Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production

ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■

Contents lists available at ScienceDirect

Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

1 2 3 4 5 6

Q2 Original Articles

Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production Q1 Hyang Sook Seol a, Sang Eun Lee a, Joon Seon Song b, Hye Yong Lee a, Sojung Park a,

Inki Kim a, Shree Ram Singh c,*, Suhwan Chang d,**, Se Jin Jang e,***

7

a

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Asan Institute for Life Science, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea c Basic Research Laboratory, Stem Cell Regulation and Animal Aging Section, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA d Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea e Asan Institute for Life Science, Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, South Korea b

A R T I C L E

I N F O

Article history: Received 21 June 2016 Received in revised form 30 August 2016 Accepted 31 August 2016 Keywords: Hepatocellular carcinoma Drug repositioning Riluzole ROS

A B S T R A C T

Liver cancer is one of the common malignancies in many countries and an increasing cause of cancer death. Despite of that, there are few therapeutic options available with inconsistent outcome, raising a need for developing alternative therapeutic options. Through a drug repositioning screening, we identified and investigated the action mechanism of the Riluzole, an amyotrophic lateral sclerosis (ALS) drug, on hepatocellular carcinoma (HCC) therapy. Treatment of the Riluzole leads to a suppression of cell proliferation in liver primary cancer cells and cancer cell lines. In addition, Riluzole induced caspasedependent apoptosis and G2/M cell cycle arrest in SNU449 and Huh7 cell lines. In a line with the known function of glutamate release inhibitor, we found Riluzole-treated cells have increased the level of inner cellular glutamate that in turn decrease the glutathione (GSH) level and finally augment the reactive oxygen species (ROS) production. We confirm this finding in vivo by showing the Riluzole-induced GSH and ROS changes in a Huh7 xenograft cancer model. Altogether, these data suggest the anti-cancer effect of Riluzole on hepatocellular carcinoma and the suppression of glutamate signaling might be a new target pathway for HCC therapy. Published by Elsevier Ireland Ltd.

41 Introduction

42 43 44 45 46 47 48 49 50 51 52 53 54 55

Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer, and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection [1]. Surveillance can lead to diagnosis at early stages, when the tumor might be curable by resection, liver transplantation, or percutaneous treatment [1]. One typical drug of HCC, Sorafenib is a multikinase inhibitor and targets both angiogenesis (the serine-threonine kinases Raf-1 and B-Raf) and proliferation (the vascular endothelial growth factor (VEGF) receptors and the platelet-derived growth factor beta (PDGFB) receptor) on tumor cells [2]. In vitro, Sorafenib has an anti-proliferative effect on liver cancer cell lines and on xenograft models. It was shown that sorafenib inhibits tumor growth

56 57 58 59 60 61 62 63

Q3

* Corresponding author. E-mail address: [email protected] (S.R. Singh). ** Corresponding author. Fax: +82 2 3010 8165. E-mail address: [email protected] (S. Chang). *** Corresponding author. Fax: +82 2 472 7898. E-mail address: [email protected] (S.J. Jang).

rather than induces tumor shrinkage [3,4]. Although these results are encouraging, the use of sorafenib is hampered by two phenomena. First, up to 80% of patients treated with sorafenib suffer from side effects such as hand-foot syndrome, diarrhea, hypertension and fatigue [5,6]. The second phenomenon is that 50–60% of patients who initially respond to therapy eventually will show recurrence [7]. The time for radiologic progression is delayed by sorafenib, but only a small percentage of patients show a partial response with rare complete response. Therapy is most often stopped at recurrence, because it is known from the literature that tumor growth is even more rapid after abrogation of anti-angiogenic therapy [8,9]. At present, there is no efficient drug for liver cancer therapy. Therefore, there has been a need to discover a new drug for HCC therapy [10]. In this research, we aimed to find new anti-HCC drug from the Prestwick Drug library. Through drug screening in primary and immortalized liver cancer cells, we identified Riluzole that showed significant inhibition of liver primary cancer cell proliferation. In addition, Riluzole inhibited glutamate release into outer cell that caused induction of ROS and apoptosis in liver cancer cells. Altogether, we propose the Riluzole as a new drug for HCC therapy.

http://dx.doi.org/10.1016/j.canlet.2016.08.028 0304-3835/Published by Elsevier Ireland Ltd.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

2

86

Materials and methods

87

Primary cancer cell culture-derived patient tissues

88 Q5 Primary cancer cells originated from surgically resected primary liver cancer 89 tissues collected by Asan Bio Resource Center under ethical approval by the insti90 tutional review board of Asan Medical Center (IRB-20120112). Prior to freezing of 91 the liver specimens for banking, a small piece of tumor tissue was separated and 92 processed to establish primary cancer cell lines. Briefly, the tumor tissues were minced 93 with scissors and subsequently digested using 1 mg/mL of type IV collagenase (Sigma 94 Chemical Co., St. Louis, MO) in DMEM/F12 for 60 minutes at 37 °C. After incuba95 tion, the tissues were washed with medium containing 10% fetal bovine serum (1600096 044; Life Technologies). To promote the adhesion and growth of the epithelial tumor 97 cells, Hepatocyte Basal Medium (HBM; Lonza, Walkersville, MD) containing human 98 epidermal growth factor (hEGF), hydrocortisone, insulin, transferrin, GA-1000, ascor99 bic acid, BSA-FAF, and 10% FBS was used to culture the primary liver cancer cells, 100 which were plated onto a collagen type 1 dish in a humidified incubator at 37 °C 101 under a 5% CO2 atmosphere [11]. 102

In vivo tumor generation in NOD-SCID mouse

103 All four primary cultured cell lines were implanted to 6- to 8-week-old NOD104 SCID mice (Charles River Laboratories, Wilmington, MA). Briefly, 5 × 105 patient105 derived primary cancer cells were suspended in 100 μL of Matrigel (BD Biosciences, 106 San Jose, CA) and injected into the subcutaneous layer on the backs of NOD/SCID 107 mice. After 2–3 months, when the tumor size reached above 1 cm3, the mice were 108 anesthetized via an intra-peritoneal injection of 40 mg/kg Zoletil (Virbac, Virbac Labo109 Q6 ratories BP 27-06511 Carrism France)-5 mg/kg Rumpum (Bayer, Korea, South Korea) 110 mixture, and the tumors were surgically removed. 111

Cell line culture

112 113 114

Two liver cancer cell lines (SNU449 and Huh7) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/ mL streptomycin.

115

Morphology and immunohistochemistry assessment

116 117 118 119 120 121 122 123 124 125 126 127 128 129

Morphological comparisons between the original and xenograft tumors obtained from the primary cancer cells were performed by 2 pathologists. On H&E staining, the identification of pathologic type, differentiation grading, and tumor architecture were evaluated. For IHC comparisons between the original and xenograft tumors, immunohistochemical staining of formalin-fixed paraffin-embedded tissue sections was performed using an automatic immunohistochemical staining device (Benchmark XT; Ventana Medical Systems, Tucson, AZ). Briefly, 4-μm-thick 2D cultured tissue sections were transferred onto poly-L-lysine-coated adhesive slides and dried at 74 °C for 30 minutes. After epitope retrieval by heating for 1 hour in ethylene diaminetetraacetic acid (pH 8.0) in the autostainer, the samples were incubated with the indicated antibodies (see supplementary Table). The sections were subsequently incubated with secondary antibodies, and then visualized using an ultraView Universal DAB Detection kit (Ventana Medical Systems, Inc). The nuclei were counterstained with Harris hematoxylin.

130

Cell proliferation assay

131 132 133 134 135 136 137

The proliferation of primary cancer cell and liver cancer cell lines was examined using CCK-8 kit (Tongren Shanghai Co., China) according to the manufacturer’s instructions. Briefly, the cells were seeded into 96-well plates with 2 × 103 cells/ well and the next day, treated drug for 72 h. Then 10 μL CCK-8 solution was added to each well and incubated for 2 h. The absorbance (A) at 450 nm was measured using a microplate reader. Results were representative of three individual experiments in triplicate.

138

FACS analysis

139 140 141 142 143 144

SNU449 and Huh7 cells (105 cells/mL) were incubated with Sorafenib or Riluzole for 24 h. The cells were collected, washed with cold PBS, fixed in cold 100% ethanol, treated with DNase-free RNase, and stained with 40 μg/mL of propidium iodide (PI). The distribution of the cells between phases of the cell cycle was deduced from the DNA content on a FACScan flow cytometer (BD FACSCanto II; BD, USA). For each sample, 10,000 gated events were acquired.

145

Annexin-V/PI staining for cell death detection

146 147 148 149 150 151

Apoptosis was determined by staining cells with Annexin-V-fluorescein isothiocyanate (FITC, Ex/Em of 488 nm/519 nm; Invitrogen Molecular Probes) and propidium iodide (PI, Ex/Em of 488 nm/617 nm; Sigma-Aldrich). In brief, 1 × 106 cells in 60-mm culture dishes (Nunc) were incubated with or without riluzole and sorafenib. Cells were washed twice with cold PBS and then resuspended in 500 μL binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 × 106

152 153 154 155 156 157 158 159

cells/mL. Annexin-V-FITC (5 μL) and PI (1 μg/mL) were then added to these cells, which were analyzed with a FACScanto flow cytometer (Becton-Dickinson). Viable cells were negative for both PI and Annexin-V; apoptotic cells were positive for Annexin-V and negative for PI, whereas late apoptotic dead cells displayed both high Annexin-V and PI labeling. Non-viable cells, which underwent necrosis, were positive for PI and negative for Annexin-V. Western blotting

160 161 162 163 164 165 166 167 168 169 170 171 172

Cells were collected and lysed with EZ-RIPA lysis buffer (ATTO, WSE-7420, Japan). The cell lysates were collected after centrifugation and the protein concentration was quantified by bovine serum albumin (BSA) method. Equal amount of protein was loaded and separated on 10–15% SDS–PAGE, and then transferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked in 5% BSA for 1 h and then incubated with antibody (see supplementary table) at 4 °C overnight. After three times of washing with PBS-T, the membranes were incubated with HRPconjugated secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h at room temperature. The membranes were developed using ECL kit (Amersham, GE Healthcare Life Sciences, USA) and exposed to X-ray film. β-actin was used as loading control. Glutamate measurement

173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193

Standard metabolites and internal standard were purchased from SigmaAldrich. All solvents including water were purchased from J. T. Baker. Cell number would be ~1 million. Cells were harvested using 1.4 mL of cold methanol/H2O (80/ 20, v/v) after sequential wash with PBS and H2O. Then cells were lysed by vigorous vortexing and 100 μL of 5 μM of internal standard (Gln-d4) was added. Metabolites were extracted from aqueous phase by liquid–liquid extraction after adding chloroform. The aqueous phase was dried using vacuum centrifuge, and the sample was reconstituted with 50 μL of 50% methanol prior to LC-MS/MS analysis. Glutamate was analyzed with LC-MS/MS equipped with Ultimate 3000 HPLC (Dionex), Orbitrap XL (Thermo Fisher Scientific), and a HILIC column (Zorbax HILIC 100 × 2.1 mm). 10 mL was injected into the LC-MS/MS system. H2O and acetonitrile were used as mobile phases A and B, respectively. The separation gradient was as follows: hold at 60% B for 10 min, 60%–10% B for 0.1 min, hold at 10% for 4.9 min, 10%–60% B for 0.1 min, then hold at 60% B for 4.9 min. LC flow was 150 μL/min, and column temperature was kept at 23 °C. Selective reaction monitoring (SRM) was used in negative ion mode, and the selected values for a precursor and a fragment ion were m/z 146.045 and m/z 128.035 with 10 ppm mass accuracy. The extracted ion chromatogram (EIC) was used for quantitation after normalization using internal standard, and the calibration range was 0.1−100 μM (r2 ≥ 0.99). Detection of intracellular glutathione (GSH) levels Cells were washed twice with PBS and fixed with 4% formaldehyde (Sigma F877525ML) for 30 min at room temperature, washed again 3 times with PBS, and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 minutes. For FACS and immunofluorescence, cells were incubated with murine monoclonal antibodies against GSH (5 μg/mL; Abcam, Cambridge, MA) for 2 h at room temperature. After washing with PBS, cells were incubated with Alexa 488-conjugated antimouse IgG (1:200, Molecular Probes, Eugene, OR) for 1 h in the dark, and then washed. Flow cytometry analysis was performed on BD FACS CantoTM (BD Biosciences) at the Asan Life Science Lab and Flow Cytometry Core Facility of Asan Medical Center. In addition, images were acquired using ZEN 2012 software and an ×40 oil immersion objective lens. ROS measurement Cells cultured in 60 cm2 dish were treated with H2O2 or Riluzole alone as indicated in each figure legend. Cells were then stained for 30 minutes with 5 μM of H2O2-sensitive fluorescent dye CM-H2DCFDA, washed 3 times with PBS, and subsequently assayed by FACScan flow cytometer (Beckman Coulter cell, Brea, CA, USA). For immunofluorescence, cells were washed twice with HBSS and stained with fluorescent dye CM-H2DCFDA in HBSS for 30 minutes at room temperature, and washed again 3 times with PBS. Images were acquired using ZEN 2012 software and an ×40 oil immersion objective lens.

Q7

194 195 196 197 198 199 200 201 202 203 204 205 206

Xenografts in immunodeficient nude mice

207 208 209 210 211 212 213 214 215 216

All animal studies were approved by the institutional review board for animal care and facilities committee of Asan Life Science Laboratory. Huh7 cells were injected subcutaneously into the right leg side of 6-week-old male athymic nude mice. The tumor diameter was measured and documented every day. The tumor volume (mm3) was estimated by measuring the longest and shortest diameters of the tumor and calculated as follows: volume = (shortest diameter)2 × (longest diameter)/2. Treatment with either vehicle (DMSO) or 10 mg/kg Riluzole was given every other day via p.o. gavage. After 28 days of treatment, tumors were collected, weighed, and processed for preparation of FFPE sample protein.

217 218 219 220 221 222 223 224 225

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

226

Statistical analysis

227 228 229 230 231

All data express the means ± SD. For paired data sets, a two-sided Student’s t-test was used. A P-value < 0.05 was considered statistically significant. Associations between categorical variables were analyzed by Pearson’s chi-square and Fisher’s exact tests. P-values less than 0.05 were considered statistically significant.

Riluzole inhibits growth of HCC primary cell and cell lines

232 Results Xenograft model using patient derived primary cancer cell has similar histology with patient’s original tumors In order to conduct a drug repositioning screening, we first established primary cultures from four HCC patients. The primary cells were all attached on collagen type 1 coated dish and grew rapidly in HBM with 5% FBS (Fig. 1A). We performed immunohistochemical analysis of the original and xenograft tumors formed in mice. As shown in the H&E staining results (Fig. 1B, left panels), the xenograft tumor retains similar morphological features of the original tumor. The similarity was confirmed by immunohistochemical staining for tumor-specific markers: CK7, CK19, p-CEA and HePpar1 (Fig. 1B, right panels). In addition to the tumor markers, the immunohistochemistry result of Ki67, CD34, CD56 and CD117 also showed similar expression pattern between the xenograft and patient’s original tumors (Supplementary Fig. S1). These data support that the xenograft model and primary HCC primary cell retain characteristic of original tumors.

HCC 2

HCC 2

CK7

CK19

p-CEA

HePpar1

PDX

253

H&E

To investigate whether the anti-proliferative effect of Riluzole on liver cancer cell lines was triggered by a cell cycle arrest, we measured cell cycle progression by flow cytometry after propidium iodide (PI) staining. As shown in Fig. 3, the Riluzole treatment induced G2/ M-phase accumulation in both of the cell lines, compared to control

Primary

HCC 1

HCC 1

B

Primary PDX Primary

HCC 4 254 255 256

PDX

HCC 3

HCC 3

HCC 4

257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275

Riluzole induces G2/M cell cycle arrest by regulating cell cycle regulator proteins

PDX

A

Using the primary cancer cells, we next aimed to discover antiHCC drug. To this purpose, we introduced Prestwick Chemical Library, which contains cancer drugs approved by FDA. The four primary cancer cells were treated with 240 kinds of candidate drugs from the library and cell proliferation was measured by CCK-8 assay. The raw data are shown in Supplementary Fig. S2. We found three out of four primary cancer cells responded and showed sensitivity to the Riluzole hydrochloride (Supplementary Fig. S2A,B,D). To confirm this result, we tested different doses of Riluzole and included sorafenib as a control drug. The Riluzole showed inhibition of cell proliferation for 3 primary HCC cells (Fig. 2B,C,D). The inhibitory effect of the Riluzole or sorafenib was also tested in liver cancer cell lines, SNU449 and Huh7 (Fig. 2E,F). We next examined the combinatory effect of the two drugs (sorafenib and Riluzole) on liver cancer cell lines. The result in Fig. 2G,H shows additive but no synergistic effect on the cell proliferation by combinatory treatment.

Primary

233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252

3

Fig. 1. Characterization of primary cancer cells from 4 hepatocarcinoma (HCC) patients. (A) Representative pictures from primary cultured cells derived from the 4 HCC patient’s specimens. Scale bar, 100 μM. (B) Histological features of primary HCC and xenografts derived from the cells. Each of the tumors was stained with one of the H&E, CK7, CK9, p-CEA and anti-HepPar antibodies.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

276 277 278 279 280 281 282 283

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

4

HCC 1

1.2

B

1

1

0.8

0.8

0.6 0.4 0.2

0.6 0.4 0.2

0.2

30uM

10uM

5uM

50uM

HCC 4

0.8 0.6 0.4 0.2

Ctrl

Riluzole

F

SNU 449

30uM

10uM

5uM

Sorafenib

1.2

1

50uM

30uM

5uM

100uM

50uM

30uM

10uM

5uM

50uM

30uM

10uM

Sorafenib

10uM

0 5uM

Riluzole

Huh 7

1 CCK-8 Absorbance (450nm)

CCK-8 Absorbance (450nm)

100uM

0.4

1.2

0.8 0.6

0.4 0.2

0.8 0.6 0.4 0.2

0

0

5uM 10uM 5uM 10uM 30uM 50uM100uM Ctrl

Sorafenib

SNU 449

120

5uM 10uM 5uM 10uM 30uM 50uM100uM

Riluzole

Ctrl

H

Sorafenib

Riluzole

Huh 7

120

100

CCK-8 Absorbance (450nm)

100

CCK-8 Absorbance (450nm)

100uM

0.6

Ctrl

80

60

40

80

60

40

20

20

0

0

R 5uM R 10uM R 30uM

R 5uM R 10uM R 30uM Ctrl

284 285 286 287

50uM

0.8

0

G

Riluzole

1

1

CCK-8 Absorbance (450nm)

CCK-8 Absorbance (450nm)

1.2

E

Sorafenib

D 1.2

HCC 3

1.4

30uM

5uM

100uM

50uM

Ctrl

Riluzole

50uM

Sorafenib

30uM

10uM

5uM

50uM

30uM

10uM

5uM Ctrl

10uM

0

0

C

HCC 2

1.2

CCK-8 Absorbance (450nm)

CCK-8 Absorbance (450nm)

A

S 3uM R 5uM R 10uM R 30uM

Sorafenib 3uM

Ctrl

S 3uM R 5uM R 10uM R 30uM

Sorafenib 3uM

Fig. 2. The effect of Riluzole or Sorafenib on primary HCC cell growth. Cells were treated with increasing concentrations of Riluzole or Sorafenib (up to 100 μM) onto HCC1 (A), HCC2 (B), HCC3 (C) and HCC4 (D). Cell proliferation was determined by CCK-8 assay. The inhibitory effects were normalized to Ctrl (in DMSO) condition. (E, F) The effect of Riluzole and Sorafenib on liver immortalized cancer cell growth (SNU449 (E) and Huh7 (F)). Cells were treated with increasing doses of the Riluzole or Sorafenib (up to 100 μM). (G, H) The combinatory effect of the Riluzole and Sorafenib in liver cancer cell lines. Cell proliferation was determined by CCK-8 assay.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

A

B

SNU 449 120

120

100

100

Percentage of total cells (%)

Percentage of total cells (%)

5

80 G2

60

S G1

40 20

Huh 7

80

G2

60

S G1

40 20

0

0 Ctrl

C

Sorafenib

Riluzole

Ctrl

Ctrl

Sorafenib

Sorafenib

Riluzole

Riluzole

SNU449

Huh7

SNU449

100 90 80 70 60 50 40 30 20 10 0

Ctrl

Sorafenb Riluzole

Late apotosis cell

Huh7

Ctrl

Riluzole

Ctrl

Sorafenib

SNU449

F

Necrosis

Ctrl Sorafenib Riluzole

Live cell

G Riluzole

Early apototsis cell

Sorafenib

Live cell

Huh7

E

100 90 80 70 60 50 40 30 20 10 0

c-caspase 9/Actin

D

3

2 1 0 Ctrl

Sorafenib Riluzole

Control

SNU449

Actin

c-caspase 3/Actin

Huh7

1.5 1 0.5 0 Ctrl

Sorafenib Riluzole

Control

Q12 Q14

c-PARP/Actin

Sorafenib Riluzole Huh7

8 6 4 2 0 Ctrl

Sorafenib Riluzole SNU449

288 289 290 291 292 293 294

Sorafenib Riluzole

2

SNU449

c-PARP

Necrosis

4

c-caspase 9

c-caspase 3

Early apototsis Late apotosis cell cell

Control

Sorafenib Riluzole Huh7

Fig. 3. Cell cycle distribution and apoptotic response in HCC cells after treatment with Riluzole or Sorafenib. (A) SNU449 and (B) Huh-7 cells were incubated in the presence of Riluzole (30 μM) or sorafenib (10 μM) for 24 hours, and fixed, stained with PI and analyzed for cell cycle distribution by flow cytometry analysis .(C) Annexin V/PI staining assay was carried out to explore cell apoptosis induced by Riluzole or Sorafenib. SNU-449 and Huh7 cells were treated with 30 μM Riluzole or 10 μM Sorafenib for 24 h, and stained with Annexin V and PI for analysis using flow cytometry. The data shown are representative of three independent experiments with similar findings. (D and E) Increased apototic cells in SNU449 and Huh7 were quantified from the data in (C). (F) Expression of apoptotic marker proteins in SNU449 and Huh7 cells after treatment with Riluzole or Sorafenib. SNU-449 and Huh7 cells were treated in the presence of the indicated concentrations of Riluzole (30 μM) or Sorafenib (10 μM) for 24 hours. Whole cell lysates were analyzed by western blotting using indicated antibodies.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

6

B

*

SNU449

*

120

100 80 60 40

120

120

100

100

*

80 60

Ctrl

Riluzole

Ctrl

SNU449

C

80 60

40

40

20

20

20 0

0

0

Riluzole

Huh7

140

140

GSH content (% of control)

140

GSH content (% of control)

Percentage of Glutamate contents (%)

A

Ctrl

Ctrl

Riluzole

Riluzole

Huh7

Ctrl

NAC

BSO

Riluzole

SNU449

Huh7

Ctrl

H2O2

E

Riluzole

F SNU449

SNU449 ROS content (Relative control FITC-A Mean)

18

Huh7

Huh 7

16 14

***

12 10 8 6

4 2

1.8 1.6 1.2 1 0.8 0.6 0.4 0.2 0

Ctrl

Ctrl

H 2O 2

Riluzole

H2O2 Riluzole

H

I

SNU449 6

SNU449

Huh7 2.5

**

295 296 297 298Q13 Q15

4 3

2

*

Intensity (DCFA/Mito Tracker)

Intensity (DCFA/Mito Tracker)

5

Huh7

**

1.4

0

G

*

2

***

ROS content (Relative control FITC-A Mean)

D

2

1.5 1

1

0.5

0

0

Fig. 4. Riluzole increased cellular glutamate and decreased GSH contents in SNU449 and Huh7 cells. Measurement of cellular glutamate (A) and cellular GSH contents (B) in two cell lines. (C) Representative fluorescence microscopy images of SNU449 and Huh7 treated with 30 μM Riluzole for 24 hrs. (D and E) Cellular ROS levels in SNU449 (D) or Hun7 (E) cell lines. Data are expressed as mean ± SD for three independent experiments. (F) Representative fluorescence microscopy images of the two cell lines treated with 30 μM Riluzole for 24 hrs.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

A

7

B

2500

Percentage of Tumor growth ratio (% of control tumor)

* **

2000

1500

* *

1000 Ctrl 500

Riluzole

0 4d

6d

8d 10d 12d 14d 16d 18d 20d 22d 24d

D 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

E 120 100

* GSH content (% of control)

Tumor weight (g)

C

*

80 60 40 20 0

Ctrl

Riluzole

Ctrl

Riluzole

ROS content (Relative control FITC-A Mean)

2d

160

*

140 120

100 80 60 40 20 0 Ctrl

Riluzole

F

Glutamate

Glutamate

299 300 301 302

Fig. 5. Riluzole exerts antitumor activities in vivo. (A) A total of 1 × 106 cells were injected into the right leg side of nude mice (n = 6). When tumor reached a volume of 100 mm3, the mice were treated with 10 mg/kg Riluzole or vehicle on every other day for 24 days. The sizes of tumors from Riluzole- and vehicle-treated mice were measured. (A) Tumor growth rate. (B) Picture of the sacrificed mouse with tumors. (C) Average tumor weight. The tumor weights were calculated as described in Materials and Methods. (D and E) Measurements of GSH (D) and ROS contents (E) in tumors. (F) Schematic diagram of the suggested action mechanism of Riluzole.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

ARTICLE IN PRESS 8

H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

303 (Fig. 3A,B). In contrast, sorafenib treatment showed marginal effect 304 on SNU449 whereas it showed antitumor effect (G2/M arrest) on Huh7 cells, comparable to the Riluzole treatment. To understand 305 the molecular basis by which Riluzole inhibits G2/M transition 306 in tumor cells, we analyzed the expression of proteins involved 307 in cell cycle regulation by western blot analysis (Supplementary 308 Fig. S3). As a result, we observed that treatment of Riluzole 309 marginally elevated the expression of cyclin B1 whereas p21 and 310 p-cdc2 expressions were decreased. These data suggest that Riluzole 311 induces the arrest of cells in the G2/M phase, thereby inhibits cell 312 proliferation. 313 On the other hand, we analyzed Riluzole-induced apoptosis 314 as a cause of anti-tumor effect, using Annexin V-FITC/PI double315 staining assay. As shown in Fig. 3C,E, the percentage of apoptotic 316 cells (including early and late apoptotic cells) was increased sig317 nificantly with Riluzole treatment, which seems more evident than 318 the sorafenib-treated group (Fig. 3D,E, compare red with black bars). 319 (For interpretation of the references to color in this text, the reader 320 is referred to the web version of this article.) Moreover, as an apototic 321 marker, we analyzed cleaved-caspase by western blot analysis. 322 Cleaved caspase-3, and -9 and PARP were clearly increased in the 323 Huh7 and SNU449 (Fig. 3F). Therefore, we concluded the Riluzole 324 confers antitumor effect by inducing apoptosis in HCC cells. 325 326 Riluzole caused ROS by reduction of GSH as a result of inhibited 327 328 Q8 release of glutamate to outer cells 329 Riluzole was known to play a role in the inhibition of gluta330 mate release in melanoma cell [12]. Therefore, we measured 331 glutamate contents in cells after treatment of Riluzole. We found 332 the cellular glutamate content was significantly increased after the 333 334 Q9 Riluzole treatment (Fig. 4A). As a result of the accumulation of glutamate, the GSH production was decreased (Fig. 4B), which is caused 335 by the shrinking of cysteine uptake into cells [13]. Through confo336 cal microscope analysis, we could confirm the reduced GSH in 337 SNU449 and Huh7 cells after treatment of Riluzole (Fig. 4C). Next, 338 we examined ROS production by FACS (Fig. 4D,E) and confocal anal339 ysis (Fig. 4F) as a potential consequence of the reduced GSH 340 production. As a result, we observed an increased ROS production 341 and accumulation of ROS in the Riluzole-treated cancer cells com342 pared to control. In addition, we detected the Riluzole induced ROS 343 production in primary liver cancer cells that showed sensitivity to 344 Riluzole in Fig. 2 (Supplementary Fig. S4). 345 Altogether, these data suggest an increased glutamate accumu346 lation in cell causes a decreased GSH level and ROS induction, which 347 finally result in cancer cell apoptosis. 348 349 Inhibition of HCC xenograft growth by Riluzole 350 351 We next examined our findings in vivo, xenograft model. To do 352 this, 106 Huh7 cells were inoculated subcutaneously into nude mice 353 on right leg. Afterwards, the mice were treated with 10 mg/kg 354 Riluzole daily by oral gavage for 28 days. The tumor sizes were mea355 sured every other day with a caliper. A significant reduction in tumor 356 volume was observed from 16 days compared to the vehicle357 treated controls (Fig. 5A,B). The tumor weight was also decreased 358 in Riluzole-treated cases than vehicle-treated controls (Fig. 5C). From 359 these tumors, we measured ROS and GSH levels. In line with the 360 results observed in HCC cells, we found the Riluzole-treated xeno361 graft tumor showed reduced GSH and increased ROS levels (Fig. 5D,E). 362 These data demonstrate the antitumor effect of Riluzole on HCC. 363 364 Discussion 365 366 Aberrant glutamate signaling has been shown to participate in 367 the transformation and maintenance of various cancer types, in368

cluding glioma, melanoma, breast cancer, and prostate cancer [14–16]. Glutamine is preferred as a fuel in tumor cells over glucose Q10 because of its ability to fulfill both energy requirements and macromolecule synthesis [9]. Riluzole, marketed by Sanofi-Aventis as Rilutek, is classified as an anti-excitotoxic, neuroprotective drug that blocks cellular release of glutamate. It is approved by FDA for the treatment of amyotrophic lateral sclerosis (ALS). Riluzole has an absolute bioavailability of approximately 60%, which is relatively high, and is also able to cross the blood–brain barrier. Its mechanism of the glutamate blockade is thought to be in part due to the inactivation of voltage-gated sodium channels on glutamatergic nerve terminals and/or the activation of a G-protein dependent signaling cascade [17]. Riluzole has also been shown to block some postsynaptic glutamatergic effects by the noncompetitive inhibition of NMDA receptors [17]. By a drug repositioning screening in patient-derived HCC primary cells, we identified the Riluzole plays a role in anti-proliferative effect on the primary HCC cell and cell lines. We found the Riluzole induces G2/M cell cycle arrest and caspase-dependent apoptosis pathway. Mechanistically, our data presented here suggest that the Riluzole inhibits release of cellular glutamate that triggers decreased cellular GSH production and increased ROS production (Fig. 5F). In this study, we showed Riluzole induces two different anticancer effects on HCC, increasing ROS production with caspase cascade (apoptosis) and G2/M cell cycle inhibition. Interestingly, accumulating evidence support these two events are linked by the modification of Cdc25A and C, which control the activity of Cdk1 and Cdk4/6 [18,19]. Further study will reveal this drug can trigger such regulation in HCC. On the other hand, our results suggest the combinatory treatment of Riluzole and Sorafenib results in stronger inhibition of cell proliferation than single treatment (Fig. 2G,H). Considering the Riluzole itself has low side effect, it will be meaningful to add this drug in other therapeutic treatment for various cancers.

369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403

Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2015R1A1A3A04001354), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) (grant numbers: HI06C0868, HI13C1538), funded by the Ministry Q11 of Health &Welfare, Republic of Korea.

404 405 406 407 408 409 410 411 412 413

Conflict of interest The authors declare no competing financial interests.

414 415 416 417

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.08.028.

418 419 420 421 422

References [1] J.M. Llovet, A. Burroughs, J. Bruix, Hepatocellular carcinoma, Lancet 362 (2003) 1907–1917. [2] S.M. Wilhelm, C. Carter, L. Tang, D. Wilkie, A. McNabola, H. Rong, et al., BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis, Cancer Res. 64 (2004) 7099–7109. [3] L. Liu, Y. Cao, C. Chen, X. Zhang, A. McNabola, D. Wilkie, et al., Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5, Cancer Res. 66 (2006) 11851–11858.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

423 424 425 426 427 428 429 430 431 432 433 434

ARTICLE IN PRESS H.S. Seol et al. / Cancer Letters ■■ (2016) ■■–■■

435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461

[4] G.K. Abou-Alfa, L. Schwartz, S. Ricci, D. Amadori, A. Santoro, A. Figer, et al., Phase II study of sorafenib in patients with advanced hepatocellular carcinoma, J. Clin. Oncol. 24 (2006) 4293–4300. [5] D. Strumberg, J.W. Clark, A. Awada, M.J. Moore, H. Richly, A. Hendlisz, et al., Safety, pharmacokinetics, and preliminary antitumor activity of sorafenib: a review of four phase I trials in patients with advanced refractory solid tumors, Oncologist 12 (2007) 426–437. [6] T.T. Batchelor, A.G. Sorensen, E. di Tomaso, W.T. Zhang, D.G. Duda, K.S. Cohen, et al., AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients, Cancer Cell 11 (2007) 83–95. [7] A.S. Makarova, N.L. Lazarevich, Deregulation of signaling pathways involved in sorafenib resistance of hepatocellular carcinoma, Klin. Lab. Diagn. 66–68 (2013) 34–67. [8] M.R. Mancuso, R. Davis, S.M. Norberg, S. O’Brien, B. Sennino, T. Nakahara, et al., Rapid vascular regrowth in tumors after reversal of VEGF inhibition, J. Clin. Invest. 116 (2006) 2610–2621. [9] R.J. DeBerardinis, J.J. Lum, G. Hatzivassiliou, C.B. Thompson, The biology of cancer: metabolic reprogramming fuels cell growth and proliferation, Cell Metab. 7 (2008) 11–20. [10] M. Peck-Radosavljevic, Drug therapy for advanced-stage liver cancer, Liver Cancer 3 (2014) 125–131. [11] H.S. Seol, S.E. Lee, J.S. Song, J.K. Rhee, S.R. Singh, S. Chang, et al., Complement proteins C7 and CFH control the stemness of liver cancer cells via LSF-1, Cancer Lett. 372 (2016) 24–35. [12] M.N. Le, J.L. Chan, S.A. Rosenberg, A.S. Nabatian, K.T. Merrigan, K.A. Cohen-Solal, et al., The glutamate release inhibitor Riluzole decreases migration,

[13]

[14]

[15]

[16]

[17] [18]

[19]

9

invasion, and proliferation of melanoma cells, J. Invest. Dermatol. 130 (2010) 2240–2249. Y. Deng, Z.F. Xu, W. Liu, B. Xu, H.B. Yang, Y.G. Wei, Riluzole-triggered GSH synthesis via activation of glutamate transporters to antagonize methylmercury-induced oxidative stress in rat cerebral cortex, Oxid. Med. Cell. Longev. 2012 (2012) 534705. A. Herner, D. Sauliunaite, C.W. Michalski, M. Erkan, T. De Oliveira, I. Abiatari, et al., Glutamate increases pancreatic cancer cell invasion and migration via AMPA receptor activation and Kras-MAPK signaling, Int. J. Cancer 129 (2011) 2349–2359. H. Hu, N. Takano, L. Xiang, D.M. Gilkes, W. Luo, G.L. Semenza, Hypoxia-inducible factors enhance glutamate signaling in cancer cells, Oncotarget 5 (2014) 8853–8868. S.S. Willard, S. Koochekpour, Glutamate signaling in benign and malignant disorders: current status, future perspectives, and therapeutic implications, Int. J. Biol. Sci. 9 (2013) 728–742. A. Doble, The pharmacology and mechanism of action of riluzole, Neurology 47 (1996) S233–S241. G. Pozo-Molina, A. Ponciano-Gomez, G.C. Rivera-Gonzalez, A. Hernandez-Zavala, E. Garrido, Arsenic-induced S phase cell cycle lengthening is associated with ROS generation, p53 signaling and CDC25A expression, Chem. Biol. Interact. 238 (2015) 170–179. L. Li, B.B. Zheng, L.S. Ma, X. Sun, J.J. Chang, W.D. Xie, et al., Telekin suppresses human hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via the p38 MAPK signaling pathway, Acta Pharmacol. Sin. 35 (2014) 1311–1322.

Please cite this article in press as: Hyang Sook Seol, et al., Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.08.028

462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487