ABSTRACT
Menin is a scaffold protein encoded by the multiple endocrine neoplasia type 1 (MEN1) gene in humans, and it interacts with a variety of transcriptional proteins to control active or repressive cellular processes. Here, we show that heterozygous ablation of Men1 in female mice reduces chemical carcinogen-induced liver carcinogenesis and represses the activation of the inflammation pathway. Using ChIP-on-chip screens and ChIP assays, we find that menin occupancy frequently coincides with H3K4me3 at the promoter of many liver cancer-related genes, such as Yes-associated protein (Yap1). Increased menin and Yap1 expression in human hepatocellular carcinoma specimens was associated with poor prognosis. Our findings reveal that menin plays an important epigenetic role in promoting liver tumorigenesis, and support the notion that H3K4me3, which is regulated by the menin-mixed-lineage leukemia complex, is a potential therapeutic target in hepatocellular carcinoma.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Phosphoproteins/genetics , Proto-Oncogene Proteins/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Carbon Tetrachloride/toxicity , Carcinogenesis/chemically induced , Carcinogenesis/genetics , Carcinogenesis/metabolism , Carcinoma, Hepatocellular/metabolism , Cell Cycle Proteins , Cell Line, Tumor , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/metabolism , Chromatin Immunoprecipitation , Diethylnitrosamine , Epigenesis, Genetic , Female , Hep G2 Cells , Humans , Kaplan-Meier Estimate , Liver Neoplasms/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Oligonucleotide Array Sequence Analysis , Phosphoproteins/metabolism , Proto-Oncogene Proteins/deficiency , RNA Interference , Up-Regulation , Xenograft Model Antitumor Assays , YAP-Signaling ProteinsABSTRACT
BACKGROUND & AIMS: The alterations of histone modification may serve as a promising diagnostic biomarker of hepatocellular carcinoma (HCC), but the clinical and mechanistic relatedness of the histone H3 lysine 27 and 4 trimethylation (H3K27me3 and H3K4me3) in HCC remains poorly understood. Here we propose that the combination of H3K27me3 and H3K4me3 is a more precise predictive/prognostic value for outcome of HCC patients. METHODS: We used chromatin immunoprecipitation (ChIP) assays and a ChIP-on-chip screen to analyse HCC. RESULTS: We found that the EZH2 occupancy coincides with the H3K27me3 at promoters and directly silences the transcription of target genes in HCC. The H3K27me3-related gene network of EZH2 contains well-established genes, such as CDKN2A, as well as previously unappreciated genes, including FOXO3, E2F1, and NOTCH2, among others. We further observed independently increasing profiles of H3K27me3 and H3K4me3 at the promoters of certain target genes in HCC specimens. Importantly, Kaplan-Meier analysis reveals that 3-year overall and tumour-free survival rates are dramatically reduced in patients that simultaneously express EZH2 and menin, compared to rates in the EZH2 or menin under expressing patients. Furthermore, an inhibitor of H3K27me3 alone, or in combination with an H3K4me3 inhibitor, effectively blocked the aggressive phenotype of HCC cells. CONCLUSIONS: Our results indicate that a combined analysis of both H3K27me3 and H3K4me3 may serve as powerful diagnostic biomarkers of HCC, and targeting both might benefit anti-HCC therapy.
Subject(s)
Carcinoma, Hepatocellular , Histones , Polycomb Repressive Complex 2/genetics , Proto-Oncogene Proteins/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Histones/analysis , Histones/metabolism , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Methylation , Middle Aged , Neoplasm Staging , Prognosis , Protein Processing, Post-Translational/geneticsABSTRACT
MEN1, which encodes the nuclear protein menin, acts as a tumor suppressor in lung cancer and is often inactivated in human primary lung adenocarcinoma. Here, we show that the inactivation of MEN1 is associated with increased DNA methylation at the MEN1 promoter by K-Ras. On one hand, the activated K-Ras up-regulates the expression of DNA methyltransferases and enhances the binding of DNA methyltransferase 1 to the MEN1 promoter, leading to increased DNA methylation at the MEN1 gene in lung cancer cells; on the other hand, menin reduces the level of active Ras-GTP at least partly by preventing GRB2 and SOS1 from binding to Ras, without affecting the expression of GRB2 and SOS1. In human lung adenocarcinoma samples, we further demonstrate that reduced menin expression is associated with the enhanced expression of Ras (p < 0.05). Finally, excision of the Men1 gene markedly accelerates the K-Ras(G12D)-induced tumor formation in the Men1(f/f);K-Ras(G12D/+);Cre ER mouse model. Together, these findings uncover a previously unknown link between activated K-Ras and menin, an important interplay governing tumor activation and suppression in the development of lung cancer.
Subject(s)
Adenocarcinoma/metabolism , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , Oncogene Protein p21(ras)/metabolism , Proto-Oncogene Proteins/biosynthesis , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation/genetics , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Mutant Strains , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Oncogene Protein p21(ras)/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins/genetics , SOS1 Protein/genetics , SOS1 Protein/metabolismABSTRACT
Hypoxia-inducible factor α proteins (HIF-αs) 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-1α expression and activity were inhibited by knocking down APPBP1 E1 enzyme for NEDD8 conjugation but enhanced by ectopically expressing NEDD8. HIF-1α and HIF-2α were identified to be covalently modified by NEDD8. NEDD8 stabilized HIF-1α 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-1α-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-1α. Furthermore, given the positive role played by HIF-αs in cancer promotion, the NEDD8 conjugation process could be a potential target for cancer therapy.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Protein Subunits/metabolism , Reactive Oxygen Species/metabolism , Ubiquitins/metabolism , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/metabolism , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , NEDD8 Protein , Neoplasms/metabolism , Procollagen-Proline Dioxygenase/metabolism , Protein Binding/physiology , Protein Stability , Protein Subunits/genetics , RNA, Small Interfering , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Ubiquitins/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
BACKGROUND: The prostate contains extremely high concentrations of zinc, but survives and grows without apparent injury. This begs the question as to how prostate cells avoid the toxic effects of zinc. In a previous study, the authors found that; HIF-1alpha is expressed concomitantly with the accumulation of zinc in the epithelial cells of normal rat prostates, the zinc ion stabilizes HIF-1alpha in prostate cells, and that HIF-1alpha protects prostate cells from zinc toxicity. In the present study, the authors addressed the mechanism responsible for the protective effect of HIF-1alpha in a high zinc environment. METHODS: Immunofluorescent staining, immunoblotting, reverse transcription-polymerase chain reaction, reporter assay, and cell cycle analysis. RESULTS: Survivin was induced by ZnCl(2) in a HIF-1 dependent manner in both DU-145 and PNT2 prostate cells. Furthermore, HIF-1 induced survivin expression at the transcriptional level and the induction of survivin was abolished by HIF-1alpha knock-down. In addition, HIF-1-dependent survivin overexpression promoted prostrate cell survival and prevented cell arrest in the presence of high zinc concentrations, and si-survivin transfected cells under zinc rich conditions contained markedly higher levels of cleaved caspase-9 and PARP than si-con transfected cells. Finally, survivin expression patterns well matched rat prostate proliferation statuses. CONCLUSION: Under zinc rich conditions, prostate epithelial cells HIF-1-dependently express survivin, which promotes prostate cell proliferation, and prevents apoptosis and cell cycle arrest. Accordingly, the HIF-1alpha-survivin pathway appears to facilitate prostate cell survival and growth in zinc rich environments, and this pathway could be a therapeutic target for the treatment of prostate hyperplasia.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Microtubule-Associated Proteins/metabolism , Prostate/metabolism , Zinc/toxicity , Animals , Apoptosis/drug effects , Apoptosis/physiology , Cell Line, Tumor , Chlorides/administration & dosage , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Mitosis/drug effects , Mitosis/physiology , Prostate/cytology , Prostate/drug effects , RNA/chemistry , RNA/genetics , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Survivin , Transcriptional Activation/drug effects , Transcriptional Activation/physiology , Zinc/metabolism , Zinc Compounds/administration & dosageABSTRACT
Hypoxia-inducible factor (HIF)-1 plays a key role in tumor promotion by inducing approximately 60 genes required for tumor adaptation to hypoxia; thus, it is viewed as a target for cancer therapy. For this reason, YC-1, which down-regulates HIF-1alpha and HIF-2alpha 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-1alpha, 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 Asn803 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-1alpha via the FIH-dependent CAD inactivation as well as via the protein down-regulation. Given these results, we suggest that the functional inactivation of HIF-alpha contributes to the YC-1-induced deregulation of hypoxia-induced genes.
Subject(s)
Enzyme Activators/pharmacology , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Indazoles/pharmacology , Repressor Proteins/metabolism , p300-CBP Transcription Factors/metabolism , Amino Acids, Dicarboxylic/pharmacology , Cell Line, Tumor , Genes, Reporter , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Indazoles/chemistry , Mixed Function Oxygenases , Models, Biological , Plasmids/metabolism , Protein Biosynthesis , RNA, Small Interfering/metabolism , Transcriptional ActivationABSTRACT
Miniature pigs are worth notice as candidate donors for xenotransplantation. However, donor organs are inevitably subjected to hypoxia, which causes vascular endothelial dysfunction. In this respect, hypoxia-inducible factor 1alpha (HIF-1alpha), the key factor for hypoxic adaptation, should be expressed in grafts. However, some immunosuppressive drugs have been reported to suppress HIF-1alpha in rat cells. Here, we first identified the cDNA and protein structures of miniature pig HIF-1alpha, and next investigated the effects of cyclosporine and FK506 on HIF-1alpha expression in endothelial cells of miniature pig. Thus, we conclude that FK506, rather than cyclosporine, may be recommended for xenotransplantation using miniature pig organs.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunosuppressive Agents/pharmacology , Swine, Miniature/metabolism , Swine/metabolism , Tacrolimus/pharmacology , Transplantation, Heterologous , Amino Acid Sequence , Anaerobiosis/drug effects , Anaerobiosis/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cloning, Molecular , Cyclosporine/pharmacology , DNA, Complementary , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Molecular Sequence Data , Protein Biosynthesis/drug effects , Protein Conformation , Swine/genetics , Swine/immunology , Swine, Miniature/genetics , Swine, Miniature/immunologyABSTRACT
Guided by bioisosterism and pharmacokinetic parameters, we designed and synthesized a series of novel benzamide derivatives. Preliminary in vitro studies indicated that compounds 10b and 10j show significant inhibitory bioactivity in HepG2 cells (IC50 values of 0.12 and 0.13 µM, respectively). Compounds 10b and 10j induced the expression of HIF-1α protein and downstream target gene p21, and upregulated the expression of cleaved caspase-3 to promote tumor cells apoptosis.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Benzamides/chemical synthesis , Benzamides/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Hep G2 Cells , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
To identify novel therapeutic agents to treat cancer, we synthesized a series of diaryl ether derivatives. Structure-activity relationship studies revealed that the presence of a chlorine or hydroxyl at the para-position on the phenyl ring (5h or 5k) significantly enhanced antitumor activity. Compound 5h had stronger growth inhibitory activity in HepG2, A549, and HT-29 cells than compound 5k, with IC50 values of 2.57, 5.48, and 30.04 µM, respectively. Compound 5h also inhibited the growth of other cells lines, including Hep3B, PLC/PRF5, SMMC-7721, HeLa, and A375, with IC50 values of 2.76, 4.26, 29.66, 18.86, and 10.21 µM, respectively. The antitumor activity of compound 5h was confirmed by a colony forming assay. Further, our results indicated that the antitumor activity of compound 5h may be mediated by enhancing expression of p21 and cl-caspase3, and leading to apoptosis of cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Ethers/pharmacology , Neoplasms/drug therapy , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Caspase 3/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Ethers/chemical synthesis , Ethers/chemistry , Gene Expression Regulation, Neoplastic/drug effects , HT29 Cells , HeLa Cells , Humans , Inhibitory Concentration 50 , Neoplasms/pathology , Structure-Activity RelationshipABSTRACT
In this study, a series of novel N-(piperidine-4-yl)benzamide derivatives was designed, synthesized, and evaluated for antitumor activity. Some compounds were found to have potent antitumor activity. In particular, compound 47 showed the most potent biological activity against HepG2 cells, with an IC50 value of 0.25 µm. Western blot analysis demonstrated that compound 47 inhibited the expression of cyclin B1 and p-Rb and enhanced the expression of p21, p53, Rb, and phospho-adenosine monophosphate-activated protein kinase (p-AMPK). Further, cell cycle arrest was observed by flow cytometry (FCM). In summary, compound 47 was screened to have potential activity for the treatment of hepatocarcinoma via the induction of cell cycle arrest by a p53/p21-dependent pathway.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/chemical synthesis , Benzamides/pharmacology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Piperidines/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Benzamides/chemistry , Carcinoma, Hepatocellular/pathology , Cell Cycle Checkpoints/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Drug Design , Drug Screening Assays, Antitumor , Hep G2 Cells , Humans , Inhibitory Concentration 50 , Liver Neoplasms/pathology , Piperidines/chemical synthesis , Piperidines/chemistry , Tumor Suppressor Protein p53/metabolismABSTRACT
OBJECTIVE: To study the effect of Boschniakia rossica extract on free radicals in the brain of D-galactose induced senile rats. METHODS: Sixty Wistar rats were randomly divided into normal group, model group (48 mg.kg(-1).d(-1) D-galactose, SC), Boschniakia rossica group (100, 150, 200 mg/kg ig and 48 mg.kg(-1).d(-1) D-galactose, SC). After 40 days, the activities of SOD, MAO and the content of MDA were measured with colorimetric method, and the histological changes were synchronously observed by electronic microscope. RESULTS: Boschniakia rossica extract significantly increased the SOD activity, decreased the MDA content, and inhibited the MAO activity in the brain tissue. It was observed under microscope that Boschniakia rossica extract could retrieve the degeneration of mitochondrion. CONCLUSION: Boschniakia rossica extract can clear the free radicals for D-galactose induced senile rats.
Subject(s)
Aging/drug effects , Brain/drug effects , Drugs, Chinese Herbal/pharmacology , Free Radicals/metabolism , Orobanchaceae , Aging/metabolism , Animals , Brain/metabolism , Female , Galactose/pharmacology , Male , Monoamine Oxidase/drug effects , Monoamine Oxidase/metabolism , Plant Extracts/pharmacology , Rats , Rats, Wistar , Superoxide Dismutase/drug effects , Superoxide Dismutase/metabolismABSTRACT
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.
Subject(s)
Ginsenosides/therapeutic use , Panax/chemistry , Plant Extracts/therapeutic use , Prostatic Hyperplasia/drug therapy , Prostatic Hyperplasia/metabolism , Receptors, Androgen/metabolism , Testosterone/adverse effects , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Humans , Male , Prostate-Specific Antigen/metabolism , Prostatic Hyperplasia/chemically induced , Rats , Signal Transduction/drug effectsABSTRACT
Hypoxia inevitably develops in rapidly growing tumors and acts as an important microenvironment that forces changes in tumor behavior. Hypoxia-inducible factor 1α (HIF-1α) is activated during hypoxia and promotes the progression of malignancy by stimulating angiogenesis and by augmenting the ability of tumors to survive. In aerobic conditions, HIF-1α is destabilized by the PHD prolyl-hydroxylases that target HIF-1α for proteolysis via the von Hippel-Lindau protein (pVHL) and further inactivated by the FIH asparaginyl-hydroxylase that precludes the recruitment of transcription coactivators. Although HIF-1α degradation is well understood, little is known about how its transcriptional activity increases gradually in response to decreasing oxygen. In particular, it has been questioned how FIH having a high affinity for oxygen regulates the HIF-1α activity in moderate hypoxia. We here found that the HIF-1α-FIH interaction is disrupted in 1-5% oxygen. Both in vitro and in vivo binding analyses revealed that pVHL acts as an adaptor for FIH to bind HIF-1α. Furthermore, because the pVHL-FIH interaction depends on oxygen tension, the FIH-mediated inactivation of HIF-1α can be exquisitely regulated according to the severity of hypoxia. Based on these findings, we propose that pVHL fine-tunes the transcriptional activity of HIF-1α in graded oxygen tensions.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mixed Function Oxygenases/metabolism , Oxygen/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Animals , Carcinoma, Renal Cell/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/chemistryABSTRACT
Benign prostatic hyperplasia (BPH) is a disease that impairs the well-being of many aged men. To alleviate BPH symptoms or to find a cure for this disease, key molecules should be identified that control prostate cell proliferation. Recently, HIF-1alpha has attracted attention in this context, because it is highly expressed in hyperplasic prostates and prevents prostate cell death. Thus, given that vitamin C inhibits HIF-1alpha expression in several malignant tumors, we examined its therapeutic potential in BPH. HIF-1alpha was noticeably induced by testosterone in prostate cells, and this HIF-1alpha induction was abolished by vitamin C. Vascular endothelial growth factor (VEGF) promoter activity reporter assays and semi-quantitative RT-PCR revealed that vitamin C inhibited HIF-1-dependent VEGF expression. Furthermore, HIF-1alpha suppression by vitamin C was rescued by knocking down HIF-prolyl hydroxylase-2, suggesting that vitamin C destabilizes HIF-1alpha via prolyl hydroxylation. Moreover, vitamin C treatment abolished cell proliferation induced by testosterone treatment to the control level. These results suggest that vitamin C inhibits testosterone-induced HIF-1alpha expression and by so doing effectively prevents prostate hyperplasia. In male rats, testosterone treatment for 4 weeks induced prostate hyperplasia. Furthermore, HIF-1alpha and VEGF levels were significantly elevated in hyperplasic prostates. In vitamin C-treated rats, however, most prostate hyperplasia parameters and prostrate HIF-1alpha/VEGF levels were markedly reduced. Accordingly, our findings indicate that vitamin C could be further developed clinically for use as an anti-BPH agent.
Subject(s)
Ascorbic Acid/therapeutic use , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Prostatic Hyperplasia/drug therapy , Animals , Ascorbic Acid/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Down-Regulation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Male , Prostate/drug effects , Prostate/metabolism , Prostatic Hyperplasia/prevention & control , Rats , Rats, Sprague-Dawley , Testosterone/antagonists & inhibitors , Testosterone/pharmacology , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia-inducible factor (HIF)-1alpha and nuclear factor-kappaB (NF-kappaB) are key transcription factors of stress response genes. An NF-kappaB inhibitor, inhibitor of NF-kappaB alpha (IkappaB alpha), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. However, FIH did not functionally regulate IkappaB alpha, and the consequence of the FIH-IkappaB alpha interaction thus remains uncertain. In the present study, we tested the possibility that IkappaB alpha regulates FIH. FIH-IkappaB alpha binding was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. Functionally, IkappaB alpha expression further enhanced the transcriptional activity of HIF-1alpha under hypoxic conditions. Furthermore, IkappaB alpha knockdown repressed HIF-1alpha activity. Mechanistically, IkappaB alpha derepressed HIF-1alpha activity by inhibiting the FIH-mediated Asn803 hydroxylation of HIF-1alpha. It was also found that IkappaB alpha activated HIF-1alpha by sequestering FIH from HIF-1alpha. However, the effect of IkappaB alpha on HIF-1alpha activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor-alpha treatment, IkappaB alpha downregulation, Asn803 hydroxylation and HIF-1alpha inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that IkappaB alpha plays a positive regulatory role during HIF-1-mediated gene expression. Therefore, IkappaB alpha, owing to its interactions with NF-kappaB and HIF-1alpha, may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses.