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.

Hypoxia-inducible factor ␣ proteins (HIF-␣s) are regulated oxygen dependently and transactivate numerous genes essential for cellular adaptation to hypoxia. NEDD8, a member of the ubiquitin-like family, covalently binds to its substrate proteins, and thus, regulates their stabilities and functions. In the present study, we examined the possibility that the HIF signaling is regulated by the neddylation. HIF-1␣ expression and activity were inhibited by knocking down APPBP1 E1 enzyme for NEDD8 conjugation but enhanced by ectopically expressing NEDD8. HIF-1␣ and HIF-2␣ were identified to be covalently modified by NEDD8. NEDD8 stabilized HIF-1␣ even in normoxia and further increased its level in hypoxia, which also occurred in von Hippel-Lindau (VHL) protein-or p53-null cell lines. The HIF-1␣-stabilizing effect of NEDD8 was diminished by antioxidants and mitochondrial respiratory chain blockers. This suggests that the NEDD8 effect is concerned with reactive oxygen species driven from mitochondria rather than with the prolyl hydroxylase (PHD)/VHL-dependent oxygen-sensing system. Based on these findings, we propose that NEDD8 is an ancillary player to regulate the stability of HIF-1␣. Furthermore, given the positive role played by HIF-␣s in cancer promotion, the NEDD8 conjugation process could be a potential target for cancer therapy. Hypoxia-inducible factors (HIFs)3 play crucial roles in tumor adaptation to hypoxia and angiogenesis by up-regulating numerous genes (1). HIF family members are composed of ␣ (HIF-1␣ and HIF-2␣) and ␤ (also known as aryl hydrocarbon receptor nuclear translocator (ARNT)) subunits. The HIF-␣s are tightly regulated by oxygen tension and function as prime transactivating factors, whereas aryl hydrocarbon receptor nuclear translocator is constitutively expressed and assists HIF-␣ binding to DNA. Under normoxic conditions, two proline residues (Pro-402 and Pro-564) in the oxygen-dependent degradation domain (ODDD) of HIF-1␣ are hydroxylated by prolyl hydroxylases (PHD1-3) (2); subsequently, HIF-1␣ is ubiquitinated by von Hippel-Lindau protein (pVHL) and finally degraded by 26 S proteasome (3). However, this hydroxylation is limited under hypoxic conditions, which stabilizes HIF-␣s. Given the essential roles played by HIFs in tumor promotion, the HIF inhibition has become a frontline topic in research on new cancer therapies (4).NEDD8 (neural precursor cell-expressed developmentally down-regulated 8) is conserved in eukaryotes and is ubiquitously expressed in most mammalian tissues. As NEDD8 is structurally similar to ubiquitin, it is classified as a member of the ubiquitin-like family (5). Furthermore, like ubiquitin, NEDD8 conjugates to its substrate proteins, which is named "neddylation," via a sequential process involving activation, conjugation, and ligation. Neddylation requires a unique set of conjugating enzymes, namely NEDD8-activating E1 complex, which is composed of APPBP1 and UBA3, NEDD8-conjugating E2 enzyme (UBC12), and various NEDD8-ligase E3 enzymes (6). Functionally, neddylation...

Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia‐inducible factor (HIF)‐1α and nuclear factor‐kappaB (NF‐κB) are key transcription factors of stress response genes. An NF‐κB inhibitor, inhibitor of NF‐κBα (IκBα), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. However, FIH did not functionally regulate IκBα, and the consequence of the FIH–IκBα interaction thus remains uncertain. In the present study, we tested the possibility that IκBα regulates FIH. FIH–IκBα binding was confirmed by yeast two‐hybrid and coimmunoprecipitation analyses. Functionally, IκBα expression further enhanced the transcriptional activity of HIF‐1α under hypoxic conditions. Furthermore, IκBα knockdown repressed HIF‐1α activity. Mechanistically, IκBα derepressed HIF‐1α activity by inhibiting the FIH‐mediated Asn803 hydroxylation of HIF‐1α. It was also found that IκBα activated HIF‐1α by sequestering FIH from HIF‐1α. However, the effect of IκBα on HIF‐1α activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor‐α treatment, IκBα downregulation, Asn803 hydroxylation and HIF‐1α inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that IκBα plays a positive regulatory role during HIF‐1‐mediated gene expression. Therefore, IκBα, owing to its interactions with NF‐κB and HIF‐1α, may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses.

Since prostate growth is governed by the androgen signaling pathway, blockade of the pathway is regarded as an appropriate strategy for the treatment of benign prostatic hyperplasia (BPH). Panax ginseng is known to have various pharmacological activities. Of several products of its root, red ginseng, having many bioactive ginsenosides, is most popularly used in Korea, and recently has been reported to control the proliferation of cancer cells. We here tested the effect of a water extract of Korean red ginseng (WKRG) on testosterone-induced prostate hyperplasia. WKRG (daily intraperitoneal injection) prevented prostate overgrowth and epithelial thickening induced by testosterone in rats, and suppressed a rat prostate kallikrein-S3. In human prostate cells, WKRG inhibited testosterone-induced cell proliferation, arrested cell cycle by inducing p21 and p27, and induced apoptosis. Testosterone-induced expression of human kallikrein-3 mRNA and activation of androgen receptor (AR) were effectively inhibited by WKRG. Of the major ginsenosides included in WKRG, 20(S)-Rg3 was identified to repress AR activity and to attenuate prostate cell growth during testosterone stimulation. Moreover, 20(S)-Rg3 downregulated AR by facilitating the degradation of AR protein. WKRG and 20(S)-Rg3 were found to have new pharmacological activities against testosterone-induced prostate overgrowth. Given that red ginseng has been used safely in Asia for 1000 years, red ginseng and 20(S)-Rg3 could be potential therapeutic regimens for treating BPH.