Zinc Downregulates HIF-1α and Inhibits Its Activity in Tumor Cells In Vitro and In Vivo

Hypoxia 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.Here 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.These 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

Effect of zinc on HIF-1-induced VEGF in glioblastoma and tube formation.

<p>(<b>A, upper panel</b>) U373MG glioblastoma cells were transfected with VEGF-luc reporter and 16 h thereafter treated with 100 µM ZnCl₂ and 2% O₂ for, respectively 24 and 16 h, before luciferase activity was assayed. Data are the mean ±S.D. of three independent experiments performed in duplicate. RLU: relative luciferase unit. *, P<0.005. (<b>A, lower panel</b>) Total cell extracts of cells treated as above were assayed for Western immunoblotting. Anti-tubulin was used as protein loading control. (<b>B</b>) U373MG glioblastoma cells were transfected with VEGF-luc reporter and 16 h thereafter treated with 100 µM ZnCl₂ and 200 µM CoCl₂ for, respectively 24 and 16 h, before luciferase activity was assayed. Data are the mean ±S.D. of three independent experiments performed in duplicate. RLU: relative luciferase unit. *, P<0.005. (<b>B, lower panel</b>) Total cell extracts of cells treated as in (A) for Western immunoblotting. (<b>C</b>) Total mRNAs were reverse transcribed from U373MG cells treated as in (A), for semi-quantitative RT-PCR analysis of VEGF expression. Aldolase (ald-A) was used as internal control. (<b>D</b>) Total mRNAs were reverse transcribed from U373MG cells treated as in (B), for semi-quantitative RT-PCR analysis of VEGF expression. Aldolase (ald-A) was used as internal control. (<b>E</b>) Serum-starved U373MG cells were cultured for 24 h with 100 µM ZnCl₂, and cell conditioned media were analyzed by ELISA assay for VEGF secretion. ELISA results (mean ±S.D.) for duplicates from four independent experiments are shown. *, P<0.005 compared with the untreated control. (<b>F</b>) HUVECs were incubated at 37°C on Matrigel with CM form U373MG cells untreated or treated with ZnCl₂ (100 µM) in normoxia or under hypoxia (CoCl₂ 200 µM for 24 h). The number of tube networks from triplicate wells (10 fields/well) was quantified at ×20 magnification after 3 hours of differentiation. (<b>G</b>) U373MG cells were treated with 100 µM ZnCl₂ and 200 µM CoCl₂ for, respectively 24 and 16 h and nuclear cell extracts were assayed for Western immunoblotting. Anti-Hsp70 was used as protein loading control. (<b>H</b>) U373MG cells were transfected with HIF-1α dominant negative vector (DN-HIF-1α, 2 µg) and 16 h after transfection treated with 100 µM ZnCl₂ and 200 µM CoCl₂ for, respectively 24 and 16 h. Luciferase activity was assayed 36 h thereafter and normalized by β-galactosidase activity. Data represent mean ±S.D. of three independent experiments performed in duplicate. *, P<0.001.</p

Zinc decreases tumor cell invasiveness <i>in vitro</i> and induces HIF-1α downregulation <i>in vivo</i>.

<p>(<b>A</b>) Serum-starved U373MG cells were treated with 100 µM ZnCl₂ and 200 µM CoCl₂ for, respectively 24 and 16 h and cell invasion was measured using a Boyden's chamber invasion assay. Cell invasion results (mean ±S.D.) for quadruplicates from four independent experiments are shown. *, P<0.0001. (<b>B</b>) Serum-starved C27 cells were treated with 100 µM for 24 h, under basal “hypoxic” condition and cell invasion was measured as in (A). Cell invasion results (mean ±S.D.) for quadruplicates from four independent experiments are shown. *, P<0.0001. (<b>C</b>) Representative tumors derived from human U373MG cells transfected with HIF-1α-ODD-luc and pcDNA3-luc control vectors marked with luciferase were imaged using the IVIS imaging system 200 series at day 6 after tumor cell injection and at day 10 following 4 days of zinc daily administration. Four mice/group are shown. (<b>D</b>) RNA samples from explanted tumors, at day 10 after tumor cell injection and after 4 days of zinc treatment, were used for reverse-transcription (RT)-PCR. The mRNA levels were normalized to GAPDH expression. (<b>E</b>) Tissue samples as in (D) were used for Western immunoblotting of VEGF and HIF-1α levels and anti-tubulin and anti-Hsp70 were used, respectively, as protein loading control. Similar results were obtained with different tissue samples.</p

HIFs-α destabilization and inhibition of HIF-1 transcriptional activity by zinc.

<p>(<b>A</b>) Proliferating C38 and C27 prostate cancer cells were treated with 100 µM ZnCl₂ and 2% O₂ for, respectively 24 and 16 h. Equal amount of nuclear cell extracts were assayed for Western immunoblotting. Anti-Hsp70 was used as protein loading control. (<b>B</b>) Proliferating C38 and C27 prostate cancer cells were treated with 100 µM ZnCl₂ and 200 µM CoCl₂ for, respectively 24 and 16 h. Equal amount of nuclear cell extracts were assayed for Western immunoblotting. Anti-Hsp70 was used as protein loading control. (<b>C</b>) ChIP analysis with anti-HIF-1α antibody or no antibody as control was performed in C38 cells treated with 100 µM ZnCl₂ and 2% O₂ for, respectively 24 and 16 h and in C27 cells treated with100 µM ZnCl₂ for 24 h under basal “hypoxic” condition. Recruitment of HIF-1α onto the VEGF promoter was detected by qRT-PCR, using primers spanning the HRE region. Relative enrichment of HIF-1α compared to no antibody onto VEGF promoter is shown. The data represent the mean of 2 independent experiments ±S.D. (<b>D, upper panel</b>) C38 and C27 cells were transfected with VEGF-luc reporter and 16 h after transfection treated with 100 µM ZnCl₂ and 200 µM CoCl₂ for, respectively 24 and 16 h, before luciferase activity was assayed. Data represent the mean ±S.D. of three independent experiments performed in duplicate. RLU: relative luciferase unit. *, P<0.005. (<b>D, lower panel</b>) Total mRNAs were reverse transcribed from C38 and C27 cells treated as above for semi-quantitative RT-PCR analyses of HIF-1 target genes. Aldolase (ald-A) is shown as internal control.</p

Silencing of GSTP1, a prostate cancer prognostic gene, by the estrogen receptor-β and endothelial nitric oxide synthase complex

We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-β and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ERβ/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ERβ antagonist or its natural ligand 5α-androstane-3β,17β-diol, eNOS inhibitors or ERβ small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ERβ/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ERβ/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ERβ/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ERβ/eNOS function by 3β-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications