Induction of apoptosis by quercetin is mediated through AMPKα1/ASK1/p38 pathway
Introduction
Apoptosis is a programmed cell death process that controls normal development and homeostasis in organisms [1], [2]. The loss of apoptotic control contributes to the survival of tumor cells, and the enhancement of cancer cell apoptosis is one approach of controlling cancer by anticancer agents. Although there seems to be remarkable progresses of the detailed mechanism of the mitochondrial cytochrome c-induced apoptosis, the overall regulatory signaling pathways whereby apoptosis is targeted by anticancer agents have not been clearly presented [3], [4], [5]. Providing the detailed pathways leading to apoptosis is an important goal for establishing effective tumor prevention and treatment strategies using chemopreventive materials such as quercetin. One of the well-known intracellular signaling pathways for apoptosis is the kinase cascade, which has been identified as a transducing pathway of apoptotic signals initiated by outside stimuli, and the mitogen-activated protein (MAP) kinases and their upstream kinases such as MAP kinase kinases [6], [7]. Apoptosis signal-regulating kinase (ASK)-1 is a member of the ROS-sensitive MAP kinase kinases and it acts as a redox sensor of cells [6], [8], [9], [10]. ASK1 is commonly regulated through protein–protein interactions, and numerous proteins have been shown to bind ASK1. The binding of ASK1 to TNF receptor-associated factor (TRAF) or death domain-associated protein (Daxx) stimulates ASK1 function, whereas the ASK1 inhibitory proteins such as thioredoxin (Trx) downregulate pro-apoptotic activity of ASK1 [11], [12], [13], [14], [15].
In this study, we investigated the involvement of ASK1 in the apoptotic process of cancer cells treated with a chemopreventive agent, quercetin. We previously reported that quercetin-induced apoptosis through the generation of ROS, which was responsible for the activation of AMPKα1, and was found to be an indispensable element of apoptosis [16]. Here, we demonstrated that quercetin strongly generated ROS and that this oxidative stress induced apoptosis through activation of AMPKα1 and ASK1, which are critical upstream signals for p38 MAP kinase activation in MCF-7 breast cancer cells. Thus, we suggest that AMPKα1 is a master regulator of ASK1-mediated apoptotic pathway.
Section snippets
Cell culture and reagents
MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO2 atmosphere. Quercetin, hydrogen peroxide, and N-acetyl-cystein were purchased from Sigma (St. Louis, MO, USA). Specific antibodies that recognize the phosphorylated forms of AMPKα1Thr172 and ACC were obtained from Cell Signaling Technology (Danvers, MA, USA) and p-ASK1Ser83, ASK1 and p-ERK were
Quercetin-generated ROS in MCF-7 breast cancer cells
To examine whether quercetin stimulates ROS generation in MCF-7 breast cancer cells, we measured the intracellular ROS levels after treatment of quercetin (0–400 μM) at the indicated time series. As shown in Fig. 1A, quercetin continuously increased ROS levels from 30 min to 9 h after treatment, and the levels were maintained at concentrations above 200 μM. These effects were completely blocked by combined treatment with NAC, a ROS scavenger (Fig. 1B). We also observed intracellular ROS by
Discussion
Our results demonstrate that activation of AMPKα1 is required for ASK1 to induce apoptosis. Additionally, a signaling pathway of AMPKα1/ASK1/p38 is responsible for MCF-7 cells to undergo apoptosis initiated by ROS when the cancer cells were treated with quercetin. Previously, we have shown that quercetin regulates cancer cell proliferation and apoptosis through AMPKα1/cyclooxygenase-2 (COX-2) pathway [16]. AMPK is known to phosphorylate several downstream proteins that control cell growth and
Conflicts of interest
None declared.
Acknowledgments
This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-20131-0).
References (27)
- et al.
Cell death in health and disease: the biology and regulation of apoptosis
Semin. Cancer Biol.
(1995) - et al.
Targeting apoptosis as an approach for gastrointestinal cancer therapy
Drug Resist. Update
(2009) - et al.
Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling
Biochim. Biophys. Acta
(2008) - et al.
ASK1 is essential for JNK/SAPK activation by TRAF2
Mol. Cell.
(1998) - et al.
Thioredoxin signaling as a target for cancer therapy
Curr. Opin. Pharmacol.
(2007) - et al.
Thioredoxin and protein kinases in redox signaling
Semin. Cancer Biol.
(2006) - et al.
Ceramide induces p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated pathway
Blood
(2008) - et al.
ROS stress in cancer cells and therapeutic implications
Drug Resist. Update
(2004) - et al.
AMP-activated protein kinase contributes to UV- and H2O2-induced apoptosis in human skin keratinocytes
J. Biol. Chem.
(2008) - et al.
Programmed cell death and apoptosis: origins of the theory
Nat. Rev. Mol. Cell Biol.
(2001)
Chemotherapeutic approaches for targeting cell death pathways
Oncologist
Programmed cell death pathways and current antitumor targets
Pharm. Res.
Molecular mechanisms of the decision between life and death: regulation of apoptosis by apoptosis signal-regulating kinase 1
J. Biochem.
Cited by (76)
The regulatory and signaling mechanisms of the ASK family
2017, Advances in Biological Regulation7-O-geranylquercetin-induced autophagy contributes to apoptosis via ROS generation in human non-small cell lung cancer cells
2017, Life SciencesCitation Excerpt :Alteration of ROS level plays a crucial role in cancer initiation and progression, and ROS are recognized as the potential target for cancer treatment [46,47]. Lee et al. demonstrated that ROS were essential in quercetin-induced apoptosis of MCF-7 cells [15]. Our previous study indicated GQ triggered ROS generation in MCF-7 cells, but ROS played no role in GQ-induced apoptosis [19].
3′,5-dihydroxy-3,4′,7-trimethoxyflavone-induces ER-stress-associated HCT-116 programmed cell death via redox signaling
2017, Biomedicine and PharmacotherapyCitation Excerpt :Transcriptional control of all pro- and anti-apoptotic bodies involves MLK/JNK, PI3K/Akt, and ras/MAPK pathways [20]. Quercetin illustrates AMPK/p38 pathway-mediated mitochondrial apoptosis against various carcinoma cell lines and plays role in oncogene-induced cell senescence [21]. To enhance its potential oral bioavailability against metabolic efflux and explore the alternative signaling mechanism against colon cancer, we developed a semi-synthetic novel derivative, 3′,5-dihydroxy-3,4′,7-trimethoxyflavone (DTMF; C18H16O7), via reaction between quercetin and corresponding alkyl halides.