Sinapine Thiocyanate Inhibits the Proliferation and Mobility of Pancreatic Cancer Cells by Up-Regulating GADD45A

Abstract

Background: Sinapine thiocyanate (ST), an alkaloid isolated from the seeds of cruciferous species, has exhibited anti-inflammatory, anti-malignancy, and anti-angiogenic effects in previous studies. However, the effects and molecular mechanisms of action of ST in pancreatic cancer (PC) are still limited. Materials and methods: PC cells were treated with different concentrations (0, 20, 40, and 80 μM) of ST. The proliferative ability of PC cells in vitro was determined using cell count kit-8 (CCK-8), 5-ethynyl-2' deoxyuridine, colony formation, and flow cytometry assays. The mobility of PC cells in vitro was analyzed using wound healing assay, transwell assay, Western blotting, and immunofluorescence. High-throughput sequencing followed by bioinformatics analysis, reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), and Western blotting were performed to identify the key targets of ST. Finally, CCK-8 assay, wound healing assay, and xenograft tumor model were used to determine the relationship between ST and growth arrest and DNA damage-inducible alpha (GADD45A; the key target of ST) and malignant biological properties of PC in vitro and in vivo. Results: ST significantly repressed the PC cell proliferation rate and colony formation in vitro and arrested cells in the G2/M phase. ST inhibited PC cell mobility in vitro and increased E-cadherin expression (an epithelial biomarker). GADD45A was considered the key target of ST in PC and was elevated in PC cells treated with ST. The inhibition of GADD45A significantly alleviated the suppressive effects of ST on PC cell proliferation and mobility in vitro. ST suppressed PC cell proliferation in vivo and increased GADD45A expression in tumor tissues. Conclusion: ST exhibited significant anti-tumor effects on PC cells by upregulating GADD45A. ST may be a potential drug for PC treatment.

Keywords: Pancreatic cancer (PC); Sinapine thiocyanate (ST); growth arrest and DNA damage inducible alpha (GADD45A).

Figure 1

ST inhibited PC cell proliferation in vitro. A. Cell proliferation rate of normal human pancreatic epithelial cells treated with different concentrations (0, 20, 40, 80, 160 and 320 μM) of ST at 24 and 48 hours, as determined by the CCK-8 assay. B-C. PANC-1, MIA PaCa-2, and AsPC-1 cells were treated with different concentrations (0, 20, 40, and 80 μM) of ST. CCK-8 assay was performed to assess cell proliferation in each group at 24 and 48 hours. D. EdU assay was performed to detect the EdU-positive PC cells after treatment with DMSO and ST. White lines indicated 20 μm. Arrow indicates cell with nuclear atypia. E-F. Colony formation assay was performed to analyze the colony formation of PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. Stereograms and representative micrographs were both exhibited, and white lines on micrographs indicate 100 μm.*, P < 0.05; **, P < 0.01.

Figure 2

ST induced PC cell arrest in the G2/M phase in vitro. A-B. PANC-1, MIA PaCa-2, and AsPC-1 cells were treated with different concentrations (0, 20, 40, and 80 μM) of ST. Flow cytometry analysis was performed to determine the cell distribution in each group. C-E. Western blotting was performed to analyze the expression levels of CCNB1 and CDK1 in PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. *, P < 0.05; **, P < 0.01.

Figure 3

ST suppressed the motility of PC cells in vitro. A-B. Wound healing assay was performed to analyze the migration rate of PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. C-D. Transwell assay was performed to analyze the invasion rate of PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. E-F. Western blotting was performed to analyze the expression levels of E- and N-cadherins in PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. G. Immunofluorescence was used to analyze the expression of E-cadherin in PC cells treated with DMSO and ST. White lines indicate 20 μm. **, P < 0.01.

Figure 4

GADD45A is the key target of ST. A-B. Differentially expressed genes in PC cells treated with DMSO and ST were identified. C. KEGG analysis was performed to determine the pathways in which differentially expressed genes enriched. D. Protein-protein interaction network analysis was performed for differentially expressed genes. GADD45A had strong relationship with other proteins encoded by differentially expressed genes. E. RT-qPCR was performed to analyze the mRNA levels of GADD45A in PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. F-G. Western blotting was performed to analyze the protein levels of GADD45A in PC cells treated with different concentrations (0, 20, 40, and 80 μM) of ST. *, P < 0.05; **, P < 0.01.

Figure 5

Silencing of GADD45A reversed the inhibitory effects of ST on PC cells. PC cells were treated with DMSO + NC siRNA, ST + NC siRNA, DMSO + GADD45A siRNA and ST + GADD45A siRNA. A-B. Western blotting was performed to analyze the expression levels of GADD45A in each group. C-D. CCK-8 assay was performed to analyze the proliferation rate of PC cells in each group at 24 and 48 hours. E-H. Wound healing assay was performed to analyze the migration rate of PC cells in each group. **, P < 0.05.

Figure 6

ST repressed the proliferation rate of PANC-1 cells in vivo and increased the expression of GADD45A. A. Schematic representation of animal experiments. B-C. Proliferation of tumor tissues in the DMSO and ST treatment groups. D. Tumor weight in the DMSO and ST treatment groups. E-G. Expression of GADD45A and KI67 in tumor tissues in the DMSO and ST treatment groups. Black lines in the left bottom indicate 100 μm. *, P < 0.05; **, P < 0.01.