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

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