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PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer.

Su Y, Loos M, Giese N, Hines OJ, Diebold I, Görlach A, Metzen E, Pastorekova S, Friess H, Büchler P - Br. J. Cancer (2010)

Bottom Line: The effects of PHD3 in tumour growth are largely unknown.The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany.

ABSTRACT

Purpose: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.

Experimental design: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.

Results: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.

Conclusion: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

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Related in: MedlinePlus

Modulation of PHD3 expression in human pancreatic cancer cell lines and hypoxic gene expression: PHD3 mRNA levels in pancreatic cancer cell lines were measured by qRT-PCR (A). PHD3 transfection increased PHD3 protein levels in MIA PaCa-2 and PANC-1 cells (B). HIF-1 western blot analysis of PHD3 and control tranfected cells after 16 h of hypoxia (C). Targeting PHD3 expression by siRNA. PHD3 western blot of siPHD3 or control siRNA-treated Capan-1 and Capan-2 cells (D). Whole-cell extracts were resolved on 10% SDS polyacrylamide gels, proteins were transferred to nitrocellulose membranes and probed with antibodies against PHD3. Incubation with the γ-tubulin monoclonal antibody was performed as a loading control (B, D). VEGF secretion determined by enzyme-linked immunosorbent assay (E). Capan-1 and -2 cells were treated by siRNA to suppress PHD3 expression, whereas MIA PaCa-2 and PANC-1 cells were transfected with a PHD3 expression vector to restore PHD3 expression (F). *P<0.05 indicates statistical significance of hypoxic vs normoxic treatment; #P<0.05 indicates statistical significance within the hypoxic treatment panel.
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fig2: Modulation of PHD3 expression in human pancreatic cancer cell lines and hypoxic gene expression: PHD3 mRNA levels in pancreatic cancer cell lines were measured by qRT-PCR (A). PHD3 transfection increased PHD3 protein levels in MIA PaCa-2 and PANC-1 cells (B). HIF-1 western blot analysis of PHD3 and control tranfected cells after 16 h of hypoxia (C). Targeting PHD3 expression by siRNA. PHD3 western blot of siPHD3 or control siRNA-treated Capan-1 and Capan-2 cells (D). Whole-cell extracts were resolved on 10% SDS polyacrylamide gels, proteins were transferred to nitrocellulose membranes and probed with antibodies against PHD3. Incubation with the γ-tubulin monoclonal antibody was performed as a loading control (B, D). VEGF secretion determined by enzyme-linked immunosorbent assay (E). Capan-1 and -2 cells were treated by siRNA to suppress PHD3 expression, whereas MIA PaCa-2 and PANC-1 cells were transfected with a PHD3 expression vector to restore PHD3 expression (F). *P<0.05 indicates statistical significance of hypoxic vs normoxic treatment; #P<0.05 indicates statistical significance within the hypoxic treatment panel.

Mentions: PHD3 mRNA expression in cultured pancreatic cancer cell lines Capan-1, PANC-1 and MIA PaCa-2 was also determined by qRT-PCR. PHD3 mRNA expression was detected in the well-differentiated tumour cell lines Capan-1 (83±8copies per μl) and Capan-2 (145±24copies/μl). The less-differentiated PANC-1 cell line expressed only low levels of PHD3 mRNA (16±3copies per μl), whereas the undifferentiated MIA PaCa-2 cell line was entirely devoid of PHD3 mRNA expression (Figure 2A).


PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer.

Su Y, Loos M, Giese N, Hines OJ, Diebold I, Görlach A, Metzen E, Pastorekova S, Friess H, Büchler P - Br. J. Cancer (2010)

Modulation of PHD3 expression in human pancreatic cancer cell lines and hypoxic gene expression: PHD3 mRNA levels in pancreatic cancer cell lines were measured by qRT-PCR (A). PHD3 transfection increased PHD3 protein levels in MIA PaCa-2 and PANC-1 cells (B). HIF-1 western blot analysis of PHD3 and control tranfected cells after 16 h of hypoxia (C). Targeting PHD3 expression by siRNA. PHD3 western blot of siPHD3 or control siRNA-treated Capan-1 and Capan-2 cells (D). Whole-cell extracts were resolved on 10% SDS polyacrylamide gels, proteins were transferred to nitrocellulose membranes and probed with antibodies against PHD3. Incubation with the γ-tubulin monoclonal antibody was performed as a loading control (B, D). VEGF secretion determined by enzyme-linked immunosorbent assay (E). Capan-1 and -2 cells were treated by siRNA to suppress PHD3 expression, whereas MIA PaCa-2 and PANC-1 cells were transfected with a PHD3 expression vector to restore PHD3 expression (F). *P<0.05 indicates statistical significance of hypoxic vs normoxic treatment; #P<0.05 indicates statistical significance within the hypoxic treatment panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990580&req=5

fig2: Modulation of PHD3 expression in human pancreatic cancer cell lines and hypoxic gene expression: PHD3 mRNA levels in pancreatic cancer cell lines were measured by qRT-PCR (A). PHD3 transfection increased PHD3 protein levels in MIA PaCa-2 and PANC-1 cells (B). HIF-1 western blot analysis of PHD3 and control tranfected cells after 16 h of hypoxia (C). Targeting PHD3 expression by siRNA. PHD3 western blot of siPHD3 or control siRNA-treated Capan-1 and Capan-2 cells (D). Whole-cell extracts were resolved on 10% SDS polyacrylamide gels, proteins were transferred to nitrocellulose membranes and probed with antibodies against PHD3. Incubation with the γ-tubulin monoclonal antibody was performed as a loading control (B, D). VEGF secretion determined by enzyme-linked immunosorbent assay (E). Capan-1 and -2 cells were treated by siRNA to suppress PHD3 expression, whereas MIA PaCa-2 and PANC-1 cells were transfected with a PHD3 expression vector to restore PHD3 expression (F). *P<0.05 indicates statistical significance of hypoxic vs normoxic treatment; #P<0.05 indicates statistical significance within the hypoxic treatment panel.
Mentions: PHD3 mRNA expression in cultured pancreatic cancer cell lines Capan-1, PANC-1 and MIA PaCa-2 was also determined by qRT-PCR. PHD3 mRNA expression was detected in the well-differentiated tumour cell lines Capan-1 (83±8copies per μl) and Capan-2 (145±24copies/μl). The less-differentiated PANC-1 cell line expressed only low levels of PHD3 mRNA (16±3copies per μl), whereas the undifferentiated MIA PaCa-2 cell line was entirely devoid of PHD3 mRNA expression (Figure 2A).

Bottom Line: The effects of PHD3 in tumour growth are largely unknown.The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, University of Heidelberg, Im Neuenheimer Feld 110, Heidelberg 69120, Germany.

ABSTRACT

Purpose: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown.

Experimental design: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model.

Results: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis.

Conclusion: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

Show MeSH
Related in: MedlinePlus