Tetrahydrocurcumin extends life span and inhibits the oxidative stress response by regulating the FOXO forkhead transcription factor

Abstract

The O-type forkhead domain transcription factor (FOXO) is involved in many biological processes such as aging, the oxidative stress response, and growth regulation. FOXO activity is tightly controlled within cells. In particular, growth factor signaling pathways and the oxidative stress response can both stimulate nuclear translocation of this transcription factor. Here, we show that tetrahydrocurcumin (THC), a curcumin metabolite, regulates the oxidative stress response and aging via FOXO. In NIH3T3 cells, THC induced nuclear accumulation of FOXO4, a member of the FOXO family of transcription factors, by inhibiting phosphorylation of protein kinase B (PKB)/Akt. In Drosophila melanogaster, THC attenuated the oxidative stress response, an effect that was blocked in a foxo mutant background. THC also extended the life span of Drosophila under normal conditions, and loss of either foxo or Sir2 activity eliminated this effect. Based on these results, THC may regulate the aging process via an evolutionarily conserved signaling pathway that includes both foxo and Sir2.

Figure 1. THC-induced nuclear accumulation of FOXO4

(A) Dose-dependent effects of THC on nuclear accumulation of FOXO4 in NIH-3T3-FOXO4 cells. Cy3 (red) and DAPI (blue) label FOXO4 and nuclei, respectively. (B) FOXO4 levels increase in nuclear fractions from NIH-3T3-FOXO4 cells. Lamin B was used as a nuclear marker and the Akt inhibitor LY294002 (LY) was used as a positive control. Anti-AFX (N-19) primary antibody was used in both analyses.

Figure 2. Time course of FOXO4 localization in response to THC

(A) FOXO4 protein levels increase in the nuclear fraction of NIH-3T3-FOXO4 cells within minutes of THC treatment. Lamin B was used as a nuclear marker, DMSO as a negative control. (B) Time course of FOXO4 nuclear translocation in NIH-3T3-FOXO4 cells following administration of THC. Red indicates FOXO4 protein. (C) The number of cells with nuclear FOXO was quantified at the indicated times following THC administration.

Figure 3. THC inhibits Akt phosphorylation

Dose-dependence (A) and time-course (B) analyses indicate that THC inhibits phosphorylation of Akt^Ser473. LY294002 (LY) was used as a positive control, Akt and GAPDH antibodies as loading controls.

Figure 4. THC regulates oxidative stress response in Drosophila via foxo activity

Effects of THC, RES, and C on the life span of wild-type (WT, Oregon-R) (A) and foxo mutant (yw background) (B) flies under oxidative stress conditions. Oxidative stress was induced by adding 7.5 mM paraquat to the food. Life span studies were carried out at 25°C with newly eclosed males (M) in each group (20 per vial; 3-4 vials were counted). Survivors were transferred every 2-3 days to fresh vials containing THC, RES, or C (50-150 μM). The statistical analysis of the data is summarized in Table 1.

Figure 5. THC, but not C, extends life span in Drosophila under normal conditions

The effect of THC and RES (A) was analyzed in a yw background, and C (B) was analyzed in Oregon-R (WT) flies. Life span studies were carried out at 25°C with newly eclosed male (M) or female (F) flies for each longevity experiment (20 per vial; 4 vials were counted). Survivors were transferred to fresh vials containing THC, RES, or C (20—150 μM) every 2—3 days. The statistical analysis of the data is summarized in Table 1.

Figure 6. THC and RES do not extend the life span of foxo or d4E-BP mutants

Effects of THC or RES (50 μM) on the life span of foxo−/− (A) or d4E-BP−/− (B) mutant Drosophila under natural conditions. Life span studies were carried out at 25°C with a total of around 80 newly eclosed male (M) flies (yw background). The statistical analysis of the data is summarized in Table 1.

Figure 7. THC and RES do not extend the life span of Sir2 mutants

Effects of THC or RES (50 μM) on the life span of Sir2-/df mutant Drosophila under natural conditions. Life span studies were carried out at 25°C with a total of around 80 newly eclosed male (M) flies (yw background). The statistical analysis of the data is summarized in Table 1.