Silvestrol inhibits nasopharyngeal carcinoma cells and synergizes with CX-5461: Insights from a proteomics study

Abstract

Nasopharyngeal carcinoma (NPC) is a cancer arising from the epithelial cells of the nasopharynx, which is rare in Western countries but extremely prevalent in Borneo and the Southern China region. Present-day hurdles in NPC treatment that lead to poor quality of life and poor survival include distant metastasis and resistance to chemoradiotherapy. Silvestrol and its 5'''-epimer, episilvestrol, are compounds isolated from the plant Aglaia stellatopilosa, which is endemic to Borneo. Silvestrol, a protein synthesis inhibitor, preferentially inhibits the translation of cancer-associated mRNAs. CX-5461 is an inhibitor of RNA polymerase I that catalyzes rRNA synthesis. The present study sought to evaluate the potential of silvestrol as a new therapeutic for NPC with two authenticated NPC cell lines, C666-1 and HK1, and patient-derived xenografts. Concentration-response profiles were generated for silvestrol, episilvestrol, or CX-5461 as a single agent or in combination. Cell death and cell cycle progression studies were performed, followed by proteomics profiling. The findings of the present study showed that silvestrol and episilvestrol, in the nM range, inhibited NPC cells and patient-derived xenografts. While CX-5461 alone induced S and G₂/M phase arrest in NPC cells, silvestrol or episilvestrol synergized with CX-5461 to arrest NPC cells at G₀/G₁ and increase NPC cell death. Differential protein expression profiling revealed that silvestrol-CX-5461 combination principally targeted processes critical to protein synthesis and downregulated proteins commonly upregulated in NPC, including eukaryotic initiation factor 5A (eIF5A), c-myc, and Cyclin D1. Combining a translation inhibitor with an rRNA synthesis inhibitor may synergize to prevent the synthesis of proteins that NPC cells depend on for growth and survival. Given that c-myc, Cyclin D1 and eIF5A are essential in cell cycle progression and cell proliferation, this finding puts forward a promising new synergistic treatment approach for NPC.

Keywords: CX-5461; NPC; PDX; c-myc; cyclin D1; eIF5A; episilvestrol; silvestrol; synergism.

Figure 1

Flow cytometry analysis of HK1 cells treated with silvestrol, episilvestrol, CX-5461, or combinations, at 3 days post-treatment. (A) Percentage sub-G₁ in HK1. The data shows mean ± SEM. Values above each bar refer to the percentage sub-G₁. (B) Histograms of DNA content of HK1. Events below the G₀/G₁ peak were labelled as sub-G₁ and encompass dead cells. The x-axis annotated by FL2-Area represents DNA content. ^*P<0.05.

Figure 2

Flow cytometric analysis of cell cycle progression. (A) Percentage cell cycle progression of HK1 cells in the presence of CX-5461, silvestrol, episilvestrol, or combinations, at 2 days post-treatment. The data shown is the mean. Values rounded off to the nearest whole number within each bar refer to the percentage of cells in a specific cell cycle phase. (B) Representative histograms displaying the proportion of cells in G₁, S and G₂ phases.

Figure 3

Flow cytometric apoptosis assay of HK1 cells treated with silvestrol, episilvestrol, CX-5461, or combinations, at 3 days post-treatment. (A) Percentage of apoptosis in HK1 measured using FITC annexin V and propidium iodide double staining. The data show mean ± SEM. Values above each bar refer to the percentage of total apoptosis. (B) Dot plots showing apoptotic, necrotic, dead and healthy events. ^*P<0.05.

Figure 4

Identification of novel targets of silvestrol-CX-5461 combination-treatment by differential protein expression profiling. (A) Venn diagram depicting the number of proteins significantly downregulated following combination-treatment in individual cell lines and overlaps between cell lines, if any. Fold change <0.625; P<0.05. (B) Gene Ontology terms, according to biological process, cellular component, and molecular function, of the seven key proteins significantly downregulated in nasopharyngeal carcinoma cells by combination-treatment.

Figure 5

Validation of selected candidate protein from differential expression profiling. (A) Western blotting was used to detect eIF5A in nasopharyngeal carcinoma compared with nasopharyngeal epithelial cells. β-actin served as an internal control. The ratio of eIF5A expression to β-actin expression is indicated for each cell line. (B) Western blotting of eIF5A in HK1 and C666-1 treated with silvestrol, CX-5461, or combinations. β-actin served as an internal control. The fold change of eIF5A expression in HK1 and C666-1 relative to their respective control is indicated. (C) Western blotting of c-myc and cyclin D1 in HK1 and C666-1 treated with silvestrol, CX-5461, or combinations. β-actin served as an internal control. The fold change of c-myc and cyclin D1 expression in HK1 and C666-1 relative to their respective control is indicated. DMSO, dimethyl sulfoxide; eIF5A, eukaryotic translation initiation factor 5A; NaH₂PO₄, sodium dihydrogen phosphate.