Interleukin-32 regulates downstream molecules and promotes the invasion of pancreatic cancer cells

Abstract

Pancreatic cancer is a malignant neoplasm with high invasiveness and poor prognosis. In a previous study, a highly invasive pancreatic cancer cell line was established and found to feature enhanced interleukin-32 (IL-32) expression. However, whether IL-32 promotes the invasiveness by enhancing or suppressing the expression of IL-32 through regulating downstream molecules was unclear. To investigate the effect of IL-32, cells were established with high levels of expression or downregulated IL-32; their invasive ability was measured using a real-time measurement system and the expression of some candidate downstream molecules involved in invasion was evaluated in the two cell types. The morphological changes in both cell types and the localization of IL-32 expression in pancreatic cancer tissues were studied using immunohistochemistry. Among the several splice variants of IL-32, cells transfected with the ε isoform had increased invasiveness, whereas the IL-32-suppressed cells had reduced invasiveness. Several downstream molecules, whose expression was changed in the two cell types, were monitored. Notably, changes of E-cadherin, CLDN1, CD44, CTGF and Wnt were documented. The morphologies of the two cell types differed from the original cell line. Immunohistochemically, the expression of IL-32 was observed only in tumor cells and not in normal pancreatic cells. In conclusion, IL-32 was found to promote the invasiveness of pancreatic cancer cells by regulating downstream molecules.

Keywords: E-cadherin; epithelial mesenchymal transition; interleukin-32; invasion; pancreatic cancer cell.

Figure 1.

Expression of IL-32 mRNA and protein. (A and B) Upregulation of IL-32, (C and D) downregulation of IL-32 by knockdown with siRNA, and (E and F) by knockout.

Figure 2.

Time-course changes of the invasiveness in IL-32. (A) Upregulated, (B) knockdown, and (C) knockout cells.

Figure 3.

Inverted microscopic findings. (A) Original parental cells, (B) cells with upregulated IL-32ε and (C) IL-32β, and (D) IL-32 knockout cells. Yellow arrows indicate characteristic structure of protrusion and white arrows indicate spindle-shaped cells with poor protrusions. Scale bar indicates 100 µm.

Figure 4.

Immunohistochemical findings in resected tissues. (A) Normal pancreatic duct, (B) cancer cells, and (C) invading cells in the invasive front.

Figure 5.

Western blot analysis of the invasion-associated proteins, IL-32-upregulated cells (ε, β) and IL-32 knockout cells (ko). Expression pattern were affected by altered expression of IL-32, especially, the expression pattern of E-cadherin was inversely associated with the expression of IL-32.