The inhibition of HIF-1 alpha activity in tumors from YC-1-treated mice is associated with blocked angiogenesis and an inhibition of tumor growth. YC-1 has the potential to become the first antiangiogenic anticancer agent to target HIF-1 alpha.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of HIF-1␣ and aryl hydrocarbon receptor nuclear translocator (ARNT), plays a key role in cell survival and angiogenesis in hypoxic tumors, and many efforts have been made to develop anticancer agents that target HIF-1␣. However, although ARNT is also required for HIF-1 activity, ARNT has been disregarded as a therapeutic target. Curcumin is a commonly used spice and coloring agent with a variety of beneficial biological effects, which include tumor inhibition. In the present study, we tested the possibility that curcumin inhibits tumor growth by targeting HIF-1. The effects of curcumin on HIF-1 activity and expression were examined in cancer cell lines and in xenografted tumors. We found that curcumin inhibits HIF-1 activity and that this in turn down-regulates genes targeted by HIF-1. Moreover, of the two HIF-1 subunits, only ARNT was found to be destabilized by curcumin in several cancer cell types, and furthermore, ARNT expression rescued HIF-1 repression by curcumin. We also found that curcumin stimulated the proteasomal degradation of ARNT via oxidation and ubiquitination processes. In mice bearing Hep3B hepatoma, curcumin retarded tumor growth and suppressed ARNT, erythropoietin, and vascular endothelial growth factor in tumors. These results suggest that the anticancer activity of curcumin is attributable to HIF-1 inactivation by ARNT degradation.

Tumor angiogenesis is required for tumor development and is stimulated by angiogenic inducers like VEGF (vascular endothelial growth factor). Our previous study demonstrated that STAT3 (signal transducer and activator of transcription 3) up-regulates HIF-1alpha (hypoxia inducible factor-1alpha) protein stability and enhances HIF-1-mediated VEGF expression in hypoxic solid tumor cells, thus suggesting that the inhibition of STAT3 signaling may have clinical applications. In this study, we examined in vitro and in vivo, whether caffeic acid (CA) or its derivative CADPE [3-(3,4-dihydroxy-phenyl)-acrylic acid 2-(3,4-dihydroxy-phenyl)-ethyl ester] exert anticancer activity by targeting STAT3. It was found that CA or CADPE significantly inhibit STAT3 activity, and that this in turn down-regulates HIF-1alpha activity. Consequently, sequential blockade of STAT3 and HIF-1alpha resulted in the down-regulation of VEGF by inhibiting their recruitment to the VEGF promoter. In mice bearing a Caki-I carcinoma, both CA and CADPE retarded tumor growth and suppressed STAT3 phosphorylation, HIF-1alpha expression, vascularization and STAT3-inducible VEGF gene expression in tumors. Taken together, our results demonstrate that CA and CADPE are potential inhibitors of STAT3 and that they suppress tumor angiogenesis by inhibiting the activity of STAT3, the expression of HIF-1alpha and VEGF.

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Hypoxia-inducible factor (HIF)-1 plays a key role in tumor promotion by inducing f60 genes required for tumor adaptation to hypoxia; thus, it is viewed as a target for cancer therapy. For this reason, YC-1, which downregulates HIF-1A and HIF-2A at the post-translational level, is being developed as a novel anticancer drug. We here found that YC-1 acts in a novel manner to inhibit HIF-1. In the Gal4 reporter system, which is not degraded by YC-1, YC-1 was found to significantly inactivate the COOH-terminal transactivation domain (CAD) of HIF-1A, whereas it failed to inactivate CAD(N803A) mutant. In coimmunoprecipitation assays, YC-1 stimulated factor inhibiting HIF (FIH) binding to CAD even in hypoxia, whereas it failed to increase the cellular levels of hydroxylated Asn 803 of CAD. It was also found that YC-1 prevented p300 recruitment by CAD in mammalian two-hybrid and coimmunoprecipitation assays. The involvement of FIH in YC-1-induced CAD inactivation was confirmed in EPO-enhancer and Gal4 reporter systems using FIH small interfering RNA and dimethyloxalylglycine FIH inhibitor. Indeed, FIH inhibition rescued HIF target gene expressions repressed by YC-1. In cancer cell lines other than Hep3B, YC-1 inhibits HIF-1A via the FIHdependent CAD inactivation as well as via the protein down-regulation. Given these results, we suggest that the functional inactivation of HIF-A contributes to the YC-1-induced deregulation of hypoxia-induced genes.