In order for any solid tumor to grow beyond 1-2mm, it must recruit a new blood supply, which typically occurs through angiogenic processes (32
). In this study, we provide evidence that ABT-510 may be effective in treating EOC through both antiangiogenic and antitumor effects. Daily i.p. administration of ABT-510 inhibited tumor growth, reduced ascites formation and decreased dissemination of secondary tumors in an orthotopic injected syngeneic mouse model of ovarian cancer.
The vasculature of rapidly growing tumors has been shown to be abnormal, with the presence of large, tortuous vessels, lack of anastomosis and numerous blind ends (33
). Endothelial cells of tumor vessels often are not stabilized by adjacent pericytes, which provide vessel support and are an indicator of mature blood vessels (34
). As a result of this rapidly-growing abnormal vasculature, tumor tissue has high interstitial pressure, reduced perfusion and areas of tissue hypoxia (35
). It has been proposed that combinational therapy of antiangiogenic agents combined with cytotoxic drugs can enhance tumor growth inhibition (34
). Antiangiogenic compounds appear to prune distorted tumor vessels, normalizing the vasculature, which improves tissue perfusion, allowing for improved delivery of cytotoxic chemotherapy drugs to the tumor tissue (34
). ABT-510 is more effective at inhibiting growth of neuroblastoma xenografts in combination with valproic acid than as a single agent (36
). Administration of ABT-510 in our model reduced the presence of large tortuous vessels, increased the proportion of mature vessels with pericyte coverage and decrease tissue hypoxia. These morphological changes by ABT-510 included smaller diameter vessels and an overall decrease in tumor vascular area, leading to a somewhat normalized vasculature. Although the tumor tissue of ABT-510 treated animals appeared to be better perfused, the decreased proliferation and increased apoptosis seen in these animals was likely due to the direct pro-apoptotic effect of ABT-510 on the ovarian tumor cells. Thus ABT-510 appears to affect ovarian tumors by stimulating tumor cell apoptosis and by limiting abnormal vessel growth and reducing tissue hypoxia. By reducing tumor hypoxia, ABT-510 also likely contributed to the reduced VEGF levels seen in the mice treated with the peptide. VEGF is potently expressed in response to tissue hypoxia in an attempt to stimulate angiogenesis and provide adequate oxygenation (37
). With reduced hypoxia, there would be lower stimulus for VEGF expression and a reduction in the abnormally high VEGF levels seen in untreated mice. Used in combination therapy, ABT-510 may be an effective agent in normalizing blood vessels and increasing tissue perfusion, which would facilitate intra-tumor delivery of chemotherapeutic drugs. ABT-510 has been shown to inhibit VEGF-induced endothelial cell migration (21
) and can stimulate endothelial apoptosis (38
). ABT-510 has also been shown to restore low wild type levels of circulating endothelial cells in TSP KO mice, where these are elevated ~5-fold (39
) when injected over 5 days. TSP-1-induced endothelial apoptosis is mediated by Fyn, p38 MAPK and caspases-3 pathways (16
) that leads to increased of FasL expression (40
). Furthermore, TSP-1 has been shown to block VEGF mediated nitric oxide synthesis, which is essential for angiogenesis (41
). The effects of TSP-1 appear ultimately to lead to reduced microvascular density (MVD). We have previously shown that full length TSP-1 can bind and internalize VEGF through the low-density lipoprotein receptor-related protein 1 (17
). Other reports have shown that other members of the TSR superfamily can inhibit VEGF induced angiogenesis by direct protein to protein interactions. Heparin affin regulatory peptide (HARP) and connective tissue growth factor (CTGF) both contain the TSR motif and this domain binds the 165 splice variant of VEGFA (42
). In human bladder cancer and Lewis lung carcinoma models (36
), ABT-510 increased endothelial cell apoptosis and reduced tumor vessel density (38
). Our results are consistent with these observations as mice receiving 100 mg/kg ABT-510 exhibited a significant reduction in vasculature area and perimeter.
As EOC progresses into later stages, the primary tumor sheds cells, which disseminate throughout the abdominal cavity to form secondary lesions. Although the mechanism behind dissemination is unclear (44
), ABT-510 treatment significantly inhibited the formation of secondary lesions. As the formation of secondary lesions are usually only seen at later stages, ABT-510 may have prevented their formation by delaying progression and development of the primary tumors, not allowing it to reach an advanced stage.
A hallmark in EOC is the accumulation of malignant ascites, which accounts for many of the non-specific symptoms experienced during late stage disease. Generally, increased morbidity is associated with the building of fluid within the abdominal and pelvic cavities. Ascites may arise from decreased clearance from abdominal lymphatics that become blocked with secondary lesions and increased vascular permeability that correlates with neovascularization of primary tumors (45
). Tumor production of VEGF is implicated in this increased permeability (46
) as VEGF protein levels are dramatically increased in malignant ascites fluid of EOC patients (47
). If the primary tumor is responsible for elevated VEGF levels, then reducing tumor volume would potentially lead to lower VEGF expression, a decrease in tumor vasculature and reduced ascites. In addition to decreasing tumor volume, ABT-510 may have reduced ascites formation by normalizing the tumor vasculature. Tumor vessels are known to be leaky, with increased fenestrations and fluid extravasation, which contributes to the accumulation of abdominal ascites in EOC (48
). The reduction in proportion of these large, leaky vessels likely contributed to decreased ascites in the ABT-510 treated animals.
The ability of ABT-510 to decrease tumor size, ascites formation and the presence of secondary lesions demonstrates its effectiveness as a single-agent therapy. Metronomic administration of chemotherapeutics along with antiangiogenic compounds has shown that cytotoxic drugs and antiangiogenic agents can have a synergistic effect (22
). Metronomic chemotherapy involves the administration of low-dose chemotherapeutics at close intervals and has been shown to inhibit tumor angiogenesis (50
). One mechanism of metronomic chemotherapy is to increase expression of pro-apoptotic Fas in endothelial cells (51
) and we showed that ABT-510 treatment increases FasL expression in ovarian tumor cells in vitro
, and others have demonstrated an increase in FasL in response to ABT-510 (40
). Quesada et al., illustrated that frequent low dose of doxorubicin sensitizes endothelial cells to FasL and ABT-510 was able to synergistically inhibit tumor growth in bladder and prostate xenograft models (22
). Activation of CD36 by TSP-1 phosphorylates p38 and JNK, which results in an increased expression of FasL and apoptosis (52
), which we saw in our tumor cells in response to ABT-510.
Tumors from ABT-510-treated mice had a significant increase in apoptosis in epithelial and endothelial tissues. Decreased phosphorylation of both phosphatidylinositol 3-kinase and Akt would lead to loss of antiapoptotic signals, which sensitizes cells to apoptosis (53
). This coupled with increased expression of FasL would create a proapoptotic environment. The stress-activated p38 MAPK was dephosphorylated in response to ABT-510 treatment when compared with PBS-treated mice. Although TSP-1 has been shown to lead to increased in p38 MAPK, which leads to apoptosis (16
), other cells types have shown that p38 MAPK can have a cytoprotective effects (54
); thus, p38 MAPK may act in cell-specific contexts. We showed in vitro
that ABT-510 treatment results in an increased expression of proapoptotic FasL and that expression of CD36 is involved in this pathway. The exact mechanism of tumor cell apoptosis in our model of EOC remains to be elucidated.
Protein collected from ABT-510-treated tumors show a decrease in expression of VEGF, VEGFR-2, and PCNA compared with PBS-treated controls. As VEGF and VEGFR-2 are expressed on both endothelial and tumor cells in our model, reduced expression would be expected to affect both cellular compartments. Reduced VEGF expression would have multiple effects in an ovarian tumor such as decreased angiogenesis, reduced vascular permeability, and increased susceptibility to apoptosis of endothelial and tumor cells. Decreased vascularization and ascites formation were evident in our models as was an increase in epithelial and endothelial apoptosis. Currently, the mechanisms behind the decreased VEGF expression are unknown but likely involve the decrease observed in primary tumor volume, reducing the secretion of VEGF (and likely other growth factors) from the ovarian tumor tissue. In addition to the apoptotic effect of ABT-510, the decrease in VEGF and VEGFR-2 may also have contributed to tumor due to their cytoprotective function in endothelial cells (55
). We have shown a strong extravascular cytoprotective effect for VEGF and VEGFR-2 in ovarian cells (56
) and have identified a survival role for these molecules in ovarian epithelial tumor cells (data not shown). In the ovarian tumors in this study, ABT-510 significantly increased apoptosis of both endothelial and tumor cells, which would explain the significantly decreased volume in our ABT-510-treated ovaries. In mice lacking TSP-1, the tumor growth rate was accelerated, and there were fewer apoptotic tumor cells. In contrast, when ABT-510 was added, there was a significant increase in tumor cell death, reduced tumor growth rate, and lower tumor burden. These results suggest that TSP-1 is an important component of the tumor microenvironment and acts to combat tumor cell proliferation.
In conclusion, ABT-510 appears to be effective as single-agent therapy in an orthotopic, syngeneic model of EOC. Our model provides evidence that ABT-510 can delay tumor progression, normalize tumor vasculature, and increase survival. Further studies administering ABT-510 in combination with commonly used chemotherapeutic drugs such as valproic acid, paclitaxel, and cisplatin are warranted.