Lappaol F regulates the cell cycle by activating CDKN1C/p57 in human colorectal cancer cells

Abstract

Context: Lappaol F (LAF), a natural lignan from Arctium lappa Linné (Asteraceae), inhibits tumor cell growth in vitro and in vivo. The underlying mechanism involves the suppression of the Yes-associated protein. However, the specific role of LAF in cell cycle regulation remains unknown.

Objective: This study determined the molecular mechanism by which LAF regulates cell cycle progression.

Materials and methods: Various colon cancer cell lines (SW480, HCT15, and HCT116) were treated with LAF (25, 50, and 75 μmol/L) for 48 h. The effects of LAF on cell proliferation and cell cycle were determined using sulforhodamine B and flow cytometry assays. Differentially expressed proteins (DEPs) were identified using quantitative proteomics. Bioinformatic analysis of DEPs was conducted via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Expression levels of DEPs in the cell cycle pathway were analyzed using RT-qPCR and western blotting.

Results: LAF suppressed the proliferation of SW480, HCT15, and HCT116 cells (IC₅₀ 47.1, 51.4, and 32.8 μmol/L, respectively) and induced cell cycle arrest at the S phase. A total of 6331 proteins were identified and quantified, of which 127 were differentially expressed between the LAF-treated and untreated groups. GO and KEGG enrichment analyses revealed that DEPs mainly participated in the cell cycle. CDKN1C/p57 showed the most significant differential expression, with the highest fold-change (3.155-fold). Knockdown of CDKN1C/p57 attenuated the S phase cell cycle arrest and proliferation inhibition induced by LAF.

Conclusion: LAF exerts antitumor effects via S phase arrest by activating CDKN1C/p57 in colorectal cancer cells.

Keywords: Cyclin-dependent kinase inhibitor 1C/p57; cyclin; cyclin-dependent kinase.

Figure 1.

LAF inhibits proliferation and induces the S phase arrest of human colorectal cancer cells. (A) Structure of LAF. (B) Human colon cancer cell lines (HCT15, HCT116, and SW480) were treated with LAF (0, 25, 50 or 75 μmol/L) for 48 h. Cell viability was assessed via sulforhodamine B assay. (C) HCT15, HCT116, and SW480 cells were treated with 50 μmol/L LAF for 48 h and then analyzed via flow cytometry after propidium iodide staining. All data are expressed as the mean ± standard deviation (SD) (n = 6). *p < 0.05, **p < 0.01, significantly different from the control without LAF treatment.

Figure 2.

Proteome profiling. SW480 cells were treated with 50 μmol/L LAF for 48 h. Proteins were quantified via tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Fold change ≥ 1.5 and p value < 0.05 were considered to indicate differential expression. There were three biological replicates in each group. (A) Quantitative overview. (B) Differentially expressed proteins (DEPs). (C) Gene Ontology (GO) enrichment analysis of DEPs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEPs.

Figure 3.

LAF inhibits colorectal cancer cell proliferation by upregulating the expression of cyclin-dependent kinase inhibitor 1 C (CDKN1C/p57). (A) Protein–protein interaction (PPI) network of differentially expressed proteins in cell cycle pathway (red indicates upregulation, green indicates downregulation). (B) Expression levels of CDKN1C/p57, cyclin-dependent protein kinases (CDKs), and cyclins in SW480 cells after treatment with 25 or 50 μmol/L LAF for 48 h determined via western blotting (*p < 0.05, **p < 0.01, significantly different from the control without LAF treatment; n = 3). (C) Role of CDKN1C/p57 in LAF-induced inhibition of CDK and cyclin expression. (D) Role of CDKN1C/p57 in LAF-induced cell proliferation inhibition. (E) Role of CDKN1C/p57 in LAF-induced cell cycle arrest. Cells infected with lentiviruses containing CDKN1C/p57-small interfering RNA (siRNA) or negative control (NC)-siRNA were treated with or without LAF (50 μmol/L) for 48 h. Protein levels, cell viability, and cell cycle distributions were assessed via western blotting, sulforhodamine B assay, and flow cytometry after propidium iodide staining, respectively (*p < 0.05, **p < 0.01, significantly different between the NC-siRNA- and CDKN1C/p57-siRNA-transfected cells). Western blotting and cell cycle analysis, n = 3; cell viability, n = 6.