Soluble Flk-1, a soluble vascular endothelial growth factor (VEGF) receptor, is a potent inhibitor of angiogenesis, which could restrain growth and metastasis of some experimental tumors. However, antiangiogenic agents alone cannot eradicate tumor completely, and should be combined with other therapy to enhance their effects. In this study, we evaluated the antitumor activity of the combination therapy in the immunocompetent BALB/c mice bearing H22 hepatoma and Meth A fibrosarcoma, respectively. Mice were treated with either msFlk-1 i.m. at 100 μg/mouse once every 3 days for four times from day 3 after the tumor cell injection, cisplatin cycled twice (2 mg/kg i.p. on days 4 and 11 after the tumor cell inoculation), or both agents together. Tumor growth and survival time were continually observed. Antiangiogenesis in vivo was determined by CD31 immunohistochemistry. Assessment of apoptotic cells and histological analysis was also conducted in tumor tissues. Our results showed that the combination therapy could evidently improve antitumor efficacy, including tumor growth suppression, mice survival prolongation, tumor cell apoptosis augmentation as well as neovascularization inhibition as compared with controls, without serious adverse effects. Our data suggest that the combination of DDP with msFlk-1 is more effective to suppress tumor growth in mice than either agent alone, and this combination regimen showed its potential for future clinical application.
Cytotoxic chemotherapy has become one of the mainstays in medical approaches to the therapy of solid cancers. Among the various kinds of chemotherapeutic drugs, DDP remains the most widely used first line anticancer agent in clinic, which can fall into the class of DNA-damaging agents and produce interstrand, intrastrand and monofunctional adduct crosslinking in DNA of tumor cells.1, 2 However, because DDP is not cancer-specific and always used in high dose, the regular scheme of DDP in clinic easily leads to severe toxic side effects and acquired resistance, which would impede its clinical success. Antiangiogenic drugs are relatively nontoxic and may postpone or even solve problems of acquired drug resistances because they aim at the genetically stable endothelial cells of newborn tumor blood vessels, rather than genetically unstable tumor cells that are inclined to mutate and develop resistance. Thus, they are likely to work well in combination with chemotherapy.3, 4, 5
Antiangiogenesis therapy for cancer can effectively inhibit tumor growth by inhibiting tumor-related angiogenesis, and thus deprive tumors of essential nutrients and oxygen, which lead to a ‘dormant’ state in which tumor cell proliferation and metastasis are halted.6, 7, 8, 9, 10 Although various proangiogenic factors, including VEGF, transforming growth factor, basic fibroblast growth factor and epidermal growth factor are related to the process of angiogenesis, VEGF is of special importance in the development of angiogenesis of tumors. It is an endothelial cell-specific mitogen, which is secreted in most tumors and has pivotal effects in tumor growth, invasion and metastasis.11, 12, 13, 14, 15, 16, 17, 18, 19
The recognition of VEGF as a primary stimulus of angiogenesis in pathological condition has led to the generation of many strategies to block VEGF activity. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF, and low molecular weight VEGF receptor tyrosine kinase inhibitors have all been developed to interfere with VEGF signaling.20, 21, 22, 23, 24, 25 msFlk-1, the entire seven globulins of extracellular domain of mouse VEGFR-2, is a promising angiogenesis inhibitor, which complexes VEGF directly and may also function in a dominant-negative manner by heterodimerizing with the extracellular ligand-binding region of the membrane spanning Flt-1 and Flk-1 VEGF receptors, thus preventing receptor tyrosine transphosphorylation and activation of downstream signal pathway. Recently, several studies have reported that not only angiogenesis but also tumor growth was suppressed by the soluble form of the VEGF receptors.26, 27, 28, 29 For example, Lin et al.28 reported that a recombinant form of the soluble Flk-1 protein inhibited tumor growth and vascular density in a cutaneous tumor window chamber. Although angiogeneic inhibitor can regress tumor growth through inhibiting angiogenesis, it cannot eradicate tumor completely, and better antitumor effects can be reached only by combining other antitumor therapy targeting directly the tumor cells. Moreover, some laboratories have reported that the combination of antiangiogenic drug with conventional antitumor strategies such as chemotherapy, radiotherapy would enhance the antitumor efficiecy.30, 31, 32
These lines of evidence led us the hypothesis that combining the msFlk-1 with DDP could produce better inhibition of tumor growth. We decided to assess the efficacy of the combination of msFlk-1 and DDP on the mice bearing H22 or Meth A, two tumors that are dependent on the VEGFR-2 pathway, and are responsible to DDP, as reported previously.33, 34 The observation includes survival of mice, occurrence of tumor growth suppression, angiogenesis inhibition, tumor cell apoptosis, and chemo-related side effects.
Materials and methods
An antiangiogenic plasmid expressing the msFlk-1 gene was bought from Invivogen Company (Invivogen, CA). This plasmid vector, named pBLAST45-msFLK-1, has the entire seven globulins of extracellular domain of Flk-1. It was purified by using two rounds of passage over Endofree columns (Qiagen, Chatsworth, CA), as reported previously.35
The mouse Meth A and H22 cells were maintained in suspended cultures in RPM1640 supplemented with 10% heat-inactivated FBS, at 37°C in a humidified atmosphere containing 5% CO2.
Tumor models and treatment
Female syngeneic BALB/c mice 6–8 weeks of age were used as model hosts for H22 and Meth A. Mice were injected subcutaneously each on day 0 with an aliquot of 5 × 105 live H22 or Meth A cells. After 3 days, mice were randomly divided into the following groups of 10 each: pBLAST45-msFLK-1 plus DDP, DDP, pBLAST45-msFLK-1, or 0.9% NaCl solution (N.S.). As our preliminary experiment showed that 100 μg pBLAST45-msFLK-1 and 2 mg DDP had optimal antitumor effects with lower toxicity in the two tumor models as compared with other dose schemes, we chose this scheme in our study. Treatment regimens were as follows: group 1, 0.1 ml pBLAST45-msFLK-1 (100 μg/mouse) intramuscularly (i.m.) once every 3 days for four times from day 3 after tumor cell injection; group 2, DDP (2 mg/Kg) intraperitoneally (i.p.) once every week for two times from day 4 after tumor cell injection; group 3, the combination of the above treatments; group 4, untreated group, mice received N.S. i.m. as the scheme of pBLAST45-msFLK-1 in the group 1 and i.p. as the scheme of DDP in the group 2. Thereafter, all tumor-challenged mice would be under continuous observation for experiment-associated modulation in behavior or physical appearance. The tumor volume was determined by the following formula: tumor volume (mm3)= 0.52 × length (mm) × width2 (mm2). The experiment was repeated twice independently. All animals were housed in standard microisolator conditions free of pathogens in accordance with institutional guidelines under approved protocol. All procedures in our study were reviewed and approved by the Institute's Animal Care and Use Committee.
Fresh tumor tissues were excised from tumor bearing mice at week 4 after tumor cell implantation and dissected into several pieces of approximately 2 mm thickness and then fixed in 4% neutral buffered paraformaldehyde for 24 h. Paraffin-embedded sections were treated by standard deparaffinization and frozen sections in OCT by fixation in acetone and chloroform, Immunohistochemical analysis were performed as follows. Briefly, endogenous peroxidase activity was inactivated by incubating slides in 3% H2O2 in methanol for 15 min. Antigen retrieval was performed by heating slides in an autoclave with 10 mM pH 6.0 ethylene diamine tetra acetate citrate buffer for 10 min after pressure gaining. Nonspecific antibody binding was blocked with 5% bovine serum albumin in phosphate buffered saline (PBS) for 30 min at room temperature (RT) before incubation with the appropriate dilution (1:100) of rat antimouse monoclonal CD31 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After the samples were rinsed three times with PBS (5 min each time), they were incubated with the appropriate dilution (1:100) of biotin-conjugated secondary goat antirat antibodies for 30 min at 37°C. Slides were washed twice with PBS and then stained with peroxidase-labeled streptavidin biotin reagents (Dako LSAB kit, Dako, Carpinteria, CA) for 30 min at 37°C; then washed twice and incubated with DAB, observed under microscope and stopped the reaction timely following the manufacturer's instructions. Negative control slides were obtained by omitting the primary antibody. Cell nuclei were counterstained with reformative Gill's hematoxylin. Quantification was performed as described previously.36 Microvessel counting was performed at × 200. The results regarding angiogenesis were expressed as the mean of absolute number of the microvessels per high-power field of five hpfs in each tumor.
Terminal deoxy transferase uridine triphosphate nick end-labelling (TUNEL) and Hoechst assays for apoptotic cells
Tumor species were prepared as described above. Paraffin-embedded tissues were cut into sections of 5 μm and mounted on Vectabond Reagent slides, deparaffinized and rehydrated through xylene, graded ethanol to distilled water. Then the tissue sections were pretreated with 20 mg/l proteinase K for 30 min, and analyzed with an in situ Cell Death Detection Kit, POD (Roche, USA), according to the manufacturer's guidelines. It is based on the enzymatic addition of digoxigenin-nucleotide to the nicked DNA by terminal deoxynucleotidyl transferase. Briefly, cells were fixed for 1 h in 4% paraformaldehyde in PBS at RT. Endogenous peroxidase was inactivated by incubation with 2% H2O2 dissolved in methanol for 10 min at RT. Cells were permeabilized with 0.1% Triton X-100 for 8 min on ice. After that, cells were labeled by incubation with TUNEL reaction mixture at 37°C for 1 h. Slides were washed three times with PBS and incubated with peroxidase converter at 37°C for 30 min.37 Then samples were analyzed in a drop of PBS under a fluorescence microscope (Nikon TE2000-U inverted fluorescent microscope). As far as Hoechst-33 258 analysis, tissue sections were deparaffinized with xylene and rehydrated through a series of graded ethanols to distilled water. Samples were rinsed three times with PBS (5 min each time) and then incubated with 1 μmol/l DNA-specific fluorochrome Hoechst-33 258 compounds (Sigma) in a dark locomotive chamber at room temperature for 10 min.38 After removal of the compounds by inversion, the sections were washed three times with PBS. The slides were mounted with antifade solution (Sigma), and analyzed by fluorescence microscopy using excitation 348 nm/emission 480 nm wavelengths. The apoptotic cells are featured as blebby cytoplasmic appearance with pyknotic and fragmented nuclei emitting intense fluorescence. Apoptosis index was evaluated by analyzing the average fractions of apoptotic cells in five equal-sized high-power fields chosen randomly from tissue sections.
To clarify potential side effects in the treated mice, the tissues of heart, liver, spleen, lung, kidney, brain, etc., were fixed in 4% neutral buffered paraformaldehyde solution and embedded in paraffin. Sections of 3–5 μm were stained with hematoxylin and eosin (HE), and observed by two pathologists in a blinded manner. Other drug toxicity indexes such as weight loss, ruffled fur, diarrhea, anorexia, cachexia, skin ulceration, or toxic deaths were continuously observed during the whole treatment.
Data analysis and statistics
For comparison of individual time points, analysis of variance and an unpaired Student's t-test were used. Survival analysis was computed by the Kaplan–Meier method and compared by the log-rank test. P<0.05 was considered statistically significant.
Improved inhibition of growth of tumor with the combined treatment
Mice bearing H22 and Meth A were treated with different treatments, including pBLAST45-msFLK-1 plus DDP, DDP alone, pBLAST45-msFLK-1 alone, or N.S. Assay of tumor volume and lifespan of mice showed that both pBLAST45-msFLK-1 and DDP individually resulted in effective suppression of tumor growth. Combined treatment had a superior antitumor effect, resulted in apparent tumor inhibition versus N.S. control (P<0.01), pBLAST45-msFLK-1 or DDP alone (Figure 1a and b). (P<0.05). In the two tumor models of H22 and Meth A, the combination of pBLAST45-msFLK-1 with DDP resulted in a significant increase in lifespan as compared with N.S. control (P<0.05) (Figure 2a and b). The data come from the compilation of two independent experiments.
Augment of angiogenesis inhibition with the combination treatment
To identify the mechanism of the antitumor effects, we examined the angiogenesis of tumor sections by immunohistochemical staining. Angiogenesis within tumor tissue was estimated by counting the number of microvessels on the section staining with an antibody reactive to CD31. The most highly vascularized areas of each tumor were identified on low power, and five high-powered fields were counted in this area with greatest vessel density. Angiogenesis could be inhibited in the treatment with pBLAST45-msFLK-1 alone, compared with N.S. control. There was an apparent inhibition of angiogenesis in tumors treated with pBLAST45-msFLK-1 plus DDP, compared with treatment of DDP alone or N.S. (P<0.05). No statistically significant differences were obtained between DDP and N.S. groups (Figure 3).
Increased apoptosis with the combination treatment
We further examined the apoptosis of tumor tissues in the four groups with different treatments. Sections of tumor tissues from different treated groups were submitted to TUNEL and Hoechst-33258 assay for respective determination of apoptotic index. pBLAST45-msFLK-1 or DDP alone treatment could affect the apoptosis rate of tumor cells, whereas the density of apoptotic cancer cells obviously increased with the combined therapy (Figure 4a–h). Although single pBLAST45-msFLK-1 or DDP treatment could affect the ratio of tumor cells with condensed and fragmented nuclei, we found a more significant increase in condensed and fragmented cell nuclei in combined therapy group, whereas only a few positive pyknotic nuclei were noted in N.S. controls. Both methods, staining with Hoechst 33258 and the TUNEL assay, revealed a significant increase in nuclei with fragmented DNA upon treatment with pBLAST45-msFLK-1 plus DDP. Data describe the average apoptotic index±s.d. of tumor cells as a percentage normalized to apoptotic index of tumor cells (Figure 4i).
Tumor tissues were fixed using 4% neutral buffered paraformaldehyde solution for paraffin sectioning of H&E. DDP or pBLAST45-msFLK-1 alone treatment showed several focal necrosis. N.S. control displayed little or no necrosis, and had rich normal capillaries. Analysis of the extent of tumor necrosis showed that the co-administration of the two agents was clearly more potent, eliciting increase in tumor necrosis relative to single-agent treatment. The difference between the four groups was obvious. Representative sections of each group from H22 (Figure 5a–d) and Meth A (Figure 5e–h) were depicted.
Observation of toxicity
To evaluate the health status of mice treated with DDP and pBLAST45-msFLK-1, weight of mice was monitored every 3 days in the whole experiment. Weight was plotted at regular intervals and considered a surrogate for evaluation of systemic well-being, anorexia or cachexia. As shown in Figure 6, no significant differences in weights were found among the four groups. The weight curves of the pBLAST45-msFLK-1 and N.S. groups run parallels closely to each other. The DDP group revealed some weight gain delay, but there was no significant difference from N.S. control and pBLAST45-msFLK-1 group. Combination treated group had similar weight curve to the single treated groups and N.S. control. No adverse consequences in other gross measures such as ruffled fur, skin ulcerations, or toxic death were found in the combination group. Furthermore, no obvious pathologic changes in liver, lungs, kidneys, spleen, brain, heart, pancreas, intestine, or bone marrow were detected by microscopic examination.
Angiogenesis can be defined as the development of new vasculature from pre-existing blood vessels and capillaries.9 It is critical for many physiological processes such as embryogenesis, endometrial and placental proliferation, wound healing and some pathological events, including cancer, psoriasis, arthritis, and ocular neovascularization. It is well established that the growth and progression of most solid cancers are angiogenesis dependent. Antiangiogenesis therapy for cancer can effectively inhibit tumor growth by inhibiting tumor-related angiogenesis, and thus deprives tumors of essential nutrients and oxygen, which lead to a ‘dormant’ state in which tumor cell proliferation and tumor metastasis are halted.10, 11, 12 However, although angiogeneic inhibitor can retard tumor growth through inhibiting angiogenesis, it cannot eradicate tumor completely and better antitumor effects can be reached only by combining other antitumor agents.30, 31, 32
It has been reported that the adenovirus-mediated delivery of msFlk-1 or gene therapy-mediated expression by tumor cells of flk-1 delays the growth of tumor in mice and potently inhibits angiogenesis in mouse models of pancreatic adenocarcinoma or neuroblastoma.39, 40 Although msFlk-1 could regress tumor growth through inhibiting angiogenesis, antitumor agents of other mechanisms should be included to enhance antitumor effects. In the present study, we evaluated the efficacy of the combination of msFlk-1 and DDP as a therapeutic regimen for cancer therapy. Several observations have been made in our study. In the group of the combination of msFlk-1 and DDP, the tumor bearing mice showed obvious tumor growth suppression, and the survival was prolonged compared with other groups, which demonstrated the efficiency of this regimen. Although the exact mechanism by which the combination of pBLAST45-msFLK-1 with DDP can enhance the antitumor activity remained to be determined, the increased antitumor efficacy in vivo may in part result from the increased induction of the apoptosis of tumor cells in the combined treatment. This suggestion is supported by the present findings. There were more apoptotic cells in the combination group compared with the treatment with pBLAST45-msFLK-1 or DDP alone or N.S. control, as well as the MVD of the combination group was lower than other groups. The mechanism leading to this result may be speculated as follows: on one hand, msFlk-1 potently inhibits tumor angiogenesis, and thus deprives tumor of essential oxygen and nutrients. On the other hand, DDP is effective in inducing tumor cell apoptosis by forming interstrand, intrastrand and monofunctional adduct crosslinking in DNA. Thus, the potent antiangiogenesis activity by pBLAST45-msFLK-1 may play an important role in retarding or preventing adequate nourishment of tumors during their regrowth after a chemotherapeutic insult, resulting in tumor growth stasis. The inhibition of angiogenesis by pBLAST45-msFLK-1, therefore, is complementary to antitumor chemotherapy.
In this study, we demonstrated that pBLAST45-msFLK-1, which absorbs VEGF and may function as a dominant-negative receptor, as a single agent, has antitumor activity against H22 and Meth A in vivo. The data also showed that the combination of pBLAST45-msFLK-1 with DDP led to an enhanced antitumor effect compared with either agent alone. Furthermore, no overt toxicity effects such as ruffled fur, cachexia, anorexia, skin ulcerations, or toxic death were found in the combination group. To our knowledge, this is the first time that msFlk-1 and DDP have been tested together and found to have improved inhibitory effects on H22 and Meth A in mice.
In summary, our data suggested that the combination of chemotherapy with antiangiogenic drugs is effective in the treatment of H22 and Meth A in mice, and that the enhanced antitumor efficacy in vivo may in part result from the increased induction of the apoptosis of tumor cells and suppression of angiogenesis in the combined treatment. The present findings may lead to the further exploration of the potential application of this combined approach in the treatment of malignant tumor. However, until now, selecting the optimal antiangiogenic and chemotherapeutic therapy doses and application schedule may remain to be elucidated. Further studies are underway to understand the molecular mechanism of antitumor effects on this combination therapy.
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This work was supported by National Basic Research Program of China (2001CB510001, 2004CB518800), the projects of National Natural Science Foundation of China, and National 863 Program.
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