In vitro study on anti-cancer properties of genistein in tongue cancer

Abstract

Purpose: Tongue cancer is an extremely aggressive disease and is characterized by a poor prognosis. It is a complex disease to treat and current therapies have produced mediocre results with many side effects. Some facts suggest that natural essences can support traditional cancer therapy by carrying out a synergistic function with chemotherapy. Therefore, we evaluated the antitumor effects of genistein on tongue carcinoma cells.

Methods: Genistein 20, 50 and 100 µM were used for 24, 48 and 72 hours on 3 tongue carcinoma cell lines. xCELLigence system was used to evaluate the effects on cell adhesion, proliferation and to calculate IC₅₀ values. Both MTT assay and Trypan blue assay were used to evaluate alterations in cell viability, scratch assay for cell migration and Western blot analysis for expression of some proteins.

Results: Cell adhesion was inhibited especially between 20 and 50 µM of genistein treatment. Proliferation was reduced by 50% for treatments with 20 µM at 24 hours, with 20 or 50 µM at 48 and 50 µM at 72 hours (P<0.0001). Viability tests confirmed a proportional reduction in concentration of genistein and duration of treatments. Even cell migration was reduced significantly (P<0.001). Genistein down-regulates vitronectin, OCT4 and survivin.

Conclusion: This in vitro study clarifies the anti-tumor effect of genistein on tongue carcinoma. In vivo studies are needed to confirm these data and develop a suitable delivery system that is capable of acting directly on tumor.

Keywords: cell adhesion; cell proliferation; genistein; tongue cancer; xCELLgence system.

Figure 1

Genistein inhibited adhesion of tongue cancer cells. Notes: All cell lines were treated with 20, 50 and 100 µM of genistein. In the graphs, there is an initial phase of cell adhesion, followed by a plateau phase prior to a gradual period of proliferation. The adhesion of (A) HSC-3 cells, (C) PE/CA-PJ15 cells and (E) PE/CA-PJ49 cells is shown. Untreated cells were used as control (red curves). We monitored in real time the adhesion for 24, 48 and 72 hours after treatment and all cell index values of HSC-3 cells (B), PE/CA-PJ15 cells (D) and PE/CA-PJ49 cells (F). The results are the mean CI for 3 replicates ± SD. ****P<0.0001.

Figure 2

Inhibition of vitronectin expression. Notes: Western blotting shows the inhibition of vitronectin expression after genistein treatment. Vitronectin is especially down-regulated at 48 hours with 100 µM of genistein and at 72 hours with 50 and 100 µM of genistein (A). Also shown is significance degree of tests used **P<0.005 (B). Abbreviation: Ctrl, control.

Figure 3

Variation in cell proliferation rate (%). Notes: Cell index (CI) values were measured and converted to percentage rate using untreated cells as control (100% adhesion). (A) This shows a 50% reduction of adhesion posttreatment with 20 µM genistein at 24 hours. (B) The same reduction is present for treatment between 20 and 50 µM of genistein at 48 hours, and for 50 µM of genistein at 72 hours (C). Also shown is significance degree of tests used (**P<0.005, ***P<0.001, ****P<0.0001).

Figure 4

IC₅₀ values. Notes: Dose-response curves are shown for HSC-3 cells (A), PE/CA-PJ15 cells (C) and PE/CA-PJ49 cells (E). In each graph, the yellow square with the numbers 1, 2 and 3 indicates, respectively, IC₅₀ values at 24, 48 and 72 hours. IC₅₀ values are expressed as the mean (M) ± standard error of the mean (n=3) and they are shown as the average of all IC₅₀ values of HSC-3 cells (B), PE/CA-PJ15 cells (D), PE/CA-PJ49 cells (F) at each time point. R² of IC₅₀ was 0.99.

Figure 5

Genistein reduces the viability of tongue cancer cells. Notes: Trypan blue test (A) and MTT assay (B) show a proportional reduction in cell viability with increasing concentrations of genistein. Experiments were performed at least 3 times and results are presented as the mean ± SD, and are statistically significant for ***P<0.001 and ****P<0.0001. Abbreviation: Ctrl, control.

Figure 6

Effects of genistein on cell migration. Notes: Significant increase of the gap size is more evident from T4 in tongue carcinoma cells compared to untreated control (Ctrl) (A). All experiments were performed at least 3 times and results are presented as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (B).

Figure 7

Genistein reduces tumorigenesis and promotes apoptosis. Notes: Variation in level of expression of OCT4 and survivin. Genistein inhibits survivin and OCT4 at all time points considered (A–C). The significance degree of tests used is **P<0.005, ***P<0.001, ****P<0.0001 (D–E). Abbreviation: Ctrl, control.