Anti-neoplastic effects of gallic acid, a major component of Toona sinensis leaf extract, on oral squamous carcinoma cells

Abstract

Extract of Toona sinensis (TS) has been reported to have various effects on cultured cell lines, including anti-proliferative activity in cancer cells. We have studied the effects of TS on various human oral squamous carcinoma cell lines (HOSCC), including UM1, UM2, SCC-4, and SCC-9. These cell lines were treated with TS leaf extract and screened for viability, apoptosis, necrosis, and apoptotic gene expression. Normal human oral keratinocytes (NHOK) served as a control for cytotoxic assays. Viability of TS-treated HOSCC was reduced, whereas that of NHOK was not affected. FACScan analysis revealed that the leaf extract induced apoptosis or a combination of apoptosis and necrosis, depending on cell type. Microarray and semi-quantitative RT-PCR analysis for apoptotic-related gene expression revealed that 3,4,5-trihydroxybenzoic acid (gallic acid, one of the major bioactive compounds purified from TS extract) up-regulated pro-apoptotic genes such TNF-α, TP53BP2, and GADD45A, and down-regulated the anti-apoptotic genes Survivin and cIAP1, resulting in cell death. This study suggests that gallic acid, the major bioactive compound present, is responsible for the anti-neoplastic effect of Toona sinensis leaf extract.

Figure 1

Effect of TS on viability of human oral squamous cell carcinoma (HOSCC) and normal human oral keratinocytes (NHOK). UM1, UM2, SCC4, and SCC9 cell lines were treated with 500 μg/mL of TSL-1 for 12, 24 or 48 hours. At the indicated time points, cells were harvested for viability assays, using trypan blue exclusion. Results are presented as mean ± SE of three independent assays (bars, SE; *, P < 0.05 compared with untreated cells). NHOK cells were treated or untreated with 250 or 500 µg/mL for 12, 24, or 48 hours. At the indicated time points, cells were harvested for viability assays. Columns, mean of the percentage of viability of cells from three independent experiment; bars, SE; #, P ≥ 0.05 compared with untreated NHOK control cells.

Figure 2

Flow cytometric analysis of TS-induced HOSCC cell death. UM1, UM2, SCC4, and SCC9 cells were grown in the absence (control) or the presence of TSL-1 (500 μg/mL) for 24 hours, stained with annexin V and propidium iodide, and analyzed by flow cytometry. The distributions of cells are illustrated in dot plots.

Figure 3

Spectra of TSL-1-5-7.

Figure 4

Relative potency of TSL-1-5-7- and gallic acid-induced UM1 cell death. UM1 were grown in the absence or presence of various concentrations of TSL-1-5-7 or gallic acid for 24 hours, and cell death was assessed by staining with annexin V-FITC and propidium iodide (PI), followed by flow cytometry analysis.

Figure 5

Microarray and semi-quantitative RT-PCR analysis of TNF-α, GADD45A, TP53BP2, Survivin, and cIAP expression in TSL-1-5-7- or gallic acid-treated or -untreated UM1 cells.