Time course changes of anti- and pro-apoptotic proteins in apigenin-induced genotoxicity

Abstract

Background: Apigenin (4',5,7-trihydroxyflavone, AP), an active component of many medicinal Chinese herbs, exhibits anticancer properties in vitro and in vivo. This study aims to investigate the genotoxic, cytostatic, and cytotoxic effects of AP and time course changes in the levels of anti- and pro-apoptotic proteins involved in the DNA damage response in HepG2 cells.

Methods: The genotoxic potential of AP was determined by sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) analysis. The levels of cytostaticity and cytotoxicity were evaluated by the proliferation rate and mitotic indices, respectively. MTT was used to study cytotoxicity, while the induction of apoptosis and the expression of apoptosis-related proteins were determined by ELISA.

Results: At concentrations greater than 10 μM, AP decreased cell survival in a dose- (48 h: 10 vs. 20 μΜ, P < 0.001 and 20 vs. 50 μΜ, P = 0.005; 72 h: 10 vs. 20 μΜ, P < 0.001 and 20 vs. 50 μΜ, P = 0.001) and time-dependent manner (20 μΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P = 0.003; 50 μΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P < 0.001; 100 μΜ: 24 vs. 48 h, P < 0.001 and 48 vs. 72 h, P < 0.001). SCEs rates, cell proliferation, and mitotic divisions were also affected in a dose-dependent manner (P < 0.001). There was no change in the frequency of aberrant cells (1 μΜ ΑP: P = 0.554; 10 μM AP: P = 0.337; 20 μΜ AP: P = 0.239). Bcl-2 levels were reduced 3 h after AP administration (P = 0.003) and remained reduced throughout the 48 h observation period (6 h, P = 0.044; 12 h, P = 0.001; 24 h, P = 0.042; 48 h, P = 0.012). Bax and soluble Fas exhibited a transient upregulation 24 h after AP treatment. The Bax/Bcl-2 ratio was also increased at 12 h and remained increased throughout the 48 h observation period.

Conclusion: AP exhibited dose-dependent genotoxic potential in HepG2 cells. The protein levels of sFas, Bcl-2, and Bax were affected by AP to promote cell survival and cell death, respectively.

Figure 1

The effect of apigenin on cell viability and induction of apoptosis. A. The cytotoxic and antiproliferative effect of apigenin in HepG2 cells. B. The apoptotic potential of apigenin (20 μΜ) after 24 h of treatment. The star (*) indicates statistical significance compared with control cultures.

Figure 2

The effect of apigenin on apoptosis-related proteins. A. The effect of apigenin (20 μΜ) treatment on sFas protein levels. Control cultures: 6 vs. 12 h, P = 0.005; 12 vs. 24 h, P = 0.001. Apigenin treatment: 3 vs. 6 h, P = 0.021; 6 vs. 12 h, P = 0.001; 12 vs. 24 h, P = 0.001; 24 vs. 48 h, P = 0.001. B. The effect of apigenin (20 μΜ) treatment on Bcl-2 protein levels. C. The effect of apigenin (20 μΜ) treatment on Bax protein levels. D. The effect of apigenin (20 μΜ) treatment on Bax/Bcl-2 ratio. According to linear regression analysis: Time intervals 1–12 h, R = 0.960, R² = 0.921, P = 0.040; Time intervals 12–48 h, R = -0.957, R² = 0.915, P = 0.188. The star (*) indicates statistical significance compared with the respective control cultures.