Dominant-negative hypoxia-inducible factor-1 alpha reduces tumorigenicity of pancreatic cancer cells through the suppression of glucose metabolism

Abstract

In the tumor cells exposed to hypoxia, hypoxia-inducible factor-1 (HIF-1)-mediated adaptation responses such as angiogenesis and anaerobic metabolism are induced for their survival. We have recently reported that the constitutive expression of HIF-1 alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and glucose deprivation. We then established dominant-negative HIF-1 alpha (dnHIF-1 alpha) transfectants and examined their susceptibility to apoptosis and growth inhibition induced by hypoxia and glucose deprivation in vitro and their tumorigenicity in SCID mice. We further examined the expressions of aldolase A and Glut-1 in vitro and Glut-1 expression and glucose uptake in the tumor tissues and microvessel counts in the tumor tissues. As a result, dnHIF-1 alpha rendered the pancreatic cancer cells sensitive to apoptosis and growth inhibition induced by hypoxia and glucose deprivation. Also it abrogated the enhanced expression of Glut-1 and aldolase A mRNAs under hypoxia and reduced the expression of Glut-1 and the glucose uptake in the tumor tissues and consequently in vivo tumorigenicity. We found no significant difference in the microvessel counts among the tumor tissues. From these results, we suggest that the disruption of the HIF-1 pathway might be effective in the treatment of pancreatic cancers.

Figure 1.

Expression of dominant-negative HIF-1α and hypoxia-inducible mRNAs. A: Structures of HIF-1α and dominant-negative HIF-1α are shown. A deletion mutant of HIF-1α (amino acids 30 to 389) lacking a DNA-binding domain, transactivation domains, and an oxygen-dependent degradation domain was generated from full-length HIF-1α (amino acids 1 to 826). This deletion mutant was reported to function in a dominant-negative manner through the inhibition of functional HIF-1 formation. B: Dominant-negative HIF-1α mRNA expressions in a vector transfectant and three dominant-negative HIF-1α transfectants are shown. C: Binding of HIF-1 to HIF-1-binding sites examined by gel shift assay and HIF-1α protein expression examined by Western blot are shown. D: Glut-1 and aldolase A mRNA expressions examined by Northern blot and mRNA expression of VEGF amplified by RT-PCR in the transfectants under hypoxic (H) and nonhypoxic (N) conditions are shown. Representative results of three different experiments are shown.

Figure 2.

Proliferation of the transfectants in hypoxia and low glucose. A: After incubation in normoxia and normal glucose medium for indicated times, viable cell numbers were estimated by a colorimetric MTS assay. The data are presented as mean ± SD of three different experiments. B: After incubation in hypoxia and low glucose for indicated times, viable cell numbers were estimated by a colorimetric MTS assay. The data are presented as mean ± SD of three different experiments.

Figure 3.

Apoptosis induced by hypoxia and low glucose in the transfectants. A: After incubation under normoxia, hypoxia, and hypoxia plus low glucose for 48 hours, apoptotic cell death was analyzed by the use of a FACScaliber. A representative result of three different experiments is shown. B: Mean ± SD of three different experiments is shown.

Figure 4.

Growth of the tumors in SCID mice. Five mice in each group were inoculated with 5 × 10⁶ cells on day 0. Tumor size was measured every 3 days after inoculation.

Figure 5.

Immunohistochemical staining of the tumor tissues for CD31/PECAM. A: A representative result of immunohistochemical staining of the tumor tissues of V3 and dnH3 for endothelial cell marker (CD31/PECAM). a: V3; b: dnH3. B: Mean ± SD of microvessel counts in three different tumor tissues in each group is shown. Original magnifications, ×200.

Figure 6.

Immunohistochemical staining for Glut-1 in the tumor tissues. A representative result of immunohistochemical staining of the tumor tissues of V3 and dnH3 for Glut-1. a: H&E staining of the tumor of V3; b: HE staining of the tumor of dnH3; c: immunohistochemical staining of the tumor of V3; d: immunohistochemical staining of the tumor of dnH3. Original magnifications, ×200.

Figure 7.

FDG uptake in the tumor tissues. A: Blood glucose levels in the mice. No significant difference was observed among the mice. B: FDG uptake in blood, tumor tissue, liver, and muscle is shown. Mean ± SD of FDG uptake in five mice is shown. C: Immunohistochemical staining of tumor tissues with anti-human p53 antibody is shown. As the antibody is specific to human p53 protein, p53-positive cells are judged as human pancreatic tumor cells. Strong staining indicated p53-positive cells in the tumor tissues. a: V3; b: dnH3; c: dnH7; d: dnH10. Original magnification, ×40.