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(NRP1) Gene Correlated with Poor Prognosis in Human Glioma

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(NRP1) Gene Correlated with Poor Prognosis in Human Glioma ANTICANCER RESEARCH 24: 547-552 (2004) Overexpression of the Neuropilin 1 (NRP1) Gene Correlated with Poor Prognosis in Human Glioma HIDEO OSADA1,3, TETSUJI TOKUNAGA1,3, MASATAKE NISHI1, HIROYUKI HATANAKA1, YOSHIYUKI ABE1, ATSUSHI TSUGU2, HIROSHI KIJIMA1, HITOSHI YAMAZAKI1, YOSHITO UEYAMA1 and MASATO NAKAMURA1 1Department of Pathology and 2Department of Neurosurgery, Tokai University, School of Medicine, Bohseidai, Isehara, Kanagawa 259-1143; 3Self Defense Force Hospital Yokosuka, Tauraminato, Yokosuka, Kanagawa 237-0071, Japan Abstract. We studied whether the expression of the Neuropilin induced mitogenesis and angiogenesis. VEGF also promotes (NRP) gene was correlated with clinicopathological features in vascular permeability, as evidenced by the breakdown of the glioma. We examined the gene expression of vascular endothelial blood-brain-barrier with surrounding peri-tumoral edema in growth factor (VEGF)-A, Flt-1, KDR, NRP1 and NRP2 in 37 malignant gliomas (5-9). gliomas by real time reverse transcriptase PCR (real time RT- Neuropilin 1 (NRP1), which is a 140 kDa transmembrane PCR) as well as immunohistochemical analysis. The vascular protein receptor (10-12), is thought to be a VEGF165 co- counts of each tumor were evaluated by anti-CD34 antibody. receptor that specifically enhances the interaction of NRP1 mRNA overexpression was significantly higher in neoplastic VEGF165 with VEGFR-2 / KDR to promote the mitogenic tissue compared to normal brain tissue samples. The higher grade activity of VEGF (13). NRP1 is expressed in most tissue, of glioma overexpressed the NRP1 gene significantly (p=0.0015). though it is reported to exist in relatively low amounts in the The glioma patients with NRP1 overexpression showed a poorer brain (13). NRP1 has been postulated to play an important prognosis (p=0.0202) than those without such overexpression. role in modulating VEGF-mediated stromal angiogenesis NRP1 was observed in the glioma cells by immunohistochemical (13-15), while the specific role of VEGF-mediated stromal analyses. VEGF-A and VEGFR overexpression did not show any angiogenesis, and the specific function of NRP1 and NRP2 correlation with the clinicopathological features, including NRP in angiogenesis and tumor progression, are not fully known expression. These results suggest that NRP1 overexpression, rather in human glioma. In this study, we examined NRP1 and than VEGF-A or VEGFR, contributes to tumor progression and NRP2 expression with regard to their clinicopathological has clinical significance for glioma. correlation in human glioma. Gliomas are well known primary brain tumors, which are Materials and Methods classified by increasing grades of malignancy based on histopathological characteristics. The clinical outcome is Tissue samples. Thirty-seven specimens of glioma were obtained by very poor even with multimodality treatment, including surgical resection between December 1990 and December 1996, at surgery, radiation, chemotherapy and immunotherapy. Tokai University Hospital, Japan, and 4 normal brain tissue Among the many positive angiogenic factors, vascular specimens, which were resected with glioma cells, were obtained with informed consent. All glioma patients were histologically classified by endothelial growth factor (VEGF)-A and its tyrosine kinase two independent pathologists according to the WHO grade (1993). receptors (VEGFR-1 / Flt-1 and VEGFR-2 / KDR) are The surgical specimens were rapidly frozen and stored at -80ÆC until known to be highly expressed in human malignant gliomas (1- analyses. Total cellular RNA was prepared from the frozen specimens. 7). Especially VEGFR-2 / KDR largely mediates VEGF-A Real time RT-PCR of NRP1 and NRP2 gene expression. To evaluate the expression level of NRP1 and NRP2, real-time quantitative RT- PCR was performed with total RNA from the 37 surgical glioma Correspondence to: Masato Nakamura, M.D., Department of specimens and the 4 normal brain tissue specimens, with TaqMan Pathology Tokai University School of Medicine, Bohseidai, Isehara, Reverse Transcription Reagents and the TaqMan PCR Core Reagent Kanagawa 259-1193, Japan. Tel: +81-463-93-1121 ext. 2570, Fax: Kit (Perkin-Elmer, Brunchburg, NJ, USA), following the +81-463-91-1370, e-mail: [email protected] manufacturer’s recommendation. Briefly, the total RNA was extracted by a single-step procedure using guanidium isothiocyanate Key Words: Neuropilin 1, glioma, VEGF, Flt-1, KDR, prognosis. and acid phenol / chloroform and treated with TRizol (GIBCO/BRL, 0250-7005/2004 $2.00+.40 547 ANTICANCER RESEARCH 24: 547-552 (2004) NY, USA). Reverse transcription was performed at 42ÆC for 60 Table I. Average values of the normalized NRP1 and NRP2 with glioma minutes (1 Ìg total cellular RNA; 100 pM random primers, WHO grade. Boehringer Mannheim, Mannheim, Germany; 40 U reverse transcriptase, GIBCO/BRL). The cDNA products were amplified WHO grade Number NRP1 NRP2 by 40 cycles of PCR, with each cycle consisting of a first denaturation at 95ÆC for 10 minutes, denaturation at 95ÆC for 15 Grade 1 2 0.97±0.014 1.26±0.127 seconds, annealing at 55ÆC for 15 seconds and extension at 72ÆC Grade 2 9 0.89±0.125 1.10±0.165 for 30 seconds. The primers and probes for real time RT-PCR were Grade 3 9 1.87±0.492 1.08±0.168 designed as follows: Grade 4 17 2.00±0.749 1.15±0.247 NRP1: 5’-AAATGCGAATGGCTGATTCAG-3’ (forward primer, Normal brain 4 0.89±0.110 0.72±0.040 157-177bp) 5’-CTCCATCGAAGACTTCCACGTAGT-3’ (reverse primer, 254- 277bp) 5’-TCAACCCTCACTTCGATTTGGAGGACA-3’ (probe, 212- 238bp) NRP2: 5’-GGATGGCATTCCACATGTTG-3’ (forward primer, ocular, 0.739 mm2 per field) using a computerized image analyzer 648-667bp) (Interactive Build Analysis System, Zeiss, Hallbergmoos, Germany) 5’-TGTGAAAGGTCAGGGAGAGGAT-3’ (reverse primer, 730- (19-21). 751bp) 5’-CACCCTCTGAACTTCGTTCATCGACGG-3’ (probe, 701- Statistical analysis. Differences in survival between the subgroups 727bp) of patients were compared with the Log-rank test and survival The human ‚2-microglobulin (‚2-M) primers and probe were curves were plotted according to the method of Kaplan and Meier. purchased from Perkin-Elmer (Boston, USA). The cDNA The Mann-Whitney U-test, Fisher’s exact test and ¯2 test, were samples prepared from another glioma surgical specimen were applied for comparisons. P<0.05 was considered to indicate a 2 3 4 5 diluted (1:4, 1:4 , 1:4 , 1:4 and 1:4 ) to construct a standard significant difference (Stat View-J5.0. Power PC Version, Abacus curve. Experiments were run in duplicate and the average value Concepts, Inc.). of the threshold cycle (CT) was calculated for NRP1, NRP2 and ‚2-M, respectively. The result of the relative increase in reporter fluorescent dye emission was analyzed by an ABI PRISM 7700 Results sequence detector (Perkin-Elmer). Analysis of the signal and the construction of the standard curve, was performed using Sequence v1.6.3 software (Perkin-Elmer). NRP1 and NRP2 gene expression. We calculated the mean amplification values of NRP1 and NRP2 from the CT Expression pattern of VEGF-A and VEGF-A receptors. We evaluated divided by the mean amplification values of human ‚2M, the isoform patterns of VEGF-A and VEGF-A receptor (Flt-1, given from the standard curves, respectively. We then KDR) expression by RT-PCR under the conditions reported standardized the NRP1 and NRP2 values to normal brain previously (16-19). as follows: (tumor mean NRP1 (or2) / ‚2M value) / (mean of adult normal NRP1 (or 2) / ‚2M values). Immunohistochemistry. Immunohistochemical analysis was The mean adult normal NRP1 / ‚2M and NRP2 / ‚2M performed using the CSA (Catalyzed Signal Amplification) system (DAKO Co. Ltd., CS, USA) according to the manufacturer’s were 0.89±0.110 (from 0.77 to 1.0) and 0.72±0.040 (from recommendations. Specific rabbit polyclonal anti-human NRP1 and 0.68 to 0.76), respectively (Table I). The average value of NRP2 antibodies (Santa Cruz Biotech, CA, USA) and mouse anti- the standardized NRP1 in the glioma showed 0.97±0.014 human CD34 monoclonal antibody (NCL-end, Novo Castra, (range from 0.96 to 0.98) in grade 1, 0.89±0.125 ( range Newcastle, UK), were purchased. The tissue sections were from 0.75 to 1.17) in grade 2, 1.87±0.492 (range from deparaffinized and dehydrated through a xylene and alcohol series. 1.25 to 2.86) in grade 3, and 2.00±0.749 (range from 0.97 After antigen retrieval (autoclaving at 121ÆC for 10 minutes in 0.01 to 3.15) in grade 4. The average value of NRP2 was M citrate buffer), blocking of endogenous peroxidase activity (3% 1.26±0.127 (from 1.17 to 1.35) in grade 1, 1.10±0.165 H2O2 for 5 minutes) and blocking of nonspecific binding (serum- free protein in phosphate-buffered saline for 5 minutes), sections (from 0.91 to 1.36) in grade 2, 1.08±0.168 (from 0.91 to were incubated with the primary antibody (anti-NRP1, NRP2 x 40 1.38) in grade 3, and 1.15±0.247 (from 0.7 to 1.56) in and anti-CD34 x 20). The immune complex on the sections was grade 4 (Table I). Overexpression of the NRP1 gene was then amplified with biotinylated secondary antibodies (rabbit anti- demonstrated in the higher grade of glioma (p<0.01, mouse immunoglobulin), streptavidin-biotin complex and Mann-Whitney U-test, Table IIa), while the expression of streptavidin peroxidase (Nichirei, Tokyo, Japan). The amplified NRP2 was not (Table IIb). We confirmed the gene products were visualized by a 3,3’-diaminobenzidine tetrahydrochloride (DAB) reaction. Light microscopy was used to expression of NRP1, NRP2 and VEGF-A receptor (Flt- identify two regions within or immediately adjacent to the tumor 1, KDR) by RT-PCR (Figure 1) The cellular localization containing the highest numbers of vessels. The microvessel counts of NRP1 was proven in the glioma cells by immuno- were evaluated at x 200 magnification (x 20 objective and x 10 histochemical analysis (Figure 2).
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