AAV-mediated human PEDF inhibits tumor growth and metastasis in murine colorectal peritoneal carcinomatosis model
- Qin Jie Wu†1,
- Chang Yang Gong†1,
- Shun Tao Luo1,
- Dong Mei Zhang1,
- Shuang Zhang1,
- Hua Shan Shi1,
- Lian Lu1,
- Heng Xiu Yan1,
- Sha Sha He1,
- Dan Dan Li1,
- Li Yang1Email author,
- Xia Zhao2 and
- Yu Quan Wei1
© Wu et al; licensee BioMed Central Ltd. 2012
Received: 4 October 2011
Accepted: 30 March 2012
Published: 30 March 2012
Angiogenesis plays an important role in tumor growth and metastasis, therefore antiangiogenic therapy was widely investigated as a promising approach for cancer therapy. Recently, pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis. Adeno-associated virus (AAV) vectors have been intensively studied due to their wide tropisms, nonpathogenicity, and long-term transgene expression in vivo. The objective of this work was to evaluate the ability of AAV-mediated human PEDF (hPEDF) as a potent tumor suppressor and a potential candidate for cancer gene therapy.
Recombinant AAV2 encoding hPEDF (rAAV2-hPEDF) was constructed and produced, and then was assigned for in vitro and in vivo experiments. Conditioned medium from cells infected with rAAV2-hPEDF was used for cell proliferation and tube formation tests of human umbilical vein endothelial cells (HUVECs). Subsequently, colorectal peritoneal carcinomatosis (CRPC) mouse model was established and treated with rAAV2-hPEDF. Therapeutic efficacy of rAAV2-hPEDF were investigated, including tumor growth and metastasis, survival time, microvessel density (MVD) and apoptosis index of tumor tissues, and hPEDF levels in serum and ascites.
rAAV2-hPEDF was successfully constructed, and transmission electron microscope (TEM) showed that rAAV2-hPEDF particles were non-enveloped icosahedral shape with a diameter of approximately 20 nm. rAAV2-hPEDF-infected cells expressed hPEDF protein, and the conditioned medium from infected cells inhibited proliferation and tube-formation of HUVECs in vitro. Furthermore, in CRPC mouse model, rAAV2-hPEDF significantly suppressed tumor growth and metastasis, and prolonged survival time of treated mice. Immunofluorescence studies indicated that rAAV2-hPEDF could inhibit angiogenesis and induce apoptosis in tumor tissues. Besides, hPEDF levels in serum and ascites of rAAV2-hPEDF-treated mice were significant higher than those in rAAV2-null or normal saline (NS) groups.
Thus, our results suggest that rAAV2-hPEDF may be a potential candidate as an antiangiogenic therapy agent.
KeywordsAAV PEDF Tumor Antiangiogesis Gene therapy
Cancer is a major public health problem in the world and causes millions of death each year. More than 1.5 million new cancer cases and 560,000 deaths from cancer are projected to occur in 2010 in the USA . Surgical resection, chemotherapy, and radiotherapy are conventional therapeutic strategies, which were widely used in clinic. Unfortunately, surgical resection is not sufficiently effective for advanced cancer, and chemotherapy and radiotherapy always induce severe side effects [2, 3]. Obviously, it is necessary to develop novel therapeutic strategies to optimize available non-surgical approaches. Gene therapy was proposed as a new approach to eradicate malignant cells, which may be beneficial to cancer patients at all stages .
Both tumor growth and systemic metastasis are highly dependent on angiogenesis . Tumor less than 1 mm3 could receive necessary nutrients and oxygen through diffusion, however, a tumor is unable to grow above approximate 1 mm3 without neovascularization . Angiogenesis is a complex multistep process consisting of proliferation and migration of endothelial cells, degeneration of basement membrane, and formation of new lumen, which is tightly regulated by both positive and negative factors . Microvascularity of normal tissue is regulated by the proangiogenic and antiangiogenic factors to maintain a quiescent state. Tumor growth and metastasis require neovascularization, therefore, tumors use various mechanisms to shift the balance to an angiogenic inductive environment. In this process, overexpression of proangiogenic factors is accompanied by down regulation of antiangiogenic factors . As angiogenesis is essential for tumor growth and metastasis, antiangiogenesis has been proposed as a therapeutic strategy for cancer treatment .
Pigment epithelium-derived factor (PEDF), a 50-kDa secreted glycoprotein, is a member of the serpin superfamily of serine protease inhibitors, which was first identified in a conditioned medium of cultured primary human fetal retinal pigment epithelial cells . To data, it was found that PEDF is widely expressed in human tissues and is involved in many physiological and pathological processes . PEDF is a multifunctional protein and proved to be neurotrophic, neuroprotective, and antiangiogenic. And PEDF has recently been shown to be a more potent inhibitor of angiogenesis than other endogenous angiogenic inhibitors, including angiostatin, endostatin, and thrombospondin-1 [12, 13]. Antiangiogenic activity of PEDF was associated with induction of endothelial cell apoptosis via the Fas/FasL death pathway and down-regulation of vascular endothelial growth factor (VEGF) expression. Previous works suggested that PEDF is a potential tumor suppressor [6, 14–17].
Although gene therapy is an effective way to treat cancers and many other chronic diseases, the safety and efficacy of gene therapy depends on the development of safe and effective delivery systems [18–23]. The recombinant adeno-associated virus (rAAV) vectors appear to be the promising potential candidate as safe and effective delivery system for gene therapy due to its attractive features [24–27]. rAAV vectors show low immunogenicity and ability to infect both dividing and nondividing cells. Besides, rAAV can maintain long-term undiminished transgene expression in vivo. The most important, rAAV vectors are nonpathogenic, which are not associated with any known human diseases. Until now, several different serotypes of AAV have been identified by serological analysis [28–30], among which rAAV serotype 2 vectors (rAAV2) are mostly studied and used in animal experiments and clinical trials [31, 32]. Most of the previous studies about rAAV-mediated gene transfer of PEDF mainly focused on eye diseases, however, rAAV-mediated gene transfer of PEDF for tumor treatment is rarely reported.
According to the BLAST analysis, the homology between human PEDF and mouse PEDF is very high. Besides, several previous contributions suggested that hPEDF was effective on mouse tumor models. Therefore, in this work, we constructed rAAV2 encoding human PEDF (rAAV2-hPEDF) and investigated its anti-tumor efficacy in colorectal peritoneal carcinomatosis (CRPC) mouse model. Our results demonstrated that rAAV2-hPEDF may be a potent tumor inhibitor and a potential candidate for cancer gene therapy.
Cell lines and animals
CT26 cells (murine colon carcinoma cell line) were purchased from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI1640 medium (GIBCO, USA) containing 10% fetal bovine serum (FBS, GIBCO, USA) and 1% penicillin/streptomycin. Primary HUVECs were isolated from human umbilical cord veins by a standard procedure , and grew in Dulbecco's Modified Eagle's medium (DMEM, GIBCO, USA) supplemented with 20% fetal bovine serum (FBS, GIBCO, USA)and basic fibroblast growth factor (bFGF, 10 ng/ml). HUVECs at passages 2 to 3 were used for experiments. All above cells were maintained at 37°°C in a humidified incubator containing 5% CO2.
BALB/c mice weighing 20 to 22 g were purchased from the Laboratory Animal Center of Sichuan University and housed at controlled temperature of 20-22°°C, relative humidity of 50-60% and 12 h light-dark cycles. Animals were provided with standard laboratory chow and tap water ad libitum. All the animals would be in quarantine for a week before treatment. All animal procedures were performed following the protocol approved by the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, P.R. China). All mice were treated humanely throughout the experimental period.
rAAV vector construction and production
rAAV2-hPEDF infection assay
Approximately 2 × 105 CT26 cells were plated into 6-well plates in complete medium and allowed to attach for 24 hours. Subsequently, cells were gently washed with PBS, and then rAAV2-hPEDF or rAAV2-null was applied to cells at MOI (multiplicity of infection) of 105 v.g. per cell in serum-free medium, with normal saline (NS) as non-infection control. After incubation for 6 hours, complete medium with 10% FBS was added to each well. Then, the conditioned medium was collected after further culture for 72 hours for western blotting analysis and cell proliferation assay.
Western blotting analysis
Conditioned media from CT26 cells infected with rAAV2-hPEDF, rAAV2-null or NS were used for Western blotting analysis. Concentrations of protein were determined using a modified Lowry protein assay kit (ThermoScientific), and an equal amount of protein (40 μg) from each sample was loaded on to 10% SDS-PAGE gel followed by transfer to a nitrocellulose membrane sheet, probed for anti-PEDF (1:500, upstates, Lake Placid, USA). The membranes were washed, and blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were detected using an enhanced chemiluminescence (ECL) detection system. (Pierce, Rockford, Illinois, USA).
Cell proliferation assay
Cell proliferation assay was conducted using 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (methyl thiazolyl tetrazolium, MTT) method to determine the effect of hPEDF derived from rAAV2-hPEDF infected cells on HUVECs. HUVECs were plated at a density of 1 × 104 cells per well in 100 μL of medium in 96-well plates and grown for 48 hours. The conditioned media from CT26 cells infected with rAAV2-hPEDF, rAAV2-null, or NS were collected as described above, and each supernatant was two-fold serially diluted and added to each well in triplicate. After 48 hours of incubation, viability of cells was measured using MTT method. The mean percentage of cell survival relative to that of untreated cells was estimated from data from three individual experiments, and all data were expressed as the mean value ± standard deviation (S.D.).
Endothelial cell capillary-like tube formation assay
Tube formation assay was conducted as previously described . Briefly, prechilled Matrigel was pipetted into prechilled 24-well plates and polymerized at 37°°C for 30 minutes. Then, HUVECs suspended in complete medium were seeded onto the Matrigel, and they were treated with conditioned medium form cells infected with NS, rAAV2-null, or rAAV2-hPEDF, respectively. After 6 hours of incubation, the endothelial cells were photographed with a digital camera attached to an inverted microscope. Three independent experiments were performed.
Animal model and treat plan
CRPC mouse model was established and subsequently treated. Briefly, the mice were intraperitoneally injected with 200 μl of cell suspension containing 5 × 105 CT26 cells. Tumors were allowed to grow for 3 days. Tumor-bearing mice were randomly assigned to one of the following groups (n = 12 mice per group): NS, rAAV2-null (2 × 1010 v.g. per mouse), and rAAV2-hPEDF (2 × 1010 v.g. per mouse).
Treatments were administered through intraperitoneal injection with a single dose of 200 μl above mentioned viruses. For tumor growth inhibition study (6 mice per group), the number and weight of tumor nodes in each group were measured on day 18 after tumor cell inoculation. After day 18, NS and rAAV2-null-treated mice began to die. To further study the therapeutic effect against colorectal cancer, the survival time of mice treated with the protocols described above was observed (6 mice per group).
Detection of microvessel density (MVD)
The antiangiogenesis of rAAV2-hPEDF was determined by immunofluorescent analysis of neovascularization in tumor tissue as described . Briefly, frozen sections of tumors were fixed in acetone, washed with PBS, stained with rat anti-mouse CD31 (platelet endothelial cell adhesion molecule-1) polyclonal antibody (1:50; BD Pharmingen™, USA), washed twice with PBS, and followed by incubation with a Rhodamine-conjugated second antibody (Abcam, USA). MVD was determined by counting the number of microvessels per high-power field in the sections with a fluorescence microscopy as described.
Quantitative assessment of apoptosis
Tumor sections were prepared as described above. Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining was done using an in situ cell death detection kit (DeadEnd™ Fluorometric TUNEL System, Promega, Madison, USA) following the manufacturer's protocol. It is based on the enzymatic addition of digoxigenin-nucleotide to the nicked DNA by the recombinant terminal deoxynucleotidyl transferase (rTdT) . In the tissue sections, four equal-sized fields were randomly chosen and analyzed. The apoptotic index was calculated as a ratio of the apoptotic cell number to the total tumor cell number in each high-power field.
Detection of hPEDF concentrations in serum and ascites
Quantitation of hPEDF expression in serum and ascites was determined by enzyme-linked immunosorbnent assay (ELISA). A commercial PEDF ELISA kit (ADL, Biotech. Dev. Co., USA) was used following the manufacturer's instructions. Briefly, pre-diluted serum or ascites were added to wells of the pre-coated ELISA plate and incubated for one hour. After washing, HRP-conjugated secondary antibody was added. Antibody binding was detected with substrate A and B. After the reaction was stopped with H2SO4, the absorbance at 450 nm was measured by an ELISA reader (Bio-Rad 680, USA).
To investigate potential toxicity or side effects of rAAV2-hPEDF, mice were observed continuously during the treatment process, including the general conditions (the activity, energy, hair, feces, behavior pattern, and other clinical signs), body weight, and mortality. For histopathological studies, samples were obtained from major organs including heart, liver, spleen, lungs, and kidneys. All the samples were fixed in 10% neutral-buffered formalin, and embedded in paraffin. Then, the tissues were sectioned and stained with hematoxylin and eosin (H&E).
The statistic analysis was carried out using SPSS 15.0 software (Chicago, IL, USA). Comparison of numbers of tumor nodes was performed using one-way analysis of variance (ANOVA). Survival curves were generated based on the Kaplan-Meier method and statistical significance was determined by Mann-Whitney U-tests. A P value < 0.05 on a 2-tailed test was considered statistically significant.
Expression of hPEDF in colonic cancer cells after rAAV-mediated gene transfer in vitro
rAAV2-hPEDF was successfully constructed (Figure 1A). TEM was used to determine the morphological characteristics of rAAV2-hPEDF particles. As presented in Figure 1B, rAAV2-hPEDF particles were non-enveloped icosahedral shape with a diameter of approximately 20 nm. To determine whether hPEDF could be secreted by rAAV2-hPEDF-infected cells, we analyzed the conditioned medium from CT26 cells infected in vitro with either rAAV2-hPEDF or control vector rAAV2-null by Western blotting analysis using a monoclonal antibody against hPEDF. As shown in Figure 1C, in the conditioned medium from rAAV2-hPEDF-infected cells, only one band with molecular weight of 50 kDa was observed, which coincided with the lane loaded with hPEDF protein. There was no hPEDF expression in the conditioned medium from the cells infected with control vector rAAV2-null or NS control. This result suggested that rAAV2-hPEDF could transfer hPEDF gene into cultured cells and produce secretory protein.
Biological activity of hPEDF produced by rAAV2-hPEDF in vitro
To evaluate the bioactivity of secreted hPEDF from CT26 cells infected with rAAV2-hPEDF, HUVEC proliferation assay and endothelial cell capillary-like tube formation assay were conducted.
These results indicated that the hPEDF secreted by infected cells was highly bioactive and could block angiogenesis in vitro by inhibiting the proliferation and reducing the formation of tube-like structure of endothelial cells.
Inhibition of tumor growth and metastasis with rAAV2-hPEDF
Inhibition of tumor angiogenesis
Induction of tumor apoptosis
hPEDF concentrations in serum and ascites
Colorectal carcinoma is one of the most frequent cancers, and is the third leading cause of cancer-associated deaths worldwide . Besides liver metastasis and regional lymph nodes metastasis, peritoneal carcinomatosis of colon cancer occurs in 12-20% of patients whose disease recurs [38, 39]. However, therapeutic effects of conventional treatments, such as surgical resection, chemotherapy and radiotherapy, are not very encouraging. Therefore, unremitting efforts were focused on developing novel therapeutic strategies. Data presented in this study showed that AAV-mediated delivery of hPEDF could prevent tumor growth and metastasis and prolong the survival time in murine CRPC model (Figure 4A to 4D).
Angiogenesis is essential to maintain tumor growth and metastasis, therefore antiangiogenic therapy is a feasible approach for cancer therapy. In recent years, a variety of angiogenesis inhibitors have been discovered, among which PEDF is the most promising one. Based on previous studies, down-regulation of PEDF e was very prevalent in a range of tumors . There are two major pathways by which PEDF exerts its antitumor effects: antiangiogenesis and apoptosis-mediated tumor suppression. In this study, we examined biological activity of hPEDF produced by rAAV2-hPEDF in vitro and in vivo. In HUVEC proliferation assay, the conditioned medium from cells infected with rAAV2-hPEDF inhibited HUVEC proliferation by 59.9 ± 8.1% at a 1:2 dilution concentration (Figure 2). In HUVEC tube formation assay, treatment with the conditioned medium from cells infected with rAAV2-hPEDF dramatically reduced the tube formation of HUVECs by 85.8% ± 4.7% (Figure 3). Furthermore, sections of tumors stained for CD31 immunofluorescence showed that the MVD of tumors in rAAV2-hPEDF-treated mice was reduced by 62.1 ± 9.6% compared with control group. In immunofluorescent TUNEL staining assay, the apoptosis index in rAAV2-hPEDF group increased by 6.5 times compared with NS group (Figure 6). Therefore, our in vitro and in vivo results suggested that suppression of tumor growth and metastasis by rAAV2-hPEDF was associated with the decreased MVD and induction of apoptosis in tumors.
Antiangiogenic therapy requires constant therapeutic levels of antiangiogenic factors in vivo to achieve its therapeutic effect, therefore recombinant proteins are limited for widespread clinical use . It is because high therapeutic doses of recombinant proteins are needed, but resultant yield rates of recombinant proteins may be low due to denaturizing in purification process. Furthermore, owing to the short half-life of protein in vivo, maintaining therapeutically effective serum levels needs a frequent dosing regimen and high doses of expensive purified recombinant proteins. Thus, efforts should be made to develop effective and wide-applied strategy, and one potential solution for this is gene therapy. The key of successful gene therapy is gene delivery system. AAV, as one of the most promising viral vectors for human gene therapy, has many advantages compared to other viral vectors, such as non-pathogenicity, low immunogenicity, and long-term undiminished transgene expression in vivo. Since the first infectious clone of AAV2 was established in 1982, AAV2 vectors have gained increasing attention in gene therapy applications [41, 42]. AAV2 have been extensively investigated in preclinical studies for many diseases, including hemophilia, rheumatoid arthritis, cystic fibrosis, and etc [42, 43]. However, further studies suggested that most humans exposed to AAV2 would develop neutralizing antibodies to the vector, which hindered its transduction efficiency [44, 45]. Although the application of AAV2 hampers clinical potential of this approach, the use of well-studied AAV2 will provide preliminary proof of efficacy which would warrant future studies using vectors derived from other serotypes. In this study, we successfully constructed and produced rAAV2-hPEDF, and the titer of rAAV2-hPEDF was 4 × 1012 v.g./mL. Besides, the morphological characteristics of rAAV2-hPEDF were detected by TEM, which exhibited that rAAV2-hPEDF particles were non-enveloped icosahedral shape with a diameter of approximately 20 nm (Figure 1B). A single intraperitoneal administration of rAAV2-hPEDF resulted in suppression of tumor growth and metastasis in the CRPC mouse model with maximum inhibition of 75.85% (Figure 4B). Besides, our results indicated that a single intraperitoneal administration of rAAV2-hPEDF could increase hPEDF levels in serum and ascites for a long period (Figure 7), which could continue to inhibit angiogenesis in tumor. During the treatment process, no severe toxic effects were observed, and no histopathological changes were found in major organs (Figure 4E). All these data confirmed that AAV is a safe and efficient gene delivery vector.
In conclusion, our studies demonstrated that rAAV2-hPEDF could infect cells and produce secretory hPEDF protein, which was proved to be functional on inhibiting proliferation and tube-formation of HUVECs in vitro. Moreover, rAAV2-hPEDF significantly suppressed tumor growth and metastasis and prolonged survival time of treated mice in CRPC model. rAAV2-hPEDF could increase hPEDF levels in serum and ascites, inhibit angiogenesis, and induce apoptosis in tumor tissues. Therefore, our results indicated that rAAV2-hPEDF may be a potential candidate as an antiangiogenesis therapy agent for cancer gene therapy.
One-way analysis of variance
Basic fibroblast growth factor
Colorectal peritoneal carcinomatosis
Dulbecco's Modified Eagle's medium
Enzyme-linked immunosorbnent assay
Fetal bovine serum
Human pigment epithelium-derived factor
Human umbilical vein endothelial cells
Hematoxylin and eosin
Methyl thiazolyl tetrazolium
Pigment epithelium-derived factor
Recombinant adeno-associated virus
Recombinant adeno-associated virus serotype 2 vectors
Recombinant adeno-associated virus serotype 2 vectors encoding human pigment epithelium-derived factor
Terminal deoxynucleotidyl transferase-mediated nick-end labeling
Vascular endothelial growth factor.
This study was funded by the National Key Basic Research Program (973 Program) of China (2010CB529900).
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