Bortezomib represses HIF-1α protein expression and nuclear accumulation by inhibiting both PI3K/Akt/TOR and MAPK pathways in prostate cancer cells.

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.

Isolation of hypoxia-inducible factor 1 (HIF-1) inhibitors from frankincense using a molecularly imprinted polymer.

Hypoxia-Inducible Factor 1 (HIF-1), a transcriptional activator, is highly involved in the pathology of cancer. Inhibition of HIF-1 retards tumor growth and enhances treatment efficiency when used in combination with chemo- or radiation therapy. The recent validation of HIF-1 as an important drug target in cancer treatment has stimulated efforts to identify and isolate natural or synthetic HIF-1 inhibitors. In the present study, quercetin, a known inhibitor of HIF-1, was imprinted in a polymer matrix in order to prepare a Molecularly Imprinted Polymer (MIP), which was subsequently used for the selective isolation of new inhibitors from frankincense, a gum resin used as anticancer remedy in traditional medicine. The frankincense components isolated by Solid Phase Extraction on MIP (MIP-SPE), efficiently inhibited the transcriptional activity of HIF-1 and decreased the protein levels of HIF-1α, the regulated subunit of HIF-1. The selective retention of acetyl 11-ketoboswellic acid (AKBA, one of the main bioactive components of frankincense) by MIP led to the revealing of its inhibitory activity on the HIF-1 signaling pathway. AKBA was selectively retained by SPE on the quercetin imprinted polymer, with an imprinting effect of 8.1 ± 4.6. Overall, this study demonstrates the potential of MIP application in the screening, recognition and isolation of new bioactive compounds that aim selected molecular targets, a potential that has been poorly appreciated until.

Flavonoids induce HIF-1alpha but impair its nuclear accumulation and activity.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is the regulatory subunit of the transcription factor HIF-1, which is highly involved in the pathology of diseases associated with tissue hypoxia. In this study we investigated the ability of plant flavonoids to induce HIF-1alpha and regulate HIF-1 transcriptional activity in HeLa cells. We demonstrate for the first time that the flavonoids baicalein, luteolin and fisetin, as well as the previously investigated quercetin, induce HIF-1alpha under normal oxygen pressure, whereas kaempferol, taxifolin, and rutin are inactive. We further reveal that the capability of flavonoids to bind efficiently intracellular iron and their lipophilicity are essential for HIF-1alpha induction. Despite the ability of flavonoids to stabilize HIF-1alpha, the transcriptional activity of HIF-1 induced by flavonoids was significantly lower than that observed with the iron chelator and known HIF-1 inducer, desferrioxamine (DFO). Furthermore, when cells in which HIF-1 had been induced by DFO were also treated with flavonoids, the transcriptional activity of HIF-1 was strongly impaired without simultaneous reduction in HIF-1alpha protein levels. Localization of HIF-1alpha by immuno- and direct fluorescence microscopy and in vitro phosphorylation assays suggest that flavonoids inhibit HIF-1 activity by impairing the MAPK-dependent phosphorylation of HIF-1alpha, thereby decreasing its nuclear accumulation.

The flavonoid quercetin induces hypoxia-inducible factor-1alpha (HIF-1alpha) and inhibits cell proliferation by depleting intracellular iron.

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1alpha and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1alpha expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein-chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1alpha by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1alpha and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.

Cobalt induces hypoxia-inducible factor-1alpha (HIF-1alpha) in HeLa cells by an iron-independent, but ROS-, PI-3K- and MAPK-dependent mechanism.

The iron-chelator desferrioxamine (DFO) and the transition metal cobalt induce hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. DFO stabilizes HIF-1alpha from proteolysis by inhibiting the activity of iron-dependent prolyl hydroxylases, but the mechanism of action of cobalt is not fully elucidated. The purpose of this study was to examine the regulation of HIF-1alpha induction and HeLa cell proliferation by cobalt and the role of iron in these processes. Our results show that, unlike DFO, induction of transcriptionally active HIF-1alpha by CoCl2 cannot be abrogated by the addition of excess Fe3+, but involves the production of reactive oxygen species (ROS) and the operation of the phosphatidylinositol-3 kinase (PI-3K) and MAPK pathways. CoCl2, as well as DFO, decreased HeLa cell proliferation, but these effects were reversed by the addition of Fe3+. We conclude that the effect of cobalt on cell proliferation is iron-dependent, while its effects on HIF-1alpha induction are ROS- and signaling pathways-dependent, but iron-independent.

Identification of the four most common beta-globin gene mutations in Greek beta-thalassemic patients and carriers by PCR-SSCP: advantages and limitations of the method.

In the present study we investigated whether the single-strand conformational polymorphism (SSCP) method could be employed to identify (rather than simply detect) the four most common beta-globin gene mutations in the Greek population: IVS-I-110, Cd39, IVS-I-1, and IVS-I-6. Using DNA from 50 beta-thalassemic patients and carriers, we amplified by PCR the appropriate 238-bp region of the human beta-globin gene, analyzed the reaction products by nondenaturing polyacrylamide gel electrophoresis, and visualized the bands by silver staining. Single-stranded DNA (ssDNA) fragments showed a reproducible pattern of bands that was characteristic of the mutations present. With the use of control samples containing six of the 10 possible combinations of the four most common beta-globin gene mutations, we were able to predict the mutations present in a quarter of the patients studied. Our predictions were confirmed independently by the amplification refractory mutation system (ARMS) method. We conclude that this non-radioactive PCR-SSCP method can be used to reliably identify mutations in patients, provided that suitable controls are available. Moreover, the method is easy to apply to the identification of mutations in carriers, which makes it particularly useful for population screening.

Cobalt induces hypoxia-inducible factor-1alpha expression in airway smooth muscle cells by a reactive oxygen species- and PI3K-dependent mechanism.

Cobalt can mimic hypoxia and has been implicated as a cause of lung defects. However, the effect of cobalt on airway smooth muscle (ASM) cells has not been analyzed in detail. In this article, we use primary cultures of ASM cells from rabbit trachea and show that exposure to cobalt chloride causes a rapid increase of the intracellular levels of hypoxia-inducible factor-1alpha, which is detected predominantly inside the nucleus. With the use of specific inhibitors, we demonstrate that induction of hypoxia-inducible factor-1alpha by cobalt depends on active protein synthesis but not transcription. Furthermore, wortmannin, LY294002, and N-acetyl-L-cysteine inhibit the effect of cobalt, suggesting that it involves the phosphatidylinositol 3 kinase pathway and production of reactive oxygen species. Interestingly, cobalt chloride attenuates the contractile response of rabbit airways induced by potassium chloride, but not by acetylcholine, suggesting a link between the cellular response to hypoxic stimuli and the contractile properties of ASM cells.