The IGF signaling system has become a target of increasing interest in cancer therapy research. A variety of approaches to disrupting this system have been investigated, including use of anti-receptor antibodies, anti-sense nucleotides, ligand mimicking compounds, IGF-binding proteins, and small molecule inhibitors [Dake et al., 2004
; Foulstone et al., 2005
]. A related pair of relatively specific and potent inhibitors of the IGF-IR, NVP-ADW742, and NVP-AEW541, inhibit the growth of a variety of tumors in vitro and in vivo, including fibrosarcoma and neuroblastoma [Garcia-Echeverria et al., 2004
; Mitsiades et al., 2004
; Tanno et al., 2006
]. Still, relatively few agents have been identified that have effects against the IGF-IR, and clinical studies for the highly specific NVP compounds are not anticipated. Considering the promising pre-clinical results of anti-IGF treatment in numerous malignancies, more candidate agents need to be characterized to increase the chances of finding clinically viable options that combine efficacy and low toxicity.
NDGA has had a long history of use as a lipoxygenase inhibitor before it was recently found to inhibit the tyrosine phosphorylation of the IGF-IR [Youngren et al., 2005
]. NDGA has been tested as a potential anti-cancer agent in several studies, where it induced apoptosis and suppressed mitogenesis [Vondracek et al., 2001
; Seufferlein et al., 2002
; Tong et al., 2002
; Hoferova et al., 2003
]. Some of these studies hypothesized that suppressing lipoxygenase-dependent prostaglandin synthesis may mediate inhibition of tumor growth. We find in this study that inhibition of lipoxygenases with specific inhibitors unrelated to NDGA has no appreciable effect on neuroblastoma growth. Rather, we propose that the anti-tumor effect of NDGA in neuroblastoma cells is at least partly mediated through inhibition of the IGF-IR.
Neuroblastoma cells are highly dependent upon paracrine and autocrine IGFs for growth [Martin and Feldman, 1993
; Meghani et al., 1993
; Leventhal et al., 1995
; Kiess et al., 1997
], and thus it is logical that agents like NVP-AEW541 and NDGA that are capable of blocking IGF-IR activation would inhibit neu-roblastoma tumorigenesis. Neuroblastoma cell lines that secrete IGF-II are capable of serum-independent growth [Martin and Feldman, 1993
; Leventhal et al., 1995
] and cell lines that express high levels of the IGF-IR are more aggressively tumorigenic [Singleton et al., 1996b
]. We find that NDGA at low doses (15–30 μM) completely blocks neuroblastoma growth over a period of several days in vitro, both in serum, and in serum-free conditions where added and autocrine IGFs support neuroblastoma growth. NDGA prevents IGF-I-mediated activation of both the IGF-IR and ERK 1 and 2 in neuroblastoma cells at the same doses that inhibit growth in vitro. The growth of Kelly neuroblastoma tumor xenografts in nude mice is also suppressed by NDGA. Additional studies with NDGA in xenografted animals are in preparation to further characterize its effi-cacy, impact on survival, and ability to inhibit putative target signaling pathways in vivo. In addition to neuroblastoma, NDGA inhibits the in vitro and in vivo proliferation of other cancers that are highly responsive to IGFs, including lung [Moody et al., 1998
] and breast [Youngren et al., 2005
IGFs are also potent stimulators of neuro-blastoma survival, causing strong activation of Akt while suppressing caspase-3 activation [Van Golen and Feldman, 2000
; Van Golen et al., 2000
]. In neuroblastoma, NDGA causes inhibition of IGF-stimulated Akt activation and is strongly apoptotic, causing caspase-3 activa-tion and a large increase in sub-G0
cells. Similar results are seen in breast cancer cell lines treated with NDGA, where Akt activation is suppressed and BAD activation is increased [Youngren et al., 2005
]. It is possible that disruption of other unknown targets of NDGA could lead to apoptosis in neuroblastoma cells. However, 10 nM IGF-I is known to completely prevent caspase-3 activation when the stressor that induces apoptosis is unrelated to IGF signaling (e.g., osmotic stress [Van Golen and Feldman, 2000
; Van Golen et al., 2000
]). In our experiments caspase-3 activation remained strong following administration of 10 nM IGF-I, suggesting that IGF-stimulated rescue is impaired by NDGA treatment.
IGF-I stimulates neuroblastoma cells to undergo organized actin polymerization and lamellipodium extension, resulting in increased cell motility [Kim and Feldman, 1998
; Meyer et al., 2001
]. Increased cell motility, along with the ability to digest extracellular matrix, affords cancer cells greater ability to invade tissues and blood vessels, leading to metastasis and diffuse tissue dissemination. This is of particular concern with neuroblastoma, where tumor invasion of bone, a site of high IGF production, is associated with poor response to therapy. We find that NDGA effectively inhibits IGF-I stimulated motility of neuroblastoma.
NDGA does not show high selectivity for a single receptor, in contrast to NVP-ADW742 and NVP-AEW541, and should not be viewed as solely an IGF-IR inhibitor. NDGA likely works on a subset of receptor tyrosine kinases, including the IGF-IR, InsR, and her2/neu receptor [Youngren et al., 2005
]. NDGA inhibits the activation of the PDGF receptor and PDGF-stimulated DNA synthesis [Domin et al., 1994
]. However, Seufferlein et al. 
, found no affect of NDGA on EGF receptor phosphoryla-tion. More work is needed to characterize which additional receptors may be affected by NDGA treatment.
As the IGF-I and insulin receptors are highly homologous, part of the effect of NDGA against neuroblastoma tumorigenesis might be mediated through InsR inhibition. However, insulin is 200 times less potent than IGF-I at stimulating SH-SY5Y proliferation, and at least one-third of insulin’s effect on proliferation is mediated by activation of the IGF-IR, not InsR [Meghani et al., 1993
]. Moreover, plasma concentrations of IGF-I are 100–1,000 times more concentrated than insulin (nanomolar for IGF-I, picomolar for insulin). Thus, inhibition of InsR activity is unlikely to account for significant anti-tumor effects of NDGA in neuroblastoma.
Of note, the anti-InsR activity of NDGA could be predicted to cause a diabetic phenotype. Paradoxically, NDGA has an anti-diabetic effect on rats, decreasing serum glucose and triglycerides without affecting insulin levels [Luo et al., 1998
; Gowri et al., 1999
; Scribner et al., 2000
]. NDGA was previously considered for treatment of diabetes because of its inhibition of prostaglandin synthesis. Thus, NDGA’s inhibition of insulin receptors may not result in a diabetes-like toxicity because of its concomitant effects on prostaglandin synthesis. NDGA analogs are being developed in an attempt to achieve better specificity (J. Youngren and I. Goldfine, unpublished work), and some have been tested for efficacy against lung cancer [Moody et al., 1998
]. Further characterization of these analogs may lead to the discovery of agents more specific for individual receptor tyrosine kinases.
An important potential advantage held by NDGA over other available agents with high IGF-IR specificity is its apparent safety in humans. A phase I trial of orally administered NDGA in prostate cancer patients at the University of California, San Francisco, is nearing completion with no observed dose limiting toxicities (personal communication, Charles Ryan, UCSF Urology). Thus, it is anticipated NDGA will soon be available for study in children, and its anti-IGF signaling and anti-tumorigenic effects in neuroblastoma warrant study of this agent’s potential for the treatment of neuroblastoma.
In summary, NDGA effectively suppresses neuroblastoma growth in vitro and in vivo, and inhibits the motility and promotes the apoptosis of neuroblastoma cells in culture. These effects appear to be mediated, at least in part, through inhibition of IGF-IR signaling. Future studies will investigate whether NDGA might be even more effective in combination with treatments that affect other aspects of neuroblas-toma tumorigenesis, such as anti-myc agents or radiation. NDGA could also be used in combination with other agents that target the IGF-IR, modulate ligand-receptor interactions, or that target downstream components of the IGF signaling pathway, such as IGF binding proteins or anti-PI-3K agents.