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* This work was supported by National Institutes of Health NCI Grant RR16440 and American Heart Association Grant 0160166B (to B. H. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β/aryl hydrocarbon nuclear translocator subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. In response to hypoxia, HIF-1 activates the expression of many genes including vascular endothelial growth factor (VEGF) and erythropoietin. HIF-1 and VEGF play an important role in angiogenesis and tumor progression. Vanadate is widely used in industry, and is a potent inducer of tumors in humans and animals. In this study, we demonstrate that vanadate induces HIF-1 activity through the expression of HIF-1α but not HIF-1β subunit, and increases VEGF expression in DU145 human prostate carcinoma cells. We also studied the signaling pathway involved in vanadate-induced HIF-1α and VEGF expression and found that phosphatidylinositol 3-kinase/Akt signaling was required for HIF-1 and VEGF expression induced by vanadate, whereas mitogen-activated protein kinase pathway was not required. We also found that reactive oxygen species (ROS) were involved in vanadate-induced expression of HIF-1 and VEGF in DU145 cells. The major species of ROS responsible for the induction of HIF-1 and VEGF expression was H2O2. These results suggest that the expression of HIF-1 and VEGF induced by vanadate through PI3K/Akt may be an important signaling pathway in the vanadate-induced carcinogenesis, and ROS may play an important role.
hypoxia-inducible factor 1
vascular endothelial growth factor
reactive oxygen species
aryl hydrocarbon nuclear translocator
mitogen-activated protein kinase
mitogen-activated protein kinase kinase
mammalian target of rapamycin
minimum essential medium
fetal bovine serum
enzyme-linked immunosorbent assay
Hypoxia-inducible factor 1 (HIF-1)1 is a heterodimer of HIF-1α and HIF-1β subunits, which contain basic helix-loop-helix PAS domains (
). HIF-1α interacts with tumor suppressor Von Hippel-Lindau protein. The mutation of Von Hippel-Lindau protein in human cancers results in the constitutive expression of HIF-1 under nonhypoxic conditions (
). Vanadium exists in oxidation states ranging from −1 to +5. Among these oxidation states, the pentavalent state is the most stable form. Previous studies provide evidence that in mammalian systems vanadium(V) is more toxic than vanadium(IV). The data on the carcinogenic activity of vanadium is limited. Epidemiological studies have shown a correlation between vanadium exposure and the incidence of cancer in humans (
). It has been reported that upon reduction by cellular reactants, vanadate is able to generate a whole spectrum of ROS, i.e. O, H2O2, and ·OH. It is well known that ROS play an important role in carcinogenesis induced by a variety of carcinogens. Through ROS-mediated reaction, vanadate is able to induce activation of activator protein-1 expression (
). Therefore, we tested whether vanadate is able to induce the expression of HIF-1α and VEGF in DU145 human prostate carcinoma cells and evaluated the role of individual ROS. The following specific questions were addressed: (a) whether vanadate is able to induce HIF-1 and VEGF expression; (b) which signaling pathway(s) is/are involved in vanadate-induced expression of HIF-1α protein; (c) whether ROS species are involved in vanadate-induced HIF-1α and VEGF expression; and (d) which species of ROS play a critical role.
The results obtained from this study show that vanadate was able to induce HIF-1α and VEGF protein expression in a dose- and time-dependent manner in DU145 cells, whereas HIF-1β protein expression was not affected by vanadate treatment. It has been reported that HIF-1β is not significantly affected by cellular oxygen tension (
). Because vanadium has been found to act in an insulin-like manner, vanadate may increase HIF-1α expression by a similar signal pathway as insulin and hypoxia. It is known that HIF-1 activates VEGF expression by directly binding to VEGF promoter in response to hypoxia (
, 59–66). It is possible that the induction of HIF-1 and VEGF may play an important role in vanadate-induced carcinogenesis.
The present study also indicates that the PI3K/AKT/FRAP signaling pathway was required for induction of HIF-1 and VEGF expression induced by vanadate, whereas the mitogen-activated protein kinase/ERK pathway was not involved. The following results provide the evidence to support this conclusion: (a) PI3K inhibitors, LY294002 and wortmannin, inhibited HIF-1α expression induced by vanadate, whereas MEK inhibitor, PD98059, did not; (b) vanadate-induced PI3K activity in a dose- and time-dependent manner, and LY294002 and wortmannin inhibited the vanadate-induced PI3K activity; (c) vanadate induced Akt phosphorylation in a dose- and time-dependent manner, and LY294002 and wortmannin inhibited the Akt phosphorylation; (d) rapamycin, a mTOR/FRAP inhibitor, inhibited HIF-1α expression induced by vanadate in a dose-dependent manner; and (e) LY294002 and wortmannin decreased the VEGF protein level induced by vanadate in a dose-dependent manner, whereas the MEK inhibitor, PD98059, did not exhibit any effect. These data suggest that activation of the PI3K/AKT/mTOR signaling pathway and induction of HIF-1 and VEGF expression could be an important mechanism to understand the vanadate-induced carcinogenesis.
Our study also indicates that ROS are involved in vanadate-induced HIF-1α and VEGF expression. Among them, H2O2plays a critical role based on the following evidence: (a) exposure of cells to vanadate-generated H2O2 as determined by quantitative H2O2 assay; (b) ESR spin trapping measurements show that cells pretreated with vanadate generated ·OH radical using H2O2 as a precursor; (c) vanadate increased the rate of cellular oxygen consumption; (d) catalase, a specific scavenger of H2O2, decreased the generation of ROS induced by vanadate; (e) catalase inhibited HIF-1α and VEGF expression and Akt phosphorylation induced by vanadate, whereas sodium formate, a scavenger of ·OH, showed a minor effect; and (f) superoxide dismutase, a scavenger of O, did not inhibit vanadate-induced HIF-1α and VEGF expression or AKT phosphorylation. It may be noted that catalase, a scavenger of H2O2, abolished the hypoxia- and CoCl2-induced stabilization of HIF-1α, and exogenous H2O2 stabilized HIF-1α expression during normoxia, suggesting that H2O2 acts as a signaling element in this response (
). The mechanism of ROS generation induced by vanadate could be that in the presence of NADPH and several cellular flavoenzymes (such as glutathione reductase, lipoyl dehydrogenase, ferredoxin-NADP+, and NADPH oxidase) the mitochondria electron transport chain is able to reduce vanadate to vanadium(IV) (
). During the reduction process, molecular oxygen was consumed to generate O, which was subsequently converted to H2O2 through superoxide dismutase dismutation (Fig. 11). Vanadium(IV) is also able to generate ·OH radical from H2O2via a Fenton-like reaction (
Molecular oxygen is the original source of ROS generation in DU145 cells under vanadate stimulation as demonstrated by the oxygen consumption assay. The major pathways involved in ROS generation are both the flavoprotein-containing NADPH oxidase complex and the mitochondrial electron transport chain. This conclusion is supported by the inhibition of ROS generation by DPI, a flavoprotein inhibitor, as well as rotenone, an inhibitor of the mitochondrial electron chain as determined by ESR measurements. In conclusion, this work demonstrates that vanadate is able to induce HIF-1α and VEGF expression via the PI3K/AKT/mTOR/FRAP signaling pathway. H2O2generated during the cellular reduction of vanadate is the major species responsible for vanadate-induced expression of HIF-1α and VEGF. Further study of the role of HIF-1α and VEGF expression in vanadate-induced carcinogenesis will be the next important challenge.