4-Hydroxyderricin Promotes Apoptosis and Cell Cycle Arrest through Regulating PI3K/AKT/mTOR Pathway in Hepatocellular Cells

Abstract

4-hydroxyderricin (4-HD), as a natural flavonoid compound derived from Angelica keiskei, has largely unknown inhibition and mechanisms on liver cancer. Herein, we investigated the inhibitory effects of 4-HD on hepatocellular carcinoma (HCC) cells and clarified the potential mechanisms by exploring apoptosis and cell cycle arrest mediated via the PI3K/AKT/mTOR signaling pathway. Our results show that 4-HD treatment dramatically decreased the survival rate and activities of HepG2 and Huh7 cells. The protein expressions of apoptosis-related genes significantly increased, while those related to the cell cycle were decreased by 4-HD. 4-HD also down-regulated PI3K, p-PI3K, p-AKT, and p-mTOR protein expression. Moreover, PI3K inhibitor (LY294002) enhanced the promoting effect of 4-HD on apoptosis and cell cycle arrest in HCC cells. Consequently, we demonstrate that 4-HD can suppress the proliferation of HCC cells by promoting the PI3K/AKT/mTOR signaling pathway mediated apoptosis and cell cycle arrest.

Keywords: Angelica keiskei; anti-tumor; apoptosis; cell cycle; chalcone; mechanism.

Figure 1

4-HD inhibits the proliferation and metastasis of HCC cells. (A,B) Determination of the survival rate of HepG2 cells and Huh7 cells treated with 4-HD (0–100 µM) after 24 h or 48 h by CCK-8 assay; (C,D) Evaluation of effects for 4-HD on cell migration by wound healing assay; (E,F) Assessment of effects for 4HD-treated cell invasion by Transwell assay. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Effects of 4-HD on apoptosis of HepG2 and Huh7 cells. (A,B) TUNEL (green) and DAPI (blue) double-positive cells were elevated after treatment with various concentrations of 4-HD (magnification, 400×); (C,D) Cells were treated with 4-HD for 48 h, stained with annexin V-FITC/PI, and then analyzed by flow cytometry; (E,F) The effects of 4-HD on the expressions of Bax, Bcl-2, cytochrome c, caspase-9, caspase-3 and PARP proteins were evaluated via Western blot. Relative expressions of the proteins were normalized to GAPDH. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

The effect of 4-HD on HCC cell cycle distribution. (A,B) Phase distribution of HepG2 treated with 4-HD for 48 h by flow cytometry analysis; (C,D) The effects of 4-HD on the expression of Cyclin B1 and CDK1/CDC2 in HepG2 cells were evaluated by Western blot; (E,F) Phase distribution of Huh7 treated with 4-HD for 48 h by flow cytometry analysis; (G,H) The effects of 4-HD on the expressions of Cyclin D1, CDK4 and CDK6 in Huh7 cells were evaluated by Western blot. Relative expressions of the proteins were normalized to GAPDH. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

4-HD inhibited the proliferation and metastasis of HCC cells by regulating the PI3K/AKT/mTOR signaling pathway. (A,B) Relative proteins expressions of PI3K/AKT/mTOR pathway in HepG2 cells and Huh7 cells treated with 0 μM, 20 μM, and 40 μM of 4-HD for 48 h; (C) Immunofluorescence was employed to quantify the expression of p-AKT protein in HepG2 cells (magnification: 400×); (D,E) Relative proteins expressions of PI3K/AKT/mTOR pathway in HepG2 cells were treated with PBS (control), 4-HD (40 μM 4-HD), LY294002 (10 μM LY294002) and 4-HD + LY294002 (40 μM 4-HD + 10 μM LY294002) for 48 h; (F) immunofluorescence was performed to quantify the expression of p-AKT protein in HepG2 cells treated with LY294002 (magnification: 400×); (G,H) wound healing assay was carried out to conduct the effect of LY294002 on the migration of HepG2 cells; (I,J) Transwell assay was implemented to detect the effect of LY294002 on the invasion of HepG2 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control (0 μM); ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. 40 μM 4-HD.

Figure 5

4-HD induced apoptosis and cycle arrest of HCC cells by regulating the PI3K/AKT/mTOR signaling pathway. (A,B) Cell apoptosis proportion treated with 4-HD+LY294002 was detected by flow cytometry; (C,D) The effects of LY294002 on the expressions of Bax, Bcl-2, cytochrome c, caspase-9 and caspase-3 and PARP in HepG2 cells treated with 4-HD were evaluated by Western blotting; (E,F) Cell cycle distribution proportion treated with 4-HD+LY294002 was determined by flow cytometry; (G,H) Effects of LY294002 on the expressions of cyclin B1 and CDK1/CDC2 proteins in HepG2 cells treated with 4-HD were assessed by Western blotting. Relative expressions of the proteins were normalized to GAPDH. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (0 μM); ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs. 40 μM 4-HD.

Figure 6

Proposed mechanism for 4-HD inducing apoptosis and cycle arrest in HCC cells through the PI3K/AKT/m-TOR signaling pathway. ⊥ indicates an inhibitory effect and → indicates a promoting effect.