Sesamin inhibits cervical cancer cell proliferation by promoting p53/PTEN-mediated apoptosis

Abstract

Background: Sesamin is a major bioactive compound in sesame seeds and has various biological properties, including anti-inflammatory and anticancer activities. Here, we explored whether sesamin activates p53, which is widely inhibited in cervical cancer cells, thereby inducing p53-mediated apoptosis. Methods: Human HeLa and SiHa cervical cancer cells and normal Hs68 dermal cells were used as cell models. Cell proliferation, cell cycle distribution, and apoptosis were evaluated by the CCK-8 assay and flow cytometry using PI/Annexin V staining, respectively. Protein expression and phosphorylation were determined using western blotting. The involvement of p53 in the apoptotic cascade was assessed by a specific inhibitor. Results: Sesamin (75 and 150 μM) clearly inhibited SiHa and HeLa cell proliferation in a dose-dependent fashion, but did not affect the proliferation of Hs68 cells. Meanwhile, sesamin increased the sub-G1 phase ratio and apoptosis, up to approximately 38.5% and 37.8%, respectively. Furthermore, sesamin induced p53 phosphorylation at serine-46 and serine-15 and upregulated the levels of PUMA, Bax, and PTEN, while inhibiting AKT phosphorylation at serine-473. Inhibition of p53 by pifithrin-α significantly reduced the levels of PUMA, Bax, and PTEN but restored AKT phosphorylation in SiHa cells exposed to sesamin. Pifithrin-α also reduced apoptosis and restored the proliferation of HeLa and SiHa cells exposed to sesamin. Conclusions: These findings indicate that sesamin inhibits cervical cancer cell proliferation, and its mechanism may be attributed to the induction of p53/PTEN-mediated apoptosis. This suggests that sesamin might be useful as an adjuvant in promoting anti-cervical cancer treatments.

Keywords: Apoptosis.; Cervical cancer cell; PTEN; PUMA; Sesamin; p53.

Figure 1

Effects of sesamin on cell proliferation of human cervical cancer cell HeLa and SiHa and normal dermal fibroblast Hs60. Cells were treated with sesamin at 15, 30, 75, 150, or 300 μM for 24 h (upper panel) or 48 h (lower panel), and then the cell proliferation was determined using CCK-8 assay. Quantitative data were presented as mean ± SD. Three independent experiments were performed for statistical analysis. *, **, and ***, P<0.05, 0.01, and 0.005 as compared to DMSO control.

Figure 2

Sesamin interfered cell cycle progression and induced apoptosis of human cervical cancer cell HeLa and SiHa. Cells were treated with sesamin at 75 or 150 μM for 24 h, followed by staining with (A) PI for cell cycle distribution, or (B) PI/Annexin V for cell apoptosis assay, and then analyzed using flow cytometry. Quantitative data were presented as mean ± SD. Three independent experiments were performed for statistical analysis. **, and ***, P<0.01, and 0.005 as compared to DMSO control.

Figure 3

Sesamin induced p53 activation, upregulated expression of PUMA, Bax, and PTEN, and inhibited AKT activation in SiHa cells. Cells were treated with sesamin at 75 or 150 μM for 24 h, collected, and then lysed for protein extraction and the subsequent Western blotting. Protein and phosphorylation level were detected using specific antibodies against (A) p53, phosphorylated p53, PUMA, and Bax; and (B) PTEN, AKT, and phosphorylated AKT. Chemiluminescence signal of β-actin was used as internal control. Molecular weights of signals were indicated. Three independent experiments were performed for statistical analysis. *, and **, P<0.05, and 0.01 as compared to DMSO control.

Figure 4

Involvement of p53 activation in sesamin-induced apoptotic cascade in SiHa cells and sesamin-induced cell apoptosis of HeLa and SiHa cells. Cells were pretreated without or with pifithrin-a at 30 μM for 2 h, and then treated with sesamin at 150 μM for 24 h. The treated cells were collected, and then (A) lysed for protein extraction and the subsequent Western blotting using specific antibodies as indicated; (B) stained with PI/Annexin V for apoptosis assay; and (C) reacted with CCK-8 for cell proliferation assay. Chemiluminescence signal of β-actin was used as internal control. Molecular weights of signals were indicated. Quantitative data were presented as mean ± SD. Three independent experiments were performed for statistical analysis. *, **, and ***, P<0.05, 0.01, and 0.005 as compared to DMSO control. # and ###, P<0.05 and 0.005 as compared to sesamin alone.