Arecoline induces epithelial-mesenchymal transformation and promotes metastasis of oral cancer by SAA1 expression

Abstract

Arecoline, the main alkaloid of areca nut, is well known for its role in inducing submucosal fibrosis and oral squamous cell carcinoma (OSCC), however the mechanism remains unclear. The aim of this study was to establish an arecoline-induced epithelial-mesenchymal transformation (EMT) model of OSCC cells and to investigate the underlying mechanisms. CAL33 and UM2 cells were induced with arecoline to establish an EMT cell model and perform RNA-sequence screening. Luminex multiplex cytokine assays, western blot, and RT-qPCR were used to investigate the EMT mechanism. Arecoline at a concentration of 160 μg/ml was used to induce EMT in OSCC cells, which was confirmed using morphological analysis, transwell assays, and EMT marker detection. RNA-sequence screening and Luminex multiplex cytokine assays showed that many inflammatory cytokines (such as serum amyloid A1 [SAA1], interleukin [IL]-6, IL-36G, chemokine [CCL]2, and CCL20) were significantly altered during arecoline-induced EMT. Of these cytokines, SAA1 was the most highly upregulated. SAA1 overexpression induced EMT and promoted the migration and invasion of CAL33 cells, while SAA1 knockdown attenuated arecoline-induced EMT. Moreover, arecoline enhanced cervical lymph node metastasis in an orthotopic xenograft model of the tongue established using BALB/c nude mice. Our findings revealed that arecoline induced EMT and enhanced the metastatic capability of OSCC by the regulation of inflammatory cytokine secretion, especially that of SAA1. Our study provides a basis for understanding the mechanism of OSCC metastasis and suggests possible therapeutic targets to prevent the occurrence and development of OSCC associated with areca nut chewing.

Keywords: SAA1; arecoline; epithelial-mesenchymal transition; inflammatory cytokines; oral squamous cell carcinoma.

FIGURE 1

Arecoline‐induced EMT and enhanced migration and invasion of OSCC cells. A, B, OSCC cells lost cell‐cell adhesion and their morphology changed from a polygon shape to a spindle‐like shape after treatment with arecoline. OSCC cells enhanced migration (C, D) and invasion (E, F) capabilities after treatment with arecoline. G, H, RT‐qPCR and western blots were performed to detect the expression of EMT markers such as E‐cad, N‐cad, Snail, and vimentin. The expression levels of N‐cad, Snail, and vimentin were upregulated and E‐cad was downregulated. Data are shown as mean ± SD of 3 independent experiments. Are, Arecoline. *P value < .05, **P value < .01, ***P value < .001

FIGURE 2

Arecoline regulated global mRNA changes in OSCC cells. A, Volcano plot depicts upregulated and downregulated genes in CAL33‐Arecoline (CAL33_A) vs CAL33‐Control (CAL33_C) (log₂ fold change > 1.0, P value adjusted (padj) < .05). B, Bubble plots of GO analysis showed that upregulated genes were predominantly enriched in the inflammatory response signaling pathway. C, KEGG pathway enrichment analysis of transcriptome sequencing revealed that signaling pathway changes were mainly enriched in the cytokine‐cytokine receptor pathway, and PI3K‐Akt signaling pathway. GSEA of differentially expressed genes might be enriched in chemokine signaling pathway (D) and cell adhesion molecules (E)

FIGURE 3

Arecoline enhanced secretion of pro‐inflammatory cytokines in OSCC cells. A, Cytokine profiles of CS and CL from CAL33 cells treated with or without arecoline were screened using the Luminex multiplex cytokine assays. B, Most cytokines, such as SAA1, CCL2, S100A8, IL‐6 and IL‐36G, were significantly increased, while CCL20 was decreased. C, mRNA levels of 6 pro‐inflammatory cytokines in OSCC cells were measured using RT‐qPCR. Data are shown as mean ± SD of 3 independent experiments. *P value < .05, **P value < .01, ***P value < .001

FIGURE 4

SAA1 promoted migration and invasion of OSCC cells. A, B, The morphology of OSCC cells changed from a polygon shape to a spindle‐like shape and cells lost cell‐cell adhesion after treatment with recombinant SAA1. OSCC cells were enhanced in migration (C, D) and invasion (E, F) after treated by recombinant SAA1. G, H, RT‐qPCR and western blot were used to detect the expression of EMT‐associated molecules. Expression levels of N‐cad, Snail, and vimentin were increased, while E‐cad was inhibited. Data are shown as mean ± SD of 3 independent experiments. *P value < .05, **P value < .01, ***P value < .001

FIGURE 5

SAA1 promoted migration and invasion of OSCC cells were inhibited by TLR2/4 inhibitor. A, B, The morphology of OSCC cells changed from a spindle‐like shape to a polygon shape when TLR2/4 inhibitor sparstolonin B (SsnB) was used. The capabilities of OSCC cells were attenuated in migration (C, D) and invasion (E, F) after treatment with recombinant SAA1 combined with SsnB. G, H, RT‐qPCR and western blots were used to detect the expression of EMT‐associated molecules. Expression levels of N‐cad, Snail, and vimentin were inhibited, while E‐cad was elevated in OSCC cells treated with SAA1 combined with SsnB. Data are shown as mean ± SD of 3 independent experiments. *P value < .05, **P value < .01, ***P value < .001

FIGURE 6

SAA1 knockdown inhibited arecoline‐induced EMT in OSCC cells. RT‐qPCR and western blot were used to verify the expression of SAA1 in SAA1‐knockdown CAL33 and UM2 cells. B, C, Effect of cell migration (B) and invasion (C) in OSCC cells after SAA1 knockdown. D, E, Effect of cell migration (D) and invasion (E) in arecoline‐exposed OSCC cells treated with or without SAA1 knockdown. F, G, Relative mRNA expression levels of EMT‐associated molecules in OSCC cells after SAA1 knockdown (F), or in arecoline‐exposed OSCC cells treated with or without SAA1 knockdown (G). H, Protein expression levels of EMT‐associated molecules in OSCC cells or arecoline‐exposed OSCC cells. Data are shown as mean ± SD of 3 independent experiments. *P value < .05, **P value < .01, ***P value < .001. Are, Arecoline

FIGURE 7

Arecoline promoted metastasis of OSCC in vivo. CAL33 cells treated with or without arecoline were inoculated into the tongues of nude mice. A, Luminescence was observed in the tongue and cervical LNs using an in vivo imaging system (IVIS) at 3 wk after cell injection. B, Representative photographs of tongue tumors and cervical LNs were shown. C, Expression of SAA1 in metastatic lymph nodules was examined by IHC. D, Representative immunohistochemical images showing the expression of cytokeratin and HLA class I in metastatic lymph nodules

FIGURE 8

Arecoline induces EMT and promotes metastasis of oral cancer by upregulation of SAA1. As shown in the graph, arecoline induces EMT and promotes metastasis through regulation of the secretion of inflammatory markers, especially the secretion of SAA1 in OSCC