Conversion of epithelial-to-mesenchymal transition to mesenchymal-to-epithelial transition is mediated by oxygen concentration in pancreatic cancer cells

Abstract

Tumor metastasis is accompanied by a two-stage process of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). Currently, the exact mechanisms underlying EMT-MET conversion are unclear. In the present study, the mechanisms by which primary sites (hypoxic) and homing sites (normoxic or hyperoxic) participate in EMT-MET conversion were evaluated. Pancreatic cancer cells were grown under different oxygenation conditions. Cell morphology and epithelial (E)-cadherin and vimentin expression were examined. Transwell chambers were used to examine tumor invasiveness, and scratch assays were performed to examine cell migration. Reverse transcription-polymerase chain reaction and western blot analysis were used to quantitate the mRNA and protein expression of E-cadherin, vimentin, Snail and hypoxia-inducible factor (HIF)-1α. BxPc-3 and Panc-1 cells grown under hypoxic conditions demonstrated increased partial EMT, reduced E-cadherin expression, and increased vimentin expression, compared with cells grown under normoxic or hyperoxic conditions. Cells grown under hypoxic conditions also indicated increased migration and invasiveness. HIF-1α mRNA and protein expression was increased in cells grown under hypoxic conditions. These changes were reversed when a specific inhibitor of the HIF-1α receptor was used to block HIF-1α signaling. Differences in oxygen concentration at primary sites and homing sites are important in the EMT-MET process, and the underlying mechanism may involve HIF-1α-Snail signaling.

Keywords: epithelial-to-mesenchymal transition; hypoxia; hypoxia-inducible factor-1α; mesenchymal- to-epithelial transition; pancreatic cancer.

Figure 1.

Effects of different oxygen concentrations on cell proliferation. Compared with the hypoxia and normoxia groups, cells grown under moderate hyperoxia demonstrated no significant differences in proliferation or cell death (P>0.05). Hypoxia, 5% oxygen; normoxia, 21% oxygen; hyperoxia, 30% oxygen.

Figure 2.

Epithelial-to-mesenchymal transition-mesenchymal-to-epithelial transition conversion in different oxygen concentrations. (A) Following hypoxic treatment, cancer cells indicated a change in morphology from a round to spindle or long-spindle shape, with a disorderly, non-directional and radial appearance (×100). (B) Cancer cells grown in a hypoxic environment demonstrated higher vimentin fluorescence and lower E-cadherin fluorescence, compared with the normoxia and hyperoxia groups (×400). (C and D) Western blot analysis results indicated that the protein expression of vimentin in the hypoxia group was higher than that in the normoxia and hyperoxia groups, and E-cadherin protein expression was lower than that in the normoxia and hyperoxia groups. Hypoxia, 5% oxygen; normoxia, 21% oxygen; hyperoxia, 30% oxygen; E-cadherin, epithelial-cadherin. *P<0.05 vs. normoxia and hyperoxia groups.

Figure 3.

Cellular migration in different oxygen concentrations. Following treatment of cells for 48 h, Panc-1 and BxPc-3 cells exposed to normoxia or hyperoxia demonstrated a reduced migration capacity, compared with hypoxia-treated cells (×100). The migration index of normoxia or moderate hyperoxia treated cells was significantly reduced than hypoxia treated cells (P<0.05). Hypoxia, 5% oxygen; normoxia, 21% oxygen; hyperoxia, 30% oxygen. *P<0.05 vs. hypoxia group.

Figure 4.

Changes in the invasive capacity of pancreatic cancer cells in different oxygen concentrations. Results of Transwell migration assays indicated that the hypoxia group had a significantly higher number of migrated cells than the normoxia and hyperoxia groups (×200). Hypoxia, 5% oxygen; normoxia, 21% oxygen; hyperoxia, 30% oxygen. *P<0.05 vs. normoxia and hyperoxia groups.

Figure 5.

Effects of different oxygen concentrations on Snail mRNA and protein expression in pancreatic cancer cells. (A) Polymerase chain reaction results demonstrated that the hypoxia group had a significantly higher level of Snail mRNA than the normoxia and hyperoxia groups. (B) Results of statistical analysis of Snail mRNA in Panc-1 cells. (C) Results of statistical analysis of Snail protein in Panc-1 cells. (D) Western blot analysis results indicated that the level of Snail protein expression was significantly upregulated in the hypoxia group, compared with the normoxia and hyperoxia groups. (E) Results of statistical analysis of Snail mRNA in BxPc-3 cells. (F) Results of statistical analysis of Snail protein in BxPc-3 cells. Hypoxia, 5% oxygen; normoxia, 21% oxygen; hyperoxia, 30% oxygen. *P<0.05 vs. normoxia and hyperoxia groups.

Figure 6.

Hypoxia regulates Snail protein through HIF-1α in pancreatic cancer cells. (A) Western blot analysis demonstrated that the hypoxia group had significantly higher Snail expression, compared with the normoxia and hyperoxia groups (*P<0.05). (B) Following treatment with the HIF-1α-specific inhibitor, YC-1, western blot analysis indicated that Snail protein expression did not differ among the different oxygen environments (P>0.05). (C) Immunofluorescence data demonstrated that Snail fluorescence intensity did not differ between cells exposed to normoxic, hyperoxic and hypoxic conditions following treatment with the HIF-1α-specific inhibitor, YC-1 (×400). Hypoxia, 5% oxygen; normoxia, 21% oxygen; hyperoxia, 30% oxygen; E-cadherin, epithelial-cadherin; HIF, hypoxia-inducible factor.