- Open Access
Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma
Journal of Experimental & Clinical Cancer Research volume 28, Article number: 75 (2009)
Angiogenesis plays an important role in tumor growth, invasion, and eventually metastasis. Antiangiogenic strategies have been proven to be a promising approach for clinical therapy for a variety of tumors. As a potent inhibitor of tumor angiogenesis, pigment epithelium-derived factor (PEDF) has recently been studied and used as an anticancer agent in several tumor models.
A recombined adenovirus carrying PEDF gene (Ad-PEDF) was prepared, and its expression by infected cells and in treated animals was confirmed with Western blotting and ELISA, respectively. Its activity for inhibiting human umbilical vein endothelial cell (HUVEC) proliferation was tested using the MTT assay. C57BL/6 mice bearing B16-F10 melanoma were treated with i.v. administration of 5 × 108 IU/mouse Ad-PEDF, or 5 × 108 IU/mouse Ad-Null, or normal saline (NS), every 3 days for a total of 4 times. Tumor volume and survival time were recorded. TUNEL, CD31 and H&E stainings of tumor tissue were conducted to examine apoptosis, microvessel density and histological morphology changes. Antiangiogenesis was determined by the alginate-encapsulated tumor cell assay.
The recombinant PEDF adenovirus is able to transfer the PEDF gene to infected cells and successfully produce secretory PEDF protein, which exhibits potent inhibitory effects on HUVEC proliferation. Through inhibiting angiogenesis, reducing MVD and increasing apoptosis, Ad-PEDF treatment reduced tumor volume and prolonged survival times of mouse bearing B16-F10 melanoma.
Our data indicate that Ad-PEDF may provide an effective approach to inhibit mouse B16-F10 melanoma growth.
Melanoma is a malignant tumor derived from melanocytes which are found predominantly in skin but also in the bowel and the eye. Approximately 160,000 new cases of melanoma are diagnosed and around 48,000 melanoma-related deaths occur worldwide each year . Despite many years of intensive research, surgical resection and systemic chemotherapy are still the main therapeutic strategies for malignant melanoma. Unfortunately, for advanced melanoma, surgical resection is insufficiently effective while chemotherapy introduces significant side effects . Compared to other type of skin cancer, melanoma is more rare but often associated with a high mortality, accounting for 75% of all deaths from skin cancer . To explore new therapeutic agents/methods with less side effects is a major initiative in melanoma research.
One hallmark of melanoma progression is angiogenesis, which is induced by angiogenic factors released by tumor cells and characterized as the formation of a new vascular network from pre-existing blood vessels. Angiogenesis facilitates tumor growth by supplying nutrients and oxygen, while promoting tumor invasion and metastases . Antiangiogenesis has been proposed as a therapeutic strategy for cancer treatment since the 1970s, but it has been limited by the unavailability of antiangiogenic agents and/or inefficient administration methods. In the past two decades, several antiangiogenic factors, such as angiostatin, endostatin, thrombospondin and pigment epithelium-derived factor (PEDF), have been found and characterized . As a new family of anti-tumor agent candidates, they are under active investigation by many researchers. Accumulating data show antiangiogenic agents have promising efficacy in tumor treatment . Because PEDF selectively and potently suppresses new vessel growth with least impact on pre-existing vessels, it is one of the top candidates for tumor therapy .
PEDF is a 50 kDa glycoprotein that was first purified from the conditioned medium of cultured primary human fetal retinal pigment epithelial cells . Later, it was found that PEDF is widely expressed in human tissues, including the adult brain, spinal cord, plasma, liver, bone, eye, heart, and lung . PEDF is a multi-functional serpin family protein. It has been reported that it activates the Fas/FasL death pathway and subsequently induces endothelial cell death, and also regulates the balance between proangiogenic and antiangiogenic factors . One prominent feature of PEDF is the selective inhibition of neovascularization, which is extremely important to minimize the side effects in tumor treatment. The underlying mechanism is still not well understood, but it has prompted scientists to apply it in cancer treatment in a variety of forms including purified, recombinant, PEDF peptide 327 to 343, and gene transfer . Adenovirus is the widely utilized gene transfer vehicle in a variety of gene therapies; however, adenovirus-mediated gene transfer of PEDF for tumor treatment is rarely reported.
In this study, we constructed a recombinant PEDF-expressing adenovirus (Ad-PEDF) and tested its anti-tumor efficacy in a mouse B16-F10 melanoma model. Our data indicate that the Ad-PEDF treatment of melanoma-bearing mice results in an increase of serum PEDF and reduction of tumor angiogenesis, growth, and animal death. The adenovirus-mediated gene transfer of PEDF is thus a promising therapeutic strategy for melanoma and other angiogenic tumors.
Recombinant adenovirus construction and viral preparation
According to the cDNA sequence of PEDF in genebank, we designed a pair of PEDF primers that contain a Pme I restriction site (underlined in the following) in both primers (5'-AGCTTTGTTTAAAC ATGCAGGCCCTGGTGCTACTCCTC-3' and 5'-AGCTTTGTTTAAAC TTAGGGGCCCCTGGGGTCCAGAATC-3'). Using these primers, we amplified human PEDF cDNA with RT-PCR. PCR product was digested with Pme I and its sequence was confirmed. Using AdEasy system, we first clone PEDF cDNA into a shuttle vector pAdenoVator-CMV5 at Pme I and Bam H I site, in which PEDF expression is under the control of the constitutive cytomegalovirus (CMV) promoter. The recombinant shuttle plasmid was used to rescue the replication-defective adenovirus . Ad-luciferase and Ad-Null was prepared as the construction of Ad-PEDF, except luciferase gene or no objective gene was inserted.
The viral particles were amplified in human embryonic kidney (HEK293) cells (ATCC Rockville Maryland, USA), which were maintained in DMEM medium (Gibico BRL, Grand Island, New York, USA) with 10% fetal bovine serum (FBS) plus 100 μg/ml amikacin in a 37°C humidified chamber with 5% CO2 atmosphere. The harvested viral particles from the cultures were purified by double cesium chloride (CsCl) gradient ultracentrifugation followed by dialysis. Final aliquots of virus were measured by absorption (A260). The virus titer was quantified using a standard TCID50 assay.
Cell culture and adenovirus infection
Primary human umbilical vein endothelial cells (HUVECs) were collected and cultured as previously described . The CT26 mouse colon carcinoma and B16-F10 mouse melanoma cell lines were purchased from the American Type Culture Collection (ATCC, Rockville Maryland, USA) and cultured in RM1640 medium (Gibico BRL, Grand Island, New York, USA) supplemented with 10% FBS and 100 μg/ml amikacin. 2.5 × 105 CT26 or B16-F10 cells were plated into 6-well plates and grew to 70%~80% confluence. The cells were washed three times gently by serum-free medium and infected with Ad-PEDF or Ad-null (both at MOI50, 2.5 × 107 pfu per 5 × 105 cells) in 0.5 ml serum-free medium, with normal saline as the non-infection control. After incubation for 90 minutes at 37°C, 1.5 mL complete medium with 10% FBS was added to wells. Supernatants were collected after further culture for 72 hours and stored at -80°C for further analysis.
Western blotting analysis
Western blot analysis was performed as described previously . Briefly, the supernatant was concentrated by super filter (5 kDa, Minipore) and mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer. The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electronically transferred onto a polyvinylidene difluoride membrane (PVDF, Bio-Rad, Richmond, CA, USA). The blots were probed with a mouse anti-human PEDF monoclonal antibody (3:1000, mAb; R&D Systems, Boston, Massachusetts, USA) plus a peroxidase-conjugated secondary antibody, goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China). The protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, Illinois, USA).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay
The MTT Assay was used to determine the effect of PEDF derived from Ad-PEDF infected cells on human umbilical vein endothelial cells (HUVECs). Three types of supernatants from B16-F10 cells infected with Ad-PEDF, Ad-Null or NS, respectively, were prepared as described above. Each type of supernatant was further diluted into a series of 1/2 dilutions in six tubes (from 1:2 to 1:64). Each supernatant dilution was added into triplicate wells (50 μl/well) of HUVECs which were seeded on 96-well plates on the previous day (2 × 103 cells in 50 μl complete medium per well). The cells were incubated at 37°C in 5% CO2 for 72 hours. Then, each well received 10 μl MTT solution (5 mg/mL). After a 4-hour incubation, the media was removed and 150 μl dimethyl sulfoxide was added. After 20 min of incubation, the OD value was determined by a microplate reader (3550-UV, BIO-RAD, USA). The following formula was used to calculate the inhibition rate of HUVEC proliferation: [1 - (experimental group OD value - negative control OD value)/(positive control OD value - negative control OD value)] × 100%.
Six to 8 week-old female C57BL/6 and BALB/c mice were purchased from the West China Experimental Animal Center of Sichuan University (Sichuan, China). Mice were permitted 1 week to acclimate to their environment before manipulation. All surgical procedures were completed in accordance with the guidelines on the care and use of laboratory animals for research purposes by the West China Hospital Cancer Center's Animal Care and Use Committee. C57BL/6 mice were inoculated with 1 × 105 B16-F10 melanoma cells s.c. in the right flank. Primary tumors usually became palpable on day 7–8 and with an average diameter of 3 mm. On day 9, the tumor-bearing mice were randomly assigned into 3 groups and each group contained 8 mice. Each mouse in Ad-PEDF group received 5 × 108 IU Ad-PEDF virus in 0.1 ml via i.v. injection on day 9, 12, 15, and 18 with a total of 4 times. The mice in the control groups received 5 × 108 IU Ad-Null or normal saline (NS), serving as vector and injection control, respectively. The details of the treatment were described previously . Tumor dimensions were measured with calipers on day 9, 12, 15, 18, 21 and 24 with a total of 6 times. The tumor volumes were calculated according to the following formula: length × width2 × 0.52. Two mice from each group were bled to collect serum on day 22, which was used to examine the PEDF concentration in serum. Surviving mice in Ad-PEDF groups were monitored up to 42 days; all other mice become moribund by day 24 and were sacrificed. Subcutaneous tumors from sacrificed mice were removed and fixed in 4% formaldehyde solution for immunochemistry staining and histological analysis.
Detection of PEDF concentration in serum
Concentrations of PEDF in serum were determined using a commercial PEDF ELISA kit (ADL, Biotech. Dev. Co., USA) following the manufacturer's instructions. Briefly, 50 μl serum and 50 μl PEDF monoclonal antibody were added to every well of the pre-coated ELISA plate and the plate was incubated at 37°C for 1 hour. After wash, 80 μl of streptavidin-HRP was added and incubated at 37°C for 30 minutes. After wash, 50 μl substrate A and B was added, respectively, and incubated for 10 minutes at 37°C, followed by 50 μl stop solution. The absorbance was read immediately at 450 nm in a spectrophotometer . There were 2 serum samples in each group, and each sample were applied to 3 replicated wells.
Luciferase assay for virus distribution
Virus distribution was analyzed using the luciferase reporting system, as reported previously . C57BL/6 mice were inoculated with 1 × 105 B16-F10 melanoma cells s.c. in the right flank. On day 9, the tumor-bearing mice were randomly assigned into 2 groups and each group contained 3 mice. Experimental group received 5 × 1010 IU Ad-luciferase and control group received 5 × 1010 IU Ad-null virus in 0.1 ml via i.v. injection. Seven days later, the mice were sacrificed. Heart, liver, spleen, lung, kidney and tumor from each mouse were collected and individually stored in liquid nitrogen. Using a luciferase assay system kit (Promega, Madison WI, USA), we analyzed luciferase expression in each type of collected organ, according to the manufacturer's instructions. Briefly, the same organs from the same group were pooled and ground to a fine powder in a mortar containing liquid nitrogen. The fine powder was dissolved and further processed in CCLB solution in the assay kit. The resultant supernatants were collected and subjected to determination of relative light units (RLU, synergy 2, Biotek, Germany), along with a group of standard samples in the kit. The amount of luciferase in each sample was calculated on the basis of the standard curve.
Detection of apoptosis and microvessel density (MVD)
On day 24 after mouse inoculation with melanoma cells, subcutaneous tumors from Ad-PEDF, Ad-null and NS treated mice were collected, fixed, embedded in paraffin, and cut into 3–5 μm sections. The apoptotic cells within the tumor tissue were evaluated using the DeadEnd Colorimetric Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) System (Promega, Corporation, Madison, Wisconsin, USA) following the manufacturer's protocol. Ten high power fields on each slide and three slides from each animal were examined. Apoptosis index was calculated by dividing the number of apoptotic cells by the total number of cells in the field.
The method reported by Weidner et al was adopted to quantify MVD in tumor tissues . Briefly, 5 μm tumor sections were stained for the epithelial cell marker, CD31. The procedure of immunostaining for CD31 was previously described in detail . The following antibodies and reagents were used: goat anti-mouse CD31 mAb (1:200, Santa Cruz Biotechnology, Santa Cruz, California, USA), biotinylated polyclonal rabbit anti-goat (1:100, Santa Cruz Biotechnology, Santa Cruz, California, USA), ABC kit (Boster biological engineering company, Wuhan, China) and DAB visualization system (ZSJQ Biotechnology, Beijing, China). The resultant sections were first examined at low magnifications (×40 and ×100) to identify the vascular-rich area in the tumor. Within this area, the CD31-positive microvessels were counted in a single high-power (×200) field. Any CD31 stained single or cluster of cells was considered a single countable microvessel. Adjacent sections were stained with hematoxylin and eosin (H&E) and examined for tissue structure and histological morphology. Each group contains 2 mice, and 3 sections from each mouse.
Alginate-encapsulated tumor cell assay
The alginate-encapsulated tumor cell assay was used to measure tumor angiogenesis in vivo, as previously described [14, 18]. Briefly, B16-F10 or CT26 cells in 1.5% (m/v) sodium alginate solution (Sigma-Aldrich, St. Louis, Missouri, USA) was dropped into a swirling 0.25 M CaCl2 solution to prepare alginate beads (1 × 105 cells/bead). Four resultant beads were implanted s.c. on both dorsal sides of BALB/c female mice (2 beads/side). On the next day, mice were randomly assigned into 3 groups (n = 2), and each group was i.v. injected with 0.1 ml Ad-PEDF (5 × 108IU/mouse), Ad-Null (5 × 108 IU/mouse), or NS, respectively. After a week, this same treatment on each mouse was repeated. On day 11 after tumor cell implantation, all mice were injected i.v. with 100 μl FITC-dextran (Sigma-Aldrich, St. Louis, Missouri, US) solution (100 mg/ml), which is a plasma-borne tracer extravasating into tissue interstitial fluid from plasma within 20 minutes. Alginate beads were exposed surgically and photographed with a digital camera (model, Canon, Japan). Then, the beads were removed and vortexed in a tube containing 2 ml NS. After centrifugation, the supernatant was collected and subjected to a fluorescence spectrophotometer for the measurement of fluorescence intensity. The amount of FITC-dextran was calculated and used to estimate the amount of blood supply and angiogenesis status.
SPSS program (version 15.0, SPSS Inc., USA) was used for statistical analysis. Log-rank test was used to compare survival rate among groups. ANOVA was used to determine statistical significances in remaining comparisons in this study. The difference is considered as significant if p < 0.05.
Recombinant Ad-PEDF virus successfully transferred PEDF gene into tumor cells and produced secretory PEDF protein in vitro
Whether an adenovirus-mediated gene transfer is successful or not mainly depends on its capacity to infect host cells and express the recombinant gene. Therefore, we first tested whether our recombinant Ad-PEDF virus is capable of infecting cells and expresses PEDF protein in vitro. CT26 and B16-F10 cell lines were infected with Ad-PEDF, Ad-null or treated with normal saline (NS). Three types of supernatant from each cell line were prepared and subjected to Western blotting analysis. As shown in Fig. 1, PEDF was detected in supernatant from both cell lines infected by Ad-PEDF virus, but neither in Ad-null infected nor NS treated cells. These results indicate that our recombinant adenovirus successfully transfers the PEDF gene into cultured cells and produces secretory protein.
PEDF protein from Ad-PEDF infected cells exhibited a potent inhibitory effect on HUVEC proliferation
Next, we tested whether Ad-PEDF from infected cell possess inhibitory bioactivity on the proliferation of epithelial cells. Using the MTT assay, we measured HUVEC cell proliferation and viability after treatment of supernatant from Ad-PEDF infected B16-F10 cells or control supernatant. As shown in Fig 2, supernatant from Ad-PEDF infected B16-F10 cells significantly inhibited the proliferation of HUVEC with an inhibitory rate of 68.8% ± 1.5% at a 1:2 dilution. Furthermore, this inhibitory rate declines with the increase of dilution, suggesting a dose-dependent effect. In contrast, the control supernatant from Ad-Null infected or NS treated B16-F10 cells had no effect on HUVEC proliferation, which did not change with the dilution. These results indicate that secretory PEDF is functional and capable of mediating a potent inhibitory effect on HUVEC proliferation.
Ad-PEDF treatment inhibited tumor growth in vivo and prolonged the survival time of the tumor-bearing mice
After confirmation of the success for PEDF gene transfer and expression of functional PEDF protein in vitro, we examined the anti-tumor efficacy of Ad-PEDF treatment in a mouse tumor model. As shown in Fig 3A, from day 21 after tumor cell inoculation, the tumor volume in Ad-PEDF treated mice started to show significant differences from those in controls (p < 0.05). Tumor volumes in the Ad-PEDF treated group was 1447.8 ± 244.4 mm3, in contrast to 2337.4 ± 365.8 mm3 in Ad-Null group and 2578.2 ± 406.7 mm3 in NS group on day 21. On day 24, the tumor size in Ad-PEDF, Ad-null and NS groups were 2195.1 ± 462.9 mm3, 4013.3 ± 518.3 mm3, and 4361.3 ± 569.6 mm3, respectively. The time of mouse death was recorded and used to calculate the survival rate. As shown in Fig 3B, the NS treated group showed 50% survival at day 13 and 0% on day 23, and the Ad-null group showed 50% survival at day 14 and 0% on day 24. In contrast, Ad-PEDF group had a 50% survival rate at day 38 and persisted up to day 42. Log-rank test indicated that survival rate in Ad-PEDF group is significantly higher than in control groups (p < 0.05)
Ad-PEDF treatment increased serum PEDF and adenovirus mainly distributed in liver
To assess whether the above observed smaller tumor volume and increased survival rate in Ad-PEDF group is associated with the effect of PEDF, we measured PEDF concentration in the serum. As shown in Fig 4A, on day 22 after tumor cell inoculation, PEDF level in Ad-PEDF group was significantly higher than control groups, 77.36 ± 3.78 ng/ml vs 33.62 ± 2.79 ng/ml in Ad-null and 36.87 ± 3.35 ng/ml in NS groups, respectively (p < 0.05). This result indicates that Ad-PEDF successfully transferred PEDF to mice and produced secretory PEDF proteins.
Next, we determined the source of PEDF by analyzing the distribution of i.v. injected virus. As shown in Fig 4B, using the luciferase reporting system, we found that the viruses mainly distributed in the liver, in agreement with many adenovirus infection models. This result suggests that while Ad-PEDF infected multiple organs, including the tumor, the liver is the major organ that adenovirus targeted and likely is the significant source of the serum PEDF.
Ad-PEDF treatment increased apoptosis and decreased MVD in tumor tissue
In the proceeding experiments, we observed the reduced tumor volume and increased serum PEDF after Ad-PEDF treatment, in comparison to control, however, the majority of the virus was entrapped in liver and did not target the tumor tissue. It is important to demonstrate whether serum PEDF indeed acts on tumor tissue and causes histological change. To address this question, we determined apoptosis in tumor tissue after Ad-PEDF treatment using TUNEL staining. As shown in Fig 5A, within a similar field of view, may more apoptotic cells (with green nuclei) in tumor tissues were observed in Ad-PEDF treated mice than in Ad-null or NS treated mice. For the quantitative comparison, the apoptosis index in each group was calculated. The apoptosis index was significantly higher in Ad-PEDF group than in Ad-Null and NS groups with values of 26.3% ± 3.3% v.s. 6.3% ± 4.7% and 5.6% ± 1.9%, respectively (p < 0.05, Fig 5B). These data suggest that decreased tumor volumes after Ad-PEDF may be caused by increased apoptosis.
To further determine whether the increase in apoptosis of Ad-PEDF treated tumor tissue was associated with the antiangiogenic effect of PEDF, we analyzed MVD of tumor tissues in each group. As shown in Fig 5C, intensive CD31 immunoreactive microvessels was observed in tumor tissue from mice treated by NS and Ad-null, but only moderate CD31 staining present in tumor tissue from mice treated by Ad-PEDF. For comparison, CD31-positive single or a cluster of cells were counted as the microvessels, and MVD was calculated for each group with the formula described in the materials and methods. MVD of tumor tissues from Ad-PEDF treated mice exhibited a significant decrease than from Ad-null or NS treated mice, (21 ± 4, 54.3 ± 7.2, 62 ± 6.5, respectively) (p < 0.05, Fig 5D). These data suggest that the decreased angiogenesis after Ad-PEDF treatment may be responsible for the increased apoptosis.
Adjacent sections were stained with H&E to evaluate the morphologic changes after Ad-PEDF or control treatments. Consistent with the results of CD31 immunochemistry staining, less vessels and remarkable necrosis areas were observed in tumor tissue from Ad-PEDF treated mice in comparison to Ad-null or NS treated mice (Fig 5E). Collectively, these data suggest that serum PEDF from infected host cells is sufficient to inhibit tumor angiogenesis, subsequently promote apoptosis, reduce tumor progression and prolong survival time.
Ad-PEDF treatment inhibited the development of tumor angiogenesis
To confirm the proceeding finding that PEDF from Ad-PEDF gene transfer is associated with the reduction of tumor angiogenesis, and to directly demonstrate the causal relationship, we performed the alginate-encapsulated tumor cell assay, which is capable of demonstrating whether the development of tumor angiogenesis is prevented by PEDF treatment. As shown in Fig 6, the intensity of blood vessels on the surface of tumor cell-containing alginate beads was noticeably less in Ad-PEDF-treated mice than Ad-null or NS treated mice (Fig 6A). The quantification results for the amount of FITC-dextran indicate that the distribution of extravasated tracer in the encapsulated tumor tissues was consistent with the distribution of blood vessels on the bead surface; the amount of FITC-dextran per beads in Ad-PEDF, Ad-null and NS group was 2.1 ± 0.3 μg/bead, 5.8 ± 0.3 μg/bead and 6.2 ± 0.6 μg/bead, respectively. Similarly, an experiment with encapsulated B16-F10 cells has been performed and its result was similar to the result for CT-26 cells. (Data were not shown). These data confirmed that Ad-PEDF-mediated PEDF gene transfer and expression is responsible for the inhibition of tumor angiogenesis in the studied tumor model.
Angiogenesis is required for a variety of physiological and pathological processes. It is a complex biological process under precise regulation of multiple factors in multiple steps . There are two groups of reciprocally antagonizing factors, proangiogenitors and antiangiogenitors. The former includes vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF) and fibroblast growth factor (FGF), and the latter includes angiostatin, endostatin, thrombospondin and PEDF. PEDF is notable for its inhibitory function responsible for the avascularity of ocular compartments by opposing the angiogenic effect of VEGF in eye. It has been shown that PEDF is the most potent endogenous inhibitor of angiogenesis; its potency is twice that of angiostatin and seven times of endostatin [5, 20]. Recently, data showed that PEDF also plays important roles in regulating normal development and tumor growth. For example, a recent study showed that the expression of PEDF is inversely proportional to the expression of VEGF at the growth plate of cartilage and is involved in the control of osteosarcoma . In addition, it has been reported that PEDF could significantly inhibit neuroblastoma and Wilms' tumor [22, 23]. A decrease of PEDF results in a tumor-permissive environment and promotes tumor growth and metastasis . It is generally thought that PEDF's anti-tumor activity is the extended function of its antiangiogenic effect, decreasing microvascularity and blood supplying.
In the past decade, researchers have prepared various forms of PEDF and demonstrated its beneficial effects in several tumor models. Doll et al reported that exogenous recombinant PEDF protein induced tumor epithelial apoptosis in mouse prostate and pancreas . Liu et al showed that a short peptide derived from the parent PEDF molecule was able to inhibit osteosarcoma growth . Streck et al reported that adeno-associated virus (AAV) delivering PEDF gene treatment successfully restricted human neuroblastoma engraftment in a dose-dependent fashion . Hase et al demonstrated that intratumoral injection of a lentivirus vector encoding PEDF resulted in inhibition of human pancreatic cancer in nude mice . Moreover, Wang et al showed that in vivo transfer of PEDF mediated by adenoviral vectors exerted a dramatic inhibition of tumor growth in athymic nude mice implanted with the human HCC and in C57BL/6 mice implanted with mouse lung carcinoma . In the present study, we investigated the adenovirus-mediated PEDF gene transfer and tested its anti-tumor effect in a mouse model of melanoma.
Melanoma, a tumor derived from neuroectoderm, has a high malignancy with poor prognosis, due to the vascular and lymphatic metastasis during the late stage . The outcomes of existing therapeutic protocols are very poor. Thus, the development of novel treatment approaches is required . Since the neovascularization is one critical underlying mechanism of vascular and lymphatic metastasis, the current study was designed to investigate whether overexpression of PEDF mediated by adenovirus gene transfer is a potential approach to suppress tumor angiogenesis and inhibit melanoma growth. Encouragingly, we constructed a recombinant PEDF adenovirus that is capable of transferring PEDF gene producing secretory PEDF protein both in vitro and in vivo. Furthermore, we showed that the secretory PEDF is a functional protein with potent inhibitory effects on HUVEC proliferation. More importantly, tumor-bearing mice exhibited significantly reduced tumor volume and prolonged survival time after Ad-PEDF treatment. Finally, we demonstrated that Ad-PEDF exerted anti-tumor activity through inhibiting angiogenesis, reducing MVD and increasing apoptosis.
Adenovirus type 5 is an established and widely used vector for the delivery of therapeutic genes . Although there is no evidence to prove Ad-PEDF has a stronger therapeutic effect on tumors than other PEDF patterns, the adenovirus vector has several properties that make it particularly promising for gene therapy. First, the adenovirus vector can efficiently transfer genes to both dividing and quiescent cells both in vivo and in vitro, and importantly possesses high stability in vivo. Additionally, adenovirus vector can be produced at high titer conveniently, which is essential for clinical utility. Finally, as opposed to the retrovirus vector such as lentivirus, adenoviral DNA does not usually integrate into host cell's genome and therefore has a very low risk of generating tumorigenic mutations. Adenovirus-related pathology is mostly limited to mild upper respiratory tract infections [30, 31].
It is very encouraging that Ad-PEDF treatment resulted in a high level of PEDF expression in serum and caused the inhibition of tumor growth. However, a few questions were left unaddressed in this study. First, this study mainly focused on the primary tumor, it is unknown whether Ad-PEDF treatment is effective in controlling late stage tumor growth, metastasis, and tumor growth in a metastasis site. Based on the assumption that Ad-PEDF may be mainly used as an option for advanced melanoma treatment, investigation of the effect of Ad-PEDF treatment for a late stage melanoma is critical to evaluate its potential clinical value, which is one of our future directions. In addition, adenovirus is highly immunogenic, which induces major humoral and cellular immune response when administered systemically . These immune responses result in a quick clearance of virus when they are re-administered. While the adenovirus-induced humoral immune response leads to the antibody-mediated neutralization of virus in circulation, the cell-mediated immune response results in lysis of adenovirus-infected cells and loss of transferred gene. To prevent this quick clearance, we treated animals with multiple injections of Ad-PEDF every 3 days in this study. Although we used a lower dose than in the literature, the optimal window for effective dose and toxicity of this treatment is still to be determined. Furthermore, consistent with previous observation, Ad5, used in the present study was mainly directed to the liver, probably, via the vitamin K-dependent coagulation zymogens or other plasma protein-directed mechanisms . We speculate that the secretory PEDF from non-tumor tissues is first released into the blood, then circulates to tumor tissue and exerts the antiangiogenesis effect. It appears not necessary to avoid the liver uptake of virus in our model and liver is probably the major source of the serum PEDF after Ad-PEDF treatment. However, because of the potential and undefined side effects and to further increase anti-tumor efficacy, modification of vector or optimization of delivery route to direct viruses into tumor tissue is critical to translate this study to an applicable therapeutic option for patients.
It has been shown that the liposome system can reduce adenoviral immunogenicity, increase localization of virus, and allow successful re-administration of the virus without loss of gene expression efficiency . Therefore, we developed an Ad-PEDF-liposome system and are under active investigation, aiming to address the above mentioned unanswered issues. In addition, to further increase efficacy and limit side effects, we are also exploring the bi-specific antibody strategy to retarget the Ad-PEDF adenovirus to melanoma tumor tissue, as Reynolds et al prepared a targetable adenovirus-mediated gene transfer to pulmonary endothelium [33, 34]
In summary, until the current study, research for experimental melanoma treated with Ad-PEDF had not been reported. Our data validate that Ad-PEDF treatment can exert an inhibitory effect on tumor angiogenesis. While the adenovirus-mediated PEDF gene therapy may provide a promising approach for primary melanoma treatment, we are still exploring the strategies for reducing its side effects and improving the tropism of Ad-PEDF to tumor.
Our study indicates that adenovirus-mediated PEDF gene transfer and expression can provide an effective approach to inhibit primary mouse B16-F10 melanoma growth.
Ries LAG: SEER Cancer Statistics Review, 1975–2000. 2003, Bethesda, MD: National Cancer Institute, Tables XVI-1-9
Hocker TL, Singh MK, Tsao H: Melanoma genetics and therapeutic approaches in the 21st century: moving from the benchside to the bedside. J Invest Dermatol. 2008, 128: 2575-95. 10.1038/jid.2008.226.
Jerant AF, Johnson JT, Sheridan CD, Caffrey TJ: Early detection and treatment of skin cancer. Am Fam Physician. 2000, 62: 357-68.
Folkman J, Shing Y: Angiogenesis. J Biol Chem. 1992, 267: 10931-4.
Ek ET, Dass CR, Choong PF: Pigment epithelium-derived factor: a multimodal tumor inhibitor. Mol Cancer Ther. 2006, 5: 1641-6. 10.1158/1535-7163.MCT-06-0107.
Folkman J: Tumor angiogenesis. Adv Cancer Res. 1985, 43: 175-203. 10.1016/S0065-230X(08)60946-X.
Tombran-Tink J, Johnson LV: Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells. Invest Ophthalmol Vis Sci. 1989, 30: 1700-7.
Volpert OV, Zaichuk T, Zhou W, Reiher F, Ferguson TA, Stuart PM, Amin M, Bouck NP: Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor. Nat Med. 2002, 8: 349-57. 10.1038/nm0402-349.
Uehara H, Miyamoto M, Kato K, Ebihara Y, Kaneko H, Hashimoto H, Murakami Y, Hase R, Takahashi R, Mega S, Shichinohe T, Kawarada Y, Itoh T, Okushiba S, Kondo S: Expression of pigment epithelium-derived factor decreases liver metastasis and correlates with favorable prognosis for patients with ductal pancreatic adenocarcinoma. Cancer Res. 2004, 64: 3533-7. 10.1158/0008-5472.CAN-03-3725.
He TC, Zhou SB, DA Costa LT, YU J, Kinzler KW, Vogelstein B: A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998, 95: 2509-2514. 10.1073/pnas.95.5.2509.
Beekhuizen H, Gevel van de JS, Olsson B, van Benten IJ, van Furth R: Infection of human vascular endothelial cells with Staphylococcus aureus induces hyperadhesiveness for human monocytes and granulocytes. J Immunol. 1997, 158: 774-82.
Wei YQ, Zhao X, Kariya Y, Fukata H, Teshigawara K, Uchida A: Induction of apoptosis by quercetin: involvement of heat shock protein. Cancer Res. 1994, 54: 4952-7.
Li Q, Wei YQ, Wen YJ, Zhao X, Tian L, Yang L, Mao YQ, Kan B, Wu Y, Ding ZY, Deng HX, Li J, Luo Y, Li HL, He QM: Induction of apoptosis and tumor regression by vesicular stomatitis virus in the presence of gemcitabine in lung cancer. Int J Cancer. 2004, 112: 143-9. 10.1002/ijc.20276.
He QM, Wei YQ, Tian L, Zhao X, Su JM, Yang L, Lu Y, Kan B, Lou YY, Huang MJ, Xiao F, Liu JY, Hu B, Luo F, Jiang Y: Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice. J Biol Chem. 2003, 278: 21831-6. 10.1074/jbc.M300880200.
Shao C, Sima J, Zhang SX, Jin J, Reinach P, Wang Z, Ma JX: Suppression of corneal neovascularization by PEDF release from human amniotic membranes. Invest Ophthalmol Vis Sci. 2004, 45: 1758-62. 10.1167/iovs.03-0882.
Ma Z, Mi Z, Wilson A, Alber S, Robbins PD, Watkins S: Redirecting adenovirus to pulmonary endothelium by cationic liposomes. Gene Ther. 2002, 9: 176-82. 10.1038/sj.gt.3301636.
Weidner N, Semple JP, Welch WR, Folkman J: Tumor angiogenesis and metastasis – correlation in invasive breast carcinoma. N Engl J Med. 1991, 324: 1-8.
Peng XC, Yang L, Yang LP, Mao YQ, Yang HS, Liu JY, Zhang DM, Chen LJ, Wei YQ: Efficient inhibition of murine breast cancer growth and metastasis by gene transferred mouse survivin Thr34-->Ala mutant. J Exp Clin Cancer Res. 2008, 27: 46-10.1186/1756-9966-27-46.
Distler JH, Hirth A, Kurowska-Stolarska M, Gay RE, Gay S, Distler O: Angiogenic and angiostatic factors in the molecular control of angiogenesis. Q J Nucl Med. 2003, 47: 149-61.
Hase R, Miyamoto M, Uehara H, Kadoya M, Ebihara Y, Murakami Y, Takahashi R, Mega S, Li L, Shichinohe T, Kawarada Y, Kondo S: Pigment epithelium-derived factor gene therapy inhibits human pancreatic cancer in mice. Clin Cancer Res. 2005, 11: 8737-44. 10.1158/1078-0432.CCR-05-1323.
Dass CR, Ek ET, Choong PF: PEDF as an emerging therapeutic candidate for osteosarcoma. Curr Cancer Drug Targets. 2008, 8: 683-90. 10.2174/156800908786733487.
Streck CJ, Zhang Y, Zhou J, Ng C, Nathwani AC, Davidoff AM: Adeno-associated virus vector-mediated delivery of pigment epithelium-derived factor restricts neuroblastoma angiogenesis and growth. J Pediatr Surg. 2005, 40: 236-43. 10.1016/j.jpedsurg.2004.09.049.
Abramson LP, Browne M, Stellmach V, Doll J, Cornwell M: Reynolds M;Arensman RM;Crawford SE. Pigment epithelium-derived factor targets endothelial and epithelial cells in Wilms' tumor. J Pediatr Surg. 2006, 41: 1351-6. 10.1016/j.jpedsurg.2006.04.038.
Doll JA, Stellmach VM, Bouck NP, Bergh AR, Lee C, Abramson LP, Cornwell ML, Pins MR, Borensztajn J, Crawford SE: Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas. Nat Med. 2003, 9: 774-80. 10.1038/nm870.
Liu H, Ren JG, Cooper WL, Hawkins CE, Cowan MR, Tong PY: Identification of the antivasopermeability effect of pigment epithelium-derived factor and its active site. Proc Natl Acad Sci USA. 2004, 101: 6605-10. 10.1073/pnas.0308342101.
Wang L, Schmitz V, Perez-Mediavilla A, Izal I, Prieto J, Qian C: Suppression of angiogenesis and tumor growth by adenoviral-mediated gene transfer of pigment epithelium-derived factor. Mol Ther. 2003, 8: 72-9. 10.1016/S1525-0016(03)00128-X.
Ota T, Maeda M, Matsui T, Kohno H, Tanino M, Odashima S: Inhibition of metastasis by a dialysable factor in fetal bovine serum in B16 melanoma cells. Cancer Lett. 1996, 110: 201-5. 10.1016/S0304-3835(96)04496-5.
Garcia M, Fernandez-Garcia NI, Rivas V, Carretero M, Escamez MJ, Gonzalez-Martin A, Medrano EE, Volpert O, Jorcano JL, Jimenez B, Larcher F, Del Rio M: Inhibition of xenografted human melanoma growth and prevention of metastasis development by dual antiangiogenic/anti-tumor activities of pigment epithelium-derived factor. Cancer Res. 2004, 64: 5632-42. 10.1158/0008-5472.CAN-04-0230.
Green NK, Morrison J, Hale S, Briggs SS, Stevenson M, Subr V, Ulbrich K, Chandler L, Mautner V, Seymour LW, Fisher KD: Retargeting polymer-coated adenovirus to the FGF receptor allows productive infection and mediates efficacy in a peritoneal model of human ovarian cancer. J Gene Med. 2008, 10: 280-9. 10.1002/jgm.1121.
Barnett BG, Crews CJ, Douglas JT: Targeted adenoviral vectors. Biochim Biophys Acta. 2002, 1575: 1-14.
Wickham TJ: Targeting adenovirus. Gene Ther. 2000, 7: 110-4. 10.1038/sj.gt.3301115.
Parker AL, Waddington SN, Nicol CG, Shayakhmetov DM, Buckley SM, Denby L, Kemball-Cook G, Ni S, Lieber A, McVey JH, Nicklin SA, Baker AH: Multiple vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes. Blood. 2006, 8: 2554-61. 10.1182/blood-2006-04-008532.
Reynolds PN, Nicklin SA, Kaliberova L, Boatman BG, Grizzle WE, Balyasnikova IV: Combined transductional and transcriptional targeting improves the specifcity of transgene expression in vivo. Nat Biotechnol. 2001, 19: 838-842. 10.1038/nbt0901-838.
Reynolds PN, Zinn KR, Gavrilyuk VD, Balyasnikova IV, Rogers BE, Buchsbaum DJ: A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo. Mol Ther. 2000, 2: 562-578. 10.1006/mthe.2000.0205.
This work is supported by the National 863 Project of China (2007AA021201). We thank Dr. Hanshuo Yang, Dr. Yongsheng Wang (State Key Laboratory of Biotherapy and Cancer Center, West China Hospital) for the manuscript revision, Dr. Xiancheng Chen (Department of Gynecology and Obstetrics, Second West China Hospital) for his immunochemistry technical support.
The authors declare that they have no competing interests.
LPY carried out transfection and viral preparation, animal experiment and histological analysis, and drafted the manuscript. PC carried out TUNEL staining and performed statistical analyses. XCP contributed to animal experiment and revised the manuscript. HSS, WHH, FYC and STL contributed to animal experiment. LY offered Adenovirus and designed the topic. YQW supervised experimental work and revised the manuscript. All authors read and approved the final manuscript.
Li-Ping Yang, Ping Cheng, Xing-Chen Peng contributed equally to this work.
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Yang, LP., Cheng, P., Peng, XC. et al. Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma. J Exp Clin Cancer Res 28, 75 (2009). https://doi.org/10.1186/1756-9966-28-75
- Human Umbilical Vein Endothelial Cell
- Normal Saline Group
- Human Umbilical Vein Endothelial Cell Proliferation
- PEDF Expression