The effect of Punicalagin on tongue carcinoma cells viability and angiogenesis by regulating TGF-β signalling pathway: an in vitro study

Abstract

Punicalagin (PUN) is a major bioactive ellagitannin derived from pomegranate and showed a potential anticancer property on several cell lines, no studies investigate its effect on tongue carcinoma cells. The present study aimed to evaluate the cytotoxic and antiangiogenic effects of PUN on human tongue carcinoma cell line. The viability of the tongue carcinoma cell line was assessed following treatment with PUN (250, 200, 150, 100, and 50 µmol) for 24 h using MTT assay. In addition, the calculated (IC 50) was used for apoptosis validation using Annexin-V/Propidium Iodide (PI) flow cytometry, angiogenesis investigation using endothelial tube formation assay, and transforming growth factor-β (TGF-β) expression on human tongue carcinoma cells after treatment with Punicalagin using quantitative real-time polymerase chain reaction (qRT-PCR). The vitality of tongue carcinoma cells treated with PUN was 61.1 ± 4.36% and the untreated cells were 98 ± 2%. Flow cytometry confirmed that treatment with 185 µmol/mL of PUN induced significant apoptosis, yielding 51.3% apoptotic cells, 2.3% necrotic cells, and 46.7% viable cells. Regarding tubules no. HPF Untreated cells had a statistically significant higher value (5.83 ± 1.94) than cells treated with PUN (3.17 ± 1.17) (p = 0.023). The junction no/HPF had a higher value in untreated cells (5.50 ± 2.43) than in cells treated with PUN (3.33 ± 1.37) yet the difference was not statistically significant (p = 0.119). The Tube length showed a statistical significance between untreated cells (5.28 ± 0.77) and cells treated with PUN (1.83 ± 0.26) (p = 0.004). The values for lobules no/HPF were higher in untreated cells (2.67 ± 0.52) than in cells treated with PUN (1.67 ± 0.82) yet the difference was not statistically significant (p = 0.053). The expression of TGF-β gene showed a significantly higher value in untreated cells (0.97 ± 0.03) than in cells treated with PUN (0.02 ± 0.01) (p = 0.004). These initial in vitro observations indicate an anticancer effect of PUN on tongue carcinoma cells through the induction of apoptosis and inhibition of cell viability and angio-mimetic tube formation, closely correlating with the downregulation of TGF-β gene expression. This provides a possible preliminary therapeutic direction to the study of tongue carcinoma.