymph node metastasis (P = 0.0173).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study the NO donor DETA NONOate induced VEGF-D mRNA and protein expression in the K1 PTC cell line, which retains wild-type p53 function and thyroglobulin synthesis (13). This induction was significantly inhibited in the presence of the NOS inhibitor L-NAME. A substantial increase in nitrate/nitrite production in the supernatants after stimulation with DETA NONOate was also observed, and this increase was also significantly inhibited by L-NAME. Our results suggest that VEGF-D expression may be regulated by NO in this PTC cell line.
In addition, nitrotyrosine was detected by immunohistochemistry in all PTC tumor samples. A previous report of nitrotyrosine formation in human PTC also supports our present observation (14). In the present study, immunohistochemistry revealed that nitrotyrosine levels were significantly correlated with VEGF-D immunoreactivity and lymph node metastasis, suggesting an association between NO production, VEGF-D expression, and metastasis in human papillary carcinoma.
In conclusion, NO induces VEGF-D expression in vitro, and levels of the NO marker nitrotyrosine are correlated with VEGF-D expression and lymph node metastasis in human PTC. NO may play an important role in the metastasis of this cancer via VEGF-D induction.
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Acknowledgments |
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Footnotes |
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First Published Online January 17, 2006
Abbreviations: DETA-NONOate, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide; NOS, NO synthase; PTC, papillary thyroid carcinoma; VEGF-D, vascular endothelial growth factor D.
Received August 8, 2005.
Accepted January 11, 2006.
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References |
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