Activation of reactive oxygen species/AMP activated protein kinase signaling mediates fisetin-induced apoptosis in multiple myeloma U266 cells
Introduction
Multiple myeloma, also known as plasma cell myeloma, is a cancer of plasma cells. Multiple myeloma proceeds through different phases: an inactive phase in which tumor cells are nonproliferating mature plasma cells, an active phase with a small percentage (1%) of proliferating plasmablastic cells, and a fulminant phase with the frequent occurrence of extramedullary proliferation and an increase in plasmablastic cells [1]. Multiple myeloma is generally incurable due to relapse or progression. Although the introduction of thalidomide, bortezomib and lenalidomide has dramatically changed the treatment paradigm of multiple myeloma, these treatments are associated with serious side effects such as peripheral neuropathy and thrombocytopenia [2].
5′ Adenosine monophosphate-activated protein kinase (AMPK) is an energy metabolism regulator that plays a critical role in the regulation of AMP to maintain cellular homeostasis [3]. AMPK responses to various stimuli such as hypoxia, oxidative stress and glucose deprivation [4] and subsequently phosphorylates its downstream substrates such as acetyl-CoA carboxylase (ACC), TSC (tuberous sclerosis complex), forkhead box O3 (FOXO3) and histone deacetyltransferases (HDACs) [5], [6]. Since many studies reported the important role of AMPK in pro-apoptotic pathway of cancer cells [7], [8], [9], AMPK has been considered as an attractive target molecule for cancer treatment.
There are evidences that fisetin derived from various plants such as Acacia greggii, Acacia berlandieri, Rhus cotinus and Gleditschia triacanthow possesses anti-oxidant [10], anti-angiogenic [11], anti-proliferative [12], anti-inflammatory [13] anti-aging [14] and anti-tumor [23] activities. The underlying mechanisms of fisetin in multiple myeloma cells remain unfully understood yet. The present study aims at investigating the roles of AMPK and reactive oxygen species (ROS) in fisetin-induced apoptosis in U266 human multiple myeloma cells, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle analysis, TUNEL assay, Western blotting and immunofluorescence assay.
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Chemicals
Fisetin (Fig. 1A) and N-acetyl-l-cystein (NAC) were purchased from Sigma–Aldrich (St. Louis, MO). Compound C was obtained from EMD biosciences (Gibbstown, NJ).
Cell culture
Human multiple myeloma U266 and MM.1S cells were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin.
Metabolic viability test
The metabolic activity change of fisetin was determined by MTT assay [15]. Cells were seeded onto a 96-well plate at a
Fisetin was cytotoxic to U266 cells
To determine the metabolic activity change by fisetin was determined by MTT metabolic viability assay. U266 cells were treated with various concentrations of fisetin (0, 10, 20, 40 or 60 μM) for 12 or 24 h, and the percentage of viable cells was analyzed. The treatment with fisetin dramatically inhibited the metabolic viability of U266 cells in a dose and time-dependent manner (Fig. 1B).
Fisetin treatment induced apoptosis in U266 cells
To examine whether fisetin can induce apoptosis in U266 cells, cells were treated with fisetin (50 μM) for 0, 3,
Discussion
The purpose of our study is to elucidate the roles of ROS production and AMPK activation in fisetin-induced apoptosis in multiple myeloma U266 cells. Fisetin was cytotoxic to U266 cells at the dose of IC50 = 40 μg/ml, in a time-dependent manner, which was similar to López-Lázaro’s study [22] with IC50 of 17.4 μg/ml in K562 leukemia cells. The cytotoxic activity of fisetin was confirmed to be exerted through apoptosis, manifested by increasing the sub-G1 apoptotic fraction and TUNEL positive cells.
Acknowledgment
The present study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea Government (MEST) (No. 2011-0006220).
References (36)
- et al.
Multiple myeloma: increasing evidence for a multistep transformation process
Blood
(1998) - et al.
Dissecting the role of 5′-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase
J. Biol. Chem.
(2006) - et al.
Management of cellular energy by the AMP-activated protein kinase system
FEBS Lett.
(2003) The AMP-activated protein kinase cascade – a unifying system for energy control
Trends Biochem. Sci.
(2004)- et al.
Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition
J. Biol. Chem.
(2010) - et al.
Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages
Biochem. Pharmacol.
(1990) - et al.
Flavonol-rich RVHxR from Rhus verniciflua Stokes and its major compound fisetin inhibits inflammation-related cytokines and angiogenic factor in rheumatoid arthritic fibroblast-like synovial cells and in vivo models
Int. Immunopharmacol.
(2009) - et al.
Fisetin, a novel flavonol attenuates benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice
Food Chem. Toxicol.
(2011) - et al.
Janus activated kinase 2/signal transducer and activator of transcription 3 pathway mediates icariside II-induced apoptosis in U266 multiple myeloma cells
Eur. J. Pharmacol.
(2011) - et al.
Activation of the AMP-activated protein kinase-p38 MAP kinase pathway mediates apoptosis induced by conjugated linoleic acid in p53-mutant mouse mammary tumor cells
Cell. Signal.
(2010)
The role of AMPK and mTOR in nutrient sensing in pancreatic beta-cells
J. Biol. Chem.
Molecular characterization of light chain 3. A microtubule binding subunit of MAP1A and MAP1B
J. Biol. Chem.
Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase
Cell Metab.
The LKB1-AMPK pathway: metabolism and growth control in tumour suppression
Nat. Rev. Cancer
Apoptotic effect of quercetin on HT-29 colon cancer cells via the AMPK signaling pathway
J. Agric. Food. Chem.
Compound K induces apoptosis via CAMK-IV/AMPK pathways in HT-29 colon cancer cells
J. Agric. Food. Chem.
Fisetin, an inhibitor of cyclin-dependent kinase 6, down-regulates nuclear factor-kappaB-regulated cell proliferation, antiapoptotic and metastatic gene products through the suppression of TAK-1 and receptor-interacting protein-regulated IkappaBalpha kinase activation
Mol. Pharmacol.
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