Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway

Abstract

Despite rapid advances in many fronts, pancreatic cancer (PC) remains one of the most difficult human malignancies to treat due, in part, to de novo and acquired chemoresistance and radioresistance. Gemcitabine alone or in combination with other conventional therapeutics is the standard of care for the treatment of advanced PC without any significant improvement in the overall survival of patients diagnosed with this deadly disease. Previous studies have shown that PC cells that are gemcitabine-resistant (GR) acquired epithelial-mesenchymal transition (EMT) phenotype, which is reminiscent of "cancer stem-like cells"; however, the molecular mechanism that led to EMT phenotype has not been fully investigated. The present study shows that Notch-2 and its ligand, Jagged-1, are highly up-regulated in GR cells, which is consistent with the role of the Notch signaling pathway in the acquisition of EMT and cancer stem-like cell phenotype. We also found that the down-regulation of Notch signaling was associated with decreased invasive behavior of GR cells. Moreover, down-regulation of Notch signaling by siRNA approach led to partial reversal of the EMT phenotype, resulting in the mesenchymal-epithelial transition, which was associated with decreased expression of vimentin, ZEB1, Slug, Snail, and nuclear factor-kappaB. These results provide molecular evidence showing that the activation of Notch signaling is mechanistically linked with chemoresistance phenotype (EMT phenotype) of PC cells, suggesting that the inactivation of Notch signaling by novel strategies could be a potential targeted therapeutic approach for overcoming chemoresistance toward the prevention of tumor progression and/or treatment of metastatic PC.

Figure 1

L3.6pl Gemcitabine-resistant (GR) cells undergo an epithelial-mesenchymal transition (EMT) phenotype compared to the epithelial phenotype of L3.6pl gemcitabine-sensitive (GS) cells. A: Morphology of GS and GR cells. B: Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify E-cadherin, vimentin and ZEB1 mRNA expression in GS and GR cells. **, P<0.01 compared with GS. C: Western blot analysis showing the expression of markers of epithelial and mesenchymal phenotypes of cells.

Figure 2

Immunofluorescence staining for the expression and cellular localization of β-catenin, E-cadherin, vimentin and F-actin.

Figure 3

Notch pathway is up-regualted in GR cells. A: Real-time RT-PCR was used to quantify Notch pathway genes at the mRNA levels in GS and GR cells. *, P<0.05, **, P<0.01 compared with GS. B: Western blot analysis showing the expression of Notch pathway related proteins. C: Immunofluorescence staining for the expression and cellular localization for Notch-2, Notch-4 and Jagged-1.

Figure 4

MMP-9 and uPA were up-regulated in GR cells. A: Real-time RT-PCR reaction was used to quantify uPA, VEGF, MMP-2 and MMP-9 mRNA expression in GS and GR cells. *, P<0.05 compared with GS. B: Western blot analysis showing MMP-9 and uPA protein levels were significantly increased in the GR cells. C: Left, MMP-9 activity assay showing that MMP-9 was up-regulated in GR cells; right, uPA assay showing that uPA level in culture medium was up-regulated in GR cells.

Figure 5

Notch pathway contributes to the regulation of molecular markers of epithelial-mesenchymal transition (EMT) in GR cells. Abbreviations: CS, control siRNA; NS, Notch-2 siRNA; JS, Jagged-1 siRNA. A: Real-time RT-PCR was used to quantify Notch-2 and Jagged-1 mRNA expression in GR cells transfected with specific siRNA. *, P<0.05, **, P<0.01 compared with CS. B: Down-regulation of Notch-2 and Jagged-1 could cause reversal of EMT phenotype of GR cells. C-D: GR cells transfected with control siRNA or Notch-2 siRNA or Jagged-1 siRNA were assessing the expression of markers of epithelial and mesenchymal phenotypes using Real-time RT-PCR and Western blot analysis, respectively.

Figure 6

Down-regulation of Notch-2 and Jagged-1 inhibited cell migration and invasion, and reduce attachment and detachment of GR cells. Abbreviations: GS, Gemcitabine-sensitive; GR, gemcitabine-resistant. CS, control siRNA; NS, Notch-2 siRNA; JS, Jagged-1 siRNA. *, P<0.05, **, P<0.01 compared with control. A: GR cells showed increased cell attachment and detached. B: Down-regulation of Notch-2 or Jagged-1 in GR cells dramatically decreased the attachment and detachment of cells. C: Transfection of GR cells with Notch-2 siRNA or Jagged-1 siRNA inhibited the migration and invasion.