Abstract:
Objective The present study aimed to examine the presence and mechanism of Fas-induced epithelial-mesenchymal transition(EMT) in human colon cancer cells.
Methods Colon cancer cells, namely, SW480 and DLD1, were treated with low-dose FasL(12.5 ng/mL) for 3 d.Total protein and total RNA levels of the experimental and control groups were extracted, and Western blot and RT-PCR were performed to measure both the transcriptional and translational levels of epithelial markers such as CDH1 and Villin, mesenchymal markers such as CHD2 and Vimentin, and EMT transcriptional factors such as Snail and Twist.Immunofluorescence was performed to observe the cellular distribution of EMT transcriptional factors on the third day of treatment.Snail and Twist stably knocked-down colon cancer cell lines were established and confirmed by Western blot.Colon cancer cells with low-expressing Snail or Twist were treated with low-dose FasL for 3 d.Western blot and RT-PCR were performed as described above to study the dependence of Fas-induced EMT on either Snail or Twist.Colon cancer cells, specifically SW480, were treated with low-dose FasL for 1 h, and Western blot was performed to test the activation status of the extra-cellular regulated protein kinases 1/2(ERK1/2) pathway and the p38 pathway at 15 min, 30 min, and 1 h.SW480 was pretreated for 2 h with the signaling pathway inhibitor, which was demonstrated efficiently by Western blot, and then treated with low-dose FasL for 3 d.Western blot and RT-PCR were performed as described above to explore the possible mechanisms of Fas-induced EMT.
Results The transcriptional and translational levels of the epithelial markers decreased, whereas those of the mesenchymal markers and of the EMT transcriptional factors increased in colon cancer cells, namely, SW480 and DLD1, after treatment of low-dose FasL for 3 d.The resulting levels were accompanied by the nucleus translocation of EMT transcription factors and cellular morphological changes from paving-stone-like shape into spindle-like shape.This result confirms the occurrence of EMT.The stable knock-down of Snail or Twist eliminated the Fas-induced EMT process mentioned above.Low-dose FasL activated the ERK1/2 pathway, whereas the ERK inhibitor weakened the FasL-induced EMT process.
Conclusion Low-dose FasL can induce the EMT process in colon cancer cells, namely, SW480and DLD1, through the activation of the ERK1/2 signaling pathway.