Inhibition of Cell Proliferation and Metastasis by Scutellarein Regulating PI3K/Akt/NF-κB Signaling through PTEN Activation in Hepatocellular Carcinoma

Abstract

Scutellarein (SCU) is a well-known flavone with a broad range of biological activities against several cancers. Human hepatocellular carcinoma (HCC) is major cancer type due to its poor prognosis even after treatment with chemotherapeutic drugs, which causes a variety of side effects in patients. Therefore, efforts have been made to develop effective biomarkers in the treatment of HCC in order to improve therapeutic outcomes using natural based agents. The current study used SCU as a treatment approach against HCC using the HepG2 cell line. Based on the cell viability assessment up to a 200 μM concentration of SCU, three low-toxic concentrations of (25, 50, and 100) μM were adopted for further investigation. SCU induced cell cycle arrest at the G2/M phase and inhibited cell migration and proliferation in HepG2 cells in a dose-dependent manner. Furthermore, increased PTEN expression by SCU led to the subsequent downregulation of PI3K/Akt/NF-κB signaling pathway related proteins. In addition, SCU regulated the metastasis with EMT and migration-related proteins in HepG2 cells. In summary, SCU inhibits cell proliferation and metastasis in HepG2 cells through PI3K/Akt/NF-κB signaling by upregulation of PTEN, suggesting that SCU might be used as a potential agent for HCC therapy.

Keywords: EMT; PI3K/Akt/NF-κB; PTEN; metastasis; proliferation; scutellarein.

Figure 1

The chemical structure of scutellarein (SCU) and the effect of SCU treatment in HCC cell lines and HaCaT cells. (a) Structure of SCU. (b) Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with SCU (0, 25, 50, 100, 150, and 200 μM) for 48 h in HaCaT cells. (c) Cell viability was measured by MTT assay with SCU (0, 25, 50, 100, 150, and 200 μM) for 24 and 48 h in HepG2 cells. (d) Cell viability was measured by MTT assay with SCU (0, 25, 50, 100, 150, and 200 μM) for 24 and 48 h in Huh-7 cells. The results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. ** p < 0.01, *** p < 0.001.

Figure 2

Effect of SCU on HepG2 cell migration and proliferation. (a) Wound-healing assay measured the migration ability changes. Cells were treated with SCU (0, 25, 50, and 100 μM) for 48 h. (b) Colony formation assay measured antiproliferation effect. Cells were treated with SCU (0, 25, 50, and 100 μM) for 2 weeks. The results obtain from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. *** p < 0.001.

Figure 3

Effect of SCU on cell cycle progression in HepG2 cells. (a) SCU induces HepG2 cell G2/M cell cycle arrest. Cells were treated with SCU (0, 25, 50, and 100 μM) for 48 h and the cell cycle was detected by flow cytometry. (b) Cdc25c, cdk1, and cyclin B1 levels were quantified. The results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Effect of SCU on PTEN expression in HepG2 cells. (a) SCU induces PTEN protein level in HepG2 cells. Cells were treated with SCU (0, 25, 50, and 100 μM) for 48 h. (b) Effect of PTEN inhibitor and SCU-induced PTEN level in HepG2 cells. Cells were treated with or without 80 μM of PTEN inhibitor: SF1670 and 100 μM of SCU for 48 h. (c) Representative immunofluorescence image images of expression of PTEN were evaluated at 0 and 100 μM (scale bars = 100 μm). The results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. * p < 0.05.

Figure 5

In silico molecular docking analysis of the ligands SCU and simvastatin (SIM) with target PTEN. (a) The 3D structure of PTEN bound efficiently with compound SCU with its interacting amino acids TYR16, THR160, ASP92, HIS93, ARG130, LYS128, GLN171, and ILE168. (b) The 3D structure of PTEN bound efficiently with the known activator compound SIM with its interacting amino acids ILE28, CYS136, PHE154, GLU150, and VAL158.

Figure 6

Effect of SCU on PI3K/Akt/NF-κB signaling pathway in HepG2 cells. SCU suppresses p-PI3K, p-Akt, and p-IκB-α levels in HepG2 cells. Cells were treated with SCU (0, 25, 50, and 100 μM) for 48 h. The results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Inhibitory effect on PI3K/Akt/NF-κB signaling pathway in HepG2 cells. Effect of PI3K inhibitor and SCU-induced inhibition of p-PI3K, p-Akt, and p-IκB-α levels in HepG2 cells. Cells were treated with or without 20 μM of PI3K inhibitor: LY294002 and 100 μM of SCU for 48 h. The results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Effect of SCU on EMT markers and migration-related proteins in HepG2 cells. (a) SCU induces EMT-related proteins E-cadherin and suppresses N-cadherin and Snail levels in HepG2 cells. Cells were treated with SCU (0, 25, 50, and 100 μM) for 48 h. (b) SCU induces extracellular inhibitor of MMPs: TIMP2 and suppresses migration-related proteins MMP-2 and MMP-9 levels in HepG2 cells. The results obtained from three independent experiments were expressed as mean ± standard deviation (SD) compared with the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 9

Schematic representation of the inhibition of HepG2 cell proliferation and metastasis by SCU. SCU inhibits proliferation and metastasis through suppression of PI3K/Akt/NF-κB signaling pathway by inducing PTEN and regulating EMT markers and migration-related proteins (E-cadherin, N-cadherin, Snail, TIMP2, MMP-2, and MMP-9) in HepG2 cells.