Quercetin regulates the sestrin 2-AMPK-p38 MAPK signaling pathway and induces apoptosis by increasing the generation of intracellular ROS in a p53-independent manner

Abstract

The induction of apoptosis in cancer cells is a therapeutic strategy for the treatment of cancer. In the present study, we investigated the regulatory mechanisms responsible for quercetin-induced apoptosis, mamely the increased expression of sestrin 2 and the activation of the 5' AMP-activated protein kinase (AMPK)/p38 MAPK signaling pathway. Our results revealed that quercetin induced apoptosis by generating the production of intracellular reactive oxygen species (ROS) and increasing the expression of sestrin 2. The induction of apoptosis by quercetin occurred through the activation of the AMPK/p38 signaling pathway and was dependent on sestrin 2. However, the silencing of sestrin 2 using small interfering RNA (siRNA) targeting sestrin 2 revealed that quercetin did not regulate AMPK or p38 phosphorylation in the cells in which sestrin 2 was silenced. On the other hand, it has been previously reported that sestrin 2 expression is not dependent on p53 expression under hypoxic conditions, whereas DNA damage is dependent on p53. We demonstrate that the increase in the expression of sestrin 2 by quercetin-generated intracellular ROS is p53-independent. The increased expression of sestrin 2 induced apoptosis through the AMPK/p38 signaling pathway in the HT-29 colon cancer cells, which are p53 mutant, treated with quercetin. Thus, our data suggest that quercetin induces apoptosis by reducing mitochondrial membrane potential, generating intracellular ROS production and increasing sestrin 2 expression through the AMPK/p38 pathway. In addition, p53 is not a necessary element for an apoptotic event induced by sestrin 2.

Figure 1

(A) Quercetin increased intracellular reactive oxygen species (ROS) production. Cells were treated with the indicated concentrations of quercetin for 6 h (left panel), pre-treated with 5 mM N-acetylcysteine (NAC) for 30 min, and then exposed to quercetin (right panel). After 6 h, teh cells were treated with 40 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 30 min, and fluorescence intensity was measured using a flow cytometer. Dashed line, quercetin 25 μM; dotted line, quercetin 50 μM. (B and C) In addition, cells were treated with the indicated concentrations quercetin for the indicated periods of time and fluorescence was detected using a fluorescence microscope.

Figure 2

(A) Cell viability was measured by MTT assay. ^*P<0.05, ^**P<0.01 and ^***P<0.001 (each experiment, n=3). (B) Cells were treated with the indicated concentrations of quercetin for 6 h. The expression of sestrin 2 and the activation of AMPKα1 and p38 were analyzed by western blot analysis. (C) Cells were treated with the indicated concentrations of quercetin for 24 h. Cells were stained with Annexin V/PI and fluorescence intensity was measured using a flow cytometer. SESN2, sestrin 2.

Figure 3

Cells were pretreated with 5 mM N-acetylcysteine (NAC) for 30 min and then exposed to quercetin for the indicated periods of time. (A) Cells were stained with Annexin V/PI and fluorescence intensity was measured using a flow cytometer. (B) cells were treated with 40 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 30 min, and fluorescence was detected using a fluorescence microscope. (C) Cells were treated with 10 μM Hoechst 33342 for 30 min, and fluorescence was detected using a fluorescence microscope. Arrows indicate apoptotic bodies, which were DNA fragments produced when apoptosis occurred. (D) The expression of sestrin 2 and the activation of AMPKα1, p38 were analyzed by western blot analysis. (E) Cells were stained with 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and fluorescence intensity was measured using a flow cytometer. (F) Cytochrome c in the mitochondrial/cytosolic fraction and Bax protein levels were analyzed by western blot analysis. Q, quercetin; SESN2, sestrin 2; Cyt. c, cytochrome c.

Figure 4

Cells were pre-treated with 10 μM SB203580 for 30 min and then exposed to quercetin for the indicated periods of time. (A) Cell viability was measured by MTT assay. ^**P<0.01 compared to control; ^##P<0.01 compared to the 50 μM quercetin-treated group. N.S., not significant (each experiment, n=3). (B) Cells were stained with Annexin V/PI and fluorescence intensity was measured using a flow cytometer. (C) The expression of sestrin and the activation of AMPKα1 and p38 were analyzed by western blot analysis. Q, quercetin; SESN2, sestrin 2.

Figure 5

(A) Cells were transfected with sestrin 2 or AMPKα1 small interfering RNA (siRNA) using DharmaFECT and treated with 50 μM quercetin for 6 h after being pre-treated with N-acetylcysteine (NAC) 5 mM for 30 min. The protein levels of sestrin 2, p-AMPKα1, AMPKα1 and p-p38 were then examined by western blot analysis. (B) Cell viability was measured by MTT assay. ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared to control, ^##P<0.01, and ^###P<0.001 compared to the 50 μM quercetin-treated group. N.S., not significant (each experiment, n=3). SESN2, sestrin 2.

Figure 6

(A) HCT116 colon cancer cells were treated with the indicated concentrations of quercetin after being pre-treated with 10 μM pifithrin-α for 30 min. (B) HT-29 cells [p53-mutant (mt)] were treated with the indicated concentrations of quercetin for 24 h. Finally, the cells were stained with Annexin V/PI and their fluorescence intensity was measured using a flow cytometer. (C) HT-29 colon cancer cells were treated with the indicated concentrations of quercetin in for 6 h. The expression of sestrin 2 and the activation of AMPKα1 and p38 were analyzed by western blot analysis. SESN2, sestrin 2.