A novel peptide (GX1) homing to gastric cancer vasculature inhibits angiogenesis and cooperates with TNF alpha in anti-tumor therapy
- Bei Chen†1,
- Shanshan Cao†1,
- Yingqi Zhang2,
- Xin Wang1,
- Jie Liu1,
- Xiaoli Hui1,
- Yi Wan2,
- Wenqi Du1,
- Li Wang1,
- Kaichun Wu1Email author and
- Daiming Fan1
© Chen et al; licensee BioMed Central Ltd. 2009
Received: 4 March 2009
Accepted: 9 September 2009
Published: 9 September 2009
The discovery of the importance of angiogenesis in tumor growth has emphasized the need to find specific vascular targets for tumor-targeted therapies. Previously, using phage display technology, we identified the peptide GX1 as having the ability to target the gastric cancer vasculature. The present study investigated the bioactivities of GX1, as well as its potential ability to cooperate with recombinant mutant human tumor necrosis factor alpha (rmhTNFα), in gastric cancer therapy.
Tetrazolium salt (MTT) assay showed that GX1 could inhibit cell proliferation of both human umbilical vein endothelial cells (HUVEC) (44%) and HUVEC with tumor endothelium characteristics, generated by culturing in tumor-conditioned medium (co-HUVEC) (62%). Flow-cytometry (FCM) and western blot assays showed that GX1 increased the rate of apoptosis from 11% to 31% (p < 0.01) by up-regulating caspase 3 expression level. A chorioallantoic membrane assay indicated that GX1 could suppress neovascularization in vivo, with the microvessel count decreasing from 21 to 11 (p < 0.05). When GX1 was fused to rmhTNFα, GX1-rmhTNFα selectively concentrated in the gastric cancer vasculature, as shown by enzyme-linked immunosorbent assay, immunofluorescence and emission-computed tomography. In vitro MTT and FCM assays showed that, compared to rmhTNFα alone, GX1-rmhTNFα was more effective at suppressing co-HUVEC proliferation (45% vs. 61%, p < 0.05) and inducing apoptosis (11% vs. 23%, p < 0.05). In a tumor formation test, GX1-rmhTNFα more effectively inhibited tumor growth than rmhTNFα (tumor volume: 271 mm3 vs. 134 mm3, p < 0.05), with less systemic toxicity as measured by body weight (20.57 g vs. 19.30 g, p < 0.05). These therapeutic effects may be mediated by selectively enhanced tumor vascular permeability, as indicated by Evan's blue assay.
GX1 had both homing activity and the ability to inhibit vascular endothelial cell proliferation in vitro and neovascularization in vivo. Furthermore, when GX1 was conjugated to rmhTNFα, the fusion protein was selectively delivered to targeted tumor sites, significantly improving the anti-tumor activity of rmhTNFα and decreasing systemic toxicity. These results demonstrate the potential of GX1 as a homing peptide in vascular targeted therapy for gastric cancer.
Ever since the essential role of angiogenesis in tumor formation and metastasis was proposed by Folkman in 1971, increasing attention has been paid to vascular targeted therapy [1–3]. The vasculature is an attractive target because vascular endothelial cells are more genetically stable than tumor parenchymal cells and less likely to acquire drug resistance, and vascular targets on endothelial cells are readily accessible to systemically delivered agents [4–6]. Based on these advantages, efforts have focused on identifying specific molecules expressed on the surface of tumor vascular endothelial and perivascular cells [7, 8]. Finding such tumor vascular targets may help make anticancer drugs more selective, through their targeted delivery, thus providing higher therapeutic efficiency while simultaneously decreasing systemic toxicity.
With this goal, we previously used in vivo screening of a phage-displayed peptide library to identify a cyclic 7-mer peptide, CGNSNPKSC, called GX1, which binds specifically to the human gastric cancer vasculature . Immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence confirmed the targeting activity of GX1 peptide, indicating that GX1 might be used as a novel vascular marker for human gastric cancer . The potential bioactivities that might accompany the targeting function of GX1, and how it might be combined with other agents for antitumor therapy, are investigated here. We conducted a series of tests to determine the effects of GX1 on vascular endothelial cells, and on tumor angiogenesis and growth. In addition, we fused GX1 to recombinant mutant human tumor necrosis factor (rmhTNFα), a variant of the TNFα cytokine that is well known for its potent antitumor activity and is less toxic than TNFα [11, 12], to see if the fusion protein could achieve synergistic therapeutic efficacy. These studies provide important preclinical evidence for the use of GX1 in targeted antitumor therapy.
GX1 inhibits endothelial cell proliferation in vitro by inducing apoptosis
GX1 inhibits angiogenesis in vivo by a chorioallantoic membrane (CAM) assay
Since GX1 could repress vascular endothelial cell proliferation in vitro, we carried out CAM assays to see if the peptide could inhibit angiogenesis in vivo. Disruption of angiogenesis was observed in GX1-treated chicken embryos, with attenuated and tortuous microvessels in the CAM and fewer angiogenic vessels contacting the disk, when compared to the PBS control group. No significant differences existed between the Pep 2 and PBS control groups, with both showing well-developed and leaf vein-like vascular nets (Figure 2).
GX1 conjugated to rmhTNFα concentrates in gastric cancer
GX1-rmhTNFα inhibits co-HUVEC proliferation in vitro by inducing apoptosis
To address whether the decrease in cell number was due to apoptosis induced by GX1-rmhTNFα, FCM was used to determine the apoptosis rate in co-HUVEC. The results showed that the apoptosis rates induced by 10 μ M GX1-rmhTNFα or rmhTNFα were 23.4% and 11.2% respectively (p < 0.01) (Figure 5B). In contrast, no significant differences in cell cycle distribution were detected between the test and control groups. These results indicated that inhibition of vascular endothelial cells by GX1-rmhTNFα might be partly caused by induction of apoptosis.
Effects of GX1-rmhTNFα on tumor growth in vivo
Since TNFα is known to alter vascular barrier function, we performed Evan's blue assay to assess the effect of GX1-rmhTNFα on tumor perfusion. Compared to rmhTNFα-treated mice, the GX1-rmhTNFα treated group showed a greater leakage of Evan's blue dye in the tumor parenchyma. Differences were statistically significant at 0.5 mg/kg (0.113 vs. 0.073, p < 0.05) (Figure 6C). We therefore hypothesize that the GX1-rmhTNFα fusion protein selectively increases tumor vascular permeability and leads to higher local drug levels, which may play an important part in the antitumor mechanism of GX1-rmhTNFα.
To improve therapeutic indices and decrease systemic toxicity, more specific and selective anticancer agents that can discriminate between tumor and nonmalignant cells are urgently needed, along with the development of antitumor radiochemotherapy [16–18]. The discovery that angiogenesis plays a crucial role in tumor formation, and that vascular targeting approaches exhibit the advantages of easy accessibility and lower incidence of drug resistance, provides a possible path to creating these new anticancer agents [19, 20]. Several studies have pursued this strategy, including the application of phage display technology to pan for peptides that bind specifically to defined tissue targets [8, 21]. Using this technique, several homing peptides have been identified, including RGD, NGR and F3, and many have showed promising results for imaging diagnosis and treatment of various tumors in preclinical or clinical investigations [22–25]. Furthermore, some of these peptides have been conjugated to bioactive agents, including drugs, cytokines, procoagulant factors, photosensitizers and radionuclides, and have been included in antineoplastic therapies. Initial results of these studies showed more selective and targeted drug delivery and fewer side effects [7, 21, 26, 27]. However, to date, no such peptide has been identified that targets human gastric cancer.
Previously, we used in vivo screening of a phage displayed peptide library to identify GX1, a cyclic 7-mer peptide CGNSNPKSC that binds specifically to the human gastric cancer vasculature . Autoradiography on different cell lines confirmed the targeting activity of GX1 toward the gastric cancer vascular endothelium, by showing that the binding affinity of GX1 was significantly higher in HUVEC cultured in tumor-conditioned medium than in HUVEC cultured in non-conditioned medium. No specific binding was observed in the human gastric carcinoma cell line SGC7901 or in the immortalized gastric epithelial cell line GES cells . Furthermore, immunohistochemical staining and immunofluorescence showed positive staining for GX1 in the vascular endothelium of human gastric adenocarcinoma, but not in heart, liver, muscle, spleen or normal gastric tissues [10, 28]. In another study, using single photon emission computed tomography (SPECT), GX1 labelled with 99TcmO4- (99Tcm -GX1) was observed to concentrate in tumor xenografts in nude mice . Collectively, these results indicate that GX1 is a novel vascular marker of human gastric cancer, and may lead to a new way of imaging diagnosis and targeted gastric cancer therapy.
Since GX1 selectively targeted the vascular endothelium of gastric cancer, we investigated whether it had specific effects on tumor angiogenesis and growth. In this study, in addition to targeting, GX1 showed bioactivity by both MTT and CAM assay, inhibiting vascular endothelial cell proliferation and hampering neovascularization. To probe into the possible mechanisms of these effects, the cell cycle distribution, cell apoptosis and the expression level of apoptosis related molecule caspase3 were detected by FCM and western blot assays. Inhibition of vascular endothelial cell proliferation by GX1 was observed, at least in part, to be through the up-regulation of caspase 3 expression and the induction of apoptosis. Further tests including RT-PCR and gene microarray are underway to investigate the precise mechanisms.
In vitro analysis showed that HUVEC cultured in tumor-conditioned media partially acquire the characteristics of tumor vascular endothelial cells, such as enhanced tubule formation, cell proliferation, and migration [13, 29]. Furthermore, some proteins like vascular endothelial growth factor receptor and the integrin αvβ3 may be up-regulated in co-HUVEC, as is the case for other cancer endothelia [6, 30]. These findings lead us to the hypothesis that GX1 receptors are up-regulated in co-HUVEC, reflecting the case in tumor vessels, and that more receptors may lead to greater selective affinity and stronger anticancer effects. This hypothesis is consistent with the MTT assay results, in which GX1 showed more significant inhibitory effects on co-HUVEC than on the parental HUVEC culture that was not exposed to tumor-conditioned medium.
To assess the possibility of using GX1 as a targeted delivery vector in combination with another antitumor molecule for treatment of gastric cancer, GX1 was conjugated to rmhTNFα. TNFα is a well-known, antitumor cytokine whose clinical application is hampered by severe systemic toxicity [12, 15]. The novel mutant cytokine rmhTNFα shows higher antitumor efficacy and has been approved for clinical use in China . Our data showed that after fusion to GX1, rmhTNFα was selectively delivered to target tumor vasculature sites. Most important, GX1-rmhTNFα delayed tumor growth in vivo, with less loss of body weight compared to rmhTNFα alone (Figure 6A, B). These results indicated that more targeted and efficient antitumor activity might be achieved by combining GX1 with other anti-tumor agents (e.g. rmhTNFα), for a significant reduction in systemic toxicity.
Despite the encouraging results, some questions are still open, such as what the receptor is for GX1 on vascular endothelial cells, and how ligand-receptor interaction interferes with tumor angiogenesis. Further studies are underway to answer these questions, and several candidate receptor molecules have been obtained. Identification of the GX1 receptor will be a great help in understanding the mechanism of GX1 and will accelerate the development of clinical applications for GX1 in diagnosis and targeted treatment of gastric cancer.
In conclusion, the data presented here, taken together with previously published results, demonstrate that GX1 is a novel vascular marker of human gastric cancer. For the first time GX1 is shown to have properties other than homing, including proapoptotic effects on vascular endothelial cells, and in vivo inhibition of neovascularization. Furthermore, when conjugated to rmhTNFα, GX1 selectively delivered the fusion protein to target tumor sites, leading to higher antitumor efficiency with less systemic toxicity. These findings demonstrate great potential for developing GX1 both as a targeted vector and as an antiangiogenic agent in the diagnosis and treatment of human gastric cancer.
Reagents, antibodies and peptides
Growth factors and Evan's blue were purchased from Sigma (St. Louis, USA). M200 basal culture media and low serum growth supplement (LSGS) were from Cascade Biologics (USA). Anti-CD31 polyclonal antibody was from ABcam (USA) and anti-TNF monoclonal antibody was from Sigma Aldrich (Saint Louis, USA).
GX1 peptide (CGNSNPKSC) was synthesized by GL Biochem (Shanghai) Ltd. A control peptide (Pep2) was created by randomly scrambling the amino acid sequence of GX1 while maintaining the disulfide bond to preserve the U-type structure (CNKSPSGNC). rmhTNFα was created by standard recombinant DNA techniques [11, 31, 32]. The GX1-rmhTNFα fusion protein was prepared as previously described .
HUVEC (Cascade Biologics, USA) and the human gastric cancer cell line SGC7901 were cultured as described . Tumor conditioned medium (TCM) was prepared by incubating SGC7901 cells in M200 (free of LSGS) (~1 × 106/ml) for 24 h. The medium was then removed, centrifuged (2000 × g, 10 min), filtered with a 0.22-μ m filter and diluted five times with M200 supplemented with LSGS. Tumor endothelial cells were generated by incubating HUVEC in TCM . All cells were cultured at 37°C in a humidified chamber with 5% CO2.
MTT, FCM and western blot assays
Proliferation of HUVEC and SGC7901 cells treated with various concentrations of tested agents was determined by MTT assay as described .
Apoptosis of co-HUVEC was detected by FCM analysis as described . Cells were treated with GX1 (50 μ M) or GX1-rmhTNFα (10 μ M) for 48 h. Pep2 (50 μ M), PBS or rmhTNFα (10 μ M) were used as controls.
The expression level of caspase 3 was measured by western blot assay. The cultured cells were lyzed in modified RIPA buffer (0.05 M Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 0.15 M NaCl, 0.001 M Na3VO4, 0.001 M EDTA and 0.5% of protease inhibitor cocktail). The lysate was centrifuged at 10,000 × g, 4°C for 10 minute, and the supernatant was collected. Protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE and were transferred to PVDF membrane. western blot analysis was carried out using the following primary antibodies: anti-cleavage caspase 3 antibody (1:500; Abnova Corporation, Taipei, Taiwan) and anti-β-actin antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were visualized using enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) according to manufacturer's instructions.
Chorioallantoic Membrane (CAM) assay
Fertilized White Leghorn chicken embryos were randomly divided into three groups with seven embryos per group, and collected on day 3 into sterile containers for subsequent incubation at 37°C, 5% CO2 for 6 days. On day 9, sterilized Whatman filter discs impregnated with 10 μ l (20 μ g) GX1 were placed on the CAM. Pep2 (20 μ g) and PBS were used as controls. On day 11, the CAM was cut, fixed by acetone and viewed under a microscope. Neovascularization around the disk was quantitated by determining the number of angiogenic vessels within the CAM around the disk.
In vivo distribution of GX1- rmhTNFα by emission computed tomography (ECT)
In vivo distribution of GX1-rmhTNFα in different organs of tumor-bearing nude mice was detected by enzyme-linked immunosorbent assay (ELISA, see below) and ECT. A suspension of SGC7901 cells was prepared at 1 × 107 cells/ml. A total of 0.2 ml of the cell suspension was implanted subcutaneously in the right upper flank of 4-6 week-old male nude BALB/c mice (animal centre of FMMU, Xi'an, Shannxi, China). Three weeks after injection, the mice were randomly divided into three groups with six mice per group, and treated with 0.25 mg/kg of GX1-rmhTNFα or rmhTNFα, or NS through the tail vein. The agents were allowed to circulate for 0.5 h, 1 h and 2 h before blood samples were taken from the eye, and tumors and major organs were removed. Tissue samples were homogenized and sonicated at 4°C, followed by centrifugation for 10 min at 12,000 × g. The supernatant was subjected to ELISA.
Simultaneously, GX1-rmhTNFα labeled with 99TcmO4 - was used for dynamic imaging in biodistribution studies. Anesthetized animals were injected intravenously with 200 μ l 99Tcm-GX1-rmhTNFα or 99Tcm-rmhTNFα at 470-540 μ Ci per mouse. Planar and single-photon emission tomography images with a low-energy collimator were obtained, with 200,000 counts acquired per image at the indicated timepoints. Time-dependent biodistribution studies were carried out by sacrificing mice at 2, 8, and 18 h after injection. Tissue samples were removed at the end of the test. The radioactivity was determined with a gamma counter and decay-corrected to the time of injection. Results were calculated as injected dose (ID) per gram of wet tissue weight (ID/g tissue), converted to percent. GX1-rmhTNFα/rmhTNFα radioactivity rates of various tissues were determined from the corresponding ID/g tissue values.
ELISA, immunohistochemical staining and immunofluorescence staining
The amount of GX1-rmhTNFα and rmhTNFα in tumor and other organs was quantified by ELISA kit (Department of Immunology, FMMU, Xi'an, Shannxi, China). The optical density at 470 nm was measured with a microplate reader (Bio-Rad Laboratories, Hercules, CA).
Immunohistochemical staining was performed as described  using anti-TNF monoclonal antibody and anti-CD31 polyclonal antibody. For immunofluorescence, tumor sections were incubated with diluted anti-TNF monoclonal antibodies and anti-CD31 polyclonal antibodies at 4°C overnight, and then treated with rhodamine-conjugated goat anti-mouse IgG and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG at 1:200 for 1 h at room temperature. Sections were analyzed by fluorescence microscopy.
Evan's blue assay
Twenty days after subcutaneous injection of SGC7901 cells, nude mice were intravenously treated with GX1-rmhTNFα, with rmhTNFα and NS as controls. Two hours later, the mice were intravenously injected with 0.1 ml Evan's blue (Sigma, St. Louis, MO; 12.5 mg/ml). After 5 min, the animals were sacrificed and the tumors were excised. Each tumor was weighed, homogenized, resuspended in cold PBS containing 1% Triton X-100 (1 ml/g), and incubated for 1 h on ice. The suspension was centrifuged (14,000 × g, 4°C, 15 min), and the supernatant mixed with trichloroacetic acid (10%, v/v). The product was centrifuged again (14,000 × g, 4°C, 15 min) and the absorbance at 405 nm of the supernatant was measured using a spectrophotometer.
In vivo tumor formation assay
Seven days after injection of SGC7901 cells as described above, nude mice were randomly divided into groups of seven mice and treated with the indicated reagents on alternate days. Tumor development was observed by sequential caliper measurements of length (L) and width (W). Tumor volume was calculated by the formula L × W2/2. After 20 days, the mice were killed and the tumors were removed and weighed. All studies were performed according to internationally recognized guidelines for animal care.
Each experiment was repeated at least three times. Numerical data are presented as mean ± standard deviation (SD). The difference between means was analyzed by ANOVA. All statistical analyses were performed using SPSS11.0 software (Chicago, IL). Differences were considered statistically significant when p < 0.05.
This work was supported by grants from the National Bio-Tech 86-3 Program (No. 2006AA022103), Medical and Healthcare Project of PLA (No. 06G087), National Key Project of Basic Research (No. 2004CB518702) and National Foundation of Natural Science, China (No. 30130260, No. 30024002 and No. 30873005).
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