Antiproliferation and apoptosis induction of paeonol in HepG2 cells

Abstract

Aim: To investigate the antiproliferative effect of paeonol (Pae) used alone or in combination with chemotherapeutic agents [cisplatin (CDDP), doxorubicin (DOX) and 5-fluorouracil (5-FU)] on human hepatoma cell line HepG2 and the possible mechanisms.

Methods: The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Morphologic changes were observed by acridine orange (AO) fluorescence staining. Cell cycle and apoptosis rate were detected by flow cytometry (FCM). Drug-drug interactions were analyzed by the coefficient of drug interaction (CDI).

Results: Pae (7.81-250 mg/L) had an inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner, with the IC50 value of (104.77 +/- 7.28) mg/L. AO fluorescence staining and FCM assays showed that Pae induced apoptosis and arrested cell cycle at S phase in HepG2 cells. Further, different extent synergisms were observed when Pae (15.63, 31.25, 62.5 mg/L) was combined with CDDP (0.31-2.5 mg/L), DOX (0.16-1.25 mg/L), or 5-FU (12.5-100 mg/L) at appropriate concentrations. The IC50 value of the three drugs decreased dramatically when combined with Pae (P < 0.01). Of the three different combinations, the sensitivity of cells to drugs was considerably different.

Conclusion: Pae had a significant growth-inhibitory effect on the human hepatoma cell line HepG2, which may be related to apoptosis induction and cell cycle arrest. It also can enhance the cytotoxicity of chemotherapeutic agents on HepG2 cells, and the S phase arrest induced by Pae may be one of the mechanisms of these interactions.

Figure 1

Structure of Pae (2-hydroxy-4-metho-xyacetophenone).

Figure 2

Dose-dependent cytotoxicity of Pae in HepG₂ cells. Data are presented as mean ± SE (error bar) of triplicate experiments.

Figure 3

Effect of Pae on apoptosis in HepG₂ cells. A: Morphological changes of HepG₂ cells treated with Pae 62.5 mg/L (× 320); B: Flow cytometry analysis of HepG₂ cells treated with Pae for 24 h. (a). Control; (b).Pae 31.25 mg/L; (c).Pae 62.5 mg/L; (d).Pae 125 mg/L.

Figure 4

Effect of Pae on cell cycle in HepG₂ cells. The distribution of cells in the sub-G₁, G₀/G₁, S, and G₂/M phases of the cell cycle were calculated and plotted. (A): Dose-dependent curve of cell cycle distribution induced by Pae. (B): Time-dependent curve of cell cycle distribution induced by Pae 31.25 mg/L. Each point represents triplicate experiments. Bars ± SE.

Figure 5

The synergistically antiproliferative effect of Pae combined with CDDP (A), DOX (B), or 5-FU (C) on HepG₂ cells. The dots represent the concentrations of chemotherapeutic drugs as 0 on the dose-response curves which means treatment with Pae alone. Data are presented as mean ± SE of triplicate experiments. ^aP < 0.05, ^bP < 0.01, vs chemotherapeutic drugs alone. And CDI for the combination treatment of Pae with CDDP (D), DOX (E), or 5-FU (F) on HepG₂ cells.