No abstract

SummaryAdaptation to hypoxia involves hypoxia-inducible transcription factors (HIFs) and requires reprogramming of cellular metabolism that is essential during both physiological and pathological processes. In contrast to the established role of HIF-1 in glucose metabolism, the involvement of HIFs and the molecular mechanisms concerning the effects of hypoxia on lipid metabolism are poorly characterized. Here, we report that exposure of human cells to hypoxia causes accumulation of triglycerides and lipid droplets. This is accompanied by induction of lipin 1, a phosphatidate phosphatase isoform that catalyzes the penultimate step in triglyceride biosynthesis, whereas lipin 2 remains unaffected. Hypoxic upregulation of lipin 1 expression involves predominantly HIF-1, which binds to a single distal hypoxiaresponsive element in the lipin 1 gene promoter and causes its activation under low oxygen conditions. Accumulation of hypoxic triglycerides or lipid droplets can be blocked by siRNA-mediated silencing of lipin 1 expression or kaempferol-mediated inhibition of HIF-1. We conclude that direct control of lipin 1 transcription by HIF-1 is an important regulatory feature of lipid metabolism and its adaptation to hypoxia.

Bortezomib represents the first proteasome inhibitor (PI) with demonstrated antitumor activity in the clinical setting, particularly for treatment of hematological malignancies. At the preclinical level, its action is shown to be mediated by induction of growth arrest and apoptosis in many tumor types, including androgen-dependent (AD) and androgen-independent (AI) prostate cancer (PCa) cells. Hypoxia-inducible factor-1α (HIF-1α), which is directly involved in tumor growth, is one of the most studied and promising molecular targets for anti-cancer therapy and is often overexpressed in PCa. Bortezomib has been reported to impair tumor growth by also inhibiting HIF-1α. In this study, we investigated the effect of bortezomib on the expression, activity and localization of HIF-1α in LNCaP (AD) and PC3 (AI) PCa cells. First, we show that hypoxic upregulation of HIF-1α protein levels and activity involves both the PI3K/Akt/mTOR and p44/42 MAPK pathways. Second, bortezomib inhibits expression of HIF-1α protein under both normoxic and hypoxic conditions, represses HIF-1 transcriptional activity and attenuates the release of vascular endothelial growth factor. These effects correlate with the ability of bortezomib to cause dephosphorylation of phospho-Akt, phospho-p70S6K, and phospho-S6RP, thus inactivating a pathway known to be required for HIF-1α protein expression at the translational level. Furthermore, bortezomib also abrogates p44/42 MAPK phosphorylation, which results to reduced nuclear translocation of HIF-1α. Taken together, these results suggest that bortezomib inhibits HIF-1α protein synthesis and its nuclear targeting through suppression of PI3K/Akt/mTOR and MAPK pathways, respectively, in both AD and AI PCa cells.

; Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, South Korea g Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infectionbased assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-␣) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCVhost cell interaction. H epatitis C virus (HCV) infection causes a wide range of clinical manifestations, from a healthy carrier state to acute and chronic hepatitis that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Nearly 3% of the world's population is chronically infected with HCV (1, 2), and current therapeutic approaches are not broadly effective (3).HCV is a positive-strand RNA virus with a 9.6-kb genome that is flanked at both termini by conserved, nontranslated regions (NTRs), required for RNA translation and replication. The 5= NTR comprises an internal ribosome entry site (IRES) that directs the expression of a polyprotein precursor (4, 5). The polyprotein is cleaved into structural (core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins that, in association with cellular factors, form a membrane-associated replicase complex. This copies the viral positive-strand RNA into a negative-strand intermediate that serves as the template for the synthesis of progeny genomes. The alternative reading frame (ARFP) or coreϩ1 and minicore proteins, with as-yet-unknown functions, appear to be synthesized from the core region by alternative translation mechanisms (6, 7).Studies of the...

Data for antiradical properties of saffron extract and its bioactive constituents (crocins, crocetin) are limited and poorly discussed in comparison with those of extracts containing potent scavengers. Further examination was sought using the Folin-Ciocalteu (F-C) reagent and various free radical species produced in cell-free or cell model systems. Oregano and turmeric methanol extracts, rich in well established scavengers, and also crocetin, rosmarinic acid, and curcumin, representing the major types of constituents in the three studied extracts, were used as "reference". On the same weight basis, saffron extract activity was found to be rather negligible in all cell-free systems with regard to that found for reference ones. On the contrary, in the human monocyte system, saffron extracts or free crocetin were found to reduce ROS production as effectively as the phenolic antioxidants. Our findings point out that saffron extracts exhibit a remarkable intracellular antioxidant activity that cannot be revealed using assays repeatedly applied to the evaluation of phenolic-type antioxidants.