Tumstatin is a recently designed endogenous vascular endothelial cell growth inhibitory factor1 originating from vascular basement membrane collagen IV. It is a polypeptide fragment of the α3 chain non-collagen (NC) domain 1 of type IV collagen, comprised of 244 amino acids with a relative molecular weight of about 28 kD. It has been found to be a strong angiogenesis inhibitory factor that selectively inhibits endothelial cell proliferation2 within the tumor bed and is currently considered to be an anti-angiogenic and anti-tumor drug with excellent prospects.
There are still problems that require solutions before tumstatin can be introduced for use in clinical trials: high molecular weight, low solubility and immunogenic factors, to name a few. In order to solve these problems and prepare it for clinical application, the alleged angiogenesis inhibition-related peptide, a small molecule peptide (herein referred to as peptide 21), was designed and synthesized using biological engineering technology to simulate the structural and functional features of the active zone of angiogenesis inhibition exhibited by tumstatin. It is assumed that, similar to tumstatin, peptide 21 acts to specifically inhibit tumorigenesis and could specifically inhibit tumor vascular endothelial cell proliferation resulting in endothelial cell apoptosis. In vitro studies and animal experiments were used to test these features, so as to lay a foundation for developing it into an antitumor drug with the characteristic of inhibiting tumor angiogenesis.
E. coli JM109 and BL-21 (DE3) (Promega, USA); human ovarian cancer cell line SKOV-3 (from the Tumor Research Institute of the Third Affiliated Hospital of Harbin Medical University), mouse embryo-derived NIH3T3 fibroblasts (from the Department of Medical Genetics of Harbin Medical University) and human umbilical vein endothelial cell line ECV304 (from the Department of Biochemistry of Harbin Medical University); RPMI1640 (GIBCO, USA); fetal bovine serum (Hangzhou Sijiqing Bio-Engineering Material Research Institute, China); T4 DNA ligase, T4 polynucleotide kinase (T4 PNK) and plasmid isolation kit (Promega, USA); pTYB2 vector, Nde I, Sma I and Xho I restriction enzymes, and chitinous affinity chromatography resin (NEB, China); MTT and DMSO (Sigma, USA); DTT and IPTG (TaKaRa, Japan); AnnexinV-FITC Apoptosis Detection Kit (BD Biosciences, USA); 30 BALB/c-nu mice (Shanghai SLAC Laboratory Animal Co., Ltd., China; immunohistochemical non-biotin two-step detection kit (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China).
Gene synthesis, recombinant expression and protein purification of peptide 21
The peptide 21 gene sequence was designed and synthesized according to 75-95 amino acid gene sequences of human tumstatin in Genebank: 5′-TATGCCGTTCTTATTCTGCAATGTTAACGATGTA-TGCAACTTCGCATCTCGTAATGATTACTCC-3’, 3′-AC-GGCAAGAATAAGACGTTACAATTGCTACATACGT-TGAAGCGTAGAGCATTACTAATGAGG-5′. The Nde I restriction endonuclease site TATG, was introduced into the 5′-end containing the translation start codon. The 3′-end was the blunt end that connected with the 5′-end of the intein gene in the infusion protein vector.
The two synthesized single-stranded DNAs were phosphorylated with T4 PNK and annealed into a double-stranded DNA. The pTYB2 plasmid vector was digested by the restriction endonuclease Nde I and Sma I. The digested vector was retrieved after electrophoresis. The peptide 21 gene and the retrieved vector, after restriction enzyme digestion, was connected to derive the recombinant fusion protein expression vector pTYB2-21T. E. coli JM109 was transformed with pTYB2-21T to isolate plasmids and gene sequencing was performed after validation by digestion with the restriction endonuclease Nde I and Xho I.
This expression of genetic engineering bacteria and purification of peptide 21 refers to the method introduced by Yu et al.3 In short, E. coli BL-21(DE3) were transformed with pTYB2-21T. The transformed BL-21(DE3) were placed in a 250 ml LB medium containing 100 mg/L ampicillin at 37°C, continually shocked and cultured to reach 0.5 OD600 and then IPTG was added until 0.1 mmol/L for 6 hours induction at 28°C. After thallus precipitation and repeated freeze thawing, the lysate supernatant was placed in the chitin affinity chromatography column balanced with buffer (20 mmol/L Tris-HCl pH 8.0, 500 mmol/L NaCl, 1 mmol/L EDTA). The first hybrid protein peak was eluted and quickly put in the buffer with 50 mmol/L dithiothreitol (DTT). The column was then transferred for preservation for 20-40 hours at 4°C. During this period, the intein in the fusion protein was separated from peptide 21 by its autolysis activity. The second eluting peak was the peptide 21 peak.
The Bradford method was used to plot standard curves to determine peptide 21concentration. The results we have another paper presented in 2005.4
The three cell types, SKOV-3, NIH3T3 and ECV304, were cultured with the 1640 medium containing 10% fetal bovine serum in a carbon dioxide incubator containing 5% CO2 at 37°C.
Cells were inoculated into a flat-bottomed 96-well plate with a density of 1×105/ml cells/well and after 24 hours culture, cells adhered to the plate; then peptide 21 of various concentrations was added. After 72 hours, the culture fluid was discarded; 100 μl MTT (5 g/L) was added for 4 hours and then the supernatant was removed; 0.1 ml DMSO was added and the plate was shaken to dissolve the cells; an ELISA reader (Bio-Rad Model 550, USA) was used to detect absorbance (reference wavelength being 630 nm) at 490 nm and the growth inhibition rate was calculated. Inhibition rate (%) = (1 — experimental well reading/control well reading) × 100%. The experiment was repeated five times and the mean value was obtained.
In vitro experimental subgroups
In vitro experimental groups were assigned to both the control group and peptide 21 group. According to the results of the MTT test, 0.03 μg/μl of peptide 21 was chosen for the experimental concentration for the peptide 21 group, when sensitive cells reached IC50, and PBS buffer was added to the control group at the same volume.
Cells were inoculated into a 24-well plate by 1.0×104 cells/well and after cells were cultured for 24 hours and adhered to the plate, peptide 21 was added to the adherent cells. Three wells were selected daily for cell counts for 6 successive days and the mean value was calculated. The experiment was repeated five times and the mean value was obtained.
FCM detection of apoptosis
After the incubation of peptide 21 with cells for 72 hours, 5×105 cells were harvested and then incubated with 5 μl annexinV-FITC and 2 μl PI for 15 minutes at room temperature in a dark place and analyzed with FCM (BD FACSAria, USA). The experiment was repeated three times and the mean value was obtained.
TEM observation of cell morphology
Peptide 21 was allowed to act on cells for 72 hours before the cells were harvested. Conventional methods were employed to process specimens, including fixation, dehydration, embedding, slicing and staining with 3% glutaric dialdehyde and 1% osmic acid. Observation under the transmission electron microscope (JEM-1220, Japanese Electronics Co. Japan) was carried out.
Animal model preparation and tumor size determination A total of 20 female nude mice, 5 to 6 weeks old and weighing 18-22 grams, were fed under specific pathogen-free (SPF) conditions. SKOV-3, with a concentration of 5×107 U/ml, was injected subcutaneously into the right scapula of the nude mouse to form a hummock, 0.2 ml for each nude mouse. Three days later, when subcutaneous nodules were palpable, animals were randomly allocated into the peptide 21 group or the control group: 10 mice to each group. Following the conversion rate of peptide 21 concentration (0.03 μg/μl) in the in vitro experiment, a peptide 21 solution, at a daily dose of 2.65 mg/kg, was injected intraperitoneally in the peptide 21 group. PBS at the same volume was given to the control group for 21 successive days. Mice were sacrificed by decapitation on the 22nd day. Body weight, tumor weight and tumor size were determined. The tumor inhibition rate was calculated as, inhibition rate = (1 — medication group tumor weight/control group tumor weight) × 100%; tumor size = a × b2 × 0.52 (a-long diameter, b-short diameter).
Immunohistochemical detection of tumor MVD and VEGF
Material was taken from the edge of tumor tissue. A solution of 4% paraformaldehyde was used for fixation. Paraffin was employed for embedding and slicing. Immunohistochemical detection was performed using an immunohistochemical non-biotin two-step detection kit in strict accordance with the manual's description. MVD determination: The region with the highest MVD was first determined under a low power lens (×10) and then the number of tumor microvessels in 10 visual fields were counted under a high-power lens (×40) with MVD indicated by the number of vessels in each high-power microscopic field.
Analysis of the results for VEGF staining: The occurrence of brown within the cytoplasm was regarded as being positive for VEGF. A double-blind test was used to randomly observe five high-power fields in each slice. These were classified according to the ratio of positive cells among the tumor cells: negative (—), no positive cells observed; positive (+), the ratio of positive cells was less than 30%; positive (++), the ratio of positive cells was 30%-60%; positive (+++), the ratio of positive cells was greater than 60%. This semi-quantitative method was employed to score: “—” a score of 0, “+” a score of 1, “++” a score of 2 and “+++” a score of 3.
SPSS 11.0 software was used to process data. The t test was used to compare mean values between the two groups. A single factor analysis of variance (ANOVA) was used to compare the mean values among multiple groups, the LST-t test was used to compare each two groups and the Wilcoxon rank sum test was used for ranked data. P <0.05 was considered statistically significant.
Results of the MTT test
Peptide 21 showed a significant inhibitory effect (P <0.01) on ECV304 cells, in a concentration-dependent manner, when compared to the NIH3T3 and SKOV-3 cells. The peptide 21concentration for IC50 was 0.026 μg/μl (Figure 1A).
Results of growth curves
In order to validate the inhibitory effect of peptide 21on the proliferation of endothelial cells, peptide 21 at the concentration of 0.03 μg/μl was used on all three cell lines for plotting the growth curves. Results from this assay conformed with those of the MTT test: Peptide 21exerted an obvious inhibitory effect on ECV304 at this concentration. Its effect was not remarkable on the other two cell lines (P <0.01) (Figure 1B).
The apoptotic rate of ECV304 in the peptide 21 group was significantly higher than that in the control group (P <0.01), but that of NIH3T3 and SKOV-3 were not altered significantly (Figure 2).
In the peptide 21 group, ECV304 cells revealed typical
apoptotic manifestations and NIH3T3 cells showed manifestations of injury to a certain extent; however, no obvious or specific changes were observed in the other groups (Figure 3).
Effect of peptide 21 on tumor growth subcutaneously grafted with SKOV-3
Tumor size: The tumor in the peptide 21 group grew slowly. After a 21-day treatment, tumor size in the peptide 21 group was obviously smaller than in the control group (P <0.05) (Figure 4A).
Tumor weight: Tumor weight in the peptide 21 group was significantly lower than in the control group (P <0.05). The average tumor inhibition rate in the peptide 21 group was 67.86%.
MVD observation in nude mouse tumor tissues
Results indicated that the MVD of tumor tissues in the peptide 21 group was significantly lower than in the control group (P <0.05) (Figure 4B and Figure 5).
Detection of the VEGF expression in nude mouse tumor tissues
A large amount of VEGF was found to be expressed in tumor cells of the control group. This was particularly evident in areas with comparatively high vascular density and actively proliferating tumor cells. VEGF was also positively expressed in microvascular endothelial cells. Compared with the control group, the peptide 21 group had fewer cells that were positive for VEGF expression and no obvious positive VEGF expression in the microvascular endothelial cells (P <0.05) (Table and Figure 6).
Neovascularization plays a key role in tumor growth and metastasis, while endothelial cell proliferation is a principal cause of tumor angiogenesis; thus, inhibition of endothelial cell proliferation may suppress tumor neovascularization and further achieve the objective of inhibiting tumor growth.
Endogenous angiogenesis inhibitors have the following advantages: they directly act on actively proliferating microvascular endothelial cells of the tumor without affecting endothelial cells that are commonly in a static state within normal tissues, so they are tumor-specific; the tumor-inhibiting effect is independent from tumor types, so they are broad-spectrum; in contrast to the genetic instability of tumor cells, genetic traits of vascular endothelial cells are stable, so endogenous angiogenesis inhibitors are not liable to engender drug resistance and their therapeutic effects are consistent; they also do not give rise to negative side effects like those of cellulotoxic drugs.1,5-10
Tumstatin is a type of endogenous vascular endothelial cell growth inhibitor, newly discovered, that has antitumor effects1 directly promoting tumor cell apoptosis and inhibiting tumor angiogenesis. It is a drug with good prospects for the treatment of tumors. In addition, when a drug is used for clinical treatment, its stability, solubility, bioavailability and the patient's immune response etc11 should all be taken into account. Tumstatin has high molecular weight and low solubility. It was initially detected as an autoantigen of Goodpasture's syndrome.12 As an antigen of Goodpasture's syndrome, it brings along with it an abundance of drawbacks when used directly for clinical treatment as a drug. Designing and synthesizing this small molecule angiogenesis inhibitory peptide has become an important field for the research and development of anti-angiogenesis drugs.
During extensive research into full-chain tumstatin, two functionally active zones were found. One consisting of 197-215 amino acids near the C-terminus inhibited tumor cell proliferation.13,14 The other consisting of 54-132 amino acids adjacent to the N-terminus (also called tum-5) exerted an obvious inhibitory effect on angiogenesis.9 The anti-angiogenesis effect of the latter functionally active zone was not affected even when the disulfide linkage or disulfide linkage alkylation was removed; that is to say, the activity of tum-5 was decided by its primary structure and changes to its secondary structure exerted no effect on its activity.9 Further study of tumstatin showed that its active domain - inhibiting angiogenesis - might be located in the T7 peptide segment;15,16 consisting of 25 amino acids within tum-5, from 74-98.
To synthesize this soluble small molecule angiogenesis-inhibitory peptide, the 25 amino acids located in the T7 peptide segment were further designed and altered to simulate the angiogenesis-inhibiting activity zone of tumstatin. As there were more hydrophobic amino acids, the three hydrophobic amino acids at 96-98 (tyrosine, tryptophan and leucine) were removed to raise solubility; then the 74 threonine was removed and the 75 threonine was used as the starting amino acid for translation, the synthesized angiogenesis inhibition-related peptide was constructed, namely, peptide 21.
During an in vitro study of the functional activity of peptide 21, the human ovarian cancer cell line SKOV-3, mouse embryo-derived NIH3T3 fibroblasts and human umbilical vein endothelial cell line ECV304 were used. The MTT test and growth curves all indicated that peptide 21 could specifically inhibit endothelial cell proliferation. It is worth mentioning that the tumor cell line, SKOV-3, had an interesting feature in the process of culturing. Its growth rate was not faster than the other two cell lines; in fact it was significantly slower. This might be related to growth characteristics of the cell line, although this has not been reported so far. As for the induction of apoptosis, TEM is by far the “gold standard” for determination of apoptosis. The occurrence of apoptosis qualitatively, in combination with apoptosis quantitatively as detected by FCM, shows that peptide 21 may specifically and significantly induce endothelial cell apoptosis. TEM detection also indicated that NIH3T3 cells exposed to peptide 21were actually injured to a certain extent. The reason and mechanism of the injury awaits further research. In short, peptide 21 has a specifically inhibitory effect on human endothelial cell proliferation in vitro and promotes endothelial cell apoptosis.
In the following animal experiments, models of mice with tumors subcutaneously grafted with human ovarian cancer cell line SKOV-3 were used to study the in vivo biological activity of peptide 21. Results indicated that peptide 21 exerted an obvious effect by inhibiting tumor growth. The average tumor inhibition rate reached 67.86% at the 21st day; peptide 21 reduced the MVD of tumor tissues and showed strong anti-angiogenesis activity. Peptide 21 did not significantly inhibit SKOV-3 proliferation or promote apoptosis during in vitro experiments. However, it significantly suppressed growth of the tumor subcutaneously grafted with SKOV-3 in vivo; indicating that peptide 21 played a role in suppressing tumor growth indirectly by inhibiting tumor-related angiogenesis, which was in accordance with the functional characteristics of tum-5.
Neovascularization is co-regulated by angiogenesis- stimulating factors (VEGF, bFGF, EGF and angiogenin, etc) and inhibitory factors (angiostatin, endostatin, tumstatin and vasostatin, etc). VEGF is one of the most specific and significant angiogenesis regulatory factors. It is also a specific mitogen for the vascular endothelium that may regulate vascular endothelial cell proliferation, basal lamina hydrolysis, migration and blood vessel construction.17 Research showed that tum-5 could inhibit VEGF-induced angiogenesis.9 To investigate whether peptide 21 also inhibits VEGF-induced angiogenesis, the VEGF expression of tumor cells and vascular endothelial cells were compared between the peptide 21 group and the control group. Results showed that VEGF expression of both tumor cells and vascular endothelial cells decreased significantly in the peptide 21 group. It can be speculated that the inhibition of endothelial cell proliferation and the induction of endothelial cell apoptosis through down- regulating VEGF expression of tumor cells and vascular endothelial cells might be the mechanism used by peptide 21 to inhibit angiogenesis.
In summary, the biological activity of peptide 21, designed and synthesized according to functional and structural features of the angiogenesis inhibition activity zone of tumstatin, was found to be similar to tum-5, which could specifically inhibit vascular endothelial cell proliferation in a dose-dependent manner and induce endothelial cell apoptosis, thus inhibiting tumor angiogenesis which indirectly suppresses tumor growth, infiltration and metastasis. Peptide 21 appears to exert its biological activities and functions through down-regulating the VEGF expression of tumor cells and vascular endothelial cells. Due to its small molecular weight and good solubility, peptide 21 may become an effective drug for antitumor treatment with potential clinical application.
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Keywords:© 2008 Chinese Medical Association
tumstatin; angiogenesis inhibitors; anti-angiogenesis; ovarian neoplasms