Taiwanin E Induces Cell Cycle Arrest and Apoptosis in Arecoline/4-NQO-Induced Oral Cancer Cells Through Modulation of the ERK Signaling Pathway

Abstract

Taiwanin E is a bioactive compound extracted from Taiwania cryptomerioides Hayata. In this research endeavor, we studied the anti-cancer effect of Taiwanin E against arecoline and 4-nitroquinoline-1-oxide-induced oral squamous cancer cells (OSCC), and elucidated the underlying intricacies. OSCC were treated with Taiwanin E and analyzed through MTT assay, Flow cytometry, TUNEL assay, and Western blotting for their efficacy against OSCC. Interestingly, it was found that Taiwanin E significantly attenuated the cell viability of oral cancer cells (T28); however, no significant cytotoxic effects were found for normal oral cells (N28). Further, Flow cytometry analysis showed that Taiwanin E induced G1cell cycle arrest in T28 oral cancer cells and Western blot analysis suggested that Taiwanin E considerably downregulated cell cycle regulatory proteins and activated p53, p21, and p27 proteins. Further, TUNEL and Western blot studies instigated that it induced cellular apoptosis and attenuated the p-PI3K/p-Akt survival mechanism in T28 oral cancer cells seemingly through modulation of the ERK signaling cascade. Collectively, the present study highlights the prospective therapeutic efficacy of Taiwanin E against arecoline and 4-nitroquinoline-1-oxide-induced oral cancer.

Keywords: Taiwanin E; apoptosis; cell cycle arrest; oral cancer; therapeutics.

Figure 1

Effect of Taiwanin E on cell viability and cell cycle progression of oral cells. Normal oral cells N28 and Cancer oral cells T28 were treated with varying concentrations of Taiwanin E (0, 1, 5, and 10 μM) for 24 h. The cell viability of N28 cells and T28 cells was assessed with MTT assay (A). **p < 0.01 represents significant differences compared with control. T28 cells were treated with varying concentrations of Taiwanin E (0, 1, 5, and 10) μM for 24 h, and the DNA content was assessed through flow cytometry. Results were represented as percentages of the cell population in G1, S, and G2 phases of the cell cycle (B).

Figure 2

Effect of Taiwanin E on cell viability and cell cycle progression of oral cells. Normal oral cells N28 and cancer oral cells T28 were treated with Taiwanin E (10 μM) for different time intervals. The cell viability of N28 cells and T28 cells was assessed with MTT assay (A). **p < 0.01 represents significant differences compared with control. T28 cells were treated with Taiwanin E (10 μM) for different time interval, and the DNA content was assessed through flow cytometry. Results were represented as a percentage of the cell population in G1, S, and G2 phases of the cell cycle (B).

Figure 3

Effect of Taiwanin E on cell cycle-related proteins in oral cancer cells. T28 cells were treated with Taiwanin E (10 μM) for different time intervals and thereafter analyzed through Western blotting. Samples were assessed for the expression of Cyclin B1, Cyclin D1, and Cyclin E (A) and p53, p21, and p27 (B), respectively. Graphs represent mean values of three independent experiment in T28 cells. *p < 0.05, **p < 0.01, and ***p < 0.001 represent significant differences compared with control.

Figure 4

Effect of Taiwanin E on the induction of cellular apoptosis in oral cancer cells. T28 cells were treated with Taiwanin E (10 μM) for different time periods The samples were stained with a TUNEL kit and analyzed through flow cytometry (A). T28 cells were treated with Taiwanin E (10 μM) for different time periods. Cell lysates were analyzed for the expressions of p-PI3K, p-Akt, Bcl-xL, Bax, Cyt C, and c Cas 3 through Western blotting (B). Graphs represents the mean value of three separate experiment in the T28 cells. *p < 0.05, **p < 0.01, and ***p < 0.001 represent significant differences compared with control group.

Figure 5

Taiwanin E modulate the ERK signaling cascade in oral cancer cells. T28 cells were treated with Taiwanin E (10 μM) for different time periods. Thereafter, cell lysates were analyzed for the expression of ERK, pERK, JNK, pJNK, p38, and p-p38 through Western blotting. Graphs represents mean values of three independent experiment in T28 cells. *p < 0.05 and ***p < 0.001 represent significant differences compared with control.

Figure 6

Effect of Taiwanin E on ERK activation in oral cancer cells. T28 cells were co-treated with ERK activator and Taiwanin E for 24 h. Thereafter, cell viability was analyzed through MTT assay (A). T28 cells were co-treated with ERK activator and Taiwanin E for 24 h. Thereafter, cell lysates were analyzed through Western blotting for the expression of Bcl-xL and Cyt C proteins through Western blotting (B). Graphs represent mean value of three separate experiment in T28 cells. ^##p < 0.01 and _###p < 0.001 represent significant differences compared with control group, whereas *p < 0.05, **p < 0.01, and ***p < 0.001 represent significant differences compared with the Taiwanin E-treated group.