NDGA directly inhibits several established anti-cancer targets, including metabolic enzymes and receptor tyrosine kinases. Our study suggests that NDGA also functions as a direct inhibitor of the serine/threonine kinase receptor, TGF β type I receptor. This conclusion is based on the following observations: a) NDGA inhibits TGF-β-induced Smad2 phosphorylation and its downstream transcription in cells; b) NDGA inhibits Smad2 phosphorylation and transcription induced by a constitutively active mutant of TGF β type I receptor in cells; c) NDGA inhibits Smad2 phosphorylation by purified TGF β type I receptor in vitro. This new activity of NDGA allows us to better understand the multiplicity of NDGA’s anti-cancer effects and enable us to further explore its clinical implications.
TGF-β signaling was well established to play a key role in carcinogenesis, and TGF-β inhibitors have been investigated as cancer treatment (Dumont and Arteaga, 2003
; Peng et al., 2005
). TGF-β neutralizing antibodies have been shown to inhibit growth and metastasis of breast tumors in mice. However, TGF-β targeting therapies have also been shown to increase proliferation and aggressiveness of other cancer models. While there are no clinically approved small molecule TGF-β inhibitors, several are currently under development despite the complicated role for this pathway in tumor biology. Considering the dichotomous role of TGF-β in cancer, NDGA and structurally related analogs represent ideal compounds for inhibition of TGF-β signaling in cancer due to their ability to inhibit additional anti-cancer targets and their consistent growth inhibitory actions across a wide range of cancer cell lines and tumor types. TGF-β signaling is also involved in several additional disorders, and TGF-β inhibitors are under investigation as therapeutics for these disorders including diffuse systemic sclerosis (Dumont and Arteaga, 2003
), glaucoma (Siriwardena et al., 2002
), and progressive fibrosis (Callahan et al., 2002
). The possibility that NDGA or related analogs could also be useful candidates for treating these disorders deserves additional study.
NDGA inhibits TGF-β signaling at micro molar concentration, which is similar to its potency for other targets. It is not clear, however, which cellular actions attributed to NDGA might take precedence in vivo
, or how these different effects might interact to produce the observed anti-cancer actions. NDGA has consistently demonstrated anti-cancer actions in a variety of animal models, with a wide range of dosing protocols, although information on target interactions in vivo
is limited. Only the IGF-1 receptor and HER2 tyrosine kinase receptors have been shown to be specifically inhibited in tumors of NDGA treated mice (Youngren et al., 2005
Relatively high doses of NDGA have been administered and well tolerated in both animal studies and clinical trials with minimal toxicity. Anticancer activities were observed in a xenograft model of pancreatic cancer when NDGA was administered at 250 mg/kg/day by gavage, although significant tumor inhibition has been observed with much lower doses (Tong et al., 2002
). Interestingly, daily consumption of NDGA estimated at over 400 mg/kg/day resulted in an increased life span of male mice, demonstrating the safety of long-term NDGA ingestion (Strong et al., 2008
). Clinically, a phase I study of prostate cancer patients indicated that up to 2500 mg per day of NDGA was well tolerated. (Ryan et al., 2008
Because of the ability of NDGA to inhibit multiple targets, we performed a preliminary investigation on the structure-potency relationship of NDGA on TGF-β inhibition to determine if this activity was specifically related to any of the structural characteristics of NDGA. We found that modification of the methyl substituents on the bridging butyl chain and the four hydroxyls of the two catechol rings dramatically change the potency of NDGA for TGF-β inhibition. More importantly, we also showed that the potency of two NDGA analogs improved after the two hydroxyl groups from one of the two catechol rings were replaced with a simple phenyl ring or alkyl-substituted phenyl. These findings indicate that the structure of NDGA can be refined to achieve higher potency against TGF-β, and that a compound with a single catechol ring is likely to show improved performance against this target. Interestingly, the asymmetrical compounds tested in the present study that showed increased potency against TGF-β signaling were reported in our previous publication to show improved growth inhibition of breast cancer cells compared to NDGA (Blecha et al., 2007
In sum, our data demonstrate a new activity of NDGA as an inhibitor of the serine/threonine kinase receptor, TGF β type I receptor. Our study also highlights the possibility that increased potency against this kinase can be achieved through modifying NDGA structure, suggesting NDGA may provide a novel structural motif to generate serine /threonine kinase inhibitors with selectivity for TGF β type I receptor. This present study suggests that NDGA and it analogs hold a great potential to be developed as effective therapeutics against cancer and other illnesses based on this novel mechanism.