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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]).

1

ACS Paragon Plus Environment

Wu


1

Abstract

2

Monacolin K, a hydrolytic product of icaritin, is the major active component in the

3

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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20

Introduction

21

Red yeast rice (RYR) is a traditional fermented product of Monascus purpureus and

22

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)

24

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,

26

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

29

The structure of monacolin K is identical to the statin drug, lovastatin. Case

30

reports and early clinical studies suggest a therapeutic potential for statins in acute

31

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

35

post-translational modification of Ras, which can lead to increased levels of

36

farnesylation of Ras by farnesyl-pyrophosphate transferase. Inhibition of mevalonate

37

synthesis leads to a depletion of its downstream intermediates, such as FPP and GGPP,

38

precursors for sterols, dolichols, Coenzyme Q, isoprenoids, and carotenoids.7 FPP and 3



39

GGPP are substrates for the post-translational modification of proteins including the

40

Ras and Rho family GTPases, well-known proto-oncogenes for control cell growth,

41

migration, survival and differentiation in mammalian cells. Statins inactivate Ras

42

translocation and downstream MEK/ERK and PI3K/Akt signalings leading to cancer

43

cell apoptosis by interrupting the biosynthesis of mevalonate.8 Genetic mutations of

44

Ras protein in cancer cells trigger dysregulation of Ras/Raf/MEK/ERK and

45

Ras/PI3K/PTEN/Akt/mTOR pathway signalings, leading to uncontrolled cellular

46

proliferation and decreased sensitivity to agents that ordinarily can trigger apoptosis.

47

It is well established that glyoxalase 1 (GLO1) plays a pivotal role in the growth

48

and survival of cancer cells. GLO1 is a key enzyme for protecting cancer cells from

49

apoptosis, and it works through the elimination of excess methylglyoxal (MG), which

50

is a toxic metabolite of the major energy source, glucose. The increased survival

51

activity of tumors, including higher glycolytic activity and a consequent rise in MG

52

production, requires an increased detoxifying action of GLO1.9 The increase of GLO1

53

expression has been shown to be associated with increased proliferative activity of

54

tumors. In antitumor agent-resistant leukemia cells, GLO1 was frequently

55

overexpressed.10 Immunohistochemical analysis confirmed the increase of GLO1

56

among patients with colon carcinoma,11 or prostate cancer.12 Significantly high

57

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

59

cells.14

60

In contrast with that in normal cells, the cholesterol content of malignant tumors

61

in the body is particularly high. The primary source of cholesterol synthesis in cancer

62

cells is glucose. Cholesterol is currently known to be synthesized from a series of

63

reactions involving the conversion of citrate in the tricarboxylic acid (TCA) cycle to

64

generate acetyl coenzyme A, which then reacts with several enzymes in the

65

mevalonate pathway to yield cholesterol.15 It is well established that HMGCR activity

66

was three-fold higher in mononuclear blood cells from patients with leukemia,

67

compared with cells from healthy subjects.16

68

We here provide a detailed mechanistic investigation of the apoptotic effect of

69

monacolin K and how it is related to the inhibition of GLO1. In the present study, we

70

hypothesize that monacolin K showed its apoptotic activity through the inhibition of

71

HMGCR, inactivation of Ras translocation, down regulation of PI3K/Akt and

72

Raf/ERK1/2 signal pathways leading to suppression of GLO1 and HMGCR in U937

73

cells.

74 75

Materials and methods

76

Chemicals and Antibodies 5



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77

Monacolin K, perchloric acid, 2-methylquinoxaline (2-MQ), o-phenylenediamine,

78

3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyltetrazolium

79

sulphoxide (DMSO) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from

80

Sigma-Aldrich (St. Louis, MO). Cell culture media were purchased from Invitrogen

81

(Carlsbad, CA). Antibodies against phospho-p44/42 MAPK (Thr202/Tyr204),

82

phospho-Akt (Ser473), ERK1/2, Akt1/PKBα and actin, were obtained from Millipore

83

(Billerica, MA). Antibodies against GLO-1, NF-κB (p65), H-Ras, Raf-1 and lamin A

84

were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against

85

K-Ras and N-Ras were obtained from GeneTex (San Antonio, TX). Alexa Fluor® 594

86

Goat Anti-Mouse IgG (H+L) was purchased from Invitrogen (Carlsbad, CA). U0126

87

(MEK1/2 inhibitor), LY294002 (PI3K inhibitor), JSH-23 (NF-κB inhibitor), IgG

88

HRP-conjugated goat anti-mouse and IgG HRP-conjugated goat anti-rabbit were

89

obtained from Chemicon (Temecula, CA).

bromide

(MTT),

dimethyl

90 91

Cell Culture

92

U937 cells were purchased from the American Type Culture Collection (Rockville,

93

MD). The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum

94

(GibcoBRL; Grand Island, NY), 5 mM L-glutamine (GibcoBRL) and 1%

95

penicillin/streptomycin (10000 units of penicillin/mL and 10 mg/mL streptomycin) at 6


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96

37 °C in a humidified 5% CO2 incubator.

97 98

Cell Viability Assay

99

Cell viability was assessed by MTT assay.17 Briefly, cells at 1×105/mL were treated

100

with various concentrations of monacolin K in 96-well plates for 48 h, followed by

101

treatment with MTT for 4 h at 37 °C. The medium was removed and DMSO was

102

added to dissolve the blue formazan residue and color intensity was measured using

103

an ELISA reader at 570 nm.

104 105

Morphological Assay

106

After treatment with various concentrations of monacolin K for 48 h, cells were

107

harvested and fixed with 4% paraformaldehyde in PBS for 20 min at room

108

temperature, and stained with 2.5 mg/mL DAPI solution for 30 min at room

109

temperature. The cells were washed two more times with PBS and analyzed via a

110

confocal microscope (CARV II Confocal Imager; BD Biosciences).

111 112

DNA Fragmentation Assay

113

After treatment with various concentrations of monacolin K for 48 h, washed with

114

ice-cold PBS, the cells were resuspended in 180 µL DNA lysis buffer (0.5% 7



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115

lauroylsarcosine, 10 mM EDTA, 0.5 mg/mL proteinase K and 50 mM Tris-HCl at pH

116

8.0) overnight at 56 °C, and treated with RNaseA (0.5 µg/mL) for 1 h at 37 °C. Total

117

DNA

118

phenol/chloroform/isoamyl alcohol (Sigma-Aldrich, St. Louis, MO). 10 µL of DNA

119

were analyzed on 1.8% agarose gel.

was

extracted

from

the

cells

with

an

equal

volume

of

120 121

Image Cytometry Analysis

122

Cell sub-G1 phase was analyzed by the DNA content using DAPI staining method.

123

After treatment with various concentrations of monacolin K for 48 h, U937 cells were

124

centrifuged at 1,500 rpm for 5 min, and the cell pellets were fixed in 70% ice-cold

125

ethanol overnight at 4 °C. Fixed cells were washed with PBS, incubated with 0.5 mL

126

hypotonic buffer containing 0.5% Triton X-100 and 0.5 mg/mL RNase A for 30 min

127

at 37 °C, and then stained with 50 µg/mL DAPI, and the sub-G1 phase content was

128

determined on a NucleoCounter NC-3000 cytometer (ChemoMetec, Allerod,

129

Denmark).

130 131

RNA Isolation and Reverse Transcription-PCR Amplification (RT-PCR)

132

Total RNA was extracted from U937 cells using a commercially available RNA

133

extraction kit (Favorgen, Pingtung, Taiwan) and following the manufacturer’s 8


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134

instruction. First-stranded cDNA was synthesized using 1 µg total RNA by RT-PCR

135

kit (Roche, Indianapolis). The relative expression of GLO1 and HMGCR was

136

analyzed using RT-PCR with GAPDH as an internal control. The primers used were

137

GLO1,

138

5’-CTACATTAAGGTTGCCATTTTGTTAG-3’, yielding a product of length 564 bp;

139

HMGCR,

140

5’-CAAGCCTAGAGACATAATCATC-3’, yielding a product of length 252 bp;

141

GAPDH,

142

5’-CAAGCCTAGAGACATAATCATC-3’, yielding a product of length 248 bp. A 10

143

µL volume reaction for PCR included 2 µL of cDNA, 1 µL each of sense and

144

antisense primers and 5 µL of PCR master mix. The conditions were: 94 °C for 5 min,

145

94 °C for 40 sec, 60 °C for 35 sec (GLO1), 58 °C for 30 sec (HMGCR) and 64 °C for

146

30 sec (GAPDH), 72 °C for 40 sec, and 72 °C for 7 min, for 26 cycles. The PCR

147

products were separated by electrophoresis on 1.5% agarose gels and the results were

148

analyzed using a Kodak Gel logic imaging system (Hyland Scientific, Stanwood,

149

WA).

sense

5’-ATGGCAGAACCGCAGCCCCCGT-3’

sense

sense

5’-TACCATGTCAGGGGTACGTC-3’

5’-TACCATGTCAGGGGTACGTC-3’

and

and

and

antisense

antisense

antisense

150 151

Western Blot Analysis

152

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

154

inhibitor cocktail at pH 7.4), followed by incubation at 4 °C for 1 h, centrifugation,

155

and collection of cell lysate. Subcellular Protein Fractionation Kit was used to

156

determine the levels of protein expression in cytosol, nucleus, and membrane. Extract

157

preparation followed instruction of the manufacturer’s published protocol (Thermo,

158

Rockford, IL). Protein concentrations were measured with a Bio-Rad protein assay

159

reagent (Bio-Rad Laboratories, Munich, Germany). Equal amounts of lysate protein

160

(30 µg/lane) were subjected to sodium dodecyl sulfate polyacrylamide gel

161

electrophoresis

162

electrotransferred to a polyvinylidene difluoride (PVDF) membranes (Millipore,

163

Billerica, MA), blocked at 37 °C for 1 h with 1% BSA in TBST (20 mM/L Tris-base;

164

125 mM/L NaCl; 0.2% Tween 20; 0.1% sodium azide), and then immunoblotted with

165

primary antibodies overnight at 4 °C, and then washed with TBST solution (0.05%

166

Tween 20 in TBS). After washing six times with TBST buffer, the blot was washed,

167

exposed to horseradish peroxidase conjugated secondary antibodies for 1 h,

168

immunoreactive proteins were visualized with an enhanced chemiluminescence (ECL)

169

detection system (GE Healthcare, Little Chalfont, UK).

(SDS-PAGE).

After

electrophoresis,

170 171

Total Cholesterol Quantitation 10


the

proteins

were

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The total cholesterol concentration was measured using Cholesterol/Cholesterol Ester

173

Quantitation Kit (Calbiochem, Novabiochem, Boston, MA). After treatment with

174

monacolin K for 48 h, cells were washed with cold PBS and centrifuged. The pellet

175

was re-suspended in 0.2 mL of chloroform:isopropanol:NP-40, homogenize for 3 min

176

and cleared by centrifugation at 14,000 rpm for 10 min. Supernatants were decanted

177

and air-dry at 50 °C, and vacuum-dry for 30 min to remove residual solvents. The

178

dried lipids were dissolved in 0.2 mL of the reaction buffer and the absorbance was

179

measured at 570 nm for total cholesterol detection.

180 181

Methylglyoxal (MG) Measurement

182

MG was measured by an o-Phenylenediamine method.18 MG was derivatized with

183

o-phenylenediamine to form the quinoxaline product 2-MQ, which is very specific for

184

MG. Briefly, the supernatant of the cell was incubated with 100 mM

185

o-phenylenediamine for 3 h at room temperature. The quantity of the quinoxaline

186

derivative of MG (2-MQ) was measured using a Hitachi L-6200 HPLC system

187

(Hitachi, Ltd., Mississauga, Ontario, Canada). The mobile phase was generally 68

188

vol% of 10 mM KH2PO4 (pH 2.5) and 32 vol% of HPLC grade acetonitrile. The

189

analysis conditions were as follows: detector wavelength, 315 nm; mobile phase flow

190

rate, 1.0 mL/min; and column temperature, 20 °C. The whole series of samples in a 11



191

single experiment was run in duplicate.

192 193

Immunofluorescence Microscopy

194

To detect the localization of NF-κB in monacolin K treated U937 cells, the U937 cells

195

were first seeded in a 12 well dish. The cells were fixed with 4% paraformaldehyde

196

for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 5 min at

197

room temperature. After specimens were blocked with 5% BSA for 1 h at room

198

temperature, and further incubated with NF-κB (p65) antibody for 2 h. After three

199

washes in TPBS, specimens were then stained with Alexa Fluor® 594 Goat

200

Anti-Mouse IgG for 1 h at room temperature. After being washed three times with

201

TPBS, cells were stained with 0.5 mg/mL of DAPI for 10 min to counter stain the

202

nucleus, and then washed three times for 5 min with TPBS. Images were collected by

203

confocal microscope.

204 205

Statistical Analysis

206

The data were analyzed using the SAS program (SAS Institute, Cary, NC). The

207

significance of the difference among mean values was determined by one-way

208

analysis of variance followed by the t-test; (*) p < 0.05 and (**) p < 0.01 were

209

considered statistically significant. Values are the mean ± SD of three independent

210

experiments. 12


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211 212

Results

213

Monacolin K Induced Apoptosis in U937 Cells

214

MTT assays were performed to evaluate the potential cytotoxic effects of monacolin

215

K. As shown in Figure 1A, U937 cells were incubated with increased concentrations

216

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

220

monacolin K treatment, the cells were stained with DAPI and analyzed by confocal

221

microscope. As shown in Figure 1B, the nuclei of U937 cells exposed to monacolin K

222

exhibited nuclear condensation and fragmented chromatin of typical apoptosis in a

223

dose-dependent manner. Apoptosis was also detected by DNA fragmentation assay.

224

DNA fragmentation was significantly increased after treatment with monacolin K in a

225

dose-dependent manner (Figure 1C). Furthermore, the degree of apoptosis was

226

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

230

fragmented DNA (sub-G1) to 68%. Taken together, these findings indicated that 13



231

exposure to monacolin K resulted in the inhibition of cell proliferation and a striking

232

induction of apoptosis in U937 cells.

233 234

Monacolin K Downregulated HMGCR Expression and Reduced Cholesterol

235

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.

241

The suppression of HMGCR mRNA expression correlated to Western Blot analysis.

242

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,

245

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

249

To understand the critical roles of GLO1 in apoptosis, expression of GLO1 in U937 14


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250

cells was monitored following treatment with monacolin K. U937 cells were treated

251

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

263

Pathways Regulation

264

Ras is considered a key enzyme for proliferative, survival, and oncogenic signals. We

265

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,

267

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



269

the H-Ras, K-Ras, N-Ras and Raf-1 levels in cytosolic fraction were significantly

270

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

273

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.

286 287

Inhibitory Effects of Several Specific Inhibitors on Expression of Akt, ERK, and 16


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288

GLO1 and on Cell Viability in U937 Cells

289

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



307

associated with cell survival and proliferation of cancer cells. In the study, the

308

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

325

motility.4 We hypothesize that monacolin K may induce apoptosis in U937 cells by 18


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326

inhibiting the mevalonate pathway and impairing Ras localization to the plasma

327

membrane. It interrupted downstream Akt and ERK pathways leading to the

328

decreased HMGCR and GLO1 proteins expression.

329

Tumors, including leukemia, transformed from normal cells alter metabolism.

330

Mutations of oncogenes and tumor-suppressor genes are believed to switch the

331

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



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


Page 20 of 45

Page 21 of 45


364

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



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


Page 22 of 45

Page 23 of 45


402

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


reductase;

ERK,


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

428

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429

Constituents of red yeast rice, a traditional Chinese food and medicine. J. Agric. Food

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Chem. 2000, 48, 5220-5225.

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Langabeer, S. E.; Kottaridis, P. D.; Moorman, A. V.; Burnett, A. K.; Linch, D. C. RAS

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582 583 584 585 586 587 588 589 590 591 592 32


Page 32 of 45

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593

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



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


Page 34 of 45

Page 35 of 45


631

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



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

660

expression through both Ras/Raf/ERKNF-κB and Ras/PI3K/Akt/NF-κB pathways

661

following monacolin K exposure in U937 cells. Farnesyl-PP, farnesyl pyrophosphate;

662

FPTase, farnesyl prenyl transferase; GEF, guanine nucleotide exchange factor; GAP,

663

GTPase-activating protein.

36


Page 36 of 45

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Figure graphics Figure 1

37



Figure 2

38


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Figure 3

39



Figure 4

40


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Figure 5

41



Figure 6

42


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Figure 7

43



Figure 8

44


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Table of Contents Graphic:

45


Akt Pathways by

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