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Article
Blockade of the Ras/Raf/ERK and Ras/PI3K/Akt pathways by monacolin K reduces the expression of GLO1 and induces apoptosis in U937 cells Chun-Chia Chen, Mei-Li Wu, Chi-Tang Ho, and Tzou-Chi Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505275s • Publication Date (Web): 08 Jan 2015 Downloaded from http://pubs.acs.org on January 13, 2015
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Journal of Agricultural and Food Chemistry
Blockade of the Ras/Raf/ERK and Ras/PI3K/Akt pathways by monacolin K reduces the expression of GLO1 and induces apoptosis in U937 cells
Chun-Chia Chen†, Mei-Li Wu†, Chi-Tang Ho‡, Tzou-Chi Huang*,†
†
Department of Food Science, National Pingtung University of Science and
Technology, Pingtung 91201, Taiwan ‡
Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA
*
Correspondence: Dr. Tzou-Chi Huang, Department of Food Science, National
Pingtung University of Science and Technology, 1, Shuehfu Road, Neipu, Pingtung 91201, Taiwan E-mail:
[email protected] Tel: +886-8-7703202 ext: 5196 Fax: +886-8-7740582
Coauthors,
Chun-Chia
Chen
(
[email protected]),
Mei-Li
(
[email protected]), and Chi-Tang Ho (
[email protected]).
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Abstract
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Monacolin K, a hydrolytic product of icaritin, is the major active component in the
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traditional fermented Monascus purpureus. Monacolin K inhibits the proliferation of
4
acute myeloid leukemia (AML) and underlying mechanisms remain to be identified.
5
In the present study, we demonstrated that Monacolin K inhibits the proliferation of
6
human AML cell line U937, in a dose-dependent manner. Importantly, morphological,
7
DNA fragmentation and image cytometry analyses indicated that monacolin K
8
induced U937 cells apoptosis. Monacolin K could inactivate Ras translocation from
9
cell membranes to cytosol. Monacolin K could also reduce the Ras-dependent
10
phosphorylation of ERK and Akt, and the subsequent translocation of nuclear factor
11
kappa B (NF-kB) from cytosol to nucleus in U937 cells. The underlying mechanisms
12
of apoptotic activity of monacolin K were associated with inhibition of the
13
Ras/Raf/ERK and Ras/PI3K/Akt signals and downregulation of HMG-CoA reductase
14
and Glyoxalase 1. Based on results obtained using specific inhibitors, U0126,
15
LY294002 and JSH-23, the Ras/Raf/ERK/NF-κB/GLO1 and Ras/Akt/NF-κB/GLO1
16
pathways were proposed for the apoptotic effect of monacolin K in U937 cells.
17 18
Keywords: monacolin K / acute myeloid leukemia / glyoxalase 1 / HMG-CoA
19
reductase / inactivate Ras translocation / apoptosis 2
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Introduction
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Red yeast rice (RYR) is a traditional fermented product of Monascus purpureus and
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white rice.1 It is popular in some Asian countries and is known to have properties of
23
lowering total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C)
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levels.2 Accumulating evidence also showed that RYR inhibited proliferation of
25
various cancer cells in vitro.3,4 The major bioactive compound in RYR, monacolin K,
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also showed tumor-suppressive activity in animal models.5 RYR is a potentially
27
useful over-the-counter cholesterol-lowering agent. Typical RYR dose used in clinical
28
trials is 1200-2400 mg/day which contains approximately 5 mg monacolin K.2
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The structure of monacolin K is identical to the statin drug, lovastatin. Case
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reports and early clinical studies suggest a therapeutic potential for statins in acute
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myeloid leukemia (AML). Statins are 3-hydroxy-3-methylglutaryl-coenzyme A
32
reductase (HMGCR) inhibitors that act on the mevalonate pathway and inhibit
33
synthesis of cholesterol, geranylgeranyl pyrophosphate (GGPP) and farnesyl
34
pyrophosphate (FPP).6 It is well established that mevalonate is essential for
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post-translational modification of Ras, which can lead to increased levels of
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farnesylation of Ras by farnesyl-pyrophosphate transferase. Inhibition of mevalonate
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synthesis leads to a depletion of its downstream intermediates, such as FPP and GGPP,
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precursors for sterols, dolichols, Coenzyme Q, isoprenoids, and carotenoids.7 FPP and 3
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GGPP are substrates for the post-translational modification of proteins including the
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Ras and Rho family GTPases, well-known proto-oncogenes for control cell growth,
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migration, survival and differentiation in mammalian cells. Statins inactivate Ras
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translocation and downstream MEK/ERK and PI3K/Akt signalings leading to cancer
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cell apoptosis by interrupting the biosynthesis of mevalonate.8 Genetic mutations of
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Ras protein in cancer cells trigger dysregulation of Ras/Raf/MEK/ERK and
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Ras/PI3K/PTEN/Akt/mTOR pathway signalings, leading to uncontrolled cellular
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proliferation and decreased sensitivity to agents that ordinarily can trigger apoptosis.
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It is well established that glyoxalase 1 (GLO1) plays a pivotal role in the growth
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and survival of cancer cells. GLO1 is a key enzyme for protecting cancer cells from
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apoptosis, and it works through the elimination of excess methylglyoxal (MG), which
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is a toxic metabolite of the major energy source, glucose. The increased survival
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activity of tumors, including higher glycolytic activity and a consequent rise in MG
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production, requires an increased detoxifying action of GLO1.9 The increase of GLO1
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expression has been shown to be associated with increased proliferative activity of
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tumors. In antitumor agent-resistant leukemia cells, GLO1 was frequently
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overexpressed.10 Immunohistochemical analysis confirmed the increase of GLO1
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among patients with colon carcinoma,11 or prostate cancer.12 Significantly high
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glyoxalase activity has been detected during differentiation of leukemia cells.13 4
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Endogenously produced MG has been shown to cause apoptosis in cultured leukemia
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cells.14
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In contrast with that in normal cells, the cholesterol content of malignant tumors
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in the body is particularly high. The primary source of cholesterol synthesis in cancer
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cells is glucose. Cholesterol is currently known to be synthesized from a series of
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reactions involving the conversion of citrate in the tricarboxylic acid (TCA) cycle to
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generate acetyl coenzyme A, which then reacts with several enzymes in the
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mevalonate pathway to yield cholesterol.15 It is well established that HMGCR activity
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was three-fold higher in mononuclear blood cells from patients with leukemia,
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compared with cells from healthy subjects.16
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We here provide a detailed mechanistic investigation of the apoptotic effect of
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monacolin K and how it is related to the inhibition of GLO1. In the present study, we
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hypothesize that monacolin K showed its apoptotic activity through the inhibition of
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HMGCR, inactivation of Ras translocation, down regulation of PI3K/Akt and
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Raf/ERK1/2 signal pathways leading to suppression of GLO1 and HMGCR in U937
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cells.
74 75
Materials and methods
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Chemicals and Antibodies 5
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Monacolin K, perchloric acid, 2-methylquinoxaline (2-MQ), o-phenylenediamine,
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3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyltetrazolium
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sulphoxide (DMSO) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from
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Sigma-Aldrich (St. Louis, MO). Cell culture media were purchased from Invitrogen
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(Carlsbad, CA). Antibodies against phospho-p44/42 MAPK (Thr202/Tyr204),
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phospho-Akt (Ser473), ERK1/2, Akt1/PKBα and actin, were obtained from Millipore
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(Billerica, MA). Antibodies against GLO-1, NF-κB (p65), H-Ras, Raf-1 and lamin A
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were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against
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K-Ras and N-Ras were obtained from GeneTex (San Antonio, TX). Alexa Fluor® 594
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Goat Anti-Mouse IgG (H+L) was purchased from Invitrogen (Carlsbad, CA). U0126
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(MEK1/2 inhibitor), LY294002 (PI3K inhibitor), JSH-23 (NF-κB inhibitor), IgG
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HRP-conjugated goat anti-mouse and IgG HRP-conjugated goat anti-rabbit were
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obtained from Chemicon (Temecula, CA).
bromide
(MTT),
dimethyl
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Cell Culture
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U937 cells were purchased from the American Type Culture Collection (Rockville,
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MD). The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum
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(GibcoBRL; Grand Island, NY), 5 mM L-glutamine (GibcoBRL) and 1%
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penicillin/streptomycin (10000 units of penicillin/mL and 10 mg/mL streptomycin) at 6
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37 °C in a humidified 5% CO2 incubator.
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Cell Viability Assay
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Cell viability was assessed by MTT assay.17 Briefly, cells at 1×105/mL were treated
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with various concentrations of monacolin K in 96-well plates for 48 h, followed by
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treatment with MTT for 4 h at 37 °C. The medium was removed and DMSO was
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added to dissolve the blue formazan residue and color intensity was measured using
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an ELISA reader at 570 nm.
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Morphological Assay
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After treatment with various concentrations of monacolin K for 48 h, cells were
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harvested and fixed with 4% paraformaldehyde in PBS for 20 min at room
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temperature, and stained with 2.5 mg/mL DAPI solution for 30 min at room
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temperature. The cells were washed two more times with PBS and analyzed via a
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confocal microscope (CARV II Confocal Imager; BD Biosciences).
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DNA Fragmentation Assay
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After treatment with various concentrations of monacolin K for 48 h, washed with
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ice-cold PBS, the cells were resuspended in 180 µL DNA lysis buffer (0.5% 7
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lauroylsarcosine, 10 mM EDTA, 0.5 mg/mL proteinase K and 50 mM Tris-HCl at pH
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8.0) overnight at 56 °C, and treated with RNaseA (0.5 µg/mL) for 1 h at 37 °C. Total
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DNA
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phenol/chloroform/isoamyl alcohol (Sigma-Aldrich, St. Louis, MO). 10 µL of DNA
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were analyzed on 1.8% agarose gel.
was
extracted
from
the
cells
with
an
equal
volume
of
120 121
Image Cytometry Analysis
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Cell sub-G1 phase was analyzed by the DNA content using DAPI staining method.
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After treatment with various concentrations of monacolin K for 48 h, U937 cells were
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centrifuged at 1,500 rpm for 5 min, and the cell pellets were fixed in 70% ice-cold
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ethanol overnight at 4 °C. Fixed cells were washed with PBS, incubated with 0.5 mL
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hypotonic buffer containing 0.5% Triton X-100 and 0.5 mg/mL RNase A for 30 min
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at 37 °C, and then stained with 50 µg/mL DAPI, and the sub-G1 phase content was
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determined on a NucleoCounter NC-3000 cytometer (ChemoMetec, Allerod,
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Denmark).
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RNA Isolation and Reverse Transcription-PCR Amplification (RT-PCR)
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Total RNA was extracted from U937 cells using a commercially available RNA
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extraction kit (Favorgen, Pingtung, Taiwan) and following the manufacturer’s 8
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instruction. First-stranded cDNA was synthesized using 1 µg total RNA by RT-PCR
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kit (Roche, Indianapolis). The relative expression of GLO1 and HMGCR was
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analyzed using RT-PCR with GAPDH as an internal control. The primers used were
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GLO1,
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5’-CTACATTAAGGTTGCCATTTTGTTAG-3’, yielding a product of length 564 bp;
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HMGCR,
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5’-CAAGCCTAGAGACATAATCATC-3’, yielding a product of length 252 bp;
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GAPDH,
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5’-CAAGCCTAGAGACATAATCATC-3’, yielding a product of length 248 bp. A 10
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µL volume reaction for PCR included 2 µL of cDNA, 1 µL each of sense and
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antisense primers and 5 µL of PCR master mix. The conditions were: 94 °C for 5 min,
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94 °C for 40 sec, 60 °C for 35 sec (GLO1), 58 °C for 30 sec (HMGCR) and 64 °C for
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30 sec (GAPDH), 72 °C for 40 sec, and 72 °C for 7 min, for 26 cycles. The PCR
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products were separated by electrophoresis on 1.5% agarose gels and the results were
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analyzed using a Kodak Gel logic imaging system (Hyland Scientific, Stanwood,
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WA).
sense
5’-ATGGCAGAACCGCAGCCCCCGT-3’
sense
sense
5’-TACCATGTCAGGGGTACGTC-3’
5’-TACCATGTCAGGGGTACGTC-3’
and
and
and
antisense
antisense
antisense
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Western Blot Analysis
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The whole cells protein were extracted via the addition of 100 µL of gold lysis buffer 9
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(50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1% protease
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inhibitor cocktail at pH 7.4), followed by incubation at 4 °C for 1 h, centrifugation,
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and collection of cell lysate. Subcellular Protein Fractionation Kit was used to
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determine the levels of protein expression in cytosol, nucleus, and membrane. Extract
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preparation followed instruction of the manufacturer’s published protocol (Thermo,
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Rockford, IL). Protein concentrations were measured with a Bio-Rad protein assay
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reagent (Bio-Rad Laboratories, Munich, Germany). Equal amounts of lysate protein
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(30 µg/lane) were subjected to sodium dodecyl sulfate polyacrylamide gel
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electrophoresis
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electrotransferred to a polyvinylidene difluoride (PVDF) membranes (Millipore,
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Billerica, MA), blocked at 37 °C for 1 h with 1% BSA in TBST (20 mM/L Tris-base;
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125 mM/L NaCl; 0.2% Tween 20; 0.1% sodium azide), and then immunoblotted with
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primary antibodies overnight at 4 °C, and then washed with TBST solution (0.05%
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Tween 20 in TBS). After washing six times with TBST buffer, the blot was washed,
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exposed to horseradish peroxidase conjugated secondary antibodies for 1 h,
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immunoreactive proteins were visualized with an enhanced chemiluminescence (ECL)
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detection system (GE Healthcare, Little Chalfont, UK).
(SDS-PAGE).
After
electrophoresis,
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Total Cholesterol Quantitation 10
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The total cholesterol concentration was measured using Cholesterol/Cholesterol Ester
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Quantitation Kit (Calbiochem, Novabiochem, Boston, MA). After treatment with
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monacolin K for 48 h, cells were washed with cold PBS and centrifuged. The pellet
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was re-suspended in 0.2 mL of chloroform:isopropanol:NP-40, homogenize for 3 min
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and cleared by centrifugation at 14,000 rpm for 10 min. Supernatants were decanted
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and air-dry at 50 °C, and vacuum-dry for 30 min to remove residual solvents. The
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dried lipids were dissolved in 0.2 mL of the reaction buffer and the absorbance was
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measured at 570 nm for total cholesterol detection.
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Methylglyoxal (MG) Measurement
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MG was measured by an o-Phenylenediamine method.18 MG was derivatized with
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o-phenylenediamine to form the quinoxaline product 2-MQ, which is very specific for
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MG. Briefly, the supernatant of the cell was incubated with 100 mM
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o-phenylenediamine for 3 h at room temperature. The quantity of the quinoxaline
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derivative of MG (2-MQ) was measured using a Hitachi L-6200 HPLC system
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(Hitachi, Ltd., Mississauga, Ontario, Canada). The mobile phase was generally 68
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vol% of 10 mM KH2PO4 (pH 2.5) and 32 vol% of HPLC grade acetonitrile. The
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analysis conditions were as follows: detector wavelength, 315 nm; mobile phase flow
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rate, 1.0 mL/min; and column temperature, 20 °C. The whole series of samples in a 11
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single experiment was run in duplicate.
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Immunofluorescence Microscopy
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To detect the localization of NF-κB in monacolin K treated U937 cells, the U937 cells
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were first seeded in a 12 well dish. The cells were fixed with 4% paraformaldehyde
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for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 5 min at
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room temperature. After specimens were blocked with 5% BSA for 1 h at room
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temperature, and further incubated with NF-κB (p65) antibody for 2 h. After three
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washes in TPBS, specimens were then stained with Alexa Fluor® 594 Goat
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Anti-Mouse IgG for 1 h at room temperature. After being washed three times with
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TPBS, cells were stained with 0.5 mg/mL of DAPI for 10 min to counter stain the
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nucleus, and then washed three times for 5 min with TPBS. Images were collected by
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confocal microscope.
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Statistical Analysis
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The data were analyzed using the SAS program (SAS Institute, Cary, NC). The
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significance of the difference among mean values was determined by one-way
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analysis of variance followed by the t-test; (*) p < 0.05 and (**) p < 0.01 were
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considered statistically significant. Values are the mean ± SD of three independent
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experiments. 12
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Results
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Monacolin K Induced Apoptosis in U937 Cells
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MTT assays were performed to evaluate the potential cytotoxic effects of monacolin
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K. As shown in Figure 1A, U937 cells were incubated with increased concentrations
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of monacolin K for 48 h. Exposure of monacolin K to U937 cells resulted in a
217
significant decrease in viable cells in a dose-dependent fashion. Treatment with 20
218
µM monacolin K achieved 57% inhibition (p < 0.01). To determine whether an
219
increase in apoptosis was associated with the observed decrease in cell number after
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monacolin K treatment, the cells were stained with DAPI and analyzed by confocal
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microscope. As shown in Figure 1B, the nuclei of U937 cells exposed to monacolin K
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exhibited nuclear condensation and fragmented chromatin of typical apoptosis in a
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dose-dependent manner. Apoptosis was also detected by DNA fragmentation assay.
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DNA fragmentation was significantly increased after treatment with monacolin K in a
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dose-dependent manner (Figure 1C). Furthermore, the degree of apoptosis was
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quantified by analyzing the amount of sub-G1 DNA content in the U937 cells treated
227
with monacolin K using an image cytometer. As shown in Figure 1D, in untreated
228
control, the percentage of untreated control cells with fragmented DNA (sub-G1) was
229
only 3%. Treatment with 20 µM monacolin K increased the percentage of cells with
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fragmented DNA (sub-G1) to 68%. Taken together, these findings indicated that 13
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exposure to monacolin K resulted in the inhibition of cell proliferation and a striking
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induction of apoptosis in U937 cells.
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Monacolin K Downregulated HMGCR Expression and Reduced Cholesterol
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Generation
236
In order to elucidate the effects of monacolin K on HMGCR production on the
237
transcriptional level, cells were treated with various concentrations of monacolin K
238
for 24 h. The cDNA was amplified by PCR with primers specific for HMGCR or
239
GAPDH (as a control gene), and the results indicated that HMGCR mRNA
240
expression was suppressed in the presence of monacolin K as shown in Figure 2A.
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The suppression of HMGCR mRNA expression correlated to Western Blot analysis.
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Treatment of U937 cells with monacolin K inhibited HMGCR protein expression also
243
in a dose-dependent manner (Figure 2B). After incubation with 20 µM monacolin K,
244
we measured the intracellular total cholesterol content. As shown in Figure 2C,
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monacolin K showed significant inhibitory effect on cholesterol level at
246
approximately 36% (p < 0.01), as compared with control cells.
247 248
Monacolin K Downregulated GLO1 Expression and Increase MG Levels
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To understand the critical roles of GLO1 in apoptosis, expression of GLO1 in U937 14
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cells was monitored following treatment with monacolin K. U937 cells were treated
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with various concentrations of monacolin K for 48 h or 20 µM of monacolin K for
252
various times. The cDNA was amplified by PCR with primers specific for GLO1 or
253
GAPDH (as a control gene). As shown in Figure 3A, monacolin K induced time- and
254
dose-dependent downregulation of GLO1 mRNA levels. Additionally, monacolin K
255
administration resulted in a marked decrease in the GLO1 protein level as compared
256
with control (Figure 3B). To investigate the effect of GLO1 expression blockage on
257
MG level, U937 cells were treated with monacolin K at a concentration of 20 µM for
258
48 h. A significant increase in MG level was observed in comparison to levels
259
produced by untreated cells (Figure 3B). Monacolin K caused an increase in MG
260
accumulation by 2.7-fold as compared with that of control (p < 0.05).
261 262
Effects of Monacolin K on Ras Protein Expression and Downstream Signaling
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Pathways Regulation
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Ras is considered a key enzyme for proliferative, survival, and oncogenic signals. We
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examined the regulatory effect of monacolin K on membrane protein Ras and its
266
downstream signaling proteins, i.e., Raf-1, Akt and ERK. As shown in Figure 4A,
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after incubation with 20 µM monacolin K for 24 h, monacolin K significantly
268
decreased the levels of H-Ras, K-Ras, N-Ras and Raf-1 in cell membrane. In contrast, 15
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the H-Ras, K-Ras, N-Ras and Raf-1 levels in cytosolic fraction were significantly
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higher in the monacolin K treatments. To assess whether monacolin K mediated
271
and/or inhibited the phosphorylation of Akt and ERK. U937 cells were treated with
272
various concentrations of monacolin K for 24 h. Figure 4B showed that monacolin K
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significantly inhibited the activation of Akt and ERK as shown by the decrease in the
274
phosphorylation of Akt and ERK.
275 276
Monacolin K Abrogated NF-κB Signaling by Blocking the Nuclear Translocation
277
of NF-κB (p65)
278
To explore the underlying mechanism of NF-κB inactivation by monacolin K, we
279
analyzed the protein expression of p65 by immunoblotting. As shown in Figure 5A,
280
treatment of the U937 cells with 20 µM monacolin K for 24 h resulted in a significant
281
decreased level of p65 in cell nucleus. In contrast, the p65 levels in cytosolic fraction
282
were significantly increased. Confocal microscope analysis revealed that monacolin K
283
inhibited p65 translocation from cytosol to nucleus (Figure 5B). Therefore, it was
284
possible that the inhibitory effect of monacolin K on the NF-κB translocation was due
285
to inactivation of Akt and ERK pathways, leading to a reduction in GLO1 expression.
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Inhibitory Effects of Several Specific Inhibitors on Expression of Akt, ERK, and 16
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GLO1 and on Cell Viability in U937 Cells
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Several specific inhibitors were used to assess whether a specific signaling pathway
290
was involved in inhibition of monacolin K induced GLO1 and other protein
291
expressions. LY294002, U0126 and JSH-23 were used in the treatment of U937 cells.
292
After treatment, cell extracts were prepared to determine the levels of GLO1. As
293
shown in Figure 6A, three LY294002, U0126 and JSH-23 inhibitors could effectively
294
suppress the GLO1 protein expressions. In addition, Figure 6B showed that Akt and
295
ERK phosphorylation decreased after LY294002 and U0126 treatment for 24 h, and
296
JSH-23 had little effects on the phosphorylation of Akt and ERK (Figure 6B). Next,
297
we administered these inhibitors, to U937 cells to determine whether suppression of
298
the Ras/PI3K/Akt/NF-κB and Ras/Raf/ERK/NF-κB signaling pathways would inhibit
299
cell viability. As shown in Figure 6C, these inhibitors had a strong inhibitory effect on
300
U937 cells growth (p < 0.01). These results confirmed that the apoptotic effect of
301
monacolin K followed the Ras/Akt/NF-κB/GLO-1 and Ras/Raf/ERK/NF-κB/GLO-1
302
signaling pathways.
303 304
Mevalonate Attenuated Monacolin K Induced Apoptosis and Protein Expression
305
in U937 Cells
306
Overexpression of mevalonate, a precursor of cholesterol, has been reported to be 17
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associated with cell survival and proliferation of cancer cells. In the study, the
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treatment of mevalonate with monacolin K fully reversed its induced apoptosis in
309
U937 cells as evidenced by DNA fragmentation and cell viability analysis (p < 0.01)
310
(Figure 7A and Figure 7B). Next, co-incubation of mevalonate with monacolin K for
311
24 h reversed monacolin K triggered loss of Akt and ERK phosphorylation (Figure
312
7C). We further investigated whether mevalonate blocked monacolin K suppresses
313
GLO1 expression. After cells pre-incubated with mevalonate for 1 h and then treated
314
with monacolin K for 48 h, as shown in Figure 7D and Figure 7E, mevalonate
315
prevented the decrease in GLO1 mRNA and protein by monacolin K. These results
316
indicated that the down-regulation of mevalonate pathway is highly associated with
317
monacolin K inhibition on GLO1 as well as the induction of apoptosis in U937 cells.
318 319
Discussion
320
The anti-cancer effect of monacolin K was demonstrated by its growth inhibitory
321
activity on various cancer cell lines including human acute myeloid leukemia,19 breast
322
cancer,20 and pancreatic cancer.21 A structure activity relationship analysis showed the
323
alkyl function group increased lipophilicity of monacolin K leading to a direct
324
diffusion through the cell membrane and affected cell proliferation, survival, and
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motility.4 We hypothesize that monacolin K may induce apoptosis in U937 cells by 18
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inhibiting the mevalonate pathway and impairing Ras localization to the plasma
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membrane. It interrupted downstream Akt and ERK pathways leading to the
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decreased HMGCR and GLO1 proteins expression.
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Tumors, including leukemia, transformed from normal cells alter metabolism.
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Mutations of oncogenes and tumor-suppressor genes are believed to switch the
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glucose metabolism from oxidative phosphorylation to glycolysis in cancer cells.22
332
Previous reports showed that cancer cells tend to use glycolysis pathway to
333
metabolize glucose due to lacking or mutated p53 gene.23 Positron emission
334
tomography (PET) data demonstrated that tumor cells accumulated extraordinary
335
amounts of glucose. Elevated glucose metabolism is seen in activated lymphocytes in
336
the draining lymph nodes of Moloney Murine Sarcoma and Leukemia Virus
337
Complex-challenged mice using a 18F-FDG probe.24
338
MG can be derived non-enzymatically from phosphorylated glycolytic
339
intermediates dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. MG
340
causes DNA fragmentation at physiological concentrations on U937 cells25 and HL-60
341
cell lines.26 Recent studies have shown that MG specifically inhibits mitochondrial
342
respiration in human leukaemic leucocytes leading to a tumoricidal effect.27 We
343
postulate that U937 cancer cell may use glycolysis to get the required energy and
344
increase the GLO1 expression to eliminate excess MG for surviving and proliferation. 19
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345
Detoxification of MG is mainly activated via the glyoxalase pathway present in
346
the cytosol of all mammalian cells that use GSH as a cofactor.28 The apoptotic effects
347
of MG were found in high glucose-treated VL-17A cells29 and brain microvascular
348
endothelial cells.30 It has been shown that GLO1 expression were significantly
349
up-regulated in prostate cancer LNCaP,31 hepatocellular carcinoma,32 and human
350
gastric cancer,33 when compared with adjacent non-tumorous tissue. Recently, a novel
351
mechanism of MG cytotoxicity in prostate cancer cells was proposed.31 The silenced
352
GLO1 in prostate LNCaP and PC3 cancer cells was found to be responsible for the
353
accumulation of excess MG that led to the apoptosis via the cell survival NF-κB
354
signaling pathway.
355
Ras has been reported to be frequently mutated in patients with acute myeloid
356
leukemia (AML). Ras gene mutation leads to the expression of activated Ras proteins,
357
and triggers constitutively overactive signaling inside the cell.34 A previous study
358
found N-Ras mutations (11%) and K-Ras mutations (5%), in AML patients.35 Among
359
the Ras isoforms, only H-Ras is palmitoylated and integrated into lipid rafts.36
360
Activated Ras recruits Raf family proteins to the lipid raft and activate the subsequent
361
ERK pathway. Constitutively activated Raf/MEK/ERK pathway has been
362
characterized in both acute myeloid leukemia (AML) and acute lymphocytic leukemia
363
(ALL).37 Overexpression of activated Raf proteins is closely related to cell cycle 20
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arrest in human promyelocytic leukemia and some hematopoietic cells.38 It is well
365
established that the cross-talk between Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt
366
pathways regulates cell growth and tumorigenesis in many leukemia samples.37 Both
367
pathways may trigger the phosphorylation of downstream targets and regulate cell
368
survival and proliferation.
369
PI3K activity is another predominant growth factor activated pathway in
370
varieties of human tumors including leukemia.39 Accumulated evidence shows that
371
PI3K is heavily involved in the malignant transformation of cells through the
372
activation of the downstream Akt.40 Blalock et al.41 reported that MEK-mediated
373
transformation of certain hematopoietic cells requires the activation of the PI3K
374
pathway. Combined treatment with the Chk1 inhibitor 7-hydroxystaurosporine
375
(UCN-01) and lovastatin was found to synergistically disrupt Ras prenylation and its
376
translocation to membrane leading to the inhibition of the phosphorylation of both
377
ERK 1/2 and Akt and enhanced apoptosis in U937 cells.42 Using immunoprecipitation
378
analysis, Chiu et al.43 showed that Raf-1, Akt and Ras can accumulate in the
379
membrane to increase cell migration/invasion through PI3K/Akt and Raf-1/ERK
380
activation. A redox modulated up-regulation of survival signaling, involving the ERK
381
and PI3K/Akt phosphorylation pathways has been reported in U937 cells.44 Recent
382
studies demonstrated that monacolin K can inhibit the Akt and ERK pathways leading 21
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383
to the decreased Bcl-2, while increased Bax expression in human colon carcinoma
384
HCT-116 cell line.45
385
The expression and activity of the transcription factor, NF-κB, have been shown
386
to be regulated by the Ras/PI3K/AKT and RAS/MAPK pathways.46 The transcription
387
factor NF-κB promotes cell proliferation and prevents apoptosis through its regulation
388
of various gene products. Monacolin K and other natural statins were found to reduce
389
NF-κB DNA-binding activity in TNF-α (tumor necrosis Factor-α)-stimulated human
390
myeloid leukemia KBM-5 cells.47
391
She et al.48 reported that farnesyltransferase inhibitor manumycin A (manumycin)
392
induced nitric oxide production and apoptosis in two myeloid leukemia cell lines
393
(U937 and HL-60). Manumycin reduced the amount of functional Ras localized at the
394
cytoplasmic membrane, thus blocking Raf-1 association with Ras. Manumycin
395
inhibits the phosphorylation of both ERK1/2 and Akt and interferes with the
396
association of PI3K and Ras in HepG2 cells.49 Monacolin K and other statins
397
stimulate cell death, which was accompanied by the collapse of the mitochondrial
398
membrane potential (MMP) in isolated Sprague-Dawley rats hepatocytes.18 This
399
observation indicates that cholesterol levels play a major role in the integrity of cell
400
membranes, and the inactivation of HMGCR activity by monacolin K may be
401
responsible for the apoptotic effect. 22
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Our results reveal that the translocation of both Ras and Raf-1 to membrane and
403
the levels of p-Akt in the PI3K/AKT/mTOR signaling pathway as well as p-ERK in
404
the MAPK signal pathway were down-regulated by monacolin K in the U937 cell line.
405
We postulate that monacolin K, a HMG CoA reductase inhibitor, blocks the
406
biosynthesis of mevalonate and the subsequent farnesyl pyrophosphate through the
407
inactivation of HMG CoA reductase. Lack of farnesyl pyrophosphate results in the
408
inhibition of the isoprenylation of Ras protein, and leading to the repression of
409
accumulation
410
phosphorylation of both Akt and ERK pathways will be thus abrogated. Overall, these
411
results provided evidence of the specific molecular pathways by which monacolin K
412
induces apoptosis: it decreases the activation of GLO1 and lowers cholesterol levels
413
through the inhibition of Ras/Raf/ERK/NF-κB and Ras/Akt/NF-κB pathways (Figure
414
8). These findings provide evidence that it will be worthwhile to investigate the
415
potential of monacolin K for the treatment of leukemia and potentially other types of
416
human cancers.
of
putative
lipid
raft-associated
proteins.
The
subsequent
417 418
Abbreviations Used
419
GLO1,
420
extracellular-signal-regulated kinases; Akt, protein Kinase B; NF-κB, nuclear factor
421
kappa-B ; MG, methylglyoxal; RYR, red yeast rice
glyoxalase
1;
HMGCR,
HMG-CoA
23
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reductase;
ERK,
Journal of Agricultural and Food Chemistry
422 423
Acknowledgments
424
This work was supported by the Ministry of Science and Technology (Taiwan) (NSC
425
103-2313-B-020-008-).
426 427
Reference
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Figure captions
594
Figure 1. Inhibition of cell viability and apoptosis-inducing effects of monacolin K on
595
U937 cells. (A) U937 cells were seeded (1 × 105 cells/well) in a 96-well plate, the cells
596
were treated with variable concentrations of monacolin K for 48 h and cell viability
597
was measured by MTT assay. (B) Nuclear condensation in apoptotic cells detected by
598
DAPI staining. U937 cells were treated with various concentrations of monaclin K for
599
48 h. (C) DNA fragmentation was detected by gel electrophoresis following
600
stimulation with 2.5-20 µM monacolin K for 48 h. (D) Image cytometry analysis
601
shows the percentage of sub-G1 phase in U937 cells treated with monacolin K for 48 h.
602
All data represent means ± SD of triplicates (n = 3). In panel A, **p < 0.01, compared
603
with untreated cells (control groups).
604 605
Figure 2. Monacolin K specifically suppresses HMGCR expression and cholesterol
606
level in U937 cells. (A) Cells were incubated with various concentrations of
607
monacolin K for 24 h, HMGCR and GAPDH mRNA levels were analyzed by RT-PCR.
608
(B) Immunoblots of whole cell lysates were probed with antibodies specific to
609
HMGCR and Actin. (C) U937 cells were treated with 20 µM monacolin K for 48 h,
610
and measured the intracellular total cholesterol content. All data represent means ± SD
611
of triplicates (n = 3). In panel C, **p < 0.01, compared with untreated cells (control 33
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612
groups).
613 614
Figure 3. Down-regulation of GLO1 and increase MG levels by monacolin K in U937
615
cells. U937 cells were incubated with various concentrations of monacolin K for 48 h,
616
or with monacolin K (20 µM) for the indicated periods. (A) GLO1 and GAPDH
617
mRNA levels were analyzed by RT-PCR. (B) Immunoblots of whole cell lysates were
618
probed with antibodies specific to GLO1 and Actin. (C) MG levels were estimated by
619
HPLC as described in Materials and methods. Monacolin K caused an increase in MG
620
accumulation by 2.7-fold as compared with that of control (Figure 3C). All data
621
represent means ± SD of triplicates (n = 3). In panel C, **p < 0.05, compared with
622
untreated cells (control groups).
623 624
Figure 4. Down-regulation of Ras and its downstream signaling molecules by
625
monacolin K in U937 cells. (A) After treatment with monacolin K for 24 h, membrane
626
and cytosol proteins were extracted and used for immunoblotting of Ras (H, K and N)
627
and Raf-1. (B) Cells were lysed and the total protein (30 mg) was submitted to
628
SDS-PAGE and immunodetected with anti-phospho- or anti-(ERK or Akt) antibodies.
629
Tests were performed in triplicate and all experiments repeated three times (n = 3).
630 34
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Figure 5. Monacolin K inhibits the translocation of NF-κB (p65) to the nucleus. (A)
632
U937 cells were incubated for 24 h with indicated concentrations of monacolin K and
633
nuclear extracts were immunoblotted for p65 and the loading control Lamin A. (B)
634
After treated with 24 h, cells were fixed, stained with p65 antibody and Alexa Fluor®
635
594 Goat Anti-Mouse (red), and counterstained with DAPI (blue). Tests were
636
performed in triplicate and all experiments repeated three times (n = 3).
637 638
Figure 6. Down-regulation of Ras downstream signaling molecules and cell viability
639
by various inhibitors in U937 cells. (A) U937 cells were treated with 20 µM various
640
inhibitors for 48 h. Total cell extracts were generated and immunoblotted with
641
antibodies against GLO1. (B) U937 cells were treated with 20 µM of various
642
inhibitors and incubated for 24 h, immunoblots of whole cell lysates were probed with
643
antibodies specific to p-Akt and p-ERK. (C) The cell viabilities of U937 cells treated
644
with 20 µM various inhibitors for 48 h were measured by MTT assay. All data
645
represent means ± SD of triplicates (n = 3). In panel C, **p < 0.01, compared with
646
untreated cells (control groups).
647 648
Figure 7. Recover of monacolin K-induced apoptosis and suppress Ras downstream
649
signaling molecules in U937 cells by adding mevalonate. Cells were preincubated 35
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650
with 1 mM mevalonate for 1 h and then treated with 20 µM monacolin K for 48 h (A
651
and B). (C) Cells were pretreated with 1 mM mevalonate for 1 h and then treated with
652
20 µM monacolin K for 24 h, immunoblots of whole cell lysates were probed with
653
antibodies specific to anti-phospho- or anti-(ERK or Akt). (D) GLO1 and GAPDH
654
mRNA levels were analyzed by RT-PCR. (E) Immunoblots of whole cell lysates were
655
probed with antibodies specific to GLO1 and Actin. All data represent means ± SD of
656
triplicates (n = 3). In panel C, **p < 0.01, compared with untreated cells (control
657
groups).
658 659
Figure 8. Diagram depicting the hypothesis of signaling regulation on GLO1
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expression through both Ras/Raf/ERKNF-κB and Ras/PI3K/Akt/NF-κB pathways
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following monacolin K exposure in U937 cells. Farnesyl-PP, farnesyl pyrophosphate;
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FPTase, farnesyl prenyl transferase; GEF, guanine nucleotide exchange factor; GAP,
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GTPase-activating protein.
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