HIF-1alpha links beta-adrenoceptor agonists and pancreatic cancer cells under normoxic condition

Abstract

Aim: To examine whether beta-adrenoceptor (beta-AR) agonists can induce hypoxia-inducible factor (HIF)-1alpha accumulation which then up-regulate the expression of its target genes in pancreatic cancer cells at normoxia, and to further elucidate the mechanism involved.

Methods: Pulse-chase assay, RT-PCR, and Western blot were employed to detect the effects of beta-AR agonists and antagonists, siRNA as well as several inhibitors of signal transduction pathways on MIA PaCa2 and BxPC-3 pancreatic cancer cells.

Results: Treatment of pancreatic cancer cell lines with beta-AR agonists led to accumulation of HIF-1alpha and then up-regulated expression of its target genes independently of oxygen levels. The induction was partly or completely inhibited not only by beta-AR antagonists but also by inhibitors of PKA transduction pathways and by siHIF-1alpha. Both beta1-AR and beta2-AR agonists produced the above-mentioned effects, but beta2-AR agonist was more potent.

Conclusion: Activation of beta-AR receptor transactivates epidermal growth factor receptor (EGFR) and then elicits Akt and ERK1/2 in a PKA-dependent manner, which together up-regulate levels of HIF-1alpha and downstream target genes independently of oxygen level. Our data suggest a novel mechanism in pancreatic cancer cells that links beta-AR and HIF-1alpha signaling under normoxic conditions, with implications for the control of glucose transport, angiogenesis and metastasis.

Figure 1

Expressions of β1-AR and β2-AR at mRNA and protein levels in MIA PaCa2 and BxPC-3 cells. (A) Expression of mRNA for β1-AR, β2-AR, and β-actin in MIA PaCa2 and BxPC-3 cells. Total RNA was isolated and subjected to RT-PCR. The β1-AR primers amplified a 236 bp fragment, the β2-AR primers amplified a 526 bp fragment and the β-actin amplified a 179 bp fragment. Lane 1 MIA PaCa2, β2-AR primers; Lane 2 MIA PaCa2, β2-AR and β-Actin primers; Lane 3 MIA PaCa2, β-Actin primers; Lane 5 BxPC-3, β-AR2 primers; Lane6 BxPC-3, β2-AR, and β-actin primers; Lane7 BxPC-3, β-actin primers; Lane 9 MIA PaCa2, β1-AR primers; Lane 10 MIA PaCa2, β1-AR and β-actin primers; Lane 11 MIA PaCa2, β-actin primers; Lane 13 BxPC-3, β1-AR primers; Lane 14 BxPC-3, β1-AR, and β-actin primers; Lane15 BxPC-3, β-actin primers; Lane 4, 8, 12, and 16 are negative controls without MMLV reverse transcriptase. (B) Expression of protein for β1-AR, β2-AR and β-actin in MIA PaCa2 and BxPC-3 cells. Total lysate from untreated cells was subjected to Western blot with anti-β1-AR and anti-β2-AR antibody. The house keeping protein β-actin was used as a control to ensure equal loading of the protein.

Figure 2

Time course of HIF-1α protein levels following treatment with β-AR agonists. (A) MIA PaCa2 and BxPC-3 cells were treated with xamoterol, salbutamol and isoproterenol; 3% oxygen provided a positive control. Protein levels were determined using Western blotting. (B) Quantitation of Western blotting data. Data from at least 3 independent experiments with duplicate determinations are expressed as means±SEM versus controls. ^bP<0.05 vs control.

Figure 3

Pulse-chase assay and cycloheximide (Chx) inhibition test. (A) In both cells, newly synthesized HIF-1α protein declined after 20 min and was hardly detectable after 60 min in the presence of β1-AR or β2-AR agonist and hypoxia. (B) HIF-1α protein expression was reduced by cycloheximide indicating that HIF-1α accumulation is also dependent on ongoing protein synthesis.

Figure 4

siRNA inhibition assay. Both siRNAs efficiently blocked β-AR-agonists-induced enhancement of HIF-1α protein expression in both kinds of cells, whose inhibition rate ranged similarly from 47% to 61%.

Figure 5

Changes in VEGF, MMP9, GLUT-1, and CXCR4 mRNA levels in (A) MIA PaCa2 and (B) BxPC-3 cells following several treatments as indicated below the panel and 3% oxygen provided a positive control. Cells were treated with the drugs alone or in combinations as indicated below the histogram and mRNA levels were quantified by Real-time PCR. Data from at least 3 independent experiments with duplicate determinations were are expressed as means±SEM vs controls. ^bP<0.05 vs control.

Figure 6

Intracellular cAMP levels following treatment with β-AR agonists and antagonists. MIA PaCa2 and BxPC-3 cells were treated with a range of activators and inhibitors as indicated below the histogram; forskolin provided the positive control. Data from at least 3 independent experiments with duplicate determinations were expressed as means±SEM vs controls. ^bP<0.05 vs control.

Figure 7

Phosphorylation of EGFR (Tyr 1173, Tyr1608, Tyr992) in response to β-AR agonists and antagonists. (A) MIA PaCa2 and BxPC-3 cells were treated with drugs as indicated below the panel and tyrosine phosphorylation at 3 sites within EGFR was assessed using specific antibodies. EGF provided the positive control. (B) Quantitation of Western blotting. Data from at least 3 independent experiments expressed as means±SEM vs controls. ^bP<0.05 vs control.

Figure 8

HIF-1α polypeptide accumulation, ERK1/2 phosphorylation (Thr202, Tyr204), and Akt phosphorylation (Ser473) after treatment with β-AR agonists and antagonists. (A) MIA PaCa2 and BxPC-3 cells were treated with drugs as indicated below the panel and protein and phosphoprotein levels were determined using Western blotting. 3% oxygen provided the positive control. (B) Quantitation of Western blotting. Data from at least 3 independent experiments with duplicate determinations were expressed as means±SEM vs controls. ^bP<0.05 vs control.