Allicin Could Potentially Alleviate Oral Cancer Pain by Inhibiting "Pain Mediators" TNF-alpha, IL-8, and Endothelin

Abstract

To evaluate the effects of allicin on mediators of pain secreted by oral cancer cells in vitro, single-cell suspensions were prepared by enzymatic method from oral squamous cell carcinoma (OSCC). Cancer stem cells were isolated by the CD133+ selection method with magnetic cell sorting. Stemness markers were checked in both cancer cells and cancer stem cells by RT-PCR. Comparative analysis of pain mediators TNF-alpha, IL-8, and endothelin at both RNA and protein levels for normal epithelial cells, cancer cells, and cancer stem cells was carried out with and without allicin treatment. CD133 and CD44 expression levels were checked in cancer cells and cancer stem cells flow cytometrically. Allicin inhibited both gene and protein expression of TNF-alpha, IL-8, and endothelin in both cancer cells and cancer stem cells. Allicin is more likely to be a promising treatment in alleviating the levels of pain and inflammation in OSCCs.

Keywords: allicin; cancer stem cells; oral cancer; pain.

Figure 1

OSCC tissues were digested enzymatically to prepare single cell suspensions and further used for experiments and culture. (A) CD133 expression in magnetically sorted oral cancer stem cells. (B) Morphology of total cancer cell population B1 and CD133+ sorted cancer stem cells B2. Scale bar = 100 μm. (C) Fold gene expression of stemness and pluripotency related genes in CD133+ sorted cancer stem cells.

Figure 2

OSCC cells were treated with different concentrations of allicin and MTT assay was performed to check the cell viability. (A) Morphology of the OSCC cells after treatment with allicin: Control A1, 20 ng/mL A2, 50 ng/mL A3, 100 ng/mL A4. Scale bar = 100 μm. (B) MTT assay. n.s. not significant.

Figure 3

Real time PCR was performed to assess the gene expressions of pain mediators TNF-α, IL-8, and endothelin. (A) TNF-α gene expression levels. (B) IL-8 gene expression levels. (C) Endothelin gene expression levels. (D) Relative gene expressions of pain mediators. n.s. not significant, ** p < 0.05, *** p < 0.01.

Figure 4

ELISA was performed to measure the secretion levels in pg/mL of pain mediators TNF-α, IL-8, and endothelin. (A) Protein levels of TNF-α. (B) Protein levels of IL-8. (C) Protein levels of Endothelin. (D) Comparative expressions of pain mediators at protein level in pg/mL values. n.s. not significant, ** p < 0.05, *** p < 0.01.

Figure 5

OSCC stem cells were treated with the different concentrations of allicin and acquired on flow cytometer to examine the expression of cancer stem cell markers CD133 and CD44. n.s. not significant, ** p < 0.05, *** p < 0.01.