The role of hypoxia-inducible factor-1α and -2α in androgen insensitive prostate cancer cells.

The aim of this study was to investigate the effects of induction and knocking down of hypoxia-inducible factor (HIF)-1α and/or -2α on tumor biology in androgen insensitive prostate cancer cell lines. The induction patterns of HIF-1α and -2α after treatment with ZnSO4 were evaluated in PC3 and DU145 cells. Both cell lines were transfected with siRNA targeted against HIF-1α and/or -2α, and the expression patterns of these 2 HIF isoforms were examined. We next performed cell counting Kit-8 (CCK-8) assays and matrigel invasion assays. Potential additive effects of HIF blockade to chemotherapy (docetaxel) or target agents (sunitinib and sorafenib) were examined. In addition, gene expression changes were determined in ZnSO4-treated DU145 cells using Western blotting. ZnSO4 affected the expression of HIF in a dose-dependent manner. HIF expression was increased within the first 3 hours but then decreased. Cells in which HIF-1α and/or -2α had been knocked down using siRNA showed decreased cell viability. Invasion abilities were increased by ZnSO4 treatment in both cell lines overexpressing HIF. However, invasion potencies were decreased in response to treatment with HIF siRNAs. Blocking HIF prominently augmented the antitumor effects of target agents. The underlying mechanism could be associated with p21, cMET, IGF-1, and GLUT-1. Our results demonstrate that HIF-1α and -2α are important for cell proliferation and invasion ability in prostate cancer. Together, our results indicate that combinations of target agents with HIF knockdown may represent a promising strategy for the treatment of prostate cancer.

Jeong CW ，Yoon CY ，Jeong SJ ，Hong SK ，Byun SS ，Kwak C ，Lee SE ... -  《-》

Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors.

Nilsson MB ，Zage PE ，Zeng L ，Xu L ，Cascone T ，Wu HK ，Saigal B ，Zweidler-McKay PA ，Heymach JV ... -  《-》

Upregulation of HIF-2α induced by sorafenib contributes to the resistance by activating the TGF-α/EGFR pathway in hepatocellular carcinoma cells.

Sorafenib, the first-line systemic drug for advanced hepatocellular carcinoma (HCC), has demonstrated limited benefits with very low response rates. Thus it is essential to investigate the underlying mechanisms for the resistance to sorafenib and seek potential strategy to enhance its efficacy. Hypoxic cells inside solid tumors are extremely resistant to therapies as their survival ability is increased due to the cellular adaptive response to hypoxia, which is controlled by hypoxia-inducible factor (HIF)-1 and HIF-2. Sorafenib inhibits HIF-1α synthesis, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways and providing a mechanism for more aggressive growth of tumors. The present study has demonstrated that upregulation of HIF-2α induced by sorafenib contributes to the resistance of hypoxic HCC cells by activating the transforming growth factor (TGF)-α/epidermal growth factor receptor (EGFR) pathway. Blocking the TGF-α/EGFR pathway by gefitinib, a specific EGFR inhibitor, reduced the activation of STAT (signal transducer and activator of transcription) 3, AKT and ERK (extracellular signal-regulated kinase), and synergized with sorafenib to inhibit proliferation and induce apoptosis of hypoxic HCC cells. Transfection of HIF-2α siRNA into HCC cells downregulated the expression of VEGF (vascular endothelial growth factor), cyclin D1, HIF-2α and TGF-α, and inhibited the activation of EGFR. HIF-2α siRNA inhibited the proliferation and promoted the apoptosis of HCC cells in vitro, and synergized with sorafenib to suppress the growth of HCC tumors in vivo. The results indicate that targeting HIF-2α-mediated activation of the TGF-α/EGFR pathway warrants further investigation as a potential strategy to enhance the efficacy of sorafenib for treating HCC.

Zhao D ，Zhai B ，He C ，Tan G ，Jiang X ，Pan S ，Dong X ，Wei Z ，Ma L ，Qiao H ，Jiang H ，Sun X ... -  《-》

Increased activation of the hypoxia-inducible factor pathway in varicose veins.

Venous hypoxia has been postulated to contribute to varicose vein (VV) formation. Direct measurements of vein wall oxygen tension have previously demonstrated that the average minimum oxygen tensions were significantly lower in VVs compared with non-varicose veins (NVVs). Hypoxia-inducible factors (HIFs) are nuclear transcriptional factors that regulate the expression of several genes of oxygen homeostasis. This study aimed to investigate if hypoxia was associated with VVs by assessing the expression of HIF-1α, HIF-2α, HIF target genes, and upstream HIF regulatory enzymes in VVs and NVVs, and their regulation by hypoxia. VVs and NVVs were surgically retrieved and immediately snap-frozen or used for organ culture preparation. The relative expression of HIF-1α, HIF-2α, HIF target genes, and HIF regulatory enzymes in VVs and NVVs was analyzed with quantitative polymerase chain reaction (Q-PCR) and Western blot. VV and NVV organ ex vivo cultures were exposed to 16 hours of normoxia, hypoxia (oxygen tension 1%), or the hypoxia mimetic dimethyloxallyl glycine (DMOG) 1 mM in normoxia. The vein organ cultures were then analyzed for HIF-1α, HIF-2α, and their target gene expression with Q-PCR and Western blot. HIF-1α and HIF-2α mRNA were significantly upregulated in VVs compared with NVVs (89.8 ± 18.6 vs 10.4 ± 7.2 and 384.9 ± 209.4 vs 8.1 ± 4.2, respectively). HIF target gene mRNA expression was also significantly elevated in VVs compared with NVVs, namely glucose transporter-1 (GLUT-1; 8.7 ± 2.1 vs 1.0 ± 0.3), carbonic anhydrase-9 (CA9; 8.5 ± 2.1 vs 2.8 ± 1.2), vascular endothelial growth factor (VEGF; 7.5 ± 2.1 vs 0.9 ± 0.2), and BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP-3; 4.5 ± 0.7 vs 1.4 ± 0.3). The upregulation of HIF-1α, HIF-2α, and HIF target genes in VVs was also reflected at protein level. Of the HIF regulatory enzymes, the expression of prolyl-hydroxylase domain (PHD)-2 and PHD-3 was found to be elevated in VVs compared with NVVs. Exposure of VV and NVV organ cultures to hypoxia or DMOG was associated with increases in HIF-1α and HIF-2α protein and HIF target gene expression compared with normoxia only. The study concluded, we believe for the first time, an increased activation of the HIF pathway, with upregulation of the expression of HIF-1α and HIF-2α transcription factors, and HIF target genes, in VVs compared with NVVs. Exposure of VVs and NVVs to hypoxic conditions was associated with increased expression of HIF-1α and HIF-2α protein and HIF target genes. The data suggest that the HIF pathway may be associated with several pathophysiologic changes in the VV wall, and that hypoxia may be a feature contributing to VV pathogenesis.

Lim CS ，Kiriakidis S ，Paleolog EM ，Davies AH ... -  《-》

2-Methoxyestradiol synergizes with sorafenib to suppress hepatocellular carcinoma by simultaneously dysregulating hypoxia-inducible factor-1 and -2.

Sorafenib is the approved systemic drug of choice for advanced hepatocellular carcinoma (HCC), but has demonstrated limited benefits because of drug resistance. 2-Methoxyestradiol (2ME2) has been shown to be a promising anticancer drug against various types of cancers and acts by dysregulating hypoxia-inducible factor (HIF)-1. Hypoxic cancer cells are extremely resistant to therapies since they elicit strong survival ability due to the cellular adaptive response to hypoxia, which is controlled by HIF-1 and HIF-2. The present study has demonstrated that sorafenib downregulated the expression of HIF-1α, making the hypoxic response switch from HIF-1α- to HIF-2α-dependent pathways, resulting in upregulation of HIF-2α, which contributes to the insensitivity of hypoxic HCC cells to sorafenib. HIF-2α played a dominant role in regulating VEGF, thus sorafenib in turn increased the expression of VEGF (a downstream molecule of both HIF-1 and HIF-2) and cyclin D1 (a downstream molecule of HIF-2), but reduced the expression of LDHA (a downstream molecule of HIF-1), in hypoxic HCC cells. 2ME2 significantly reduced the expression of both HIF-1α and HIF-2α, and their downstream molecules, VEGF, LDHA and cyclin D1, rendering hypoxic HCC cells to increased sensitivity to 2ME2. 2ME2 also inhibited the nuclear translocation of HIF-1α and HIF-2α proteins, but had no effect on their mRNA expression. 2M2 synergized with sorafenib to suppress the proliferation and induction of apoptosis of HCC cells in vitro and in vivo, and inhibited tumoral angiogenesis. These results indicate that 2ME2 given in combination with sorafenib acts synergistically for treating HCC.

Ma L ，Li G ，Zhu H ，Dong X ，Zhao D ，Jiang X ，Li J ，Qiao H ，Ni S ，Sun X ... -  《-》