Cinobufacini Inhibits the Development of Pancreatic Cancer Cells through the TGF β/Smads Pathway of Pancreatic Stellate Cells

Abstract

This study aimed to test cinobufacini therapeutic potential for pancreatic cancer, verify its potential molecular mechanism, and evaluate the cinobufacini impact on pancreatic cancer microenvironment. First, the effect of cinobufacini-treated pancreatic stellate cells (PSCs) supernatant on the value-added ability of pancreatic cancer (PCCs) was tested. The results show that cinobufacini can effectively reduce the ability of PSCs supernatant to promote the value-added PCCs. Further results show that cinobufacini can effectively reduce the concentration of TGFβ in the supernatant of PSCs. Subsequently, the impact of cinobufacini on the transcription and translation levels of key genes in the TGFβ/Smads pathway was examined. The results showed that the impact of cinobufacini on the transcription levels of Smad2, Smad3, and Smad7 was in a concentration-dependent manner, while the transcriptional activity of collagen I mRNA was decreased with the increase of cinobufacini concentration. The results of protein expression showed that cinobufacini could upregulate the expression of inhibitory protein Smad7, inhibit the phosphorylation level of p-Smad2/3, and then suppress the expression of type I collagen (collagen I). On the one hand, this study shows that cinobufacini can inhibit the promotion of PSCs on the proliferation of PCCs. On the other hand, cinobufacini can upregulate the expression of the inhibitory molecule, Smad7, through the TGFβ/Smads pathway and reduce the phosphorylation level of p-Smad2/3, thereby inhibiting the expression of collagen I and pancreatic fibrosis. cinobufacin can inhibit the proliferation of SW1900 cells by blocking the TGFβ/Smads pathway of pancreatic stellate cells. These results provide a clinical basis for the treatment of pancreatic cancer.

Figure 1

PCC lines and proliferation index treated with different doses of cinobufacini. 0.0 mg/ml (a), 1.2 mg/ml (b), 6.0 mg/ml (c), and 30.0 mg/ml (d). (e) PCC proliferation index after treatment with different concentrations of cinobufacini. ^#Control group, ^∗∗p < 0.01.

Figure 2

Concentration of growth factors TGFβ and PDGF-BB in PSCs supernatant. (a) Concentration of growth factors TGFβ. (b) Concentration of growth factors PDGF-BB. ^#Control group, ^∗p < 0.05.

Figure 3

TGFβ/Smads pathway key gene transcription level. (a) The expression level of Smads2. (b) The expression level of Smads3. (c) The expression level of Smads7. (d) The expression level of collagen I treated with different concentrations of cinobufacini. ^#Control group, ^∗p < 0.05.

Figure 4

PSC TGFp/Smads pathway protein expression relative gray value and WB experiment results. (a–e) Smad2, Smad3, p-Smad2/3, Smad7, and collagen I protein expression relative gray value after treated with different concentrations of cinobufacini. ^#Control group, ^∗p < 0.05.