Hispidulin potently inhibits human glioblastoma multiforme cells through activation of AMPK

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Hispidulin potently inhibits human glioblastoma multiforme cells through activation of AMPK

Journal: Journal of Agricultural and Food Chemistry Manuscript ID: jf-2010-019533.R2

Manuscript Type: Article Date Submitted by the

Author: 23-Jul-2010

Complete List of Authors: Way, Tzong-Der; china medical university, Department of Biological Science and Technology

Lin, Ying-Chao; Institute of Biochemistry, College of Life Science, National Chung Hsing University, Taichung, Taiwan

Hung, Chao-Ming; Department of General Surgery, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan

Tsai, Jia-Chun; Department of Biological Science and Technology Lee, Jang-Chang; Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy

Chen, Yi-Lin; National Ilan University, Biotechnology Kao, JungYie; Instititu of Biochemistry

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1

Hispidulin potently inhibits human glioblastoma multiforme cells through 2

activation of AMPK 3

Ying-Chao Lina,b,1, Chao-Ming Hungc,1, Jia-Chun Tsaid, Jang-Chang Leee,f, Yi-Lin 4

Sophia Cheng, Chyou-Wei Weih, Jung-Yie Kaoa**and Tzong-Der Wayd* 5

a

Institute of Biochemistry, College of Life Science, National Chung Hsing University, 6

Taichung, Taiwan 7

b

Division of Neurosurgery, Buddhist Tzu Chi General Hospital, Taichung Branch 8

c

Department of General Surgery, E-Da Hospital, I-Shou University, Kaohsiung, 9

Taiwan 10

d

Department of Biological Science and Technology, College of Life Sciences, China 11

Medical University and Hospital, Taichung, Taiwan 12

e

School of Pharmacy, College of Pharmacy, China Medical University, Taichung, 13

Taiwan 14

f

Graduate institute of Pharmaceutical Chemistry, China Medical University, Taichung, 15

Taiwan 16

g

Graduate Institute of Biotechnology, National Ilan University Ilan, Taiwan

17

h

Institute of Biomedical Nutrition, College of Medicines and Nursings, Hungkuang

18

University, Taichung, Taiwan

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1

Contributed equally to this work 20

21

Running title: Hispidulin activated AMPK in human brain glioblastoma multiforms 22 cells 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

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39

Tzong-Der Way, Ph.D. 40

Department of Biological Science and Technology, College of Life Sciences, China 41

Medical University, Taichung, Taiwan 42

No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 43 Tel: +886-4-2205-3366 ext: 5209 44 Fax: +886-4-2203-1075 45 E-mail: [email protected] 46 **Co-corresponding author: 47 Jung-Yie Kao, Ph.D. 48

Institute of Biochemistry, College of Life Science, National Chung Hsing University, 49 Taichung, Taiwan 50 Tel: (886)-4-22840468 ext: 222 51 Fax: (886)-4-2285-3487 52 E-mail: [email protected] 53 54 55

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57

Glioblastoma multiforme (GBM) is the most common and lethal type of primary 58

brain tumor. Despite recent therapeutic advances in other cancers, the treatment of 59

GBM remains ineffective and essentially palliative. The current focus lies in the 60

finding of components that activate the AMP-activated protein kinase (AMPK), one 61

key enzyme thought to be activated during the caloric restriction (CR). In the present 62

study, we found that treatment of hispidulin, a flavone isolated from Saussurea 63

involucrate Kar. et Kir., resulted in dose-dependent inhibition of GBM cellular 64

proliferation. Interestingly, we show that hispidulin activated AMPK in GBM cells. 65

The activation of AMPK suppressed downstream substrates, such as the mammalian 66

target of rapamycin (mTOR) and eukaryotic initiation factor 4E-binding protein-1 67

(4E-BP1) and a general decrease in mRNA translation. Moreover, hispidulin activated 68

AMPK decreases the activity and/or expression of lipogenic enzymes, such as the 69

fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Furthermore, 70

hispidulin blocked the progression of the cell cycle at G1 phase and induced apoptosis 71

by inducing p53 expression and further up-regulating p21 expression in GBM cells. 72

Based on these results, we demonstrated that hispidulin has the potential to be a 73

chemoprevention and therapeutic agent against human GBM. 74

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Chinese Medicine; Glioblastoma multiforme 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

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95

Glioblastoma multiforme (GBM) is the most common and aggressive class of 96

malignant brain tumors. Standard therapy for GBM consists of surgical resection, 97

radiotherapy, and chemotherapy (1). Compared to the advances in the treatment of 98

other types of tumors, the overall prognosis for GBM patients with this disease 99

remains dismal, the average time for recurrence of the tumor is only 6.9 months, and 100

the 5-year survival rate for GBM patients is still less than 5% (2). Therefore, new 101

chemotherapeutic agents on the treatment of GBM are still an energetic topic. 102

Caloric restriction (CR) is a 20-40% lowering of caloric intake, known to retard 103

aging processes and to lengthen life in many organisms (3). It has been suggested that 104

both dietary restriction and decreased nutrient-sensing pathway activity can lower the 105

incidence of age-related loss of function and disease by reducing the levels of DNA 106

damage and mutations that accumulate with age (4). Cancer is an age-related disease 107

in organisms with renewable tissues, as the incidence of most cancers increase with 108

age following an accumulation of mutations. Moderate CR lowered the incidence of 109

cancer. 110

The AMP-activated protein kinase (AMPK) is a critical monitor of cellular 111

energy status, thought to be activated during CR. AMPK is a heterotrimeric 112

serine/threonine protein kinase that is composed of a catalytic α-subunit, and 113

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through phosphorylation in the activation loop of the α-subunit (5). AMPK controls 115

processes relative to tumor development, including cell growth, survival, cell cycle 116

progression, and protein synthesis. The AMPK pathway is linked to tumor growth and 117

proliferation through regulation of the mammalian target of rapamycin (mTOR) 118

pathway. AMPK activation inhibits the growth of a broad spectrum of cancers via 119

mTOR, reduces the proliferation of certain tumor cells and can cooperate with other 120

agents to induce apoptosis. The best-understood roles of mTOR in mammalian cells 121

are related to the control of mRNA translation by the 4E-BP1 (6). In the 122

hypophosphorylation form, 4E-BP1 by mTOR ultimately results in the initiation of 123

translation of certain mRNAs, including those that are needed for cell cycle 124

progression and are involved in cell cycle regulation (7). 125

Defects in fatty acid synthesis or processing contribute to the development of 126

many diseases, including insulin resistance, type 2 diabetes, obesity, non-alcoholic 127

fatty liver disease, and cancer (8). Fatty acid synthase (FASN), a key enzyme for 128

lipogenesis, provides the best opportunity for therapeutic applications because of its 129

tissue distribution and unusual enzymatic activity. FASN is downregulated in most 130

normal human tissues because of the fat in our diet, with the exception of lactating 131

breasts and cycling endometrium. In contrast, FASN is often highly expressed in 132

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stomach, lung, oral tongue, oral cavity, head and neck, thyroid and endometrium, and 134

also in mesothelioma, nephroblastoma, retinoblastoma, soft tissue sarcomas, Paget’s 135

disease of the vulva, cutaneous melanocytic neoplasms including melanoma, and 136

hepatocellular carcinoma (9). This differential tissue distribution makes FASN an 137

attractive target for cancer cells. Moreover, acetyl-CoA carboxylases (ACC) are 138

rate-limiting enzymes in de novo fatty acid synthesis, catalyzing ATP-dependent 139

carboxylation of acetyl-CoA to form malonyl-CoA. Recently, ACC up-regulation has 140

been recognized in multiple human cancers, not only in advanced breast carcinomas 141

but also in preneoplastic lesions associated with increased risk for the development of 142

infiltrating breast cancer (10). Therefore, FASN and ACC might be effective as potent 143

targets for cancer intervention, and the inhibitors developed for the treatment of 144

metabolic diseases would be potential therapeutic agents for cancer therapy. 145

Hispidulin (4’,5,7-trihydroxy-6-methoxyflavone) is a naturally occurring 146

flavone commonly found in Saussurea involucrata Kar. et Kir., a rare traditional 147

Chinese medicinal herb (11). Several in vitro studies have demonstrated its potent 148

antioxidative, antifungal, anti-inflammatory, antimutagenic, and antineoplastic 149

properties (12-14). Recently, hispidulin is identified as a potent ligand of the central 150

human BZD receptor in vitro (15). It also acts as a partial positive allosteric modulator 151

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152

anticonvulsant activity in the central nervous system (CNS) (16). Based on more 153

observations it has been found that hispidulin acts as a potential modulator of CNS 154

activity, prompted us to investigate its antineoplastic activity against GBM. In this 155

work, we examined the effects of hispidulin on GBM cells. We present here, for the 156

first time that AMPK is activated by hispidulin in GBM cells. The activation of 157

AMPK suppresses protein synthesis, lipogenesis, and cell cycle progression in GBM 158

cells. Our study suggests that hispidulin may be useful as a GBM chemopreventive or 159 therapeutic agent. 160 161 162 163 164 165 166 167 168 169 170

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171

Chemicals. MTT, compound c, PI and antibodies for β-Actin were purchased from 172

Sigma (St. Louis, MO). Hispidulin was purchased from Tocris Bioscience (Bristol, 173

UK). Antibodies for FASN, phospho-ACC (Ser 79), phospho-mTOR (Ser2448), 174

pohspho-4E-BP1 (Thr 37/46), phosphor-AMPK (Thr 172), PARP, p21 and p53 were 175

purchased from Cell Signaling Technology (Beverly, MA). Antibodies for mouse and 176

rabbit conjugated with horseradish persdish peroxidase were purchased from 177

Chemicon (Temecula, CA). Immobilon Western Chemiluminescent HRP Substrate 178

was from Milliore Corporation (Billerica, MA). 179

Cell culture. GBM8401 and GBM8901 human GBM cells were obtained from 180

Bioresources Collection and Reasearch Center (Hsin Chu, Taiwan). These cells were 181

cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and 1% 182

penicillin-streptomycin, and were grown at 37°C in a humidified atmosphere of 5% 183

CO2.

184

Cell Proliferation Assays. As described previously (17), the effects of hispidulin on 185

cell proliferation were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 186

tetrazolium bromide) method. 187

Western blot. Cells (2 × 106) were seeded onto a 100-mm tissue culture dish 188

containing 10% FBS DMEM/F12 and cultured for 24 h. Then cells were incubated in 189

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After treatment, cells were placed on ice, washed with cold PBS, and lysed in lysis 191

buffer. Western Blot was done as described previously (18). The intensityof the bands 192

was scanned and quantified with NIH image software. 193

Cell cycle analysis. Cells (5×105) were cultured in 60-mm cell culture dish and 194

incubated for 24 h. Then cells were harvested in 15 mL tube, washed with PBS, 195

resuspended in PBS, and fixed in 2 mL of iced 100 % ethanol at -20 ℃ overnight. 196

Cell pellets were collected by centrifugation, resuspended in 0.5 mL of hypotonic 197

buffer (0.5 % Triton X-100 in PBS and 0.5 µg/ml RNase), and incubated at RT for 198

30min. Then 1 mL of propidium iodide solution (50 µg/ mL) was added, and the 199

mixture was allowed to stand on ice for 30 min. Fluorescence emitted from the 200

propidium iodide-DNA complex was quantitated after excitation of the fluorescent 201

dye by FAC-Scan cytometry (BD Biosciences, San Jose, CA). 202

Short hairpin RNA. RNAi reagents were obtained from the National RNAi Core 203

Facility located at the Institute of Molecular Biology/Genomic Research Center, 204

Academia Sinica, supported by the National Research Program for Genomic 205

Medicine Grants of NSC (NSC 97-3112-B-001-016). Short hairpin RNAs (shRNAs) 206

were designed to target specific sequences of human AMPK (Clone ID: 207

TRCN000000861; Target sequence: 5’-GTT GCC TAC CAT CTC ATA ATA-3’). One 208

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20 nM AMPK shRNAs were transfected into cells by lipofectamine 2000 (Invitrogen, 210

Carlsbad, CA). Cells were incubated for an additional 24h before addition of 211

hispidulin as previously described. The effects of hispidulin on cell proliferation were 212

examined by MTT method. 213

Statistical analysis. All valves were expressed as mean ±SD. Each value is the mean 214

of at least three separate experiments in each groups, Student’s t-test was used for 215

statistical comparison. Asterisks indicate that the values are significantly different 216

from the control (*, P < 0.05; **, P < 0.01). 217 218 219 220 221 222 223 224 225 226 227

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228

Hispidulin exhibits potent antiproliferative activity against human GBM cells. To 229

investigate the bioactivity of hispidulin (Figure 1A) in human GBM cells, we treated 230

GBM8401 and GBM8901 cells with different concentrations of hispidulin at 37℃ for 231

48 h, and assessed cell proliferation by MTT assay. The results showed that 232

GBM8401 (Figure 1B) and GBM8901 (Figure 1C) cells were inhibited by hispidulin 233

in a dose-dependent manner. The IC50 values of hispidulin against GBM8401 and

234

GBM8901 cells were 60 µM and 40 µM, respectively. 235

Hispidulin suppresses protein synthesis by activating AMPK to inbibit the 236

mTOR pathway. The current focus lies in the finding of components that activate 237

AMPK. We next identify whether the antiproliferative effects of hispidulin is by 238

activating AMPK in human GBM cells. GBM8401 and GBM8901 cells were treated 239

with 60 µM and 40 µM hispidulin at 37℃ for different durations, respectively. 240

Western blot analysis indicated that hispidulin stimulates AMPK phosphorylation in a 241

time-dependent manner (Figure 2A). Those results showed that hispidulin 242

up-regulated AMPK activity and suppressed cell proliferation in human GBM cells. 243

mTOR/4E-BP1 pathway controls the protein translation/synthesis in various types of 244

cells. 4E-BP1 is phosphorylated by mTOR upon growth factor stimulation, and then 245

the cells undergo cell cycle progression and proliferation (7). To determine whether 246

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pathway, phospho-mTOR and 4E-BP1 were detected by western blotting. The 248

phosphorylation of mTOR (Figure 2B) and 4E-BP1 (Figure 2C) were decreased at 249

12h in GBM8401 and GBM8901 cells. Next, we added the compound c, an AMPK 250

inhibitor, in the absence or presence of hispidulin. The AMPK activity was suppressed 251

by compound c in the presence of hispidulin and the mTOR activity was recovered in 252

GBM8401 (Figure 3A) and GBM8901 (Figure 3B) cells. We hypothesized that 253

hispidulin, by up-regulating AMPK activity, would inhibit mTOR activation and 254

downstream events in human GBM cells. 255

Hispidulin decreases lipid synthesis by decreasing FASN expression and 256

inhibiting ACC activity. The activity of FASN and ACC were known to be 257

negatively regulated by AMPK (19). In the present study, the FASN protein level was 258

decreased (Figure 4A) and ACC was phorsphorylated (Figure 4B) in a time 259

dependent fashion when GBM8401 and GBM8901 cells were treated with 60 µM and 260

40 µM hispidulin, respectively. To further study the effect of AMPK in regulating the 261

activity of fatty acid synthesis enzymes. We added the compound c, in the absence or 262

presence of hispidulin. After the treatment of hispidulin, the protein levels of FASN 263

were decreased and phospho-ACC was increased. However, the activities of enzymes 264

of fatty acid synthesis were restored in the presence of compound c in GBM8401 265

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hispidulin inhibits the activity of fatty acid synthesis enzymes through the activation 267

of the AMPK pathway. 268

Hispidulin induces growth arrest and apoptosis. We examined effects of hispidulin 269

on the cell cycle to clarify the mechanism of hispidulin-induced inhibition of 270

proliferation. Hispidulin caused the accumulation of the G0/G1 phase followed by an 271

increase in hypodiploid cells as indicated by apoptotic cells in GBM8401 (Figure 6A) 272

and GBM8901 (Figure 6B) cells. Moreover, we examined the expression of G1 273

related cell cycle control proteins and apoptosis related proteins on western blot 274

analysis. GBM8401 and GBM8901 cells were treated with 60 µM and 40 µM 275

hispidulin for indicated durations and used 50 µg of whole-cell extracts on Western 276

blot analyses. After 12 hours of hispidulin treatment, we found increased levels of p53 277

and p21 in GBM8401 and GBM8901 cells (Figure 6C). Moreover, hispidulin showed 278

a clear apoptosis within 12 hours, showing cleavages for PARP in Western blot 279

analyses (Figure 6D). To further determine whether hispidulin induced inhibition of 280

proliferation by activating AMPK, we added the compound c, an AMPK inhibitor, in 281

the absence or presence of hispidulin. The inhibition of proliferation by hispidulin 282

was resumed in the presence of compound c in GBM8401 (Figure 7A) and 283

GBM8901 (Figure 7B) cells. In addition, we also treated cells with AMPK shRNA to 284

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proliferation by hispidulin in GBM8401 (Figure 7A) and GBM8901 (Figure 7B) 286

cells. We hypothesized that hispidulin, by up-regulating AMPK activity, would inhibit 287 GBM cells proliferation. 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303

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304

The previous study illustrates that flavone hispidulin probably acts as a partial 305

positive allosteric modulator at GABAA receptors, penetrates the BBB and possesses 306

anticonvulsant activity in the CNS (15, 16). These observations encouraged us to 307

investigate its antineoplastic activity against GBM. In the field of food and nutrition, 308

the current focus lies in the finding of components that activate AMPK. Here, we 309

show that hispidulin activated AMPK in GBM cells. The activation of AMPK 310

suppressed protein synthesis, lipogenesis, and cell cycle progression. Targeting 311

AMPK signaling by hispidulin may have potential therapeutic implications for GBM 312

and age-related diseases. 313

mTOR, a serine-threonine kinase, plays a key role in the regulation of cellular 314

growth. The mTOR pathway is aberrantly activated in many human cancers. The role 315

of mTOR in tumor acts as a sensor for energy, growth factors and nutrients, all of 316

which are required for protein translation. Thus, approaches to block the pathway are 317

being actively pursued in many laboratories and pharmaceutical companies. 318

Activation of AMPK results in a decrease of mTOR signaling. The AMPK signal 319

system contains some tumor suppressor genes including LKB1, TSC1, TSC2 and p53, 320

and suppresses tumor growth by inhibiting the activity of various proto-oncogene 321

such as PI3K, Akt and ERK (20). Both TSC1 (also named hamartin) and TSC2 (also 322

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Activation of AMPK induces activation of the TSC2-TSC1 complex to inhibit mTOR 324

(21). The Eukaryotic translation initiation factor 4E-BP1 is the downstream effector 325

of mTOR. Through this effector mTOR controls the protein translation (22). Data 326

presented here show that the inhibition of protein translation via the AMPK-mTOR 327

pathway by hispidulin in GBM cells is effective. 328

AMPK acts as a fuel gauge by monitoring cellular energy levels (23). FASN and 329

ACC are key enzymes for lipogenesis. AMPK specifically regulated both the 330

phosphorylation and dephosphorylation cycles of ACC and the expression levels of 331

FASN. Acutely activated AMPK phosphorylates and inhibits ACC. Chronically 332

activated AMPK decreases the expression of SREBP1c, thus suppressing the 333

synthesis of ACC, FASN and other lipogenic enzymes (24). A recent study identified 334

that pharmacologically inducing a ‘low-energy status’ in tumour cells can result in 335

AMPK-induced ACC phosphorylation, FASN downregulation and marked decrease 336

of endogenous lipogenesis. Cancer cells, thus, stopped proliferating and lost their 337

invasive and tumorigenic properties in vitro and in vivo (25). In this study, we show 338

that AMPK is activated by hispidulin, and is required for hispidulin suppression of 339

lipogenesis. From a clinical perspective, these findings justify further work exploring 340

the ability of ‘low-energy mimickers’ to therapeutically manage lipogenic carcinomas. 341

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view AMPK as a survival factor for cancer cells. AMPK raises energy production via 343

the activation of glucose uptake, glycolysis, and fatty acid oxidation in response to 344

ATP-depleting stresses (27). Recent study shows that AMPK is critical for cancer cell 345

adaptation in response to hypoxia or glucose deprivation (28). Solid tumors that 346

outgrow the existing vasculature are continuously exposed to a microenvironment in 347

which the supply of both oxygen and nutrition are quite limited. In accordance with 348

the aforementioned reports and the data documented herein, it seems reasonable to 349

conclude that the inhibition of AMPK in cancer cells may prove useful as an approach 350

for the increased induction of apoptosis in tumor cells after hispidulin treatment. 351

However, some have concluded that AMPK activation may be employed as a 352

component of an anticancer therapy (29). The logic of this approach is predicated on 353

recent observations that AMPK also strongly suppresses cell proliferation. This effect 354

is mediated, in part, by several tumor suppressor proteins associated with the AMPK 355

signaling network, including LKB1 and the tuberous sclerosis complex (TSC2). Jones 356

et al. recently reported that the activation of AMPK induces p53-Ser15 357

phosphorylation in response to glucose deprivation, resulting in replicative senescence 358

(30). The ability of AMPK to promote senescence or to inhibit cell proliferation in 359

response to energy starvation has been interpreted as a check point that couples 360

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activation of AMPK might promote the conservation of the remaining energy to 362

support the survival and physiological functions of the cell during cell cycle arrest. 363

Our results indicated that hispidulin inhibited the proliferation of GBM cells via the 364

activation of AMPK. Hispidulin treatment inhibited the progression of cell cycle in 365

the G1 phase. Hispidulin increased the expression level of p53 and subsequently 366

enhanced the expression level of p21 resulting in cell cycle arrest in GBM cells. It is 367

likely that induction of p21 promotes growth arrest and exerts a protective effect after 368

AMPK activation. 369

In conclusion, AMPK is activated by hispidulin in GBM cells. When this 370

occurs, a key enzyme involved in protein synthesis, mTOR, is inhibited. In addition, 371

the activity and/or expression of lipogenic enzymes, such as FASN and ACC are 372

decreased. Interestingly, hispidulin blocked the progression of cell cycle at G1 phase 373

and induced apoptosis in GBM cells (Figure 8). Taken together, our study suggests 374

that hispidulin may be useful as a GBM chemopreventive agent. Nevertheless, 375

additional studies are required to evaluate the efficacy of hispidulin in suitable 376

experimental animal systems. 377

378

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380

ACC, Acetyl-CoA carboxylase; AMPK, AMP-activated preotin kinase; CR, Caloric 381

restriction; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, Dimethyl 382

sulfoxide; 4E-BP1, Eeukaryotic initiation factor 4E-binding protein-1; FASN, Fatty 383

acid synthase; FBS, Fetal bovine serum; mTOR, Mmammalian target of rapamycin; 384

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide. 385

386

387

388

389

ACKNOWLEDGMENT This study was supported by the National Science Council 390

(NSC) Grants 97-2320-B-039-008-MY3, by the China Medical University Grant 391

(CMU98-S-13 and CMU98-OC-04). 392 393 394 395 396 397 398

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513

Figure 1. Hispidulin inhibits the proliferation of human GBM cells. (A) Chemical 514

structures of hispidulin. (B) GBM8401 and (C) GBM8901 cells were seeding into 515

24-well plates in the presence of 10% FBS and after 24 hr treated with various 516

concentrations of hispidulin at 37 o C for 48 hr. The effect on cell growth was 517

examined by MTT assay, and the percentage of cell proliferation was calculated by 518

defining the absorption of cells without of hispidulin treatment as 100%. This 519

experiment was repeated three times. Bar represents the SD. 520

Figure 2. Hispidulin up-regulates AMPK activity. GBM8401 and GBM8901 cells 521

were treated with 60 µM and 40 µM hispidulin for indicated duration, respectively. 522

After harvesting, cells were lysed and prepared for western blotting analysis using 523

antibodies against (A) phospho-AMPK (Thr172), (B) phospho-mTOR (Ser2448), (C) 524

phospho-4E-BP1 (Thr37/46) and β-actin. Western blot data presented are 525

representative of those obtained in at least three separate experiments. The values 526

below the figures represent the change in protein expression normalized to β-actin. 527

Figure 3. Hispidulin decreases the protein synthesis by activating AMPK to 528

inhibit mTOR pathway. (A) GBM8401 and (B) GBM8901 cells were incubated with 529

15 µM compound c in the absence or presence of hispidulin for 24 hr. 530

Phospho-AMPK (Thr172), phospho-mTOR (Ser2448), and β-actin were detected by 531

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least three separate experiments. The values below the figures represent the change in 533

protein expression normalized to β-actin. 534

Figure 4. Hispidulin decreases the activity of fatty acid synthesis by inhibiting the 535

expression of FASN and the activity of ACC. GBM8401 and GBM8901 cells were 536

treated with 60 µM and 40 µM hispidulin for indicated duration, respectively. After 537

harvesting, cells were lysed and prepared for western blotting analysis using 538

antibodies against (A) FASN, (B) pospho-ACC (Ser79) and β-actin. Western blot data 539

presented are representative of those obtained in at least three separate experiments. 540

The values below the figures represent the change in protein expression normalized to 541

β-actin. 542

Figure 5. Hispidulin decreases the activity of fatty acid synthesis via activating of 543

AMPK. (A) GBM8401 and (B) GBM8901 cells were incubated with 15 µM 544

compound c in the absence or presence of hispidulin for 24 hr. After harvesting, cells 545

were lysed and prepared for western blotting analysis using antibodies against FASN, 546

phospho-ACC (Ser79), and β-actin. Western blot data presented are representative of 547

those obtained in at least three separate experiments. The values below the figures 548

represent the change in protein expression normalized to β-actin. 549

Figure 6. Hispidulin induces cell cycle arrest and apoptosis in GBM cells. (A) 550

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for the indicated duration, respectively. After harvesting, cells were analyzed for 552

propidium iodide-stained DNA content by flow cytometry. The indicated percentages 553

are the mean of three independent experiments, each in duplicate. Bar represent the 554

S.D. GBM8401 and GBM8901 cells were treated with 60 µM and 40 µM hispidulin 555

for indicated duration, respectively. After harvesting, cells were lysed and prepared 556

for western blotting analysis using antibodies against (C) p53, and p21, (D) PARP and 557

β-actin. Western blot data presented are representative of those obtained in at least 558

three separate experiments. The values below the figures represent the change in 559

protein expression normalized to β-actin. 560

Figure 7. Hispidulin decreases the activity of fatty acid synthesis via activating of 561

AMPK. (A) GBM8401 and (B) GBM8901 cells were incubated with 15 µM 562

compound c in the absence or presence of hispidulin for 48 hr. GBM8401 and 563

GBM8901 cells were transfected with 50 nmol/L AMPKα1 shRNA using 564

lipofectamine. After twenty-four hour transfection, cells were treated with hispidulin 565

for 48 hr.The effect on cell growth was examined by MTT assay, and the percentage 566

of cell proliferation was calculated by defining the absorption of cells without of 567

hispidulin treatment as 100%. This experiment was repeated three times. Bar 568

represents the SD. Asterisks indicate that the values are significantly different from 569

For Review. Confidential - ACS

570

Figure 8. A schematic summary for the anti-GBM cells mechanisms of hispidulin 571

shown in the present study. 572

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Figure 1

(A)

(B)

(C)

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Figure 2

(A)

GBM8401

P-AMPK

Hispidulin

GBM8901

Hispidulin

GBM8401

P-4E-BP1

Hispidulin

GBM8901

Hispidulin

(C)

(B)

GBM8401

P-mTOR

Hispidulin

GBM8901

Hispidulin

β

β

β

β-actin

β

β

β

β-actin

β

β

β

β-actin

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(A)

Hispidulin

Compound c

(B)

GBM8401

Figure 3

P-AMPK

P-mTOR

GBM8901

P-AMPK

P-mTOR

Hispidulin

Compound c

β

β

β

β-actin

β

β

β

β-actin

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Figure 4

(A)

GBM8401

FASN

Hispidulin

GBM8901

Hispidulin

(B)

GBM8401

P-ACC

Hispidulin

GBM8901

Hispidulin

β

β

β

β-actin

β

β

β

β-actin

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(A)

Hispidulin

Compound c

(B)

GBM8401

Figure 5

FASN

P-ACC

Hispidulin

Compound c

GBM8901

P-ACC

FASN

β

β

β

β-actin

β

β

β

β-actin

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Figure 6

(A)

(B)

GBM8401

p53

Hispidulin

GBM8901

Hispidulin

(C)

p21

β

β

β

β-actin

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GBM8401

Hispidulin

GBM8901

Hispidulin

(D)

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Figure 7

(A)

(B)

*

* *

* *

* *

* *

* *

*

*

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AMPK

P-AMPK

Hispidulin

Hispidulin

Growth arrest

Apoptosis

mTOR

Protein synthesis

ACC

FASN

Fatty acid synthe

sis

Figure 8