Anti-angiogenic effects of somatostatin receptor subtype 2 on human pancreatic cancer xenografts
Manoj Kumar1,
Zheng-Ren Liu1,
Laxmi Thapa2 and
Ren-Yi Qin1,3
1 Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China and 2 Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
3 To whom correspondence should be addressed Email: ryqin{at}tjh.tjmu.edu.cn
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Abstract
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Somatostatin receptor subtypes, especially subtype 2 (SSTR2), exert their antitumor (cytostatic and/or cytotoxic) and anti-angiogenic effects. Here we aimed to investigate the anti-angiogenic effect of SSTR2 gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect. The full-length human SSTR2 complementary DNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection, and stable expression of SSTR2 was detected by immunohistochemistry and RTPCR. Athymic mice were separately xenografted with SSTR2-expressing cells (experimental group), vector control and mock control cells. Intratumoral microvessel density (MVD) was assessed by immunohistochemistry. Immunohistochemistry and RTPCR were used to determine the expression of angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase (MMP)-2 in xenograft tumors. MVD was significantly lower in the experimental group (5.16 ± 1.34) than that in the vector control (16.52 ± 2.25) and mock control (15.32 ± 2.53) (P < 0.05). The immunohistochemical assay showed a significant decrease in the expression of VEGF, bFGF and MMP-2 protein in the experimental group compared with the vector control and mock control, considering both the integral optical density and area of staining (P < 0.05). RTPCR showed a significant reduction of VEGF, bFGF and MMP-2 mRNA expression in the experimental group compared with the vector control and mock control (P < 0.05). Thus, introduction of the SSTR2 gene, the expression of which is frequently lost in human pancreatic adenocarcinoma, exerts its anti-angiogenic effects by down-regulating the expression of the factors, which are involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a promising strategy of gene therapy for pancreatic cancer.
Abbreviations: bFGF, basic fibroblast growth factor; cDNA, complementary DNA; DMEM, Dulbecco's modified Eagle's medium; EC, endothelial cell; ECM, extracellular matrix; FBS, fetal bovine serum; MMP-2, matrix metalloproteinase-2; MVD, microvesel density; MT1-MMP, membrane type 1-matrix metalloproteinase; MT2-MMP, membrane type 2-matrix metalloproteinase; SABC, streptavidinbiotin complex; SS, somatostatin; SSTR2, somatostatin receptor subtype 2; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor
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Introduction
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The anti-proliferative action of somatostatin (SS)/analogs is signaled by specific G-protein coupled receptors, and up to date, six different subtypes (SSTR-1, -2A, -2B, -3, -4 and -5) have been cloned and functionally characterized in various cell systems, including pancreas, adrenal cortex and brain tissue (13). Studies have demonstrated that somatostatin receptor subtypes (SSTRs) are strongly expressed in the normal pancreas, and even in the tissues adjacent to pancreatic cancer (4), whereas not only a desensitization or mutation of these receptors occurs in pancreatic tumors (5), but also expression of these receptors, especially SSTR2, is frequently lost in human pancreatic adenocarcinomas (48). Despite remarkable biochemical properties of SS analogs in vitro, poor therapeutic results with them in phase I/II clinical trials against the majority of cases of pancreatic cancers were due to the loss of SSTR2 gene expression in pancreatic cancers (4,810) and relatively low expressions of SSTR3 and SSTR5 (11).
Vascular endothelial growth factor (VEGF), an endothelial cell (EC)-specific mitogen and also known as a vascular permeability factor, is highly expressed in various types of tumors (12). Studies have found positive correlations between tumor cell VEGF expression, blood vessel density, tumor growth and metastasis, disease progression and poor prognosis of pancreatic carcinomas (1315), and suppression of VEGF expression attenuated pancreatic cancer cell tumorigenicity (16), suggesting that over-expression of VEGF might be associated with the aggressive phenotype of this disease and that VEGF should be an important target for anticancer and anti-angiogenic therapy.
Basic fibroblast growth factor (bFGF) and its receptor are over-expressed in various types of cancers, including human pancreatic carcinoma, and have mitogenic and angiogenic activity both in vitro and in vivo (17,18). Synergistic action between bFGF and VEGF in induction of neovascularization has been demonstrated (1921). FGFs have been shown to be responsible for production of extracellular matrix (ECM) and release of matrix metalloproteinases (MMPs) for selective degradation and organization of ECM (22). MMP-2 has been reported as the most commonly expressed MMPs in pancreatic tumor but not in normal pancreas, and is correlated with the aggressive phenotype (invasive and metastatic potential) of pancreatic carcinomas (2326). The expression of these angiogenic factors VEGF, bFGF and MMP-2 have been shown to be markedly associated with an increase in the microvessel density (MVD), and in addition, tumor hypervascularity is associated with shorter median survival time, suggesting these as prognostic factors of various tumor types, including pancreatic cancer (15,2729). Taken together, there are complex correlating regulations among these three angiogenic factors.
SS/analogs or SSTR2-selective agonists exhibit anti-secretory effects by inhibiting the release of growth factors and trophic hormones, e.g. growth hormone, insulin-like growth factor-1, insulin, gastrin (5,3032). As pancreatic cancer is characterized by over-expression of several angiogenic factors, such as VEGF, bFGF, MMP-2, etc., it is necessary to elucidate the effects of the genes or molecules with anti-angiogenic properties on pancreatic cancer. Previous studies have reported the anti-angiogenic properties of SS/analog and SSTRs in various tumors (33,34); however, the mechanisms involved in the anti-angiogenic effects of SSTR2 have been poorly elucidated in this pancreatic cancer cell line PC-3 as yet. Hence, in the present study, we evaluated the potency of introduction of exogenous SSTR2 gene, a tumor suppressor (3), as negative regulators of VEGF, bFGF and MMP-2 production in pancreatic cancer cell line PC-3.
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Materials and methods
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Materials
PC-3, a human pancreatic cancer cell line, was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), lipofectamine and geneticin (G418) were purchaesd from Gibco BRL, USA. The full-lenth complementary DNA (cDNA) of human SSTR2 was kindly provided by G.I.Bell (Howard Hughes Medical Institute, Chicago, IL). Mouse anti-SSTR2, anti-VEGF, anti-bFGF, anti-MMP-2 and anti-CD34 monoclonal antibodies, and streptavidinbiotin complex (SABC) kit and streptavidin peroxidase (SP) kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). TRIZOL® reagent, RNasin, oligo(dt) 15, dNTPs, M-MLV reverse transcriptase, Taq DNA polymerase were purchased from Promega, USA. Eukaryotic expression vector pcDNA3.1 was purchased from Invitrogen (Invitrogen, San Diego, CA).
Plasmid construction and gene transfer
Human SSTR2 cDNA was digested with EcoRI/XbaI and cloned in the EcoRI/XbaI site of pcDNA3.1 following the manufacturer's instructions. Briefly, a tube containing 3 µl of the plasmid and 100 µl of competent Escherichia coli was placed on ice for 45 min and then immersed in a 42°C water bath for 90 s without agitation. After transfer of 800 µl of LB broth, the tube was shaken at 150 r/min for 1 h at 37°C, followed by spreading 200 µl of the suspension onto each LB plate containing ampicillin and incubation at 37°C for 16 h. After formation of bacterial colonies, the colonies were picked from the plates and incubated with 5 ml of LB medium containing ampicillin for 16 h. For the extraction of plasmid, 1.5 ml of the bacteria suspension (in an Eppendorf tube) was centrifuged at 12000 r.p.m. for 1 min, then treated with Solution I (50 mmol/l glucose, 25 mmol/l TrisCl pH 8.0, 10 mmol/EDTA), Solution II (0.2 N NaOH/1% SDS) and Solution III (mixture of 5 mol/l potassium acetate, glacial acetic acid and H2O in the ratio of 6:1.15:2.85), respectively, and centrifuged at 12 000 r.p.m for 10 min. The supernatant was treated with phenol:chloroform (1:1) and centrifuged at 12000 r.p.m. for 10 min at 0°C, then placed at 20°C by adding 2 vol of alcohol for 1 h, followed by centrifugation at 12 000 r.p.m. for 10 min, removal of supernatant and drying at room temperature. Then 20 µl of RNase (100 µg/ml) was added to each tube and incubated at 65°C for 30 min. DNA thus obtained was electrophoresed on 1% agarose gel. Recombinant plasmid was purified by QIA prep spin miniprep kit (QIAGEN).
PC-3 cells were routinely cultured in DMEM media supplemented with 10% heat-inactivated FBS, 100 µg/ml penicillin and 100 µg/ml streptomycin, and incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. Gene transfer was performed according to the manufacturer's protocols. Briefly,
3x105 cells/well containing 2 ml appropriate complete growth medium were seeded in a 6-well culture plate, and incubated at 37°C in a 5% CO2 incubator until the cells were 7080% confluent. A cover slip was plated in each well before seeding. After the cells were ringed with serum-free and antibiotics-free medium, the cells were transfected separately with pcDNA3.1-SSTR2 1 µg/lipofectamine 3 µl (experimental group), pcDNA3.1 1 µg/lipofectamine 3 µl (vector control) and only lipofectamine 3 µl (mock control), followed by incubation at 37°C in a 5% CO2 incubator for 6 h. Then the medium was replaced by DMEM culture medium containing 20% FBS. After 48 h, two wells in each group were taken out to detect the transient expression of SSTR2 by immunohistochemical SABC methods, whereas others were continuously cultured for stable expression of SSTR2. G418 (500 mg/l) was added to select the resistant clones after 48 h. Six days later, when most of the cells died, the concentration of G418 was decreased to 300 mg/l and cells were cultured for another 6 days. The medium was changed every 3 or 4 days, and mixed population of G418 resistant cells were collected
2 weeks later for the examination of stable expression of SSTR2 by immunohistochemical SABC methods and RTPCR assay.
Confirmation of SSTR2 protein expression by immunohistochemical staining
The stable expression of SSTR2 in the experimental group cells was detected by using immunohistochemical SABC methods. The cover slips with attached cells were dried at room temperature and washed twice with phosphate-buffered saline (PBS) solution (pH 7.2), followed by treatment with 3% H2O2 for 10 min at room temperature. Then the cover slips were incubated with 5% bovine serum albumin in PBS solution for 20 min to block the non-specific antibody binding. The cover slips were then incubated with mouse anti-human SSTR2 antibody (diluted to 1:50 in 0.5% bovine serum albumin in PBS) for 12 h at 4°C. The bridging antibody (biotinylated goat anti-mouse IgG) and SABC complex were diluted to 1:100 and incubated with the specimens for 20 min at 30°C. Finally, 3,3'-diaminobenzidine tetrachloride (DAB) was used for color development and the cover slips were counterstained with hematoxylin. The primary antibody was replaced by non-immune serum or PBS for the negative control. The brown yellow staining of SSTR2 was observed in the cell membrane and cytoplasm (300x magnification).
Confirmation of SSTR2 mRNA expression by RTPCR
Total RNA was extracted separately from PC-3 cells of each group with TRIZOL® reagent following the manufacturer's instructions. A 2-µg (treated in 5 µl DEPC water) sample of total RNA was denaturalized by incubating at 70°C for 5 min, and the tube was placed on ice for 3 min, and then reverse-transcribed into cDNA by using M-MLV. Briefly, the denaturalized RNA (5 µl) was incubated for 60 min at 37°C and for 5 min at 95°C with 4 µl 5x reverse transcriptase buffer, 1 µl oligo(dt)15, 1 µl RNasin (50 u/µl), 1 µl dNTPs (10 mmol/l), 1 µl reverse transcriptase (200 u/µl), and 7 µl DEPC water in a total volume of 20 µl. For polymerase chain reaction (PCR), 5 µl of the resulting cDNA, 31 µl of tripled-distilled H2O, 5 µl of 10x PCR buffers, 3 µl of MgCl2 (25 mmol/l), 1 µl of dNTPs, 1 µl of each of sense and antisense primers (10 pmol/l), 1 µl of each of sense and antisense ß-actin, and 1 µl Taq DNA polymerase (3 u/µl) in a total volume of 50 µl were added. The samples were amplified through 35 cycles, each amplification cycle consisting of denaturation at 94°C for 40 s, primers annealing at 55°C for 40 s and extension at 72°C for 1 min. Cycles were preceded by incubation at 94°C for 5 min to ensure full denaturation of the target gene, followed by an extra incubation at 72°C for 10 min to ensure full extension of the products. PCR products were analyzed on 1.5% agarose gel containing ethidium bromide. The sequences of the primers for SSTR2 were 5'-CCC CAG CCC TTA AAG GCA TGT-3' (sense) and 5'-GGT CTC CAT TGA GGA GGG TCC-3' (antisense), and for ß-actin were 5'-GTG CGT GAC ATT AAG GAG-3' (sense) and 5'-CTA AGT CAT AGT CCG CCT-3' (antisense).
Animal models
Exponentially growing cells (3 x 106 cells in 0.3 ml DMEM/mouse) were inoculated subcutaneously into the right flank of 5-week-old athymic male nude mice (BALB/c nu/nu; obtained from the Animal Center of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China) weighing
1820 g. Mice were bred and maintained under pathogen-free conditions. Each group consisted of five mice. After 8 weeks of the inoculation, mice were killed and tumors were excised. A part of each tumor was embedded in paraffin, and the remaining part was preserved at 80°C until use. All of the animal experiments were carried out in accordance with institutional guidelines for animal care.
Detection of SSTR2 mRNA expression in xenografted tumors by RTPCR
Tumor tissue from each xenograft was homogenized with polytron tissue homogenizer and total RNA was extracted with TRIZOL® reagent by following the manufacturer's instructions. The RTPCR was carried out as described above.
Detection of VEGF, bFGF, MMP-2 and MVD by immunohistochemistry
Consecutive 4 µm paraffin-embedded tissue sections were subjected to immunostaining according to the SP methods. Briefly, the tissue sections were deparaffinized in xylene at 37°C for 20 min. Endogenous peroxide was blocked by incubating the slides with 3% hydrogen peroxide (H2O2) for 10 min at 37°C, then it was washed thoroughly with distilled water three times (2 min each time), the slides were then heated in the jar containing antigen retrieval solution (0.01 M citrate buffer, pH 6.0) in oven at 9298°C for 15 min for the retrieval of the antigens and cooled to room temperature. After being washed with phosphate-buffered-saline (PBS, 0.01 M, pH 7.4) for 5 min, the sections were further blocked by goat serum for 20 min at 37°C to reduce non-specific antibody binding, and then incubated with primary antibody of VEGF, or bFGF, or MMP-2 (each at 1:50 dilution) at 4°C overnight. After being washed three times (3 min each time) with PBS, the sections were incubated with the biotin-labeled goat anti-mouse immunoglobulin (IgG) at 37°C for 30 min, washed again with PBS, followed by an incubation with streptavidin peroxidase complex for 30 min at 37°C. Staining was visualized with DAB for 10 min at room temperature. Finally, the sections were counterstained for nuclei by hematoxylin solution. For immunostaining of MVD, CD34 monoclonal antibody (diluted at 1:50) was used as the primary antibody. For the microvessel counting, positive stainings for MVD, in five most highly vascularized areas (hot spots) in each slide, were counted in 200x fields and MVD was expressed as the average of the microvessel count in these areas (35). Any EC or endothelial cluster positive for CD34 (brown yellow staining) was considered to be a single countable microvessel. The primary antibodies were replaced by non-immune serum or PBS for the negative controls.
Detection of VEGF, bFGF and MMP-2 mRNA expression by RTPCR assay
Total RNA was extracted separately from the tumors of each group with TRIZOL® reagent following the manufacturer's instructions. RTPCR was carried out as described above with some changes in conditions of amplification cycles. For VEGF mRNA expression, samples of each group were subjected to PCR at an annealing temperature from 60 to 50°C decreasing by 0.5°C per cycle for 20 cycles, followed by an additional 15 cycles at an annealing temperature of 50°C for 35 s. The sequences of primers for VEGF were 5'-TTG CTG CTC TAC CTC CAC-3' (sense) and 5'-CTC CAG GCC CTC GTC ATT-3' (antisense), and for ß-actin were 5'-GTG CGT GAC ATT AAG GAG-3' (sense) and 5'-CTA AGT CAT AGT CCG CCT-3' (antisense). For mRNA expression of bFGF, samples of each group were subjected to PCR for 30 cycles, each cycle consisting of denaturation at 94°C for 2 min, primer annealing at 58°C for 30 s and extension at 72°C for 1 min. The sequences of primers for bFGF (36) were 5'-GGC TTC TTC CTG CGC ATC CA-3' (sense) and 5'-GCT CTT AGC AGA CAT TGG AAG A-3' (antisense), and for ß-actin were 5'-GTG CGT GAC ATT AAG GAG-3' (sense) and 5'-CTA AGT CAT AGT CCG CCT-3' (antisense). For mRNA expression of MMP-2, samples of each group were subjected to PCR for 33 cycles, each cycle consisting of denaturation at 94°C for 1 min, primer annealing at 55°C for 35 s and extension at 72°C for 1 min. The sequences of primers for MMP-2 were 5'-GCG GAT CCA GCG CCC AGA GAG ACA C-3' (sense) and 5'-TTA AGC TTC CAC TCC GGG CAG GAT T-3' (antisense), and for ß-actin were 5'-CCT TCC TGG GCA TGG AGT CCT G-3' (sense) and 5'-GGA GCA ATG ATC TTG ATC TTC-3' (antisense). The RTPCR for these three angoigenic factors was repeated twice with the samples of each group. Each PCR product was analyzed on 1.5% agarose gel containing ethidium bromide, and quantified by a complete gel documentation and analysis system. The mRNA expression levels of VEGF, bFGF and MMP-2 were determined by the ratio of VEGF/ß-actin, bFGF/ß-actin and MMP-2/ß-actin, respectively.
Image analysis
High-resolution pathological image analysis system-1000 (HPIAS-1000) was used for the quantitative analysis of expressions of VEGF, bFGF and MMP-2 protein. A total of 25 microscopic fields (5 fields/slide) were selected randomly from each group under 10 x 40 magnification, and both the area of staining and integral optical density of each vision were automatically measured by the computer.
Statistical analysis
Results were expressed as mean ± SD. The mean values were compared among the three groups by using the ANOVA (SNK, StudentNewmanKeuls test) in the SAS 8.1 software. A value of P < 0.05 was considered statistically significant.
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Results
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Up-regulation of SSTR2 after transfection
After 48 h of the in vitro transfection, most of the cells in the experimental group demonstrated positive staining for SSTR2 in the cell membrane and cytoplasm, whereas almost no positive staining for SSTR2 was detected in the cells of vector control and mock control as detected by immunohistochemical SABC methods. The cells in the experimental group were continuously cultured by adding G418 (500 mg/l) with 20% FBS, and then we were able to select a mixed population of PC-3 cells resistant to the toxic effects of G418. After 2 weeks, almost all of the cells expressed SSTR2 (Figure 1). RTPCR analysis of total RNA extracted from the cells and xenografted tumors of each group showed SSTR2 mRNA expression in the experimental group, but not in the vector control and mock control (Figure 2A and B). The results suggested that the exogenous SSTR2 gene was successfully expressed in PC-3 cell line devoid of SSTR2.

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Fig. 1. Immunohistochemical staining of SSTR2 expression after stable transfection. The brown yellow stainings in cell membrane and cytoplasm represent SSTR2 expression. (A) Experimental group; (B) vector control; (C) mock control (300x magnification). See online Supplementary material for a color version of this figure.
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Fig. 2. RTPCR analysis of SSTR2 mRNA expression after transfection. (A) RTPCR of total RNA extracted from PC-3 cells of each group. (B) RTPCR of total RNA extracted from xenografted tumors. Transfection with SSTR2 gene caused up-regulation of SSTR2 mRNA transcription. Lane M, DNA marker DL 2000; lane 1, experimental group; lane 2, vector control; lane 3, mock control.
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Effect of SSTR2 on VEGF mRNA, bFGF mRNA and MMP-2 mRNA expression
RTPCR analysis showed that the expression of VEGF mRNA was significantly decreased in the experimental group (0.1968 ± 0.037) compared with the vector control (0.9242 ± 0.103) and mock control (0.9505 ± 0.132) (P < 0.05, Figure 3A and D). Similarly, the expression of bFGF mRNA was markedly reduced in the experimental group (0.1438 ± 0.029) compared with the vector control (0.7452 ± 0.088) and mock control (0.7351 ± 0.043) (P < 0.05, Figure 3B and D). The expression of MMP-2 mRNA was also obviously down-regulated in the experimental group (0.3363 ± 0.065) as compared with the vector control (1.132 ± 0.115) and mock control (1.120 ± 0.12) (P < 0.05, Figure 3C and D). But no statistical differences either in VEGF mRNA or bFGF mRNA or MMP-2 mRNA expression between the vector control and mock control were observed. These results suggested that the up-regulation of the SSTR2 gene could suppress the expression of VEGF, bFGF and MMP-2 at mRNA level in vivo.

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Fig. 3. Anti-angiogenic effect of SSTR2 transfer is associated with in vivo down-regulation of (A) VEGF, (B) bFGF and (C) MMP-2 mRNA expressions as assessed by RTPCR. Summary of quantitative analysis of these factors are shown in (D). The quantitative data are shown as mean ± SD. Lane M, DNA marker DL 2000; lane 1, experimental group; lane 2, vector control; lane 3, mock control.
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Down-regulation of VEGF, bFGF and MMP-2 protein expression by SSTR2
Positive staining of VEGF, bFGF and MMP-2 was located in the cytoplasm and membrane of the tumor cells (Figure 4). The immunohistochemical staining showed a significant decrease in the expression of VEGF, bFGF and MMP-2 protein in the experimental group compared with the vector control and mock control (P < 0.05). According to HPIAS-1000 and statistical analysis, both the area of staining and integral optical density of VEGF staining were significantly reduced in the experimental group (103.88 ± 16.61 µm2 and 72.69 ± 8.53, respectively) compared with the vector control (193.93 ± 9.20 µm2 and 126.02 ± 12.59, respectively) and mock control (187.58 ± 11.60 µm2 and 122.51 ± 13.20, respectively) (P < 0.05, Figure 4C).

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Fig. 4. SSTR2 up-regulation is associated with in vivo down-regulation of VEGF, bFGF and MMP-2 protein expressions and decrease in MVD as assessed by immunohistochemistry. (A, D, G and J) represent the experimental group and (B, E, H and K) represent the mock control for VEGF, bFGF, MMP-2 and MVD stainings, respectively. The data of quantitative analysis are mean ± SD of 25 microscopic fields (5 fields/tumor sample) in each group at 300x magnification for (C) VEGF, (F) bFGF and (I) MMP-2, and at 200x magnification for (L) MVD. See online Supplementary material for a color version of this figure.
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Similarly, both the area of staining and integral optical density of bFGF staining were markedly reduced in the experimental group (71.75 ± 8.51 µm2 and 48.69 ± 8.24, respectively) compared with the vector control (103.47 ± 12.33 µm2 and 90.57 ± 9.27, respectively) and mock control (110.28 ± 13.54 µm2 and 94.81 ± 11, respectively) (P < 0.05, Figure 4F).
Both the area of staining and integral optical density of MMP-2 staining were obviously decreased in the experimental group (96.86 ± 10.06 µm2 and 81.49 ± 10.83, respectively) compared with the vector control (179.39 ± 15.86 µm2 and 144.25 ± 10.22, respectively) and mock control (175.38 ± 13.67 µm2 and 148.22 ± 12.29, respectively) (P < 0.05, Figure 4I). However, no significant differences in the expression of these three factors were observed between the vector control and mock control.
Lower MVD in the tumors with SSTR2 gene up-regulation
The MVD was significantly lower in the tumors of experimental group (5.16 ± 1.34) as compared with the vector control (16.52 ± 2.25) and mock control (15.32 ± 2.53) (P < 0.05) (Figure 4L). However, no significant difference was observed between the vector control and mock control.
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Discussion
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The properties of tumor cells to release and induce several angiogenic and anti-angiogenic factors, which play a crucial role in regulating EC proliferation, migration, apoptosis or survival, cellcell and cellmatrix adhesion through different intracellular signalings, have been thought to be the essential mechanisms during tumor-induced angiogenesis (17). Transfer of anti-oncogene or molecules with anti-oncogenic properties constitutes one of the new and promising therapeutic approaches to cancer. SSTR2 could act as an antioncogene in human pancreatic cancer cells, showing its anti-proliferative and anti-metastatic effects (3,7). To our knowledge, whether the aberrant expression of SSTR2 is associated with pancreatic tumor angiogenesis, has not been reported as yet. But several previous investigations showed that SS/analogs as well as SSTRs exhibited their antitumor effects through different pathways (5,3032). It was demonstrated, in chick chorioallantoic membrane model, that unlabeled SS analogs inhibited angiogenesis, which was proportional to the ability of the analog to inhibit growth hormone production (11). Mentlein et al. (30) reported that VEGF produced by cultured glioma cell lines constantly over-expressing SSTRs, especially SSTR2, was reduced to 2580% by co-incubation with SS or SSTR2-selective agonists (octreotide and L-054 522) in a dose-dependent manner. Interestingly, transfer of the SSTR2 gene was found to restore the responsiveness of SSTR2-negative cells to SS analogs, and inhibited the tumorigenicity of pancreatic tumor cells in vitro without administration of exogenous SSTR2 ligands (7). Similar to these, in our study, the exogenous human SSTR2 gene was successfully up-regulated in pancreatic cancer cell line PC-3 by lipofectamine-mediated stable transfection.
The most commonly found angiogenic growth factors such as VEGF could contribute to the progression of various solid tumors by promoting the angiogenic switch (37). Since VEGF, via binding to its high affinity receptors (Flt-1/VEGFR-1, Flk-1/KDR/VEGFR-2) on EC, promotes angiogenic switch, resulting in tumor neovascularization (16,17), the VEGFVEGFR system has been considered as a promising target for the development of anti-angiogenic tumor therapy (3842). In our study, we observed a marked decrease in expression of VEGF in the tumors of the experimental group (tumors with SSTR2 up-regulation) compared with the controls. Hipkin et al. (43) reported that the inhibitory effect of the SSTR2 gene was not permanent and stable because of its down-regulation or desensitization by long-term exposure to SS. However, in our experiment, we observed a significant inhibitory effect of SSTR2 on VEGF expression both at protein and mRNA levels in vivo until 2 months post-transfection. All of these anti-secretory events evoked by SSTR2 expression in PC-3 cells may explain the anti-angiogenic effect observed in vivo.
Our results showed that the expression of bFGF protein and mRNA was significantly down-regulated in the experimental group compared with the controls, suggesting that the up-regulation of SSTR2 gene in cell line PC-3 has a strong anti-angiogenic and anti-secretory action. Some other studies demonstrated that SS 14 and SMS 201-995 were able to decrease bFGF-induced corneal angiogenesis and growth-promoting effect of bFGF, respectively (31,32).
Up-regulation of MMPs activity favour proteolytic degradation of the basement membrane and ECM, thereby releasing angiogenic mitogens stored within the matrix, and has been linked to tumor growth and metastasis, as well as tumor-associated angiogenesis (17). Due to the high level expression of MMP-2 in clinical and experimental models of pancreatic cancer, inhibition of MMP-2 has shown great promise with synthetic inhibitors as antitumor agents (anti-angiogenesis, anti-proliferative and anti-metastasis) in pre-clinical models (44). In the present study, we observed that MMP-2 was obviously down-regulated both at protein and mRNA levels in vivo in the experimental group compared with the controls, suggesting that the up-regulation of SSTR2 in pancreatic cancer cell line PC-3 could decrease the aggressive phenotype of pancreatic carcinoma. A previous study reported that administration of the SS analog, octreotide, was found to inhibit the migration and invasion of gastric cancer cells in vitro and the metastasis of cancer in vivo via down-regulation of MMP-2 and VEGF expression, thereby decreasing tumor angiogenesis (34). In addition, we observed a significant decrease in MVD in the experimental group tumors compared with the controls. A recent study demonstrated that the 111In-pentetreotide, an SSTR2-preferring SS analog, treatment partially blocked initiation of the angiogenic response but significantly decreased the growth of neovessels after initiation (45).
In conclusion, our present study demonstrates that the up-regulation of the SSTR2 gene can inhibit angiogenesis in pancreatic carcinoma by decreasing endogenous levels of angiogenic factors VEGF, bFGF and MMP-2, suggesting that introduction of exogenous SSTR2 in pancreatic cancer cell line PC-3 may offer an avenue for anti-angiogenic therapy.
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Supplementary material
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Supplementary material can be found at: http://www.carcin.oupjournals.org/
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Acknowledgments
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The authors thank Dr G.I.Bell (Howard Hughes Medical Institute, The University of Chicago) for providing cDNA of human SSTR2. This research was supported by National Natural Science Foundation of China, No. 30271473.
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Received February 26, 2004;
revised May 19, 2004;
accepted June 10, 2004.