The role of PPARγ in tumors has been widely studied. PPARγ ligands have been reported to have direct effects on tumor cells. We show that, by inhibiting angiogenesis, PPARγ ligands may have clinical application in treating not only primary tumor growth but metastatic growth as well, independent of tumor cell PPARγ expression or direct TZD-induced inhibition of tumor cell proliferation.
Our proliferation studies show that endothelial and tumor cells display markedly different sensitivities to low doses of TZDs in vitro. The antiproliferative endothelial cell effects after 72 hours of treatment with 1–10 μM rosiglitazone are similar to those reported in HUVECs and choroidal endothelial cells (15
). Furthermore, at lower concentrations (0.01–0.1 μM) of rosiglitazone that correspond to the range of its affinity for binding PPARγ (30
), we observed similar endothelial cell inhibition after 7 days. These concentrations also activate PPARγ in fibroblasts and adipocytes (27
). Most PPARγ activation markers have been identified in adipocytes, such as adipocyte fatty acid–binding protein (aP2) (35
); to date, none have been described for the endothelial cell system. Consequently, we used ligand-induced PPARγ degradation as a surrogate marker of activation. These results indicate that PPARγ is activated by its ligands in endothelial cells and suggest that the inhibition of proliferation of endothelial cells by TZDs may be a direct result of this activation. Importantly, the levels of rosiglitazone that inhibit endothelial proliferation are readily achieved in patients undergoing standard antidiabetic rosiglitazone treatment (36
TZDs have been shown to inhibit tumor cell proliferation in a PPARγ-independent manner. In PPARγ-deficient embryonic stem cells, TZDs (25 μM) inhibit translation initiation by phosphorylation of eIF-2α (12
). Rosiglitazone concentrations of 5 μM and higher led to phosphorylation of eIF-2α in HUVECs, suggesting that inhibition of proliferation of endothelial cells was mediated by a PPARγ-independent pathway. However, we observed that 0.01-1 μM of rosiglitazone, the concentration range at which PPARγ was activated, had the strongest antiproliferative effect on endothelial cells in vitro. Therefore, the inhibitory activity of rosiglitazone on endothelial cells is most likely mediated through PPARγ, whereas PPARγ-independent effects on translation may become important when higher concentrations of rosiglitazone are used.
In addition to direct action on endothelium, tumor angiogenesis can also be affected by indirect mechanisms. Our results showed that TZDs decreased VEGF production by tumor cells. Antiangiogenesis can result from a decrease of stimulators (e.g., VEGF and bFGF) and/or an increase of inhibitors (e.g., thrombospondin) in the tissue. It has been shown that PPARγ activation downregulates leptin and TNF-α, both of which are angiogenic factors (30
). Other reports show that PPARγ activation upregulates the expression of the angiogenesis inhibitor maspin and CD36, the receptor for antiangiogenic thrombospondin (5
Antiangiogenic activity can also be mediated indirectly by pericytes and macrophages, which both express PPARγ. Pericytes (also known as mural cells) have recently been shown to be present in significant numbers in tumor blood vessels (40
). Therefore, PPARγ ligands, which inhibit vascular smooth muscle cell proliferation and migration (30
), may indirectly affect endothelial cell survival. Our CAM assay results suggest that PPARγ ligands have macrophage-independent effects in vivo. However, PPARγ ligands have been shown to inhibit macrophage activation and invasion, which are important modulators of tumor angiogenesis (30
). Thus, PPARγ ligands may inhibit tumor angiogenesis by indirect as well as by direct mechanisms.
Surprisingly, higher doses of rosiglitazone (e.g., 400 mg/kg/d) were less antiangiogenic than lower doses (e.g., 50 mg/kg/d). Such a biphasic effect is not unprecedented, because other angiogenesis inhibitors, such as IFN-α, have less antiangiogenic activity at higher doses (20
). This biphasic response is indicative of a negative feedback loop that operates at higher doses. One possibility is the drug-induced degradation of PPARγ, which at high dose might lead to complete depletion, and thus unresponsiveness. Alternatively, higher doses of rosiglitazone might induce enzymatic drug metabolism pathways. The results from twice-daily administration suggest that the doses of TZDs may be lowered further to achieve efficacy similar to that achieved with daily administration. Such a result is consistent with reports that frequent low doses of chemotherapy have increased antiangiogenic efficacy and that continuous administration of angiostatin increases tumor suppression at significantly reduced doses (29
). This is presumably because endothelial inhibition is more related to duration of drug exposure than to peak levels achieved.
Several cancer cell lines, including bladder, breast, and thyroid carcinoma, require higher doses (50–100 μM) for inhibition of proliferation or are relatively resistant to antiproliferative effects of TZDs in vitro (5
). Our tumor lines were minimally inhibited by rosiglitazone in vitro but were dramatically suppressed in vivo, in agreement with reports that TZDs have antitumor effects in PPARγ-negative tumors (12
). Our antitumor effect resulted from a reduction in tumor microvessel density and a decrease in endothelial cell proliferation. Importantly, no signs of tumor cell differentiation or reduction in tumor cell proliferation were observed in treated tumors. This, together with the direct effects on the endothelium, suggests that the observed antitumor activity of TZDs is not solely mediated by a cell-autonomous response of the tumor cells.
In our studies, systemic PPARγ ligand therapy prevented metastatic invasion of LLC, consistent with the report that TZD administration inhibited the metastatic spread of thyroid tumors to the liver (11
). Vascular invasion involves ECM proteolysis. Moreover, endothelial and tumor cells secrete proteinase inhibitors, such as TIMPs or plasminogen activator inhibitors (PAIs), which suppress tumor invasion and angiogenesis (26
). Our studies demonstrate that rosiglitazone increases the inhibition of MMP activity in HUVECs, and others have shown that PPARγ ligands can increase PAI-1 expression in human endothelial cells (14
). The mechanism by which PPARγ ligands such as rosiglitazone may prevent the establishment and progression of metastatic disease includes the previously described antiangiogenic effects such as inhibition of endothelial proliferation and decrease in VEGF secretion, in addition to the upregulation of negative regulators of metastatic invasion such as MMP inhibitors.
Our studies suggest that PPARγ ligands may be useful in treating angiogenic diseases such as cancer because of their effect on endothelium. Moreover, the endothelium has become an important target in the treatment of non-neoplastic diseases such as psoriasis, endometriosis, and arthritis. PPARγ ligands may represent a novel antiangiogenic therapy in rheumatoid arthritis (48
). Furthermore, it is now becoming evident that multi-drug-resistant tumors can be effectively targeted by antiangiogenic chemotherapy (also called metronomic) chemotherapy (29
). As an orally administered FDA-approved drug used in over 2 million people with minimal side effects, rosiglitazone would be ideally suited for use in combination with other antiangiogenesis regimes and holds great promise to complement conventional modalities for cancer treatment.