In order to overcome difficulties that hampered widespread application of antiangiogenesis in cancer therapy, a highly specific delivery system may be engaged in vivo to deliver and express antiangiogenic genes. We selected a strain of Bifidobacterium adolescentis (B. adolescentis) as the delivery system to transport endostatin gene to solid tumors. B. adolescentis with endostatin gene were injected into tumor-bearing mice through the tail vein. After the mice were sacrificed, the tumor and some normal tissues of the mice were examined. B. adolescentis were only found in the tumors and no bacilli were found in other normal tissues. Also, a strong inhibition of angiogenesis had been shown to inhibit local tumor growth in the administrated group. These results suggested that B. adolescentis only germinated and proliferated in solid tumors and might be a highly specific and efficient vector for transporting anticancer genes into target tumor in cancer gene therapy.
Angiogenesis is required for tumor progression and metastasis. In tumor growth process, tumor cells promote angiogenesis by the secretion of many kinds of angiogenic factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).1 At the same time, tumor cells also secrete antiangiogenic factors, including angiostatin and endostatin.2,3 These factors form a network and tumor angiogenesis is dependent upon the local balance of these positive and negative regulators. Antiangiogenesis in tumors can inhibit the growth and metastases of the tumor.4 Endostatin specially inhibits the proliferation of vessel endothelial cells stimulated by bFGF.5,6 Systemic administration of endostatin on tumor-bearing mice keeps the primary tumors in the dormant state.3
Clinical trials involving angiostatin and endostatin require large quantities of bioactive recombinant proteins, which are difficult to produce. This difficulty may be resolved by tumor-specific in vivo delivery and expression of these antiangiogenic genes.
Hypoxic regions are characteristic of solid tumors in rodents and many types of human tumors. Oxygen partial pressure in tumors of cancer patients is 10–30 mm Hg, whereas those in normal tissues range from 24 to 66 mm Hg. The hypoxic regions of solid tumors provide some species of anaerobic bacteria such as Bifidobacterium and Lactobacillus a suitable environment to germinate and grow.7
Bifidobacterium bifidum and B. longum both can selectively germinate and grow in the hypoxic regions of solid tumors after intravenous injection.8,9 It is currently being investigated if the genus of Bifidobacterium are used to achieve tumor-specific gene delivery. This provides a method and a way to transport antitumor gene expressions into tumors directly.
We selected a strain of B. adolescentis as a delivery system to transport endostatin gene to hypoxic tumors. B. adolescentis with endostatin gene were injected into the mice bearing Heps liver cancer. At 168 hours after the third injection of B. adolescentis with endostatin gene, B. adolescentis were only found in the tumors and no bacilli were found in other normal tissues. Our results indicate a strong inhibition of both the angiogenesis and the hypoxic tumor growth in the administrated group, and suggest that B. adolescentis might be a highly tumor-specific gene delivery vector for transporting anticancer genes into tumors in cancer gene therapy.
Materials and methods
BALB/c male mice (obtained from Animal Center of Nanjing Medical University, Nanjing, China) weighing 20±2 g were used in this study. Mice were fed standard rodent diet.
Heps mouse liver cancer (obtained from the Animal Center of Chinese Academy of Sciences, Shanghai, China)10 was minced thoroughly and cancer cells were obtained at the concentration of 5×106 cells/mL. A total of 1×106 tumor cells were inoculated under the skin of the right thigh for each mouse.
B. adolescentis11 was from the Chinese IFFI Microbial Center and anaerobically cultured at 37°C in TPY medium.
Plasmid construction and transformation of B. adolescentis12
A shuttle vector, pBV220,13 containing Ampr gene, was from the Institute of Virology, Chinese Academy of Preventive Medicine. Human endostatin gene was obtained by PCR method from human liver cDNA library (Clontech, Franklin Lakes, NJ) and inserted into pBV220 vector. Recombinant plasmid was purified by QIA prep spin miniprep kit (Qiagen, Hilden, Germany).
An overnight culture of B. adolescentis was inoculated in 13% milk with 0.05% cysteine–HCl and 0.5 M sucrose and anaerobically cultivated overnight at 37°C. This overnight culture was diluted 1:25 in above 13% milk buffer and cultivated at 37°C until an OD600 reached about 0.2. B. adolescentis were chilled on ice, harvested by centrifugation at 4°C, and washed twice with 0.5 M sucrose. Bacteria were resuspended in about 1/150 of the original culture volume of ice-cold 0.5 M sucrose supplemented with 1 mM ammonium citrate (pH 6.0); every 100 μL of bacteria suspension was mixed with 1.0 μg of purified pBV220/endostatin plasmid and incubated at 4°C for 3.5 hours. The above mixture was added in a precooled Gene Pulser disposable cuvette (interelectrode distance 0.2 cm; Bio-Rad, Hercules, CA). A high-voltage electric pulse (duration=1.5–3.0 milliseconds) was delivered with a Gene Pulser apparatus (Bio-Rad) by using the 25-μF capacitor and 200-Ω parallel resistance. Then the mixture was inoculated in above 13% milk buffer and cultivated at 37°C for 2.5 hours, plated on the solid TPY medium (1.5% agar) with 10% rabbit blood and containing 50 μg/mL ampicillin, and incubated at 37°C under the anaerobic condition for 72 hours.
Determination of B. adolescentis transfected with endostatin by PCR method
The plasmids of B. adolescentis transfected with endostatin gene were extracted as reported.14 PCR was performed with the following primers: N-terminal, 5′ IndexTermCCG GAA TTC ATG CAC AGC CAC CGC GAC TTC CAG CCG 3′ and C-terminal, 5′ IndexTermGCC GGA TCC CTA CTT GGA GGC AGT CAT GAA GCT 3′, 94°C for 1 minutes, 60°C for 1 minutes, 72°C for 1 minutes, 30 cycles.
Expression of endostatin from B. adolescentis and Western blot analysis
B. adolescentis with endostatin gene were incubated at 30°C for 12 hours, diluted in about 1/50 of fresh TPY medium, and incubated at 42°C for 6 hours when OD600 reached 0.5–0.7. Then the bacteria were harvested by centrifugation, resuspended in SDS sample buffer, and sonicated. The sample was subjected to SDS-PAGE using 12% polyacrylamide gels, followed by Western blot analysis. Purified endostatin (Calbiochem, Darmstadt, Germany) acted as positive control and normal B. adolescentis as negative control. The primary polyclonal mouse antihuman endostatin antibody was diluted 200 times. The secondary antibody was a 1:50 dilution of goat antimouse IgG-HRP (Sino-American Biotechnology, Shanghai, China). The membrane was stained by DAB.
Detection of antibiotics resistance and utilization of carbon source of B. adolescentis with endostatin gene
Antibiotics susceptibility of B. adolescentis with endostatin gene was detected by disc agar diffusion method. The concentration of antibiotics was 30 μg/mL. The bacteria were plated on TYP solid medium containing different antibiotics paper discs and cultured at 37°C for 72 hours, and the diameter of inhibition rings of antibiotic was measured.
B. adolescentis were inoculated in media containing different carbon source. After weekly incubation at 37°C in anaerobic condition, they were detected by the color of indicator. Normal B. adolescentis were examined as control.
Treatment on tumor-bearing mice with B. adolescentis with pBV220/endostatin through tail vein
Before injection, the B. adolescentis were washed three times with 5% glucose in 0.9% NaCl and were resuspended with this buffer. A total of 0.4 mL of the above suspension was injected into the tumor-bearing mice through tail vein (1×108 bacilli/mouse). Injections were carried out on days 3, 6, 9, 12, and 15 after inoculation of tumor cells. Mice were divided into three groups (eight mice per group). One group was injected with endostatin-carrying B. adolescentis, one group was treated with normal B. adolescentis, and one was treated with 5% glucose in 0.9% NaCl. The animals were sacrificed on day 18 and tumors were excised and weighed. The level of inhibition of tumor growth was determined by the formula as follows:
Examination of location and number of B. adolescentis with endostatin gene after the injection
After 72 hours of inoculation, the tumor-bearing mice were injected with B. adolescentis carrying endostatin gene through tail vein (1×108 bacilli/mouse), and the mice were successively treated with the same dose of B. adolescentis every 72 hours. At 1, 24, 48, 96, and 168 hours after the third injection, six tumor-bearing mice were sacrificed. Under aseptic conditions, tissue samples were obtained from the lung, liver, spleen, kidney, heart and tumor. Each tissue sample (0.1 g) was placed into a homogenizer to prepare a homogenate with 1 mL of 5% glucose in 0.9% NaCl. The homogenates were diluted and plated on the solid TPY medium (1.5% agar) with 10% rabbit blood, and incubated at 37°C under anaerobic conditions for 72 hours. Then the number of colonies per dish was counted to determine the number of viable bacteria.
Mice were sacrificed after 18 days of treatment as described above. Tumors were excised and fixed in 4% paraformaldehyde. Tissues were frozen, and the sections (20 μm in thickness) of the tissues were cut and mounted on glass slides. The sections were subsequently treated with 0.1 M PBS (pH 7.0) for 5 minutes and methanol mixed with 0.3% H2O2 for 10 minutes. The sections were blocked with 1% milk in PBS, then incubated at 37°C for 2 hours with 1:200 dilution of rat antimouse CD-31 monoclonal antibody (Pharmingen, San Diego, CA), followed by 1 hour of incubation at 37°C of 1:25 dilution of goat–antirat IgG-HRP (Kirkegaard and Perry Laboratories, Gaithersburg, CA). The sections were stained by 0.05% DAB mixed with 0.03% H2O2, and photographed. The positive cells were quantitated by 801 Morphologic Analysis software (Jieda, Jiangsu, China).
DNA fragmentation assay15
The tumor tissues were excised and homogenized thoroughly. Following centrifugation at 3000×g at 4°C for 5 minutes, the pellets were resuspended in a lysis buffer containing 10 mM Tris–HCl (pH 8.0), 10 mM EDTA, 1% SDS, 20 μg/mL DNase-free RNase, and 200 μg/mL proteinase K. After overnight incubation at 37°C, the DNA was purified by phenol/chloroform extraction, precipitated, and resuspended in TE buffer (pH 7.4). The DNA (10 μg) was subjected to electrophoresis on a 1.5% agarose gel containing 0.5 μg/mL ethidium bromide and visualized under UV light. Electrophoresis was carried out in TE buffer (pH 8.0) at 20 V for 8 hours.
The data were statistical analyzed by using the Student's t test, and P values of <.05 were considered to be significant.
Endostatin expression plasmid construction
Human endostatin gene was obtained by PCR method, and restrictive sites of EcoRI and BamHI were inserted in the PCR primers. The pBV220/endostatin plasmid was transfected into Escherichia coli DH5α, and the positive clones were selected and digested with EcoRI and BamHI. A band of about 560 bp was shown (Fig 1).
Determination of B. adolescentis with endostatin gene by PCR method
The plasmids extracted from B. adolescentis were amplified by PCR with above primers. A band of about 560 bp was obtained from the B. adolescentis with endostatin gene, and the negative result was obtained from normal B. adolescentis (Fig 2).
Expression of endostatin from B. adolescentis carrying pBV220/endostatin plasmid
The expression of endostatin from B. adolescentis was assayed by Western blot analysis using a polyclonal mouse antihuman endostatin antibody. Positive control, recombinant endostatin protein migrated at about 20 kDa. A same band about 20 kDa also was observed from transfected B. adolescentis. There was no positive antibody reactivity band from normal B. adolescentis (Fig 3).
Antibiotics resistance and carbon source utilization
Results showed that transfected B. adolescentis and wild B. adolescentis both could be killed easily by several antibiotics (Table 1). Resistance of ampicillin of transfected B. adolescentis was due to the Ampr gene in the recombinant plasmid. There was no difference between B. adolescentis with endostatin gene and normal B. adolescentis in carbon source utilization (Table 2).
Selective growth of B. adolescentis with pBV220/endostatin in tumor tissues
The tumor-bearing mice were intravenously injected with 1×108 viable transfected B. adolescentis; sacrificed at 1, 24, 48, 96, and 168 hours after the third injection; and examined for the presence of bacilli in tumors and several normal tissues. At 168 hours, about 1.2×107 bacilli/g tumor tissue were found, but no bacilli were detected in normal tissues such as the liver, spleen, kidney, and lung from the tumor-bearing mice (Fig 4). The increasing number of bacilli in tumors suggested that the transformed B. adolescentis germinated in the tumor tissue. In contrast, the number of B. adolescentis in normal tissues decreased immediately after injection, which indicated that B. adolescentis did not germinate in these normal tissues (Fig 5).
B. adolescentis with plasmid pBV220/endostatin inhibits the growth of primary tumors
The growth of primary tumors was potently suppressed by systemic therapy with B. adolescentis carrying endostatin gene. Tumor growth was inhibited by 69.9% as compared to control mice treated with 5% glucose in 0.9% NaCl. The tumor growth of mice treated by normal B. adolescentis was inhibited by 23.1%, compared with control group. The inhibitory role of normal B. adolescentis was rather weak than that of B. adolescentis carrying endostatin gene (P<.01, Fig 6). The results suggested that the role of endostatin gene was strong in the inhibition of tumor growth.
Inhibition of tumor angiogenesis by B. adolescentis with endostatin gene
Immunohistochemical analysis showed a potent inhibition of angiogenesis in the tumors treated by B. adolescentis with endostatin gene. Quantitative determination of vessel density was made by microscopic measurement of the brown areas (positive) at 432-fold magnification. One unit visual field encompassed 128×128 pixels. There were significantly less microvessels in the tumors of treated mice versus control mice after staining of the tumor tissue sections with a rat antimouse CD-31 monoclonal antibody (Fig 7). The results showed that B. adolescentis with endostatin gene blocked angiogenesis, but normal B. adolescentis did not block angiogenesis, which suggested that the inhibition of angiogenesis was caused by the introduced endostatin gene.
Induction of apoptosis in tumor cells by the introduced endostatin gene
Fragmentation of cellular DNA represents a main change in the nuclei of cells undergoing an apoptosis. Administration of B. adolescentis carrying endostatin gene led to the typical DNA ladder pattern in apoptotic tumor cells (Fig 8). However, no DNA fragmentation could be detected in tumor cells from mice treated with normal B. adolescentis or in the mice treated with 5% glucose in 0.9% NaCl. These results showed that the introduced endostatin gene blocked angiogenesis accompanied by induction of apoptosis in liver tumors.
An important obstacle for cancer gene therapy is specific gene delivery system introducing anticancer gene into target tumors. We selected a strain of B. adolescentis as gene delivery vector. After B. adolescentis with endostatin gene were intravenously injected into tumor-bearing mice, viable bacilli could be examined initially in most tissues throughout the body. However, after 96–168 hours of injection, B. adolescentis could be found only in tumor tissue. These data showed that B. adolescentis selectively germinated and proliferated in tumor tissue, and suggested that tumor tissue provided a suitable condition for B. adolescentis with endostatin gene to grow. In this method for cancer gene therapy, only the hypoxic regions of solid tumors were suitable for administration of Bifidobacterium with anticancer gene.
There are several advantages of selecting Bifidobacterium as gene delivery vector for cancer gene therapy: (1) Bifidobacterium is a domestic anaerobic bacterium in the human body that does not produce endotoxin and toxin; (2) Bifidobacterium increases immune response16 and inhibits many tumor growth in vivo such as liver cancer, breast cancer, etc.;17 (3) Bifidobacterium can be killed easily by antibiotics or in oxygen environment in vitro or in vivo. We confirmed that both wild-type and genetically engineered B. adolescentis were killed easily with kanamycin, cefoperazone, and penicillin in vitro; (4) when B. adolescentis was injected intravenously, bacilli only germinated and proliferated in solid tumor but not in other normal tissues; and (5) we demonstrated that B. adolescentis carrying endostatin gene specifically located in solid tumor and selectively inhibited angiogenesis and hypoxic tumor growth. These results strongly suggested that B. adolescentis could be used as a highly specific gene delivery vector for anticancer gene therapy.
Tumor growth and metastasis are angiogenesis-dependent. Angiogenic inducers secreted by tumor cells induce endothelial cells proliferation and migration into tumor, and form new vessels. The new intratumoral blood vessels provide nutrients and oxygen to tumor cells, and they are the way for tumor cells entering the circulation and metastasizing to distant organs. Many antiangiogenic factors detected in recent years inhibit vessel endothelial cell proliferation, induce tumor degradation and apoptosis, and limit tumor migration. Endostatin is an endogenous angiogenic inhibitor. It specifically suppresses endothelial cell proliferation, and acts as a competitor of angiogenic inducers secreted by tumor cells.3 The inhibition of tumor growth was associated with endostatin levels in tumor model and was nearly the same in different models of solid tumors.18 Other authors reported that endostatin showed no toxicity in all normal tissues, and that it could inhibit tumor metastasis with no drug resistance.3
Endostatin as an inhibitor of angiogenesis has shown an attractive new strategy in cancer gene therapy. However, poor solubility of recombinant endostatin protein and its high effective dose hampered its widespread application, although high and continuous endostatin expression can be obtained by direct induction of the endostatin gene in vivo. Many reports have shown that several vectors, such as viral vector,19,20 nonviral vector, and liposome,18 have been used in animals as gene delivery systems. However, Bifidobacterium as a gene therapy vector has its special characters. It can specifically reach tumor tissue by circulation and grow in hypoxic solid tumors. Bifidobacterium is beneficial rather than nonpathogenic for its host, immunobiologic,16,21 anti-infective,22 and antiaging,23,24 and observably inhibits the growth of tumors in mice.25 Therefore, with Bifidobacterium as a vector for gene therapy, its inherent role as antitumor makes it more effective in inhibition of tumor growth.
Endostatin induced DNA fragmentation in the nuclei of solid tumor cells. The fragmentation of nuclear DNA is one of the distinct morphological changes occurring in the nucleus of an apoptotic cell. However, DNA fragmentation in the nuclei of solid tumor cells treated with wild-type B. adolescentis was not obviously detected. These results suggested that endostatin exerted its regulatory activity and inhibition of tumor growth in transfected B. adolescentis.
In summary, transfected B. adolescentis were introduced systemically into tumor-bearing mice, and bacteria were found only in hypoxic environment of solid tumor. B. adolescentis could be used as a highly specific gene delivery vector in cancer gene therapy. These engineered B. adolescentis carrying anticancer gene specifically inhibited angiogenesis and hypoxic tumor growth, and showed a strong regulatory role on apoptosis of solid tumor cells.
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This work was supported by Grant BK20000001 from the Natural Science Foundation of Jiangsu Province, China, to GXX; Grant 30070250 from the National Natural Science Foundation of China; and a Grant-in-aid of “985 Project” from the Nanjing University to JJW. We thank Yan Chen (New York University School of Medicine) for reading this manuscript.
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