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Vascular Endothelial Growth Factor: Acting as an Autocrine Growth Factor for Human Gastric Adenocarcinoma Cell MGC803
Biochemical and Biophysical Research Communications 286, 505–512 (2001) doi:10.1006/bbrc.2001.5409, available online at http://www.idealibrary.com on
Vascular Endothelial Growth Factor: Acting as an Autocrine Growth Factor for Human Gastric Adenocarcinoma Cell MGC803 1 Xuejun Tian,* ,2 Shumei Song,* Jian Wu,* Lin Meng,* Zhiwei Dong,† and Chengchao Shou* ,3 *Department of Biochemistry and Molecular Biology, Beijing Institute for Cancer Research, Peking University School of Oncology, Beijing 100034, People’s Republic of China; and †Cancer Research Institute, Chinese Medical Science Academy, Beijing 100021, People’s Republic of China
Received July 17, 2001
Vascular endothelial growth factor (VEGF) is known to be a highly specific mitogen for endothelial cells through two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, which are almost specifically expressed in endothelial cells. However, recent findings showed that VEGF receptors may also expressed by nonendothelial cells, especially by tumor cells. To further understand the functional expression of VEGF receptors by nonendothelial cells, our preliminary screening detected the expression of VEGFR-2 in 115 different paraffin-embedded cancer specimens including 35 cases of bladder tumor, 30 cases of breast cancer, 25 cases of intestinal cancer, and 25 cases of lung cancer with immunohistochemistry. The results showed that VEGFR-2 was widely expressed in different tumor tissues. By reverse transcription PCR, NCIH23, NCI-H460, MGC803, MDA-MB-231, 293, and MCF7 cells were evaluated for the mRNA expression of both VEGF and VEGFR-2. The data indicated that all these tumor cell lines expressed detectable amounts of VEGF mRNA, but only 293, MCF7, and MGC803 cells coexpressed VEGFR-2. Immunoblot analysis also demonstrated the expression of VEGFR-2 at protein level. We further demonstrate that exogenous rhVEGF 165 could stimulate cell growth in MGC803, a tumor cell Abbreviations used: VEGF, vascular endothelial growth factor; VEGFR-1, fms-like tyrosine kinase 1; Flk-1/VEGFR-2, fetal liver kinase; RT-PCR, reverse transcription followed by polymerase chain reaction; EC, endothelial cell. 1 Supported by National Nature Science Foundation for Outstanding Youth Scientist of China (to C. S., No: 39525021). 2 Current address: Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. Fax: (617) 636-2409. E-mail: [email protected]. 3 To whom correspondence and reprint requests should be addressed at Department of Biochemistry and Molecular Biology, Beijing Institute for Cancer Research and the School of Oncology, Beijing Medical University, #1 Da-Hong-lou-Chang Street, Western District, Beijing 100034, People’s Republic of China. Fax: 86-0106617-5832. E-mail: [email protected].
line derived from gastric adenocarcinoma, in a doseand time-dependent manner. Furthermore, the antibodies against rhVEGF 165 and VEGFR-2 could block rhVEGF 165-mediated proliferation of MGC803 cells. These unexpected results provided direct evidence that VEGF may act as an autocrine growth factor to induce the proliferation of gastric adenocarcinoma cells as well as tumor angiogenic cells, thus suggesting a promising tumor therapeutic application based upon the VEGF system. © 2001 Academic Press Key Words: tumor; VEGF, VEGFR-2; stimulation.
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a potent inducer of microvascular hyper permeability (1, 2) as well as an endothelial cell mitogen (3– 6). Because of its effects on endothelial cell growth and microvascular permeability, VEGF is believed to be an important mediator of angiogenesis (7). A variety of malignant human tumors, including breast, lung, and prostate carcinomas, are known to secrete VEGF (8 –11). Two high-affinity protein tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR, human homologue; Flk-1, murine homologue), have been cloned, and their biologic effects on VEGF have been identified (12, 13). VEGFR-1 and VEGFR-2 are probably activated by all VEGF isoforms but fulfill different functions. Ligand binding studies show that human umbilical vein endothelial cells (HUVEC) possess lower levels of VEGFR-1 relative to VEGFR-2 (14). Activation of VEGFR-2 induced by VEGF in cells devoid of VEGFR-1 result in a mitogenic response, but the activation of VEGFR-1 by VEGF in cells lacking VEGFR-2 does not induce cell proliferation (14, 15). Expression of a dominant negative carboxyl-terminally truncated VEGFR-2 prevent VEGFR-2 activation and VEGFinduced mitogenesis in cultured cells and inhibits gli-
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oblastoma tumor growth in nude mice (16). These suggest that VEGFR-2 is the major transducer of VEGF signals leading to mitogenesis and other biological effects in endothelial cells. VEGFR-1 is the VEGF receptor that mediates VEGF and PIGF to stimulate cell chemotaxis and tissue production (17). Previous studies suggest that the known human VEGF receptors, VEGFR-1 and VEGFR-2, are primarily expressed in endothelial cells, and the statement is frequently made that these receptors are endothelial cell-specific. Recently, however, a few studies proved that other cell types also express these receptors. VEGFR-1 is expressed in trophoblast cells (18), monocytes (19) and renal mesangial cells (20). VEGFR-2, on the other hand, can be expressed in hematopoietic stem cells, megakaryocytes and retinal progenitor cells (21). In the retina, two functional forms of VEGFR-2 are expressed as a result of alternative splicing (22). Tumor cells such as ovarian carcinoma cells and malignant melanoma cells (23) can express high level of VEGFR-1 and VEGFR-2. Few of these studies, however, demonstrated the function of VEGF receptors expressed by no-endothelial cells especially by solid tumor cells. In our study, we investigated VEGFR-2 expression by different types of tumor using immunohistochemistry staining and we tested the expression of both VEGF and VEGFR-2 by several tumor cell lines using RTPCR. We also tested the expression of VEGFR-2 at protein level by immunoblot. Cell proliferation studies indicated that exogenous rhVEGF 165 can stimulate MGC803 cell growth in conditions of lower serum medium, and both the anti-VEGF 165 and anti-VEGFR-2 monoclonal antibodies could block the rhVEGF 165 induced MGC803 cell growth. Besides functioning in the angiogenesis of solid tumor development, these unexpected results give direct evidence that VEGF could be an autocrine growth factor for gastric adenocarcinoma tumor cell growth. MATERIALS AND METHODS Cell Cultures and Reagents Human gastric carcinoma cells MGC803 was established from a primary poorly differentiated mucoid adenocarcinoma of human stomach (24). The other cell lines were obtained from American Type Culture Collection (ATCC). All cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS). Recombinant human VEGF 165 (rhVEGF 165) was purchased from Upstate Biotech. (U.S.A.). Anti-VEGF 165 and anti-VEGFR-2 monoclonal antibodies were neutralizing antibodies. They were purchased from R&D Systems Inc. (U.S.A.). Immunohistochemistry and immunocytochemistry. One hundred fifteen paraffin-embedded tumor specimens were collected from the Department of Pathology at the Beijing Institute for Cancer Research (25 cases of lung carcinomas, 30 cases of breast cancers and 25 cases of intestinal carcinomas) and from the Institute of Urinology at Beijing Medical University (35 cases of bladder carcinomas). All
specimens were cut into two sections at consecutive 4-m each and were fixed in acetone. One section was stained with hematoxylin and eosin, and another was immunostained for VEGFR-2 with antiVEGFR-2 antibody. Immunohistochemical staining was performed by biotin–streptavidin immunoperoxidase technique followed by the blocking of native oxidase with 0.3% H 2O 2 and incubated in 0.01 M sodium citrate buffer (pH 6.0) for 10 min. The anti-VEGFR-2 antibody (0.5 g/ml), biotin-conjugated second antibody and streptavidin–peroxidase were added sequentially. The sections were colored with 3,3⬘-diaminobezidine (DAB) (0.5 mg/ml in PBS plus 1 l 30% H 2O 2 per ml DAB solution). For immunocytochemical staining of VEGFR-2, cells were plated on glass slides and grown for 36 h, then washed with PBS. Cell samples were air-dried and fixed with acetone. The antibody was raised against VEGFR-2 at a concentration of 0.5 g/ml. Immunostaining analysis was performed as described above after second antibody was added. Sections of tumor tissue or cells were immunostained with preimmune mouse IgG (1 g/ml) as negative controls.
RT-PCR Total RNA was extracted from 5 ⫻ 10 6 cultured NCI-H23, NCIH460, MGC803, MDA-MB-231, 293, MCF7 cells and endothelial cells (EC) by Trisol isolation of RNA kit (Biotech. Laboratories, Inc., U.S.A.). First-strand cDNA was synthesized using the SuperScript preamplification system for first-strand cDNA synthesis (GIBCO BRL) with 5 g total RNA in a 20-l reaction volume, following the procedure described by the manufacturer. After the reaction, 2 l first-strand cDNA was used as template in a 100-l PCR volume. The primer for VEGFR-2 reverse transcription was designed from the site of 2270 to 2255 bp (5⬘-TCC TGG GCA CCT TCT-3⬘) according to its cDNA sequence. Fragment from 1311 to 2217 bp of the VEGFR-2 cDNA sequence which encodes VEGFR-2 extracellular V-VII Ig-like domain was amplified using the following sequences primer (25): forward primer 5⬘-TAA GGA TCC CAC TCA AAC GCT GAC ATG TAC-3⬘ and reverse primer 5⬘-GGA GAA TTC ACT GCA TGC CTG GCA GGT G-3⬘. PCR for VEGFR-2 was carried out at 94°C for 45 s, 42°C for 1.5 min and 72°C for 2 min for two cycles, then changed the annealing temperature from 42 to 60°C for 1.5 min; other conditions remained the same for 35 cycles. The primer for VEGF reverse transcription was Oligo-dT (16). Fragment from 690 to 1207 bp of cDNA encoding the VEGF 165 amino acid isoform was amplified using primers as below (26): forward primer 5⬘-GGG GGA TCC GCC TCC GAA ACC ATG AAC TT-3⬘ and reverse primer 5⬘-CCC GAA TTC TGT GTG TTT TTG CAG GAA CAT T-3⬘. PCR for VEGF was carried out at 94°C 45 s, 55°C 40 s, 72°C 1 min for 36 cycles. The lines indicated the BamHI or EcoRI restriction endonuclease sites in the primers above. -Actin was used as control, primers for -actin included the reverse primer (5⬘-GGA GTT GAA GGT AGT TTC GTG-3⬘) spanning bases 2429 –2409 and the forward primer (5⬘-CGG GAA ATC GTG CGT GAC AT-3⬘) spanning bases 2107–2126. The predicted size of the amplified -actin PCR products was 214 bp. The PCR was carried out in Engine Peltier Thermal Cycler Model PTC200 (MT Research, Inc., U.S.A.).
Immunoblot Analysis Expression of VEGFR-2 was detected by immunoblot. Total protein extracts from cultured NCI-H23, NCI-H460, MGC803, MDAMB-231, 293, and MCF7 cells were obtained by lysing the cells in cold RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin). EC were used as positive control. The protein was separated by 8% SDS–PAGE and transferred to nitrocellulose. The nitrocellulose membrane was blocked in 5% milk/PBS– 0.5% Tween 20 for 1 h at room temperature, followed by incubation with primary anti-VEGFR-2 antibody at a concentration of 1 g/ml
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for 1 h at room temperature. The membranes were developed after incubation with appropriate peroxidase-conjugated secondary antibody by ECL enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
Cell Proliferation Assay Cell number counting. Proliferation of 293, MCF7 and MGC803 cells induced by exogenously added rhVEGF 165 was measured by cell number counting. 293 cells and MGC803 cells were seeded at a density of 4 ⫻ 10 4 per well, MCF7 cells were seeded at a density of 5 ⫻ 10 4 per well in 24-well plates on day 0. Cells were incubated with full growth medium (RPMI 1640 ⫹ 10% FCS) for 24 h at 37°C, then changed the serum concentration to 0.4 or 2.0% with or without rhVEGF 165 (10 ng/ml), respectively. Cells were counted in triplicate in a counter hemocytometer every day. The same medium and rhVEGF 165 were replaced every 2 days. [ 3H]Thymidine incorporation. MGC803 cells were seeded at a density of 2 ⫻ 10 4 per well in 24-well plates and incubated with full growth medium (RPMI 1640 ⫹ 10% FCS) for 24 h at 37°C. The cells were then incubated with 0.4% FCS RPMI 1640 for 24 h. The medium was replaced with 0.4% FCS RPMI 1640 containing various concentrations of rhVEGF 165 (0 –10 ng/ml) for another 35 h. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h of rhVEGF 165 treatment. Cells were washed with PBS and fixed in cold 10% trichloroacetic acid for 15 min, followed washing with 95% ethanol. Incorporated [ 3H]thymidine was extracted in 0.2 M NaOH and measured in a liquid scintillation counter. For time-dependent effects of VEGF, MGC803 cells were seeded at a density of 2 ⫻ 10 4 per well. Cells were made quiescent as above. The medium was replaced with 0.4% FCS RPMI 1640 containing 2 ng/ml rhVEGF 165 as time point 0. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h at each time point (6 –72 h). Culture medium did not change during this period of time. No rhVEGF 165 added cells were used as control. Anti-VEGF 165 and anti-VEGFR-2 antibodies were used to block the effect of VEGF 165 on MGC803 cells. rhVEGF 165 (2 ng/ml) was incubated with various concentrations of the antibody (0.001–1.0 g/ml) for 1 h at 22°C and added to the wells containing 2 ⫻ 10 4 MGC803 cells per well. Preimmune mouse IgG (1 g/ml) was used as control. After being incubated for 30 h, [ 3H]thymidine was added for another 5 h and its incorporation into DNA was measured as above. Values were expressed as the mean ⫾ SEM from 4 wells from three separate experiments. ANOVA Duncan’s Test SAS6.04 performed statistical analysis.
RESULTS 115 tumor specimens were studied by immunohistochemistry for detecting the expression of VEGFR-2. The results showed that immunoreactivity of VEGFR-2 was almost restricted to tumor cells and EC in most tumor tissues. Rare lymphocytes and vascular smooth muscles within tumor stroma appeared to be positive as well. Among different types of tumor, 35 bladder tumor tissues from transmigrate epithelium all exhibited immunoreactivity. The immunoreactive intensity was moderate or strong. EC staining in these tumor tissues was also strong (Fig. 1A-a). Immunoreactivity of breast cancer cells and intestinal tumor cells from adenoepithelium appeared to be weaker compared with bladder tumor cells, and positive rates were 92 and 90%, respectively. The immunoreactive intensity was moderate (Figs. 1A-c and 1A-d). The immunoreactivity of lung cancer from squamous epithelium is
FIG. 1. Immunochemical staining of VEGF receptor VEGFR-2. (A) Immunohistochemical staining for different human cancer tissues with VEGFR-2 antibody (a, b, c, d, e, ⫻200; f, ⫻100). a, transmigrant epithelium cancer of bladder; b, same cancer tissue with a, but preimmune mouse IgG was used as control; c, adenoepithelium cancer of breast; d, adenoepithelium cancer of intestine; e, adenoepithilium cancer of lung; f, squamous-epithelium cancer of lung. (B) Cytochemical immunostaining of adenocarcinoma cell line MGC803 from human stomach (⫻125). a, immunostaining with preimmune mouse IgG; b, immunostaining with VEGFR-2 antibody.
weaker (Fig. 1A-f) than that from adenoepithelium and transmigrate epithelium, its expression rate is about 77% (Fig. 1A-e). No immunostaining was observed on bladder cancer tissue with preimmune mouse IgG (Fig. 1A-b). Based on the results above, we investigated expression of VEGF and VEGFR-2 in tumor cells in our lab. We amplified the cDNA from NCI-H23, NCI-H460, MGC803, MDA-MB-231, 293, MCF7 and EC cell lines with VEGF-specific and VEGFR-2-specific primers. The predicted sizes for VEGF and VEGFR-2 should be 517 and 906 bp, respectively. As shown in Fig. 2, all the cell lines expressed VEGF mRNA, but only MGC803,
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FIG. 2. Identification of VEGF and VEGFR-2 expression in cultured NCI-H23, NCI-H460, MGC803, MDA-MB-231, 293, MCF7 cells, and EC using RT-PCR. Expected VEGFR-2 and VEGF amplification products of 906 and 517 bp were obtained, respectively (upper and middle panel). Lower panel shows a 214-bp band amplified with primer specific for -actin with the same cDNA.
293, MCF-7 and EC expressed VEGFR-2 mRNA. Amplification of -actin with -actin-specific primers was used as control. The predicted product was 214 bp (Fig. 2, lower panel). Next, we investigated VEGFR-2 expression by these tumor cells at protein level using immunoblot. Similar results were obtained that MGC803, 293, MCF7 cells expressed VEGFR-2 protein as well as RNA (Fig. 3). As an important part of the study, we checked whether those VEGFR-2 positive tumor cells could be stimulated by exogenous-added VEGF. When functional rhVEGF 165 was added to cultured MGC803, 293 and MCF7 cells with full growth medium, it failed to increase cell number while it increase EC cell growth (data not show). But when we lower the FCS concentration in the medium to 0.4%, we found that rhVEGF 165 increased MGC803 cell number. These effects began at day 2 after VEGF was added and the maximal stimulation was at day 3 and day 4 (Fig. 4C). rhVEGF 165 failed to increase 293 and MCF7 cell number even at lower FCS concentration medium (Figs. 4A and 4B, respectively). Based on the results above, we double-checked the expression of VEGFR-2 protein by MGC803 cells using immunocytochemistry. The results indicated that VEGFR-2 was detected on MGC803 cells (Fig. 1B-b) compared with the control (Fig. 1B-a). To confirm these unexpected findings, we examined the effect of exogenous rhVEGF 165 on DNA synthesis in
MGC803 cells using [ 3H]thymidine incorporation assay. The results showed that rhVEGF 165 increased MGC803 cell proliferation in both dose-dependent and time-dependent manners. As little as 0.5 ng/ml VEGF caused no significant increases in [ 3H]thymidine incorporation. At concentration of 2 ng/ml, VEGF could significantly increase the incorporation of [ 3H]thymidine (P ⬍ 0.01), and the effect of VEGF on MGC803 cells at this concentration made no big difference compared with its effect at concentration of 10 ng/ml (Fig. 5A). Results of the time-cause cell growth induced by VEGF indicated that the increase in [ 3H]thymidine incorporation began at 24 h and peaked at the period of 30 to 48 h after VEGF was added (P ⬍ 0.01), the effect was reduced after 72 h (P ⬍ 0.05) (Fig. 5B). To block the effect of rhVEGF 165 on MGC803 cells, rhVEGF 165 of 2 ng/ml was incubated with different concentration of either anti-VEGF 165 or anti-VEGFR-2 antibodies first, then the incubated mixtures was added to the cultured MGC803 for stimulation. The results showed that when the VEGF antibody concentration was below 0.01 g/ml, no obvious inhibition of VEGF inducing MGC803 proliferation was observed. As antibody concentration increased, the VEGF was inhibited gradually. When VEGF antibody concentration was up to 1 g/ml, the exogenous VEGF 165 was inhibited completely (Fig. 5C). Unlike VEGF antibody, the VEGFR-2 antibody showed only partly inhibiting effect on VEGF-induced MGC803 cell growth at concentration of 0.1–1.0 g/ml. No further inhibition was observed when the anti-VEGFR-2 antibody was presented at a higher concentration (Fig. 5D) DISCUSSION Recently, nonendothelial cell localization of VEGF receptors, especially on tumor cells, raises the possibility of VEGF autocrine stimulation of tumor growth (18 –21, 23). Masood et al. (27) used VEGF antisense oligonucleotides specifically blocking VEGF mRNA and inhibiting Kaposi sarcoma cell growth. For the first time, it gave direct evidences of VEGF acting as an
FIG. 3. Immunoblot analysis of VEGFR-2 protein expression by NCI-H23, NCI-H460, MGC803, MDA-MB-231, 293, MCF7 cells and EC. Total cell protein extracts were separated by 8% SDS–PAGE gel and immunoblotted by anti-VEGFR-2 antibodies. Position indicating molecular weights was according to the prestained SDS–PAGE marker.
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FIG. 4. Effects of rhVEGF 165 on proliferation of 293, MCF7, and MGC803 (A, B, and C, respectively). Quiescent cells were cultured in medium containing 0.4% FCS (}), 0.4% FCS plus 10 ng/ml rhVEGF 165 (Œ), 2% FCS (F), or 2% FCS plus 10 ng/ml rhVEGF 165 (■). Cells were counted in triplicate in a Coulter hemocytometer on the indicated day. Values represent the mean cell number from experiments in triplicate.
autocrine stimulator for tumor cell growth. Our immunohistochemistry results suggest that the expression of VEGFR-2 be not only restricted to EC but also to tumor cells in tumor tissues. Moreover, the intensity of
VEGFR-2 expression by tumor cells is sometimes as strong as that by EC. Our studies demonstrated that VEGFR-2 also expressed in vascular smooth muscle cells and other tissue cells such as macrophage in lung cancers and lymphocytes in intestinal and bladder cancer. These results suggest that VEGFR-2 might play an important role in VEGF autocrine stimulation of tumor development. Functional expression of VEGFR-2 has been demonstrated in leukemic cell (28) RT-PCR revealed that tumor cell lines MGC803, 293 and MCF7 express RNA encoding both VEGF and VEGFR-2. We further detected the expression of VEGFR-2 by these three tumor cell lines using immunoblot. The results showed that they all express VEGFR-2 protein. These results encouraged us to further investigate the responsiveness of these cell lines to VEGF. We tested the effect of exogenous added rhVEGF 165 on cells in medium with different serum concentrations. The results showed that rhVEGF 165 in 0.4% FCS medium increased MGC803 cell growth. To make sure that the primary cultured MGC803 cells were not confused with EC, we carefully identified the cells by immunocytochemistry (data not shown). We further investigated the effect of exogenous added rhVEGF 165 on MGC803 cells using [ 3H]thymidine incorporation assay. Exogenous added rhVEGF 165 could induce MGC803 cell proliferation in dose and time dependent manners. Antibodies against VEGF or against VEGFR-2 can block these effects. When excessive anti-VEGF antibodies were added to cultured cells, no further inhibition effect was observed compared with no VEGF added control. It suggests that the antibody failed to inhibit the effect of endogenous VEGF on MGC803 cell growth. There are at least two possible reasons for these phenomena: (1) the concentration of VEGF secreted by cultured MGC803 cells is lower than that in vivo. VEGF expression is known to be up-regulated by hepatocyte growth factor and other growth factors such as TGF- and PDGF in other cell types through paracrine in vivo (29). We failed to detect VEGF on MGC803 conditional medium by ELISA after the cells were incubated at different time. (2) The anti-VEGF antibody we used here was anti-VEGF 165 antibody, but tumor cells can secrete all forms of VEGF that could not be blocked by the anti-VEGF antibody. Furthermore, tumor cells could secrete many other growth factors that cause cell proliferation, thus covering the cell growth effects induced by endogenous VEGF. In their study, Boocock et al. (30) mentioned that receptor-positive primary ovarian carcinoma cells failed to respond to exogenous VEGF, although no experimental details were provided. A possible difference may be caused by to the selection of serum deprivation period to quiesce the cells before addition of VEGF. We noticed that when cells were incubated with low serum medium for a period longer than 72 h, the VEGF-
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FIG. 5. Effect of rhVEGF 165 on MGC803 proliferation using [ 3H]thymidine incorporation assay (A) Dose response of rhVEGF 165-induced increases in [ 3H]thymidine incorporation in MGC803 cells. MGC803 cells were made quiescent by incubation in 0.4% FCS RPMI 1640. The medium was replaced with 0.4% FCS RPMI 1640 containing various concentrations of rhVEGF 165 (0 –10 ng/ml) for another 35 h. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h of rhVEGF 165 treatment. Incorporated [ 3H]thymidine was measured in a liquid scintillation counter. (B) rhVEGF 165 stimulates MGC803 proliferation with time-dependent manner. MGC803 cells were seeded at a density of 2 ⫻ 10 4 per well. Cells were made quiescent as above. The medium was replaced with 0.4% FCS RPMI 1640 containing 2 ng/ml rhVEGF 165 as time point 0. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h at each time point as indicated. Culture medium did not change during the time-dependent experiments. No rhVEGF 165 added cells were used as control. (C and D) Both anti-VEGF 165 and anti-VEGFR-2 antibodies inhibit the rhVEGF 165 effects on MGC803 proliferation (bar n, no VEGF added; bar IgG, added preimmune mouse antibody, 1 g/ml). The rhVEGF 165 concentration was 2 ng/ml in C and D. EC was used as a positive control in A, C, and D.
induced cell growth effect was reduced. Even repeated the addition of VEGF, similar results were observed (Fig. 6). This suggests that under the conditional culture medium, VEGF-induced MGC803 cell growth could only last for a relatively short time that is not easily detected. Both VEGFR-1 and VEGFR-2 are VEGF-specific receptors. The distribution of these two receptors is different in various cell types, and the expression of the receptors is modulated in a dynamic manner during development. The different distribution of VEGF receptors determines the specificity of their biological functions in tumor development. VEGF can stimulate monocyte chemotaxis and migration through VEGFR-1, but not VEGFR-2 (19). Recently, new VEGF receptors such as neuropilin-1, neuropilin-2 etc. were discovered (31). In our study, we also observed that the anti-VEGFR-2 antibody partly blocks the VEGF-
induced MGC803 cell growth. We also detected the expression of VEGFR-1 on MGC803 cells by RT-PCR under different reaction conditions, but no specific PCR products were obtained (data not shown). This suggests that MGC803 cell growth caused by VEGF is mainly mediated via VEGFR-2. It may also be caused by other functional VEGF receptors besides VEGFR-2. To our knowledge, this is the first report to show that the gastric carcinoma cell line MGC803 expresses VEGF and VEGFR-2 and proliferates strongly in response to VEGF directly. Furthermore, we successfully used anti-VEGF and anti-VEGFR-2 antibodies to block the VEGF-induced tumor cell growth in vitro. These results give direct evidence that the angiogenic factor VEGF can also acts as an autocrine growth factor for gastric carcinoma. Over expression of VEGF by tumor cells could thus not only indirectly facilitate their growth and invasion, via its effects on angiogenesis,
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FIG. 6. Effects of repeated addition of rhVEGF 165 on MGC803 cell proliferation.. Two separated parts of MGC803 cells were made quiescent by incubation in 0.4%FCS RPMI 1640. The medium was replaced with 0.4% FCS RPMI 1640 containing 2 ng/ml rhVEGF 165 as time point 0. One part was incubated for 36 h, [ 3H]thymidine (0.5 Ci/ml) was added during the last 5 h. Collecting the cells and measure the [ 3H]thymidine incorporation. For the other part, adding the same concentration of rhVEGF 165 but did not change the culture medium. After incubating for another 36 h, adding [ 3H]thymidine and measure its incorporation. The results indicated that, compared with control, rhVEGF 165-induced MGC803 cell growth reduced (at 36 h, P ⬍ 0.01, at 72 h, P ⬎ 0.05).
but also directly promote tumor cell proliferation. Identification of VEGF as an autocrine growth factor expands our knowledge of cancer biology and contributes more to tumor biotherapy.
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factor/vascular endothelial growth factor: A multifunctional angiogenic cytokine. EXS 79, 233–269. Olson, T. A., Mohanraj, D., Carson, L. F., and Ramakrishnan, S. (1994) Vascular permeability factor gene expression in normal and neoplastic human ovaries. Cancer Res. 54, 276 –280. Ohta, Y., Endo, Y., Tanaka, M., Shimizu, J., Oda, M., Hayashi, Y., Watanabe, Y., and Sasaki, T. (1996) Significance of vascular endothelial growth factor messenger RNA expression in primary lung cancer. Clin. Cancer Res. 2, 1411–1416. Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Manseau, E. J., Senger, D. R., and Dvorak, H. F. (1993) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res. 53, 4727– 4735. Joseph, I. B., Nelson, J. B., Denmeade, S. R., and Isaacs, J. T. (1997) Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue. Clin. Cancer Res. 3, 2507–2511. Ferrara, N., Houck, K., Jakeman, L., and Leung, D. W. (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr. Rev. 13, 18 –32. Risau, W. (1997) Mechanisms of angiogenesis. Nature 386, 671– 674. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M., and Heldin, C. H. (1994) Different signal transduction properties of KDR and Flt-1, two receptors for vascular endothelial growth factor. J. Biol. Chem. 269, 26988 –26995. Seetharam, L., Gotoh, N., Maru, Y., Neufeld, G., Yamaguchi, S., and Shibuya, M. (1995) A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene 10, 135–147. Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, (1994) A. Glioblastoma growth inhibited in vivo by a dominantnegative Flk-1 mutant. Nature 367, 576 –579. Clauss, M., Weich, H., Breier, G., Knies, U., Rockl, W., Waltenberger, J., and Risau, W. (1996) The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J. Biol. Chem. 271, 17629 –17634. Charnockjones, D. S., Sharkey, A. M., Boocok, C. A., Ahmed, A., Plevin, R., Ferrara, N., and Smith, S. K. (1994) Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells. Biol. Reprod. 51, 524 –530. Barleon, B., Sozzani, S., Zhou, D., Weich, H. A., Mantovani, A., and Marme, D. (1996) Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87, 3336 –3343. Takahashi, T., Shirasawa, T., Miyake, K., Yahagi, Y., Maruyama, N., Kasahara, N., Kawamura, T., Matsumura, O., Mitarai, T., and Sakai, O. (1995) Protein tyrosine kinases expressed in glomeruli and cultured glomerular cells: Flt-1 and VEGF expression in renal mesangial cells. Biochem. Biophys. Res. Commun. 209, 218 –226. Katoh, O., Tauchi, H., Kawaishi, K., Kimura, A., and Satow, Y. (1995) Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation. Cancer Res. 55, 5687–5692. Wen, Y., Edelman, J. L., Kang, T., Zeng, N., and Sachs, G. (1998) Two functional forms of vascular endothelial growth factor receptor-2/Flk-1 mRNA are expressed in normal rat retina. J. Biol. Chem. 273, 2090 –2097. Cohen, T., Gitay-Goren, H., Sharon, R., Shibuya, M., Halaban, R., Levi, B. Z., and Neufeld, G. (1995) VEGF121, a vascular
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endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J. Biol. Chem. 270, 11322–11326. Wang, K. H. (1983) A in vitro cell line (MGC803) of a poorly differentiated mucoid adenocarcinoma of human stomach. Acta Biol. Exp. Sin. 16, 257–267. Terman, B. I., Dougher-Vermazen, M., Carrion, M. E., Dimitrov, D., Armellino, D. C., Gospodarowicz, D., and Bohlen, P. (1992) Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem. Biophys. Res. Commun. 187, 1579 –1586. Claffey, K. P., Shih, S. C., Mullen, A., Dziennis, S., Cusick, J. L., Abrams, K. R., Lee, S. W., Detmar, M. (1998) Identification of a human VPF/VEGF 3⬘ untranslated region mediating hypoxiainduced mRNA stability. Mol. Biol. Cell 9, 469 – 481. Masood, R., Cai, J., Zheng, T., Smith, D. L., Naidu, Y., and Gill, P. S. (1997) Vascular endothelial growth factor/vascular permeability factor is an autocrine growth factor for AIDS-Kaposi sarcoma. Proc. Natl. Acad. Sci. USA 94, 979 –984.
28. Dias, S., Hattori, K., Zhu, Z., Heissig, B., Choy, M., Lane, W., Wu, Y., Chadburn, A., Hyjek, E., Gill, M., Hicklin, D. J., Witte, L., Moore, M. A., Rafii, S. (2000) Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J. Clin. Invest. 106, 511–521. 29. Moriyama, T., Kataoka, H., Hamasuna, R., Yokogami, K., Uehara, H., Kawano, H., Goya, T., Tsubouchi, H., Koono, M., and Wakisaka, S. (1998) Up-regulation of vascular endothelial growth factor induced by hepatocyte growth factor/scatter factor stimulation in human glioma cells. Biochem. Biophys. Res. Commun. 249, 73–77. 30. Boocock, C. A., Charnock-Jones, D. S., Sharkey, A. M., McLaren, J., Barker, P. J., Wright, K. A., Twentyman, P. R., and Smith, S. K. (1995) Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma. J. Natl. Cancer Inst. 87, 506 –516. 31. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., Klagsbrun, M. (1998) Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92, 735–745.
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Vascular Endothelial Growth Factor: Acting as an Autocrine Growth Factor for Human Gastric Adenocarcinoma Cell MGC803 1 Xuejun Tian,* ,2 Shumei Song,* Jian Wu,* Lin Meng,* Zhiwei Dong,† and Chengchao Shou* ,3 *Department of Biochemistry and Molecular Biology, Beijing Institute for Cancer Research, Peking University School of Oncology, Beijing 100034, People’s Republic of China; and †Cancer Research Institute, Chinese Medical Science Academy, Beijing 100021, People’s Republic of China
Received July 17, 2001
Vascular endothelial growth factor (VEGF) is known to be a highly specific mitogen for endothelial cells through two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, which are almost specifically expressed in endothelial cells. However, recent findings showed that VEGF receptors may also expressed by nonendothelial cells, especially by tumor cells. To further understand the functional expression of VEGF receptors by nonendothelial cells, our preliminary screening detected the expression of VEGFR-2 in 115 different paraffin-embedded cancer specimens including 35 cases of bladder tumor, 30 cases of breast cancer, 25 cases of intestinal cancer, and 25 cases of lung cancer with immunohistochemistry. The results showed that VEGFR-2 was widely expressed in different tumor tissues. By reverse transcription PCR, NCIH23, NCI-H460, MGC803, MDA-MB-231, 293, and MCF7 cells were evaluated for the mRNA expression of both VEGF and VEGFR-2. The data indicated that all these tumor cell lines expressed detectable amounts of VEGF mRNA, but only 293, MCF7, and MGC803 cells coexpressed VEGFR-2. Immunoblot analysis also demonstrated the expression of VEGFR-2 at protein level. We further demonstrate that exogenous rhVEGF 165 could stimulate cell growth in MGC803, a tumor cell Abbreviations used: VEGF, vascular endothelial growth factor; VEGFR-1, fms-like tyrosine kinase 1; Flk-1/VEGFR-2, fetal liver kinase; RT-PCR, reverse transcription followed by polymerase chain reaction; EC, endothelial cell. 1 Supported by National Nature Science Foundation for Outstanding Youth Scientist of China (to C. S., No: 39525021). 2 Current address: Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. Fax: (617) 636-2409. E-mail: [email protected]. 3 To whom correspondence and reprint requests should be addressed at Department of Biochemistry and Molecular Biology, Beijing Institute for Cancer Research and the School of Oncology, Beijing Medical University, #1 Da-Hong-lou-Chang Street, Western District, Beijing 100034, People’s Republic of China. Fax: 86-0106617-5832. E-mail: [email protected].
line derived from gastric adenocarcinoma, in a doseand time-dependent manner. Furthermore, the antibodies against rhVEGF 165 and VEGFR-2 could block rhVEGF 165-mediated proliferation of MGC803 cells. These unexpected results provided direct evidence that VEGF may act as an autocrine growth factor to induce the proliferation of gastric adenocarcinoma cells as well as tumor angiogenic cells, thus suggesting a promising tumor therapeutic application based upon the VEGF system. © 2001 Academic Press Key Words: tumor; VEGF, VEGFR-2; stimulation.
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a potent inducer of microvascular hyper permeability (1, 2) as well as an endothelial cell mitogen (3– 6). Because of its effects on endothelial cell growth and microvascular permeability, VEGF is believed to be an important mediator of angiogenesis (7). A variety of malignant human tumors, including breast, lung, and prostate carcinomas, are known to secrete VEGF (8 –11). Two high-affinity protein tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR, human homologue; Flk-1, murine homologue), have been cloned, and their biologic effects on VEGF have been identified (12, 13). VEGFR-1 and VEGFR-2 are probably activated by all VEGF isoforms but fulfill different functions. Ligand binding studies show that human umbilical vein endothelial cells (HUVEC) possess lower levels of VEGFR-1 relative to VEGFR-2 (14). Activation of VEGFR-2 induced by VEGF in cells devoid of VEGFR-1 result in a mitogenic response, but the activation of VEGFR-1 by VEGF in cells lacking VEGFR-2 does not induce cell proliferation (14, 15). Expression of a dominant negative carboxyl-terminally truncated VEGFR-2 prevent VEGFR-2 activation and VEGFinduced mitogenesis in cultured cells and inhibits gli-
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oblastoma tumor growth in nude mice (16). These suggest that VEGFR-2 is the major transducer of VEGF signals leading to mitogenesis and other biological effects in endothelial cells. VEGFR-1 is the VEGF receptor that mediates VEGF and PIGF to stimulate cell chemotaxis and tissue production (17). Previous studies suggest that the known human VEGF receptors, VEGFR-1 and VEGFR-2, are primarily expressed in endothelial cells, and the statement is frequently made that these receptors are endothelial cell-specific. Recently, however, a few studies proved that other cell types also express these receptors. VEGFR-1 is expressed in trophoblast cells (18), monocytes (19) and renal mesangial cells (20). VEGFR-2, on the other hand, can be expressed in hematopoietic stem cells, megakaryocytes and retinal progenitor cells (21). In the retina, two functional forms of VEGFR-2 are expressed as a result of alternative splicing (22). Tumor cells such as ovarian carcinoma cells and malignant melanoma cells (23) can express high level of VEGFR-1 and VEGFR-2. Few of these studies, however, demonstrated the function of VEGF receptors expressed by no-endothelial cells especially by solid tumor cells. In our study, we investigated VEGFR-2 expression by different types of tumor using immunohistochemistry staining and we tested the expression of both VEGF and VEGFR-2 by several tumor cell lines using RTPCR. We also tested the expression of VEGFR-2 at protein level by immunoblot. Cell proliferation studies indicated that exogenous rhVEGF 165 can stimulate MGC803 cell growth in conditions of lower serum medium, and both the anti-VEGF 165 and anti-VEGFR-2 monoclonal antibodies could block the rhVEGF 165 induced MGC803 cell growth. Besides functioning in the angiogenesis of solid tumor development, these unexpected results give direct evidence that VEGF could be an autocrine growth factor for gastric adenocarcinoma tumor cell growth. MATERIALS AND METHODS Cell Cultures and Reagents Human gastric carcinoma cells MGC803 was established from a primary poorly differentiated mucoid adenocarcinoma of human stomach (24). The other cell lines were obtained from American Type Culture Collection (ATCC). All cells were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS). Recombinant human VEGF 165 (rhVEGF 165) was purchased from Upstate Biotech. (U.S.A.). Anti-VEGF 165 and anti-VEGFR-2 monoclonal antibodies were neutralizing antibodies. They were purchased from R&D Systems Inc. (U.S.A.). Immunohistochemistry and immunocytochemistry. One hundred fifteen paraffin-embedded tumor specimens were collected from the Department of Pathology at the Beijing Institute for Cancer Research (25 cases of lung carcinomas, 30 cases of breast cancers and 25 cases of intestinal carcinomas) and from the Institute of Urinology at Beijing Medical University (35 cases of bladder carcinomas). All
specimens were cut into two sections at consecutive 4-m each and were fixed in acetone. One section was stained with hematoxylin and eosin, and another was immunostained for VEGFR-2 with antiVEGFR-2 antibody. Immunohistochemical staining was performed by biotin–streptavidin immunoperoxidase technique followed by the blocking of native oxidase with 0.3% H 2O 2 and incubated in 0.01 M sodium citrate buffer (pH 6.0) for 10 min. The anti-VEGFR-2 antibody (0.5 g/ml), biotin-conjugated second antibody and streptavidin–peroxidase were added sequentially. The sections were colored with 3,3⬘-diaminobezidine (DAB) (0.5 mg/ml in PBS plus 1 l 30% H 2O 2 per ml DAB solution). For immunocytochemical staining of VEGFR-2, cells were plated on glass slides and grown for 36 h, then washed with PBS. Cell samples were air-dried and fixed with acetone. The antibody was raised against VEGFR-2 at a concentration of 0.5 g/ml. Immunostaining analysis was performed as described above after second antibody was added. Sections of tumor tissue or cells were immunostained with preimmune mouse IgG (1 g/ml) as negative controls.
RT-PCR Total RNA was extracted from 5 ⫻ 10 6 cultured NCI-H23, NCIH460, MGC803, MDA-MB-231, 293, MCF7 cells and endothelial cells (EC) by Trisol isolation of RNA kit (Biotech. Laboratories, Inc., U.S.A.). First-strand cDNA was synthesized using the SuperScript preamplification system for first-strand cDNA synthesis (GIBCO BRL) with 5 g total RNA in a 20-l reaction volume, following the procedure described by the manufacturer. After the reaction, 2 l first-strand cDNA was used as template in a 100-l PCR volume. The primer for VEGFR-2 reverse transcription was designed from the site of 2270 to 2255 bp (5⬘-TCC TGG GCA CCT TCT-3⬘) according to its cDNA sequence. Fragment from 1311 to 2217 bp of the VEGFR-2 cDNA sequence which encodes VEGFR-2 extracellular V-VII Ig-like domain was amplified using the following sequences primer (25): forward primer 5⬘-TAA GGA TCC CAC TCA AAC GCT GAC ATG TAC-3⬘ and reverse primer 5⬘-GGA GAA TTC ACT GCA TGC CTG GCA GGT G-3⬘. PCR for VEGFR-2 was carried out at 94°C for 45 s, 42°C for 1.5 min and 72°C for 2 min for two cycles, then changed the annealing temperature from 42 to 60°C for 1.5 min; other conditions remained the same for 35 cycles. The primer for VEGF reverse transcription was Oligo-dT (16). Fragment from 690 to 1207 bp of cDNA encoding the VEGF 165 amino acid isoform was amplified using primers as below (26): forward primer 5⬘-GGG GGA TCC GCC TCC GAA ACC ATG AAC TT-3⬘ and reverse primer 5⬘-CCC GAA TTC TGT GTG TTT TTG CAG GAA CAT T-3⬘. PCR for VEGF was carried out at 94°C 45 s, 55°C 40 s, 72°C 1 min for 36 cycles. The lines indicated the BamHI or EcoRI restriction endonuclease sites in the primers above. -Actin was used as control, primers for -actin included the reverse primer (5⬘-GGA GTT GAA GGT AGT TTC GTG-3⬘) spanning bases 2429 –2409 and the forward primer (5⬘-CGG GAA ATC GTG CGT GAC AT-3⬘) spanning bases 2107–2126. The predicted size of the amplified -actin PCR products was 214 bp. The PCR was carried out in Engine Peltier Thermal Cycler Model PTC200 (MT Research, Inc., U.S.A.).
Immunoblot Analysis Expression of VEGFR-2 was detected by immunoblot. Total protein extracts from cultured NCI-H23, NCI-H460, MGC803, MDAMB-231, 293, and MCF7 cells were obtained by lysing the cells in cold RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin). EC were used as positive control. The protein was separated by 8% SDS–PAGE and transferred to nitrocellulose. The nitrocellulose membrane was blocked in 5% milk/PBS– 0.5% Tween 20 for 1 h at room temperature, followed by incubation with primary anti-VEGFR-2 antibody at a concentration of 1 g/ml
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for 1 h at room temperature. The membranes were developed after incubation with appropriate peroxidase-conjugated secondary antibody by ECL enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
Cell Proliferation Assay Cell number counting. Proliferation of 293, MCF7 and MGC803 cells induced by exogenously added rhVEGF 165 was measured by cell number counting. 293 cells and MGC803 cells were seeded at a density of 4 ⫻ 10 4 per well, MCF7 cells were seeded at a density of 5 ⫻ 10 4 per well in 24-well plates on day 0. Cells were incubated with full growth medium (RPMI 1640 ⫹ 10% FCS) for 24 h at 37°C, then changed the serum concentration to 0.4 or 2.0% with or without rhVEGF 165 (10 ng/ml), respectively. Cells were counted in triplicate in a counter hemocytometer every day. The same medium and rhVEGF 165 were replaced every 2 days. [ 3H]Thymidine incorporation. MGC803 cells were seeded at a density of 2 ⫻ 10 4 per well in 24-well plates and incubated with full growth medium (RPMI 1640 ⫹ 10% FCS) for 24 h at 37°C. The cells were then incubated with 0.4% FCS RPMI 1640 for 24 h. The medium was replaced with 0.4% FCS RPMI 1640 containing various concentrations of rhVEGF 165 (0 –10 ng/ml) for another 35 h. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h of rhVEGF 165 treatment. Cells were washed with PBS and fixed in cold 10% trichloroacetic acid for 15 min, followed washing with 95% ethanol. Incorporated [ 3H]thymidine was extracted in 0.2 M NaOH and measured in a liquid scintillation counter. For time-dependent effects of VEGF, MGC803 cells were seeded at a density of 2 ⫻ 10 4 per well. Cells were made quiescent as above. The medium was replaced with 0.4% FCS RPMI 1640 containing 2 ng/ml rhVEGF 165 as time point 0. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h at each time point (6 –72 h). Culture medium did not change during this period of time. No rhVEGF 165 added cells were used as control. Anti-VEGF 165 and anti-VEGFR-2 antibodies were used to block the effect of VEGF 165 on MGC803 cells. rhVEGF 165 (2 ng/ml) was incubated with various concentrations of the antibody (0.001–1.0 g/ml) for 1 h at 22°C and added to the wells containing 2 ⫻ 10 4 MGC803 cells per well. Preimmune mouse IgG (1 g/ml) was used as control. After being incubated for 30 h, [ 3H]thymidine was added for another 5 h and its incorporation into DNA was measured as above. Values were expressed as the mean ⫾ SEM from 4 wells from three separate experiments. ANOVA Duncan’s Test SAS6.04 performed statistical analysis.
RESULTS 115 tumor specimens were studied by immunohistochemistry for detecting the expression of VEGFR-2. The results showed that immunoreactivity of VEGFR-2 was almost restricted to tumor cells and EC in most tumor tissues. Rare lymphocytes and vascular smooth muscles within tumor stroma appeared to be positive as well. Among different types of tumor, 35 bladder tumor tissues from transmigrate epithelium all exhibited immunoreactivity. The immunoreactive intensity was moderate or strong. EC staining in these tumor tissues was also strong (Fig. 1A-a). Immunoreactivity of breast cancer cells and intestinal tumor cells from adenoepithelium appeared to be weaker compared with bladder tumor cells, and positive rates were 92 and 90%, respectively. The immunoreactive intensity was moderate (Figs. 1A-c and 1A-d). The immunoreactivity of lung cancer from squamous epithelium is
FIG. 1. Immunochemical staining of VEGF receptor VEGFR-2. (A) Immunohistochemical staining for different human cancer tissues with VEGFR-2 antibody (a, b, c, d, e, ⫻200; f, ⫻100). a, transmigrant epithelium cancer of bladder; b, same cancer tissue with a, but preimmune mouse IgG was used as control; c, adenoepithelium cancer of breast; d, adenoepithelium cancer of intestine; e, adenoepithilium cancer of lung; f, squamous-epithelium cancer of lung. (B) Cytochemical immunostaining of adenocarcinoma cell line MGC803 from human stomach (⫻125). a, immunostaining with preimmune mouse IgG; b, immunostaining with VEGFR-2 antibody.
weaker (Fig. 1A-f) than that from adenoepithelium and transmigrate epithelium, its expression rate is about 77% (Fig. 1A-e). No immunostaining was observed on bladder cancer tissue with preimmune mouse IgG (Fig. 1A-b). Based on the results above, we investigated expression of VEGF and VEGFR-2 in tumor cells in our lab. We amplified the cDNA from NCI-H23, NCI-H460, MGC803, MDA-MB-231, 293, MCF7 and EC cell lines with VEGF-specific and VEGFR-2-specific primers. The predicted sizes for VEGF and VEGFR-2 should be 517 and 906 bp, respectively. As shown in Fig. 2, all the cell lines expressed VEGF mRNA, but only MGC803,
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FIG. 2. Identification of VEGF and VEGFR-2 expression in cultured NCI-H23, NCI-H460, MGC803, MDA-MB-231, 293, MCF7 cells, and EC using RT-PCR. Expected VEGFR-2 and VEGF amplification products of 906 and 517 bp were obtained, respectively (upper and middle panel). Lower panel shows a 214-bp band amplified with primer specific for -actin with the same cDNA.
293, MCF-7 and EC expressed VEGFR-2 mRNA. Amplification of -actin with -actin-specific primers was used as control. The predicted product was 214 bp (Fig. 2, lower panel). Next, we investigated VEGFR-2 expression by these tumor cells at protein level using immunoblot. Similar results were obtained that MGC803, 293, MCF7 cells expressed VEGFR-2 protein as well as RNA (Fig. 3). As an important part of the study, we checked whether those VEGFR-2 positive tumor cells could be stimulated by exogenous-added VEGF. When functional rhVEGF 165 was added to cultured MGC803, 293 and MCF7 cells with full growth medium, it failed to increase cell number while it increase EC cell growth (data not show). But when we lower the FCS concentration in the medium to 0.4%, we found that rhVEGF 165 increased MGC803 cell number. These effects began at day 2 after VEGF was added and the maximal stimulation was at day 3 and day 4 (Fig. 4C). rhVEGF 165 failed to increase 293 and MCF7 cell number even at lower FCS concentration medium (Figs. 4A and 4B, respectively). Based on the results above, we double-checked the expression of VEGFR-2 protein by MGC803 cells using immunocytochemistry. The results indicated that VEGFR-2 was detected on MGC803 cells (Fig. 1B-b) compared with the control (Fig. 1B-a). To confirm these unexpected findings, we examined the effect of exogenous rhVEGF 165 on DNA synthesis in
MGC803 cells using [ 3H]thymidine incorporation assay. The results showed that rhVEGF 165 increased MGC803 cell proliferation in both dose-dependent and time-dependent manners. As little as 0.5 ng/ml VEGF caused no significant increases in [ 3H]thymidine incorporation. At concentration of 2 ng/ml, VEGF could significantly increase the incorporation of [ 3H]thymidine (P ⬍ 0.01), and the effect of VEGF on MGC803 cells at this concentration made no big difference compared with its effect at concentration of 10 ng/ml (Fig. 5A). Results of the time-cause cell growth induced by VEGF indicated that the increase in [ 3H]thymidine incorporation began at 24 h and peaked at the period of 30 to 48 h after VEGF was added (P ⬍ 0.01), the effect was reduced after 72 h (P ⬍ 0.05) (Fig. 5B). To block the effect of rhVEGF 165 on MGC803 cells, rhVEGF 165 of 2 ng/ml was incubated with different concentration of either anti-VEGF 165 or anti-VEGFR-2 antibodies first, then the incubated mixtures was added to the cultured MGC803 for stimulation. The results showed that when the VEGF antibody concentration was below 0.01 g/ml, no obvious inhibition of VEGF inducing MGC803 proliferation was observed. As antibody concentration increased, the VEGF was inhibited gradually. When VEGF antibody concentration was up to 1 g/ml, the exogenous VEGF 165 was inhibited completely (Fig. 5C). Unlike VEGF antibody, the VEGFR-2 antibody showed only partly inhibiting effect on VEGF-induced MGC803 cell growth at concentration of 0.1–1.0 g/ml. No further inhibition was observed when the anti-VEGFR-2 antibody was presented at a higher concentration (Fig. 5D) DISCUSSION Recently, nonendothelial cell localization of VEGF receptors, especially on tumor cells, raises the possibility of VEGF autocrine stimulation of tumor growth (18 –21, 23). Masood et al. (27) used VEGF antisense oligonucleotides specifically blocking VEGF mRNA and inhibiting Kaposi sarcoma cell growth. For the first time, it gave direct evidences of VEGF acting as an
FIG. 3. Immunoblot analysis of VEGFR-2 protein expression by NCI-H23, NCI-H460, MGC803, MDA-MB-231, 293, MCF7 cells and EC. Total cell protein extracts were separated by 8% SDS–PAGE gel and immunoblotted by anti-VEGFR-2 antibodies. Position indicating molecular weights was according to the prestained SDS–PAGE marker.
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FIG. 4. Effects of rhVEGF 165 on proliferation of 293, MCF7, and MGC803 (A, B, and C, respectively). Quiescent cells were cultured in medium containing 0.4% FCS (}), 0.4% FCS plus 10 ng/ml rhVEGF 165 (Œ), 2% FCS (F), or 2% FCS plus 10 ng/ml rhVEGF 165 (■). Cells were counted in triplicate in a Coulter hemocytometer on the indicated day. Values represent the mean cell number from experiments in triplicate.
autocrine stimulator for tumor cell growth. Our immunohistochemistry results suggest that the expression of VEGFR-2 be not only restricted to EC but also to tumor cells in tumor tissues. Moreover, the intensity of
VEGFR-2 expression by tumor cells is sometimes as strong as that by EC. Our studies demonstrated that VEGFR-2 also expressed in vascular smooth muscle cells and other tissue cells such as macrophage in lung cancers and lymphocytes in intestinal and bladder cancer. These results suggest that VEGFR-2 might play an important role in VEGF autocrine stimulation of tumor development. Functional expression of VEGFR-2 has been demonstrated in leukemic cell (28) RT-PCR revealed that tumor cell lines MGC803, 293 and MCF7 express RNA encoding both VEGF and VEGFR-2. We further detected the expression of VEGFR-2 by these three tumor cell lines using immunoblot. The results showed that they all express VEGFR-2 protein. These results encouraged us to further investigate the responsiveness of these cell lines to VEGF. We tested the effect of exogenous added rhVEGF 165 on cells in medium with different serum concentrations. The results showed that rhVEGF 165 in 0.4% FCS medium increased MGC803 cell growth. To make sure that the primary cultured MGC803 cells were not confused with EC, we carefully identified the cells by immunocytochemistry (data not shown). We further investigated the effect of exogenous added rhVEGF 165 on MGC803 cells using [ 3H]thymidine incorporation assay. Exogenous added rhVEGF 165 could induce MGC803 cell proliferation in dose and time dependent manners. Antibodies against VEGF or against VEGFR-2 can block these effects. When excessive anti-VEGF antibodies were added to cultured cells, no further inhibition effect was observed compared with no VEGF added control. It suggests that the antibody failed to inhibit the effect of endogenous VEGF on MGC803 cell growth. There are at least two possible reasons for these phenomena: (1) the concentration of VEGF secreted by cultured MGC803 cells is lower than that in vivo. VEGF expression is known to be up-regulated by hepatocyte growth factor and other growth factors such as TGF- and PDGF in other cell types through paracrine in vivo (29). We failed to detect VEGF on MGC803 conditional medium by ELISA after the cells were incubated at different time. (2) The anti-VEGF antibody we used here was anti-VEGF 165 antibody, but tumor cells can secrete all forms of VEGF that could not be blocked by the anti-VEGF antibody. Furthermore, tumor cells could secrete many other growth factors that cause cell proliferation, thus covering the cell growth effects induced by endogenous VEGF. In their study, Boocock et al. (30) mentioned that receptor-positive primary ovarian carcinoma cells failed to respond to exogenous VEGF, although no experimental details were provided. A possible difference may be caused by to the selection of serum deprivation period to quiesce the cells before addition of VEGF. We noticed that when cells were incubated with low serum medium for a period longer than 72 h, the VEGF-
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FIG. 5. Effect of rhVEGF 165 on MGC803 proliferation using [ 3H]thymidine incorporation assay (A) Dose response of rhVEGF 165-induced increases in [ 3H]thymidine incorporation in MGC803 cells. MGC803 cells were made quiescent by incubation in 0.4% FCS RPMI 1640. The medium was replaced with 0.4% FCS RPMI 1640 containing various concentrations of rhVEGF 165 (0 –10 ng/ml) for another 35 h. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h of rhVEGF 165 treatment. Incorporated [ 3H]thymidine was measured in a liquid scintillation counter. (B) rhVEGF 165 stimulates MGC803 proliferation with time-dependent manner. MGC803 cells were seeded at a density of 2 ⫻ 10 4 per well. Cells were made quiescent as above. The medium was replaced with 0.4% FCS RPMI 1640 containing 2 ng/ml rhVEGF 165 as time point 0. [ 3H]Thymidine (0.5 Ci/ml) was added during the last 5 h at each time point as indicated. Culture medium did not change during the time-dependent experiments. No rhVEGF 165 added cells were used as control. (C and D) Both anti-VEGF 165 and anti-VEGFR-2 antibodies inhibit the rhVEGF 165 effects on MGC803 proliferation (bar n, no VEGF added; bar IgG, added preimmune mouse antibody, 1 g/ml). The rhVEGF 165 concentration was 2 ng/ml in C and D. EC was used as a positive control in A, C, and D.
induced cell growth effect was reduced. Even repeated the addition of VEGF, similar results were observed (Fig. 6). This suggests that under the conditional culture medium, VEGF-induced MGC803 cell growth could only last for a relatively short time that is not easily detected. Both VEGFR-1 and VEGFR-2 are VEGF-specific receptors. The distribution of these two receptors is different in various cell types, and the expression of the receptors is modulated in a dynamic manner during development. The different distribution of VEGF receptors determines the specificity of their biological functions in tumor development. VEGF can stimulate monocyte chemotaxis and migration through VEGFR-1, but not VEGFR-2 (19). Recently, new VEGF receptors such as neuropilin-1, neuropilin-2 etc. were discovered (31). In our study, we also observed that the anti-VEGFR-2 antibody partly blocks the VEGF-
induced MGC803 cell growth. We also detected the expression of VEGFR-1 on MGC803 cells by RT-PCR under different reaction conditions, but no specific PCR products were obtained (data not shown). This suggests that MGC803 cell growth caused by VEGF is mainly mediated via VEGFR-2. It may also be caused by other functional VEGF receptors besides VEGFR-2. To our knowledge, this is the first report to show that the gastric carcinoma cell line MGC803 expresses VEGF and VEGFR-2 and proliferates strongly in response to VEGF directly. Furthermore, we successfully used anti-VEGF and anti-VEGFR-2 antibodies to block the VEGF-induced tumor cell growth in vitro. These results give direct evidence that the angiogenic factor VEGF can also acts as an autocrine growth factor for gastric carcinoma. Over expression of VEGF by tumor cells could thus not only indirectly facilitate their growth and invasion, via its effects on angiogenesis,
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FIG. 6. Effects of repeated addition of rhVEGF 165 on MGC803 cell proliferation.. Two separated parts of MGC803 cells were made quiescent by incubation in 0.4%FCS RPMI 1640. The medium was replaced with 0.4% FCS RPMI 1640 containing 2 ng/ml rhVEGF 165 as time point 0. One part was incubated for 36 h, [ 3H]thymidine (0.5 Ci/ml) was added during the last 5 h. Collecting the cells and measure the [ 3H]thymidine incorporation. For the other part, adding the same concentration of rhVEGF 165 but did not change the culture medium. After incubating for another 36 h, adding [ 3H]thymidine and measure its incorporation. The results indicated that, compared with control, rhVEGF 165-induced MGC803 cell growth reduced (at 36 h, P ⬍ 0.01, at 72 h, P ⬎ 0.05).
but also directly promote tumor cell proliferation. Identification of VEGF as an autocrine growth factor expands our knowledge of cancer biology and contributes more to tumor biotherapy.
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