cis-Terpenones as an effective chemopreventive agent against aflatoxin B1-induced cytotoxicity and TCDD-induced P450 1A/B activity in HepG2 cells

Abstract

Aflatoxin B1 (AFB1) is a potent carcinogen, which can significantly increase the risk of hepatocellular carcinoma development through food contamination. In past decades, chemopreventive agents, such as oltipraz and chlorophyllins, have demonstrated that chemo-intervention is an effective approach to reduce hepatotoxicity by AFB1. However, because of the potential adverse effects of these agents, alternative novel mechanism-based chemopreventive agents are needed. We report here that novel cis-terpenones 1-3, which were synthesized as the precursors of natural product analogues in our laboratory, showed promising protective effects against AFB1-induced cytotoxicity in HepG2 cells. Chemo-protection was observed with increasing concentrations of cis-terpenones in the co-treatment of AFB1, and no cytotoxicity was observed with cis-terpenones alone. In addition, cis-terpenones 1-3 at 10 and microM effectively inhibited induced cytochrome P450 1A/1B activity by 50% in HepG2 cells, as indicated by an EROD assay. P450 1A/B is involved in the activation of many pre-carcinogens and is highly inducible in liver cells. These results suggested that novel terpenones 1-3 are candidates for the development of novel mechanism-based chemopreventive agents against AFB1 and other carcinogenic stimuli.

Figure 1

Chemo-protection with cis-terpenones against AFB1 induced cytotoxicity. HepG2 cells were co-treated with AFB1 (2 μM) and cis-terpenones 1-3 at various concentrations (10-40 μM) and incubated for 72 h. Cell viability was measured with the MTT assay. The percentage of viable cells was based on cells treated with medium only. Each bar represents the mean ± SD of 4 replicates. The data are representative of three independent experiments. * P<0.05, ** P<0.001 compared to treatment with AFB1 by one way ANOVA and Dunnett’s test.

Figure 2

Chemo-protection with cis-terpenone 2 against AFB1 induced apoptosis. HepG2 cells were co-treated with AFB1 (2 μM) and cis-terpenone 2 at various concentrations (10-40 μM) and incubated for 72 h. The cells were stained with PI (shown as the Y-axis of each graph) and Annexin-V FITC (shown as the X-axis of each graph), and analyzed with a flow cytometer. The numbers in each divided box represent the percent distribution of HepG2 cells within the gated areas. PI positive (at top left), both PI and annexin V positive - late apoptosis or dead (at top right), both PI and annexin V negative - viable (at lower left), and annexin V positive - early apoptosis (at lower right). The data are representative of three independent experiments.

Figure 3

Inhibition of TCDD-induced P450 1A/B activity with cis-terpenones. HepG2 cells were co-treated with TCDD (1 nM) and cis-terpenones 1-3 and incubated for 24 h. The activity of P450 1A/B was determined with the EROD assay. Bars represent the mean ± SD of EROD activity at 590 nm in 4 replicates. The data are representative of three independent experiments. C: DMSO only. * P<0.05, ** P<0.001 compared to TCDD only with one way ANOVA and Dunnett’s test.

Scheme 1

cis-Terpenones 1-3 as Precursors of Analogues of the Natural Bioactive Tingenone.

Scheme 2

Synthesis of cis-Terpenones 1-3.