5-fluorouracil suppresses stem cell-like properties by inhibiting p38 in pancreatic cancer cell line PANC-1.

Abstract

Introduction: Suppressing the phenotype of cancer stem cells (CSCs) is a promising treatment strategy for cancer. P38 mitogen-activated protein kinases (MAPK, p38) play an important role in the occurrence, development, and stemness maintenance of tumors. The aim of the current study was to investigate the effect of p38 on the stemness maintenance of CSCs in pancreatic cancer cell line PANC-1.

Material and methods: PANC-1 human pancreatic cancer cells were treated with 5-fluorouracil (5-FU) at 0.5 IC50, IC50, and 2 IC50 for 24 h. PANC-1 cells were treated for 24 h with 5-FU at 0.5IC50, IC50, and 2IC50 with or without VX-702, p38 phosphorylation inhibitor. Cells were resuspended in DMEM supplemented with 20 ng/ml epidermal growth factor, 2% B27, 5 mg/ml insulin, 20 g/ml basic fibroblast growth factor, and 10 μg/ml transferrin. Cells were seeded in ultra-low adhesion 6-well dishes to observe tumor spheroidization. The expression of CDK2, cyclin B1, cyclin D1, OCT4, SOX2, Nanog, and p38 was measured by Western blot. The mRNA expression of p38, OCT4, Nanog, and SOX2 was measured by RT-PCR. Flow cytometry was performed to evaluate the cell cycle, apoptosis, and proportion of CD44+CD133+ PANC-1 cells.

Results: 5-FU decreased cell viability and increased apoptosis. 5-FU suppressed the stemness maintenance of CSCs in PANC-1 cells, as demonstrated by the inhibition of tumorsphere formation, the decrease in CD44+CD133+ cells' fraction, and downregulation of OCT4, Nanog, and SOX2 expression. In addition, 5-FU inhibited the phosphorylation of p38 in PANC-1 cells. The phosphorylation of p38 was subsequently suppressed by VX-702, p38 mitogen-activated protein kinase inhibitor, which exhibited similar effects as those of 5-FU treatment. The effect of VX-702 on PANC-1 cells was further enhanced by 5-FU treatment. Thus, p38 inhibitor decreased the viability and increased the apoptosis of PANC-1 cells. P38 inhibitor suppressed the stemness maintenance of CSCs in PANC-1 cells, as demonstrated by the inhibition of tumorsphere formation, the decrease in CD44+CD133+ cells, and the downregulation of OCT4, Nanog, and SOX2 expression.

Conclusions: These findings indicate that the inhibition of p38 phosphorylation suppresses the stemness maintenance and 5-FU resistance of PANC-1 cells, providing a potential therapeutic target for the prevention and treatment of pancreatic cancer.