Acrolein-derived DNA adduct formation in human colon cancer cells: its role in apoptosis induction by docosahexaenoic acid

Abstract

The apoptotic effects of docosahexaenoic acid (DHA) and other omega-3 polyunsaturated fatty acids (PUFAs) have been documented in cell and animal studies. The molecular mechanism by which DHA induces apoptosis is unclear. Although there is no direct evidence, some studies have suggested that DNA damage generated through lipid peroxidation may be involved. Our previous studies showed that DHA, because it has a high degree of unsaturation, can give rise to the acrolein-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) as a major class of DNA adducts via lipid oxidation. As a first step to investigate the possible role of oxidative DNA damage in apoptosis induced by DHA, we examined the relationships between oxidative DNA damage and apoptosis caused by DHA in human colon cancer HT-29 cells. Apoptosis and oxidative DNA damage, including Acr-dG and 8-oxo-deoxyguanosine (8-oxo-dG) formation, in cells treated with DHA and omega-6 PUFAs, including arachidonic acid (AA) and linoleic acid (LA), were measured. DHA induced apoptosis in a dose- and time-dependent manner with a concentration range from 0 to 300 microM as indicated by increased caspase-3 activity and PARP cleavage. In contrast, AA and LA had little or no effect at these concentrations. The Acr-dG levels were increased in HT-29 cells treated with DHA at 240 and 300 microM, and the increases were correlated with the induction of apoptosis at these concentrations, while no significant changes were observed for 8-oxo-dG. Because proteins may compete with DNA to react with acrolein, we then examined the effects of BSA on DHA-induced apoptosis and oxidative DNA damage. The addition of BSA to HT-29 cell culture media significantly decreases Acr-dG levels with a concomitant decrease in the apoptosis induced by DHA. The reduced Acr-dG formation is attributed to the reaction of BSA with acrolein as indicated by increased levels of total protein carbonyls. Similar correlations between Acr-dG formation and apoptosis were observed in HT-29 cells directly incubated with 0-200 microM acrolein. Additionally, DHA treatment increased the level of DNA strand breaks and caused cell cycle arrested at G1 phase. Taken together, these results demonstrate the parallel relationships between Acr-dG level and apoptosis in HT-29 cells, suggesting that the formation of Acr-dG in cellular DNA may contribute to apoptosis induced by DHA.

Fig. 1

Structures of PUFAs (A) and Acr-dG (B).

Fig. 2

Apoptosis induction in HT-29 cells by DHA, AA or LA. In A and B, HT-29 cells were treated with different concentrations of PUFAs for 24h. The apoptotic responses were measured using caspase-3 activities (A) and PARP cleavage (B). In C and D, cells were treated with indicated concentrations of DHA, AA or LA for 0,4,8,12 and 24h and the apoptotic responses were measured by caspase-3 activities (C) and PARP cleavage (D).

Fig. 3

Acr-dG and 8-oxo-dG levels in HT-29 cells treated with different concentrations of PUFAs. Acr-dG adduct levels were measured with the ³²P/SPE/HPLC-based postlabeling assay (A) and 8-oxo-dG levels were measured with an HPLC/electrochemical assay (B).

Fig. 4

Comparison of apoptotic responses of HT-29 cells treated with DHA delivered with ethanol or BSA. HT-29 cells were treated with different concentrations of DHA delivered with and without BSA for 24h, then caspase-3 activities (A) and PARP cleavage (B) were measured. In another experiment, cells were treated with 200µM DHA in the presence of different concentrations of BSA for 24h, the dose-dependent effect of BSA on caspase-3 activities (C) and PARP cleavage (D) were examined. To investigate the oxidative effects of DHA on protein and DNA in the presence of BSA, the cells were treated with 0 or 250µM of DHA with and without 100µM BSA. The total protein carbonyl formation in media (E) and Acr-dG in DNA were measured (F).

Fig. 4

Comparison of apoptotic responses of HT-29 cells treated with DHA delivered with ethanol or BSA. HT-29 cells were treated with different concentrations of DHA delivered with and without BSA for 24h, then caspase-3 activities (A) and PARP cleavage (B) were measured. In another experiment, cells were treated with 200µM DHA in the presence of different concentrations of BSA for 24h, the dose-dependent effect of BSA on caspase-3 activities (C) and PARP cleavage (D) were examined. To investigate the oxidative effects of DHA on protein and DNA in the presence of BSA, the cells were treated with 0 or 250µM of DHA with and without 100µM BSA. The total protein carbonyl formation in media (E) and Acr-dG in DNA were measured (F).

Fig. 5

Comparison of apoptosis and Acr-dG formation of HT-29 cells treated with Acr. Cells were incubated with from 0 to 200µM of Acr for 24h. Then, the Annexin V-FITC / PI assay were used to measure apoptosis (A), and Acr-dG adduct levels were measured (B).

Fig. 6

DNA strand breaks in HT-29 cells treated with DHA. Cells were treated with different concentrations of DHA and DNA strand breaks were measured with an electrophoresis-based comet assay. Irradiation of 9Gy was to treated cells as positive control.

Fig. 7

Cell cycle arrest analysis of HT-29 cells treated with PUFAs. Cells treated with different concentrations of DHA, AA, or LA were harvested and stained with propidium iodide for flow-cytometry-based FACT analysis.