An RGD-Modified Endostatin-Derived Synthetic Peptide Shows

Sep 19, 2008 - It has been reported that an endostatin-derived synthetic peptide, named ES-2, that contains the amino acids 60−70 of endostatin from...
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1980

Bioconjugate Chem. 2008, 19, 1980–1986

An RGD-Modified Endostatin-Derived Synthetic Peptide Shows Antitumor Activity in ViWo Han-Mei Xu,† Runting Yin,† Luosheng Chen,† Sami Siraj,† Xiaofeng Huang,‡ Min Wang,† Huisheng Fang,† and Ying Wang*,† Department of Life Science and Technology, China Pharmaceutical University, Nanjing, 210009, and Department of Oral Pathology, Nanjing Stamotology Hospital, Affiliated Medical School, Nanjing University, 210008, P. R. China. Received April 1, 2008; Revised Manuscript Received August 22, 2008

It has been reported that an endostatin-derived synthetic peptide, named ES-2, that contains the amino acids 60-70 of endostatin from its N terminus, efficiently inhibits basic fibroblast growth factor-induced directional migration and tubular morphogenesis of microvascular endothelial cells. We found that the peptide had no effects on tumor growth in ViVo. However, when the peptide Arg-Gly-Asp (RGD) was introduced into ES-2, the modified ES-2 showed significant antitumor results in animal models. Histochemical and immunohistochemical analysis showed that RGD-modified ES-2 induced large areas of continuous necrosis within tumors and significantly reduced the vessel density compared to control. Furthermore, only the peptides with RGD were able to bind tumor cells in Vitro, suggesting that additional RGD domains may help in improving the receptor-binding ability and pharmacokinetic properties of ES-2 and preventing organic clearance, as well as enzymatic degradation of the peptide, thus enabling a greater fraction of the administered dose to be biologically available.

INTRODUCTION

structure-function relationship of endostatin to meet the urgent need for new antitumor drugs. To achieve this goal, several groups have synthesized and studied the peptides that correspond to the partial sequence of endostatin (12-17). One group has reported that an 11-amino-acid peptide (ES-2: IVRRADRAAVP) derived from the amino terminus of endostatin promoted endothelial cell adhesion, migration, and tube formation (12). However, another group obtained data indicating that a peptide containing the 11 amino acids failed to inhibit tumor growth (13). In our previous study (18), we found that ES-2 dramatically inhibited angiogenesis on the chorioallantoic membrane of the chick embryo in ViVo, but had no antitumor effect in tumorbearing mice. We speculate that this difference might be caused by a lesser distribution of the drug in the mouse model than in the chick embryo. In order to validate this hypothesis, a new strategy that increases the targeted delivery of the peptide to tumors is needed. In the past decade, many molecules with the RGD peptide have been described that efficiently deliver drugs to tumor vasculature endothelium (19, 20). Therefore, in the present study, we examined whether the addition of RGD will also lead to the pronounced antitumor activity of the peptide.

Endostatin, the C-terminal fragment of collagen XVIII with a molecular weight of 20 kDa, has been approved by the State Food and Drug Administration (SFDA1) of China for its antitumor activity. It specifically inhibits endothelial cell proliferation and migration and reduces vascularization and blood flow in gliosarcoma (1). It also dramatically inhibits growth of various primary tumors in mice. Integrins could be the potential targets for endostatin (2, 3). Endostatin binds one atom of zinc (Zn) per monomer via the three histidines in the amino terminus of the molecule (histidines 1, 3, and 11) and aspartic acid 76 (4-6). The crystal structure of mouse endostatin reveals a compact globular fold and a basic patch of 11 arginine residues, which have been suggested to act as binding sites for heparin (7). Numerous attempts have been made to produce biologically active endostatin via recombinant DNA technology (8). However, when expressed in Escherichia coli, recombinant endostatin accumulated as inclusion bodies in the cytoplasm. During the in Vitro renaturation or refolding process, the recovery of soluble and active endostatin was very low, and most of the protein precipitated during the refolding process owing to the renaturation of proteins containing multiple disulfide bonds (9-11). Further investigations are necessary to elucidate the

MATERIALS AND METHODS

* Corresponding author. Han-Mei Xu, Center of Biotechnology, Department of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, P. R. China. Tel: 8625-83271007. Fax: 86-25-85438355. E-mail: [email protected]. † China Pharmaceutical University. ‡ Nanjing University. 1 Abbreviations: Arg-Gly-Asp (RGD); Ala-Cys-Asp-Cys-Arg-GlyAsp-Cys-Phe-Cys (RGD-4C); the State Food and Drug Administration (SFDA); American type Cell Culture (ATCC); Dulbecco’s modified Eagle’s medium (DMEM); human umbilical vein endothelial cells (HUVEC); fetal bovine serum (FBS); phosphate-buffered saline (PBS); fluorescein isothiocyanate (FITC); bovine serum albumin (BSA); diaminobenzidine (DAB); basic fibroblast growth factor (bFGF); subcutaneously (s.c.); hematoxylin and eosin (H&E).

Cell Lines, Animals, And Peptides. B16F10 mouse melanoma cells (provided by Nanjing University), human hepatic carcinoma Bel-7402, human gastric cancer cell line MGC-803, and human hepatic carcinoma SMMC-7721 cell line were purchased from American type Cell Culture (ATCC, Shanghai, China) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and antibiotics. Human umbilical vein endothelial cells (HUVEC, provided by Nanjing Keygen Biotech Co. Ltd.) were routinely grown in DMEM, supplemented with 20% heat-inactivated fetal bovine serum (FBS) and 25 µg of endothelial cell growth factor. Mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. Synthesis of the peptides was performed by GL Biochem (Shanghai) Ltd. All peptides are

10.1021/bc800132p CCC: $40.75  2008 American Chemical Society Published on Web 09/19/2008

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Table 1. Sequences of Synthetic Peptides name of peptides

sequences

ES-2 P1 P2 ES-2mut1 P3

IVRRADRAAVP IVRRADRAAVPGGGGRGD RGDGGGGIVRRADRAAVP IVAAADRAAVP IVAAADRAAVPGGGGRGD

very soluble in water. The purity of the products was more than 99% by analytical high-performance liquid chromatography. Amino acid analysis and mass spectroscopy were conducted to confirm their compositions. N-terminal amino-acid sequence analysis was performed at Fudan University (Shanghai, China). Endostatin was expressed in E. coli and purified by our laboratory. Antibodies were purchased from Boster Biological Company (Wuhan, P. R. China). Preparation of Conditioned Medium. Confluent cells were washed and grown in serum-free DMEM. After 20 h of incubation, conditioned medium was collected and centrifuged at 500 × g for 10 min and then at 800 × g for 20 min to remove debris. The resultant conditioned medium was immediately used for migration and tube formation assays or stored at -70 °C until use. Cell Migration Assay. To quantify cell migration, a modified Boyden chamber assay was used as described previously (21). Polycarbonate filters with 8 µm pores in a 24-well Transwell chamber (Costar, Shanghai, China) were coated with 15 µL Matrigel (Becton-dickinson, Shanghai, China, 50 µg/pore) and placed at 37 °C for 1 h. Cells (0.5 mL, 8 × 104) suspended in serum-free medium were placed in the upper compartment of the chambers. Six hundred microliters of conditioned medium was added to the lower compartment. Following 48 h incubation at 37 °C, cells in the upper chamber and the remaining cells on the upper surface of filters were mechanically removed. After washing with phosphate-buffered saline (PBS), the membranes were fixed in 30% methanol for 10 min and stained with 0.1% methyl violet, and the cells that migrated to the lower surface were counted under inverted microscope at 200× magnification. Five random fields from each well were counted, and values represent five independent experiments. Migration activity was assessed by the average number of cells that migrated to the lower surface of the filter. Tumor Implantation. B16F10 mouse melanoma, human gastric cancer cell line MGC-803, and human hepatic carcinoma SMMC-7721 cell line were grown in culture. Cell lines were washed with PBS, dispersed in a 0.05% trypsin solution, and resuspended. After centrifugation at 800 × g for 5 min, the cell pellets were resuspended in PBS and adjusted to a concentration of 2.5 × 106 cells/mL. BALB/c nude female mice, 5-6 weeks old, were implanted subcutaneously (s.c.) on the midright side with 5 × 106 MGC-803, SMMC-7721 cells in 0.2 mL PBS. C57BL/6 female mice (5-6 weeks old) were implanted s.c on the midright side with 1 × 106 B16F10 cells in 0.2 mL PBS. C57BL/6 Female Mouse Treatment. After the B16F10 mouse melanoma tumor mean volume reached 100-300 mm3, mice were separated randomly into different groups with eight mice per group. Group 1 received recombinant human endostatin at a dose of 10 mg/kg in 0.1 mL PBS, and group 2 was injected with an equal volume of PBS. The other nine groups were treated with P1, P2, or ES-2 at doses of 5, 10, or 20 mg/kg in 0.1 mL PBS. All of the groups were injected s.c., at a site distant from the tumor, once daily for 10 d. After the first round of treatment, we carried out another round of treatment with P1 alone, at doses of 6, 3, and 1.5 mg/kg. BALB/c Nude Mouse Treatment. After the mean SMMC7721 tumor volume reached 100-300 mm3, mice were ran-

domly divided into four groups with eight mice per treatment group and twelve mice in the negative control group. Group 1 received paclitaxel (Taxol, provided by Tai ji Pharmaceutical Co. Ltd., Chendu in Sichuan province of China) at a dose of 10 mg/kg, group 2 received equal volume injections of PBS alone. Another group was treated with P1 at a dose of 3 mg/ kg. All the nude mouse treatment groups were injected intravenously with 0.1 mL of the drug, once a day for 10 d. Mice implanted with MGC-803 tumor cells were injected with 3 mg/kg/d of P1 peptide. The duration of this treatment was also 10 d. Measurement of Tumor Growth. Tumors were measured individually with a vernier caliper. Volumes were determined using the formula: tumor volume ) length × width2 × 0.52. Therapeutic effects on tumor growth were expressed as mean tumor volumes versus time, calculated as (1 - T/C) × 100%, where T ) treated tumor volume and C ) control tumor volume. For example, if treated tumors were 40% of the volume of control tumors on a given day, tumor suppression in the treated group was 60%. Western Blot Analysis. Cells were harvested, centrifuged, and the protein fractions separated. The protein was analyzed by 10% SDS-PAGE and further identified by Western blot analysis with PVDF Western blotting membranes (Roche, Shanghai, China) and mouse polyclonal antibody. Flow Cytometric Analysis of Peptide Binding. Cells were grown in a 24-well plate until 80% confluency, harvested, and washed twice with ice-cold PBS. Before labeling, cells were resuspended in PBS + 1% bovine serum albumin (BSA) for 30 min, then added with 1 µL fluorescein isothiocyanate (FITC)labeled peptides (P1, P2, RGD, and ES-2) for an additional 25 min. After labeling, cells were collected and washed again with ice-cold PBS twice, resuspended in PBS at a volume of 400 µL, and analyzed with flow cytometry (Becton Dickinson, Shanghai, China). To exclude nonviable cells, 0.5 µM of each peptide was added immediately before analysis. FITC fluorescence was measured in FL1 channel. Cells with FITC emission were gated for further analysis. Histochemistry and Immunohistochemistry. Mice were euthanized 2 weeks post-treatment, and tumor tissue was collected, fixed with 4% formaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining and immunohistochemical staining for CD31. H&E staining was performed according to standard histological procedures. Necrosis and vascularization in tumor tissue were observed under a light microscope. Vascular structures in tumors were evaluated by immunohistochemical staining of CD31 with rabbit antiCD31 polyclonal antibody (Boster Biological Company). Briefly, staining for CD31 was performed on sections using their specific primary antibodies and biotinylated goat antirabbit secondary antibody, incubated with horseradish peroxidase-labeled streptavidin, visualized with diaminobenzidine (DAB) chromogen, counterstained with hematoxylin, and observed with a microscope. Three-Dimensional Modeling. Modeling of the composed peptides’ structure was done by using the InsightII/CHARMm module (Accelrys Inc., USA). During the simulation, residues 1 to 11 of P1 and residues 8 to 18 of P2 were combined by template which is the structure of residues 50 to 60 in human endostatin (Protein Data Base, PDB ID: 1BNL). The modified protocol was performed by 500 steps of steepest descents (convergence is 0.1 kcal/mol) and 500 steps of conjugate gradients (convergence is 0.01 kcal/mol) under the CHARMm27 force field. Then, simulation of the three-dimensional structure was performed by molecule dynamics at 300 K. Statistical Methods. Data are expressed as mean ( SD. Statistical significance was assessed using the Student t test. For all statistical comparisons, treated groups were compared

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Figure 1. HPLC (A), mass spectroscopy (B), and amino acid sequencing (C) analyses of peptide P1.

with PBS-treated controls, and P < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Endostatin, after its approval for clinical use, has given a new dimension to cancer treatment by targeting the tumor vasculature; however, the exact mechanism of endostatin remains unknown. Moreover, it is relatively expensive compared to other conventional antineoplastic drugs such as chemical antiangiogenic agents. These disadvantages limit its further use. Recently, evidence has outlined the role of integrin binding in the biological effects of endostatin in the cell (22-24). In another study, it was shown that an arginine-rich peptide, ES-2, in the soluble form inhibited basic fibroblast growth factor-induced directional migration and tubular morphogenesis of microvascular endothelial cells (12). The peptide induced the loss of focal adhesions and actin stress fibers in these cells. This activity was found to be lost in mutated peptides where arginine residues were substituted with alanine. This reflects some of the properties of endostatin that are associated with the heparinbinding characteristic of endostatin. However, in one study, an endostatin peptide (hP3) that contains ES-2 failed to inhibit tumor growth in ViVo (13). The authors found that the entire antiangiogenic activity of endostatin was located in a 27-aminoacid peptide that binds Zn, and showed that Zn binding was required for antitumor and antimigration activities of endostatin. These results indicated that the mechanism of endostatin is complex and the unique chemical composition of each protein (or peptide) is important for its function. A change in just one amino acid can change the structure and function of a protein, so an endostatin-derived peptide could not reflect the true characteristic of endostatin. Therefore, protein properties and activities relate to the number and type of factors. Although certain amino acid sequences can be identified as more likely to have activity in Vitro, it is still not possible to completely predict whether the

protein will be active in ViVo. The in Vitro antimigratory activity of ES-2 has been mapped to 60-70 amino acids of endostatin (12), but the in ViVo inhibitory effect of this peptide remains poorly investigated. Peptides containing ES-2 (like hP3) cannot reflect the true characteristics of ES-2. However, ES-2 is the shortest active sequence derived from endostatin, and the results of antimigratory activity shed some light on reducing the cost of endostatin. The synthesis of a smaller peptide is much simpler and less expensive when compared to the expression and purification of endostatin, and its mechanism is clear. Still, it is necessary to identify the in ViVo activity of ES-2. We previously tested the antiangiogenic activity of ES-2 peptide. ES-2 at different concentrations was loaded onto chick embryos to validate the in ViVo antiangiogenic activity, the results of which demonstrated that ES-2 dose-dependently suppressed the embryonic neovascularization and angiogenesis (18). Furthermore, ES-2 higher than 200 ng/mL almost completely prevented blood vessel formation and was lethal to the chicken embryo. These data indicate that the ES-2 peptide is a potent inhibitor of angiogenesis. However, in a tumor-bearing mouse model, we found that ES-2 peptide did not show any significant antitumor activity (data not shown). We speculate that this inconsistency may be due to the lesser bioavailability and/or receptor binding by ES-2 peptide. A possible explanation could be that heparin may not be the only candidate for this peptide, which could mean it has to bind to another ligand and/or receptor in ViVo. Additionally, direct administration of the peptide in chick embryo assay will lead to better bioavailability of the drug, but in an animal model, the drug is distributed to many compartments. Biotransformation of these drugs could lead to its inactivation because of the changes in the elimination after metabolism or the effects of other biomolecules. If ES-2 could be effectively transferred to tumor angiogenic endothelium, it may exhibit antitumor activity. Recently, lines of evidence have shown that the addition of RGD, or RGD-4C short peptide, facilitates the molecular

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Figure 2. Western blot analysis of the expression of integrin Rν and β3 in human Bel-7402 cells using mouse antihuman integrin polyclonal antibody (A). Flow cytometry demonstrating the binding of RGD-FITC-labeled peptide to human Bel-7402 cells (B).

targeting of integrins on angiogenic endothelium, which has proven to be an effective strategy for delivering a drug to the tumor vasculature. The RGD sequence on ES-2 might have the ability to bind with the tumor endothelial cells. In view of this fact, we synthesized two peptides with RGD attached to the amino and carboxy terminals of ES-2. Four glycine residues were added between the RGD motif and ES-2 in an attempt to increase its flexibility (Table 1). The purity, molecular weight, and sequence of P1 were shown (Figure 1). First, we wanted to know whether the modified peptides could efficiently bind integrins. Western blot analysis was performed on human hepatic carcinoma Bel-7402 whole-cell lysates using integrin Rv and β3 polyclonal antibody, and expression of both subunits was observed in Bel-7402 (Figure 2A). A target analysis was performed on P1 and P2 coupled with FITC; RGD and ES-2 coupled with FITC were used as positive control and negative control, respectively. Bel-7402 cells were used as the target to determine the RGD-FITC-peptide binding with the cell surface receptors (integrins). Flow cytometric analysis of FITC-peptides binding to tumor cells was performed. A positive reaction was seen with P1, P2, and RGD, while ES-2 did not bind to cells (Figure 2B), which suggested that RGDmodified ES-2 could specifically bind to the receptor expressed by Bel-7402 tumor cells, resulting in efficient antitumor activity of P1 and P2 in ViVo. Reselection was performed with the mutant peptides. First of all, in Vitro selection has been used to find the peptides with

similar or better activity than ES-2. To investigate the role of the peptides for their antimigratory effect, transwell chamber migration assays were performed using the synthetic peptides. P1, P2, and ES-2 all displayed reduced HUVEC ability to migrate toward the basic fibroblast growth factor (bFGF) stimulus. At 1 µg/mL, P1 was most effective in inhibiting migration of HUVEC (Figure 3). Thus, P1 and P2 were used for further investigations. For the in ViVo assay, the mice (C57BL/6) implanted with B16F10 melanoma cells were injected s.c. with the peptides to perform the preliminary screening. According to published data, 5-20 mg/kg/d of endostatin has been reported to exert an antitumor effect; we used doses up to 20 mg/kg/d in the experiment. We assessed P1 and P2 at three doses of 20, 10, and 5 mg/kg/d and discovered that 5 mg/kg/d has the most effective cytostatic effect on the melanoma (data not shown). On the basis of this finding, we performed another experiment by testing three smaller doses, i.e., 6, 3, and 1.5 mg/kg/d, and found that 3 mg/kg/d of P1 or P2 was most effective in arresting the growth of the tumor (data not shown). Moreover, we also discovered that the administration of the drug twice a day had the most desirable effect. Adding RGD peptide sequences to any protein can cause its binding to antiangiogenic integrins. Hence, it is not clear that the biological effects are associated with ES-2 or RGD. It would be important to compare the active peptide with RGD and modified inactive endostatin sequence for reference. A mutated peptide, ES-2mut1, displayed reduced

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Figure 3. Inhibition of endothelial cell migration by synthetic peptides. The migration response of HUVEC was assayed by the Boyden chamber. Cells were treated with P1, P2, and ES-2. As controls, rhEndostatin and PBS were used. Columns represent mean ( SD of 5 experiments (*P < 0.05; **P < 0.01 vs control).

levels of activity compared with ES-2 (12). RGD was also introduced into the carboxy terminal of ES-2mut1 (i.e., P3). We found that P3 and RGD all exhibited moderate antitumor activity (compared with P1 and P2; data not shown), suggesting that the two domains were involved in the antitumor activity. On the basis of these findings, we selected P1 to further examine their effects on tumor-bearing nude mice (P1 and P2 were all modified by RGD and P2 has antitumor effects similar to P1, so only P1 was selected). ES-2 did not show any activity in ViVo, while endostatin expressed and purified by different laboratories had different antitumor effects because of the inclusion body’s denaturation and renaturation, so for this paper, further experiments of ES-2 and endostatin were not conducted. Taxol is a standard drug for the treatment of advanced cancer, and in order to get a reliable and comparable result, we selected Taxol as a positive control. In ViVo, the results show that intravenous injection of P1 at 3 mg/kg/d inhibited human SMMC-7721 hepatic carcinoma growth by 78% (Figure 4A). However, P1 displayed higher antitumor effects as compared to Taxol at 10 mg/kg/d, suggesting that P1 has significant antitumor effect at lower doses. We performed an additional series of experiments in a human gastric tumor model. The intravenous injection of P1 at 3 mg/kg/d inhibited human gastric cancer cell MGC-803 growth by 58% (Figure 4B). We also examined the effect of the peptides on tumor tissue. P1 displayed more severe tissue necrosis than the control (Figure 5A,B). The control displayed necrosis interspersed with viable tumor cells, whereas P1 induced large areas of continuous necrosis within tumors. We then counted the number of visible

Figure 4. Therapeutic effects of peptides on the growth of tumor. (A) represents human SMMC-7721 hepatic carcinoma suppression mediated by 3 mg/kg P1 twice a day. (B) displays the tumor in different treatment groups. (C) MGC-803 human gastric cancer suppression by 3 mg/kg P1 twice a day. Each point represents mean ( SD of each group (*P < 0.05; **P < 0.01 vs control).

blood vessels in CD31 staining, and found that P1 significantly reduced the vessel density compared to control (Figure 5C,D,E) (P < 0.05). The finding that the addition of RGD at amino and carboxy terminals has the same result calls for a justification. The peptides with an additional RGD sequence (P1, P2), regardless

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Figure 6. Modeling of the structures of the composed peptides was done by using the InsightII (Accelrys Inc.)/CHARMm module. Peptides 1 and 2 (A,B) have less secondary structure and fold in an extended manner.

Figure 5. Necrosis areas within tumor and tumor blood vessel density. The figure shown by H&E staining observed with light microscope (40× original magnifications). PBS (A) treatment exibit incomplete tissue necrosis interspersed with viable tumor cells. (B) represents P1 treatment, showing continuously extensive necrosis area. (C, D, E) Tumor blood vessel density. (C) represents PBS, and (D) represents P1 treatment. Blood vessels in CD31 staining were indicated with arrows (40× original magnifications). Results were expressed as mean ( SD of nine regions of three sections per group (*P < 0.05; **P < 0.01 vs control).

of its location at either the amino or carboxy terminal, show an effective arrest of tumor growth. This may be due to the fact that the RGD peptide, the primary recognition sequence of some integrins, has less secondary structure (Figure 6A,B), thus P1 and P2 exhibited similar binding capability to integrins. ES-2 is the smallest endostatin structure unit that has been discovered recently, but there is no further in ViVo assessment to support its effectiveness. In this study, we evaluated the effectiveness of ES-2 in ViVo for the first time, along with the determination of the most effective dosage range of modified ES-2, the most effective administration frequency, and the change in its in ViVo activity after the addition of an RGD motif at different locations. We also performed the histochemical and immunohistochemical analysis of the tumor under investigation. Moreover, safety experiments were conducted to study the possible toxic effects of P1, and a tail vein injection of 1500 mg/kg/d P1 into Kunming strain mice revealed no evidence of toxicity in the treated animals (data not shown). These results show that P1 is an effective and safe peptide as an anticancer

drug. RGD modified ES-2 (P1 and P2) has only 18 amino acids and showed remarkable suppression of growth of human gastric and hepatic cancer at doses as small as 3 mg/kg/d. The study has proven that the addition of RGD motif to peptides is an effective strategy for the delivery of the peptide to the tumor vasculature. During the past decade, RGD peptides have become a popular tool for the targeting of drugs and imaging agents to tumor vasculature expressing Rv and β3 integrins. Some of these products show impressive results in preclinical animal models, and an RGD-targeted radiotracer has already successfully been tested in humans for the visualization of Rv and β3 integrins, which demonstrates the feasibility of this approach (25-27). The study on P1 and P2 has made it possible to develop a new drug to fight solid tumors. We are currently investigating the activity of P1 and P2 on another four types of human cancer. We also plan to study the pharmacokinetic and pharmacodynamic aspects of peptide P1, to ensure a better clinical application of it.

ACKNOWLEDGMENT The authors are grateful to Zichun Hua (Department of Life Science, Nanjing University, 210008, P. R. China) for providing B16F10 mouse melanoma. We thank Dr. Yinwu (Department of Life Science, Nanjing University, P. R. China) for critical reading of the manuscript and Drs. Wanzhou Zhao, Yijun Sun, and Junshi Ma for assisting us to implant tumors.

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