Figure 1.
Hypoxia induces EMT phenotype in pancreatic cancer cells.
(A) Western blot analysis of epithelial marker (E-cadherin) and mesenchymal markers (Vimentin, N-cadherin) and HIF-1α of PANC-1, BxPC-3 cells under normoxic (N) or hypoxic (H) conditions for 48 h. A representative blot from three independent experiments was shown. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (B) Phase contrast analysis (original magnification, ×100) of morphological changes detected in pancreatic cancer cells under normoxic or hypoxic conditions for 48 h. (C) Immuofluorescence staining of E-cadherin and Vimentin in PANC-1, BxPC-3 cells under normoxic or hypoxic conditions for 48 h. Green represents E-cadherin staining, whereas red represents Vimentin staining. Blue signal represents nuclear DNA staining by DAPI. (D) The viability of cells was assessed by the Cell Counting Kit-8 assay and used to calculate the viability index. PANC-1 and BxPC-3 cells were exposed to gemcitabine (0 µmol/L) for 48 h as the baseline. *, p<0.05. (E) The proliferation of cells was measured by crystal violet assay. Figures were selected as representative scenes from three independent experiments. (F) Matrigel invasion assay. Left, photomicrographs of cells that have passed through Matrigel under normoxic or hypoxic conditions for 48 h (original magnification, ×100). Right, quantification of invasion. *, p<0.05.
Figure 2.
Hypoxia induced HIF-1α and NF-κB overexpression in pancreatic cancer cells.
PANC-1 and BxPC-3 cells were cultured under normoxic (N) or hypoxic conditions (H) for various periods (24 h, 48 h, 72 h). After each indicated incubation period, the cells were collected. Nuclear extracts and total protein extracts were prepared. (A) NF-κB p65 and HIF-1α were analyzed by Western blot analysis from respective cell homogenate. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (B) EMSA for NF-κB DNA-binding activity on respective nuclear extracts. Competitive assay confirmed the specificity of NF-κB binding to the DNA (see Materials and methods for details). Right two lanes, wt, wild-type; m, mutant. (C) VEGF was analyzed by Western blot analysis from respective cell homogenate. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (D) Western blot analysis. Effects of the NF-κB p65 siRNA on VEGF expression of PANC-1 and BxPC-3 cells under hypoxic conditions for 48 h. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05.
Figure 3.
Inhibition of NF-κB reverses EMT characteristics in pancreatic cancer cells under hypoxic conditions.
(A) Western blot analysis. Effects of the NF-κB p65 siRNA and IKK inhibitor BAY 11-7082 on N-cadherin and E-cadherin expression of PANC-1 and BxPC-3 cells under hypoxic conditions for 48 h. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (B) Matrigel invasion assays. Left, photomicrographs after cells were treated with NF-κB p65 siRNA or BAY 11-7082 (10 µmol/L) or appropriate respective controls. Original magnification, ×100. Right, quantification of invasion assay. *, p<0.05. (C) The proliferation of cells was measured by Crystal violet assay. Figures were selected as representative scenes from three independent experiments. (D) The viability of cells was assessed by the Cell Counting Kit-8 assay and used to calculate the viability index. *, p<0.05.
Figure 4.
Heightened expression of Twist in pancreatic cancer cells under hypoxic conditions is attributable to increased NF-κB activity.
(A) Western blot analysis of baseline expression of EMT-mediating transcription factors: Twist, Snail, Zeb1, and Zeb2 in pancreatic cancer cells under normoxic (N) versus hypoxic (H) conditions for 48 h. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (B) Western blot analysis. Dose-dependent effects of a 48-h exposure of the IKK inhibitor BAY 11-7082 on expression of Twist, Snail, Zeb1, and Zeb2 under hypoxic conditions. A representative blot from three independent experiments was shown. The histogram showed the average volume density corrected for the loading control (β-actin).
Figure 5.
Overexpression of HIF-1α in pancreatic cancer cells under normoxic conditions induces NF-κB activation.
(A) Western blot analysis of NF-κB p65 and HIF-1α expression of PANC-1 cells under hypoxic conditions for 48 h (H/48 h), PANC-1 cells transduced with pcDNA3.0 empty vector (pcDNA3.0/N) and pcDNA3.0/HIF-1α (HIF-1α+/N) under normoxic conditions for 48 h. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (B) EMSAs for NF-κB DNA-binding activity in H/48 h, HIF-1α+/N, and pcDNA3.0/N PANC-1 cells. Right two lanes, cold competition EMSA.
Figure 6.
NF-κB blockade reverses the EMT driven by the HIF-1α.
(A) Western blot analysis of levels of indicated proteins in PANC-1 cells transduced with pcDNA3.0 empty vector (pcDNA3.0/N) and pcDNA3.0/HIF-1α (HIF-1α+/N) under normoxic conditions for 48 h. The histogram showed the average volume density corrected for the loading control (β-actin). *, p<0.05. (B) EMT phenotype changes was confirmed with Immuofluorescence microscopy staining of E-cadherin and Vimentin. Green represents E-cadherin staining, whereas red represents Vimentin staining. Blue signal represents nuclear DNA staining by DAPI. (C) Western blot analysis. Dose-dependent effects of a 48-h exposure to the IKK inhibitor BAY 11-7082 on the indicated proteins. The histogram showed the average volume density corrected for the loading control (β-actin). (D) Matrigel invasion assays. Effects of a 48-h BAY 11-7082 (10 µmol/L) on invasion of PANC-1 cells under normoxic conditions. Left, photomicrographs at original magnification of ×100; Right, histogram to illustrate invasion assay results. *, p<0.05. (E) Crystal violet assay. Effects of a 48-h BAY 11-7082 (10 µmol/L) on gemcitabine susceptibility of PANC-1 cells under normoxic conditions. Figures were selected as representative scenes from three independent experiments. (F) The Effects of IKK inhibitor on gemcitabine susceptibility of PANC-1 cells under normoxic conditions was also measured by Cell Counting Kit-8 assay and used to calculate the viability index. *, p<0.05.