Abstract
BackgroundHypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.Methodology/Principal FindingsHere we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.Conclusions/SignificanceThese findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.
Highlights
The development of intratumoral hypoxia and angiogenesis is a hallmark of rapidly growing solid tumors [1]
As cobalt stabilized Hypoxia inducible factor-1a (HIF-1a) and to a larger extent HIF-2a levels and they were both repressed by zinc (Figure 1G), we evaluated the requirement of HIF-1a for VEGF inhibition by transducing U373MG cells with an expression vector encoding the dominant negative form of HIF-1a without DNA binding and transactivation domains (HIF-1aDN) [23]
Inhibition of HIF-1a by HIF1aDN vector strongly impaired the cobalt-induced VEGF-luc activity that was not further reduced by zinc (Figure 1H). These data show that zinc inhibited VEGF expression and tube formation induced by glioblastoma conditioned media (CM) and that HIF-1a was required for zinc-induced VEGF downregulation
Summary
The development of intratumoral hypoxia and angiogenesis is a hallmark of rapidly growing solid tumors [1]. Prolyl hydroxylation is inhibited, thereby stabilizing HIF-1a, which can translocate into the nucleus and bind to constitutively expressed HIF-1b, forming the active HIF-1 complex [7]. Transition metals such as cobaltous ions can inhibit hydroxylation of HIF-1a and induce elevated HIF-1a levels, mimicking hypoxia [3]. After dimerization with HIF-1b, HIF-1a binds to a promoter consensus sequence called hypoxiaresponsive element (HRE), and controls the expression of several target genes involved in many aspects of cancer progression, including angiogenesis (e.g., vascular endothelial growth factor VEGF), metabolic adaptation (e.g., Glut1), chemoresistance (e.g., MDR1), apoptosis resistance (e.g., Bcl2), invasion and metastasis (e.g., c-Met) [8]. We evaluated the molecular mechanisms of zinc-induced HIF-1a downregulation and whether zinc affected HIF-1a in vivo
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