Increase of phosphatase and tensin homolog by silymarin to inhibit human pharynx squamous cancer

Abstract

Silymarin is an active principle from the seeds of the milk thistle plant and is widely used as a hepatoprotective gent due to its antioxidant-like activity. In the present study, we evaluated the potential efficacy of silymarin against oral cancer and investigated its possible mechanism of action. Cell viability assay and western blotting analyses were used to identify silymarin-induced apoptotic cell death in human pharynx squamous cell carcinoma (FaDu) cells. The short interfering RNA (siRNA) is used to confirm the role of phosphatase and tensin homolog (PTEN) in silymarin-induced apoptosis. Treatment of FaDu cells with silymarin resulted in a significant decrease in cell viability (up to 70%). Silymarin inhibited the phosphorylation of Akt (over 10-fold) with an increase in expression of PTEN (five to sixfold). Consequently, the level of Bcl-2 expression was decreased five to sixfold and caspase 3 activated to induce apoptosis. Treatment with siRNA specific to PTEN gene diminished the action of silymarin. The results suggest that silymarin inhibits the Akt signaling pathway by increasing PTEN expression in FaDu cells and directly affects Bcl-2 family members. Also, we demonstrated the inhibitory activity of silymarin for oral cancer is related to cell survival. These mechanisms may in part explain the actions of silymarin and provide a rationale for the development of silymarin as an anticancer agent.

FIG. 1.

Silymarin inhibits Akt by enhancing PTEN expression and cytotoxicity in FaDu cells. Cell proliferation and viability were determined by an MTT assay. Reduced cell viability was observed with silymarin treatment (28.8–67.2 μg/mL) at 6, 12, 24, and 48 h (A). Proteins isolated from FaDu cells were probed with antibodies against PTEN (B), p-AKT, and AKT (C). The same membrane was re-probed with the antibody for β-actin to verify that equal amounts of protein were loaded. Data are expressed as the mean±SE (n=4 for each group). **P<.01 compared with control; ***P<.001 compared with control. PTEN, phosphatase and tensin homolog.

FIG. 2.

Silymarin treatment resulted in altered levels of apoptosis-associated proteins. (A) FaDu cells were treated with silymarin, and then protein samples were probed with antibodies against p-Bad (A), Bcl-2 (B), por-caspase 3, and caspase 3 (C). The same membrane was re-probed with the antibody for β-actin to verify that equal amounts of protein were loaded. Data are expressed as the mean±SE (n=4 for each group). **P<.01 compared with control; ***P<.001 compared with control.

FIG. 3.

PTEN siRNA attenuated cytotoxicity and PTEN expression in FaDu cells treated with silymarin. Cell proliferation and viability were determined by an MTT assay (A). After treated with silymarin 57.6 μg/mL for 24 h, proteins isolated from FaDu cells were probed with antibodies against PTEN, p-AKT, and AKT (B). Proteins isolated from FaDu cells were probed with antibodies p-Bad, Bcl-2, and caspase 3 and pro-caspase 3 (C). siRNA, short interfering RNA.

FIG. 4.

Comparison of cytotoxic effects of silymarin and silibinin on FaDu cells. Cell proliferation and viability were determined by an MTT assay. Reduced cell viability was observed with silymarin (57.6 μg/mL) or silibinin (96.4 μg/mL) treatment at 0–24 h (A). Proteins isolated from FaDu cells were probed with antibodies against PTEN (B). The same membrane was re-probed with the antibody for β-actin to verify that equal amounts of protein were loaded. **P<.01 compared with control; ***P<.001 compared with control.