Pterostilbene Enhances TRAIL-Induced Apoptosis through the

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Pterostilbene enhances TRAIL-induced apoptosis through the induction of death receptors and downregulation of cell survival proteins in TRAIL-resistance triple negative breast cancer cells Chao-Ming Hung, Liang-Chih Liu, Chi-Tang Ho, Ying-Chao Lin, and Tzong-Der Way J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02358 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 23, 2017

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Journal of Agricultural and Food Chemistry

1

Pterostilbene Enhances TRAIL-Induced Apoptosis through the Induction of

2

Death

3

TRAIL-Resistance Triple Negative Breast Cancer Cells

4

Chao-Ming Hung†,‡, Liang-Chih Liu§,≠, Chi-Tang Ho¢, Ying-Chao Lin#,ƒ,¥**,

5

Tzong-Der Way£,$*

6

†

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

7

‡

School of Medicine, I-Shou University, Kaohsiung, Taiwan

8

§

Department of Surgery, China Medical University Hospital, Taichung, Taiwan

9

≠

Receptors

and

Downregulation

of

Cell

Survival

Proteins

in

School of Medicine, College of Medicine, China Medical University, Taichung,

10

Taiwan

11

£

12

Department of Biological Science and Technology, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, Taiwan

13

¢

14

#

Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA

15

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

16

ƒ

School of Medicine, Tzu Chi University, Hualien, Taiwan

17

¥

Department of Medical Imaging and Radiological Science, Central Taiwan University

18 19 20

of Science and Technology, Taichung, Taiwan $

Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan

1

ACS Paragon Plus Environment


21 22

*Corresponding

23

Tzong-Der Way, Ph.D.

24

Department of Biological Science and Technology, China Medical University, Taiwan

25

Address: No. 91 Hsueh-Shih Road, Taichung, Taiwan 40402

26

Tel: +886-4-2205-3366 ext: 2509

27

Fax: +886-4-2203-1075

28

E-mail: [email protected]

author:

29 30

**Co-corresponding

31

Ying-Chao Lin, Ph.D.

32

School of Medicine, Tzu Chi University, Hualien, Taiwan

33

Address: No.701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan

34

E-mail: [email protected]

author:

35

36

37

Abbreviations:

38

ATF6, activating transcription factor 6; c-FLIP, FLICE inhibitory protein; CHOP, 2


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39

C/EBP-homologous protein; DcR, decoy receptor; DMSO, dimethyl sulfoxide; DR,

40

death receptor; ECL, enhanced chemiluminescence; eIF2, PERK/eukaryotic

41

initiation factor-2; ERK, extracellular signal-regulated kinase; ER, estrogen receptor;

42

FADD, fas-associated protein with death domain; FBS, fetal bovine serum; IRE1,

43

inositol-requiring

44

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

45

MAPKs,

46

osteoprotegerin; PARP, poly ADP-ribose polymerase; PBS, phosphate-buffered saline;

47

PERK, protein kinase R-like endoplasmic reticulum kinase; PI, propidium iodide; PR,

48

progesterone receptor; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl

49

sulfate-polyacrylamide;

50

apoptosis-inducing ligand.

kinase

1;

mitogen-activated

JNK,

protein

TRAIL,

c-Jun

kinases;

tumor

N-terminal

NAC,

necrosis

51

3


kinase;

N-acetylcysteine;

factor

MTT,

OPG,

(TNF)-related


52

ABSTRACT

53

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is nontoxic to

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normal cells and preferentially cytotoxic to cancer cells. Recent data suggest that

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malignant breast cancer cells often become resistant to TRAIL. Pterostilbene (PTER),

56

a naturally occurring analogue of resveratrol found in blueberries, is known to induce

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cancer cells undergo apoptosis. In the present study, we examined whether PTER

58

affects TRAIL-induced apoptosis and its mechanism in TRAIL-resistant triple negative

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breast cancer (TNBC) cells. Our data indicated that PTER induced apoptosis (14.68 ±

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3.78% for 40 μM PTER vs 1.98 ± 0.25% for control, p < 0.01) in TNBC cells and

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enhanced TRAIL-induced apoptosis in TRAIL-resistant TNBC cells (18.45 ± 4.65%

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for 40 μM PTER vs 29.38 ± 6.35% for combination of 40 M PTER and 100 ng/mL

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TRAIL, p < 0.01). We demonstrated that PTER induced death receptors DR5 and DR4

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as well as decreased decoy receptor DcR-1 and DcR-2 expression. PTER also

65

decreased the anti-apoptotic proteins c-FLIPS/L, Bcl-Xl, Bcl-2, Survivin, and XIAP.

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PTER induced the cleavage of bid protein and caused the pro-apoptotic Bax

67

accumulation. Moreover, we found that PTER induced the expression of DR4 and DR5

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through the reactive oxygen species (ROS)/ endoplasmic reticulum (ER) stress/ERK

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1/2 and p38/C/EBP-homologous protein (CHOP) signaling pathways. Overall, our

4


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results showed that PTER potentiated TRAIL-induced apoptosis via ROS-mediated

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CHOP activation leading to the expression of DR4 and DR5.

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KEYWORDS: TRAIL; Pterostilbene; triple negative breast cancer; death receptor;

73

ROS

74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 5



89

INTRODUCTION

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Triple-negative breast cancer (TNBC) is a heterogeneous group of diseases with

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poor prognosis. TNBC does not express estrogen receptor/progesterone receptor

92

(ER/PR) or HER-2, which can be targeted by available therapies. Therefore, TNBC

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does not respond to hormonal therapy (such as tamoxifen or aromatase inhibitors) or

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therapies that target HER2 (such as Herceptin).1 TNBC can only be treated with

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combination therapies such as radiation therapy, surgery, and chemotherapy, therefore,

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identification of novel targeted and biologic therapies for TNBC would be of great

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benefit.

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A proapoptotic molecule, tumor necrosis factor-related apoptosis-inducing

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ligand (TRAIL), selectively induces apoptosis in a variety of human tumor cell lines

100

without affecting normal cells.2 Previous studies showed that interaction of human

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TRAIL ligand with two agonistic receptors i.e., TRAIL receptors 1 (DR4) and 2 (DR5),

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two antagonistic decoy receptors i.e., TRAIL receptors 3 (DcR-1) and 4 (DcR-2), and a

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soluble receptor i.e., osteoprotegerin (OPG).3 The DR4 and DR5 can trigger apoptosis

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in TRAIL-sensitive human cancer cells,4 whereas DcR-1 and DcR-2 completely or

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partially lack functional death domains, and therefore cannot trigger apoptosis.5

6


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Moreover, compared to the other death receptors, OPG has a lower binding affinity to

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TRAIL.6

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It has been demonstrated that TRAIL has long been perceived as a potential

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chemotherapeutic agent. However, it has now emerged that many human cancer cells

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are intrinsically resistant to TRAIL.3 Therefore, it would be necessary to identify

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sensitizing agents capable of increasing TRAIL-mediated cell death. Deregulation of

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apoptotic pathways plays important roles in the resistance of human cancer cells to

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TRAIL-induced apoptosis. Mutations in DR4 and DR5 or absent of them on the cell

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surface can cause human cancer cells resistance to TRAIL-mediated apoptosis.7

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Moreover, the up-regulation of DcR-1, and DcR-2 does not have the ability to trigger

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apoptosis.8 The down-regulation of caspase 8 or Fas-associated death domain

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(FADD),7 overexpression of cFLIP or antiapoptotic Bcl-xL or Bcl-2, or loss of Bax

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pro-apoptotic function shows a correlation with TRAIL resistance in multiple

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cancers.9,10

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Recent studies indicated that DR5 is a critical mediator of endoplasmic

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reticulum (ER) stress-induced apoptosis.11,12 Several studies have suggested that

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C/EBP-homologous protein (CHOP) is one of the most highly inducible genes during

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the ER stress, and this provides an important role for the link between DR5 and ER

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stress. It has also been documented that CHOP can regulate the transcriptional 7



125

expression of DR5 through binding to the CHOP-binding site in the DR5 gene

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5'-flanking regions.13 Three ER stress transducers: protein kinase R-like endoplasmic

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reticulum kinase (PERK), inositol-requiring kinase 1 (IRE1) and activating

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transcription factor 6 (ATF6) conduct ER stress. During ER stress, the

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PERK/eukaryotic initiation factor-2 (eIF2) signaling pathway plays an important

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role in the three pathways.14,15

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Recent studies found that pterostilbene (PTER), a natural dimethylated

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analogue of resveratrol from blueberries, have biological activities including anticancer,

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anti-inflammation and so on. Previous research has indicated that PTER triggers cell

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cycle arrest or apoptosis in lung cancer,16 leukemia,17 breast cancer18 and prostate

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cancer.19 We reported recently that PTER stimulates Fas signaling, which drives

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epithelial-mesenchymal transition (EMT) through the ERK1/2 and GSK3β/β-catenin

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signaling pathway and also triggers autophagy in TNBC.20 However, it is unclear

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whether PTER can sensitize TNBC cells to TRAIL-induced apoptosis.

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We aim to identify the effect of PTER on TRAIL-induced apoptosis in

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TRAIL-resistance TNBC cells. Overall, our results demonstrate that PTER could

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sensitize the TRAIL-induced apoptosis through up-regulating the expression of DR4

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and DR5 and modulating the anti-apoptotic proteins expression. Moreover, PTER

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induces the expression of DR4 and DR5 via the ROS/ER stress/ERK 1/2 and 8


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p38/CHOP signaling pathways.

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9



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MATERIALS AND METHODS Chemicals. We purchased β-actin antibody, N-acetylcysteine (NAC), PTER,

148

propidium iodide (PI), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT)

149

from Sigma Chemical Co. (St. Louis, MO, USA). We purchased SB203580,

150

SP600125, and PD98059 from Calbiochem (San Diego, CA, USA). We purchased

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PARP, cleaved-PARP, pro-caspase-9, pro-caspase-8, pro-caspase-3, Survivin, Bcl-xL,

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Bcl-2, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-JNK1/2 (Thr 183/Tyr 185), JNK1/2,

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p-p38 (Thr180/Tyr182), p38, CHOP, p-eIF2 (Ser51) and GRP78 antibodies from Cell

154

Signaling Technology (Danvers, MA, USA). We purchased DR4 and DR5 antibodies

155

from Abcam Inc. (Cambridge, MA, USA) and Novus Biologicals (Littleton, CO, USA),

156

respectively. We purchased Bax, Bid, XIAP, c-FLIP-S, and c-FLIP-L antibodies from

157

Santa Cruz Biotechnology (Santa Cruz, CA, USA). We purchased DcR1 and DcR2

158

antibodies from ProSci Inc. (Poway, CA, USA). We purchased HRP-conjugated Goat

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anti-Mouse IgG and Goat anti-Rabbit IgG secondary antibodies from Millipore

160

(Billerica, MA, USA). We purchased recombinant human soluble TRAIL from

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PeproTech (Rocky Hill, NJ, USA).

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Cell culture and treatments. We obtained BT-20, MCF-7, MDA-MB-468,

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HL-60, SKOV3, MDA-MB-231, PC3, DU145, A549 and H1299 cells from the

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American Type Culture Collection (Manassas, VA, USA). The cells were maintained 10


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in medium supplemented with 10% fetal bovine serum (FBS), 100 μg streptomycin, 100

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U penicillin, and 2 mM L-glutamine (Invitrogen Corporation, Carlsbad, CA, USA).

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Cells were kept at 37 °C in a humidified 5% CO2 incubator.

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MTT assay. To determine the cell viability, the number of all viable cells was

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estimated by the uptake of the MTT. Approximately 1 × 104 cells were plated in

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24-well plates overnight. Cells were treated with reagents as indicated in the legend.

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Cells were then incubated with 100 μL of 1 μg/mL MTT for 2 h at 37 °C. The purple

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insoluble formazan was further dissolved using 80 μL DMSO. Readings were recorded

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at 570 nm using an enzyme-linked immunosorbent assay (ELISA) microplate reader.

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Cell clonogenic assay. Cells were plated overnight in six-well plates and then

175

treated with reagents as indicated in the legend. The colonies were cultured in an

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incubator at 5% CO2 with a controlled temperature of 37 °C. The colonies formation

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varies from 2-3 weeks for different cells. Finally, colonies were fixed for 15 mins by

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100% ice-cold methanol and stained with Giemsa staining. Colonies with >50 cells

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were counted using an inverted microscope.

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Flow cytometric analysis. Approximately 5 × 105 cells were treated with

181

different reagents as indicated in the legend. The cells were fixed with 500 μL of

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pre-cooling 70% ethanol at -20 °C overnight and then were washed, pelleted and

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resuspended in PBS containing 100 mg/mL RNase, 0.1% TritonX-100, and 0.04 11



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mg/mL of PI. The level of apoptotic cells was analyzed by FACScan and Cell Quest

185

software. For DR5 staining, live cells were incubated with DR5 antibody.

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Western blotting analysis. Protein expressions were carried out by Western

187

blotting according to our previous study.21 Cells on 10 cm culture dishes (1 × 106

188

cells/dish) were treated with reagents as indicated in the legend. Protein concentration

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was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Proteins

190

(50

191

sulfate-polyacrylamide

192

nitrocellulose membrane. The membranes were blocked in TBST contained 5% non-fat

193

milk and 0.1% NaN3. Finally, membrane was hybridized with primary and secondary

194

antibody and immunoreactive bands were presented using the ECL™ Prime Western

195

Blotting Detection Reagent (GE Healthcare UK Ltd.).

μg)

from

the

whole gel

cell

were

electrophoresis

separated

using

(SDS-PAGE)

and

sodium

dodecyl

transferred

to

196

Transfection. The shRNA and siRNA sequences used here were as follows: DR5,

197

AAGUUGCAGCCGUAGUCUUGA; CHOP, AAGAACCAGCAGAGGUCACAA.

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The cells were transiently transfected in 2 ml OPTI-MEM (GIBCO, Carlsbad, CA,

199

USA) containing 9 μL LipofectamineTM 2000 (Invitrogene, Carlsbad, CA, USA), with

200

50 nmol/L CHOP-siRNA and DR5-shRNA. After 6 h of transfection, the medium was

201

changed to complete growth medium for 24 h.

202

Determination of ROS. Approximately 5 × 104 cells were treated with different 12


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reagents as indicated in the legend for 24 h. Stained cells with 10 mM DCFDA in 1X

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Buffer for 15 min and the intensity of fluorescence was measured by flow cytometry.

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Statistical analysis. Student's t-tests can be used to detect a statistical difference

206

in means between two groups. All results were expressed as means ± standard

207

deviation (SD) of at least three independent experiments. The P values of less than

208

0.05 were considered statistically significant and highly significant (*, P < 0.05; **, P

209

< 0.01; ***, P < 0.001).

210 211

13



212

RESULTS

213

PTER

inhibits

the proliferation

and

induces

apoptosis

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in highly

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TRAIL-resistant BT20 cells. To examine the mechanisms of TRAIL resistance in

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TNBC cells, we compared the sensitivity of TRAIL in MDA-MB-468 and BT-20 cells.

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As shown in Figure 1B, MDA-MB-468 cells were less sensitive and BT-20 cells were

217

resistant to TRAIL-induced apoptosis. Our findings indicated that the BT-20 cells were

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highly resistant to TRAIL-induced apoptosis. We next examined the anti-proliferative

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activity of PTER (Figure 1A) in MDA-MB-468 and BT-20 cells. Treatment of the

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MDA-MB-468 and BT-20 cells with increasing concentrations of PTER for 48 h, and a

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marked dose-dependent inhibition of cell proliferation was consistently observed

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(Figure 1C). Interestingly, PTER inhibited the proliferation in highly TRAIL-resistant

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BT20 cells. The colony formation test also revealed that PTER inhibited the colony

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formation in highly TRAIL-resistant BT20 cells (Figure 1D). To confirm whether

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PTER induced apoptosis, we observed the distribution of apoptotic cells by PI staining

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in BT-20 cells. An apoptotic fraction of sub-diploid cells was detected as a ‘sub-G1’

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peak. We found that PTER treatment significantly induced apoptosis in a time- (Figure

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1E) and dose-dependent manner (Figure 1F). Moreover, PTER effectively enhanced

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PARP cleavage in a dose- (Figure 1G) and time-dependent manner (Figure 1H). Our

230

results provide the first set of evidence for PTER inhibits the proliferation and induces 14


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apoptosis in highly TRAIL-resistant BT20 cells.

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PTER sensitizes TNBC cells to TRAIL-induced apoptosis. Next, we identified

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whether PTER was able to sensitize TRAIL-resistant TNBC cells to TRAIL-induced

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apoptosis. We sought to identify the potential toxicity effect of PTER alone or in

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combination with TRAIL in MDA-MB-468 and BT-20 cells. The combined treatment

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with PTER and TRAIL strongly reduced cell viability (Figure 2A) and the colony

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formation in TRAIL-resistant TNBC cells (Figure 2B). Our results suggest that PTER

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plus TRAIL combined therapy effectively inhibited cell viability in TRAIL-resistant

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TNBC cells. We next examined whether the decreased cell viability from combined

240

treatment was due to induction of apoptosis. A combined treatment of 100 ng/mL

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TRAIL and 40 M PTER for 48 h significantly increased apoptosis (Figure 2C),

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whereas PTER or TRAIL alone did not. Moreover, our study indicated that the

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combined therapy significantly increased the activity of caspase-3, caspase-8,

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caspase-9, and PARP (Figure 2D). These results suggest that PTER sensitizes TNBC

245

cells to TRAIL-induced apoptosis through the activation of caspases activity.

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PTER suppresses the expression of antiapoptotic members. The antiapoptotic

247

members of the Bcl-2 family are well known for their ability to suppress

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TRAIL-induced apoptosis.9,10 Next, we investigated whether the alteration in

249

expression

levels

of

antiapoptotic

proteins

involved

15


in

PTER

enhanced


250

TRAIL-induced apoptosis. We treated BT-20 cells with PTER and then investigated

251

the expression of Bcl-2, Bcl-xL, cFLIP-L, cFLIP-S, Survivin and XIAP. The data

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indicated that expression of antiapoptotic members were suppressed by PTER in a

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dose- (Figure 3A) and time-dependent manner (Figure 3B). Next, we tested whether

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PTER affected the expression of proapoptotic proteins. The cleaved form of Bid (t-Bid)

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and the upregulation of proapoptotic member bax was also induced by PTER in a dose-

256

(Figure 3C) and time-dependent manner (Figure 3D). Our data suggest that the

257

mitochondrial pathway of apoptosis provided an important component in PTER

258

sensitizes TNBC cells to TRAIL-induced apoptosis.

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PTER up-regulates the expression of death receptors in TNBC cells. Recent

260

study found that if cancer cells lose expression of DR4 and DR5 expression may

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develop resistance to TRAIL.7 This study aim to investigate the effects of PTER on

262

DR4 and DR5 expression in TNBC cells. These data confirm that PTER significantly

263

induced the expression of DR4 and DR5 in a dose- (Figure 4A) and time-dependent

264

manner (Figure 4B). We next used flow cytometry to identify whether PTER induced

265

the expression of DR5. As shown in the Figure 4C, PTER increased the expression of

266

DR5 on the cell surface. To analyze whether the upregulation of DR5 played important

267

role in mediating TRAIL-induced apoptosis by PTER, we stably transfected BT-20

268

cells with shRNA against DR5. As shown in Figure 4D, transfection of cells with 16


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shRNA of DR5 effectively inhibited the PTER/TRAIL-induced apoptosis. Our results

270

find that PTER potentiates TRAIL-induced cytotoxicity in TNBC cells via

271

upregulating DR4 and DR5 expression.

272

PTER induces the expression of DR4 and DR5 is not restricted to only one

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cell type. We also investigated whether PTER induced the expression of DR4 and DR5

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was specific to TNBC cells or also occurred in other cell types. Figure 4E shows that

275

PTER induced the expression of DR4 and DR5 in prostate cancer cells (PC3 and

276

DU145), ovarian cancer cells (SKOV3), breast cancer cells (MCF-7 and

277

MDA-MB-231), lung cancer cells (H1299 and A549), and leukemia cells (HL60).

278

Therefore, it suggests that PTER induces the expression of DR4 and DR5 is not cell

279

type-specific.

280

PTER downregulates DcRs. Antiapoptotic receptors, DcRs can compete with

281

DRs for TRAIL binding.5 Next, we analyzed whether PTER potentiated

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TRAIL-induced apoptosis by downregulating DcRs expression. As expected, PTER

283

treatment resulted in downregulating DcR-1 and DcR-2 expression in a dose- (Figure

284

4F) and time-dependent manner (Figure 4G). Thus, it is clear that PTER might

285

potentiate TRAIL-induced apoptosis by downregulating DcR-1 and DcR-2 expression.

286

PTER upregulates DR4 and DR5 expression appear to be dependent on the

287

formation of ROS. Recent studies have shown that increased oxidative stress will 17



288

induce the expression of DRs.22 Therefore, we examined whether PTER increased the

289

intracellular ROS levels. After treatment with PTER, we used dichlorofluorescein

290

diacetate (DCFH-DA) dye to detect the levels of intracellular ROS produced. We

291

found that ROS induction by PTER in a time-dependent manner (Figure 5A). We next

292

used the N-acetylcysteine (NAC), a thiol antioxidant which is known to function as a

293

reactive oxygen intermediate scavenger, to test whether ROS involved in

294

PTER/TRAIL-induced apoptosis. Our study found that NAC pretreatment reduced the

295

upregulation of ROS by PTER treatment (Figure 5B). NAC also suppressed

296

PTER-induced DR4 and DR5 up-regulation (Figure 5C). Moreover, PTER

297

significantly enhanced TRAIL-induced PARP cleavage in BT-20 cells, and NAC

298

pretreatment significantly attenuated PTER/TRAIL-induced PARP cleavage (Figure

299

5D). A combination of 40 M PTER and 100 ng/mL TRAIL significantly enhanced

300

the accumulation of cells in the sub-G1 phase (Figure 5E), and NAC pretreatment

301

significantly attenuated PTER/TRAIL-induced apoptosis. Therefore, our data find that

302

the generation of ROS and caspases activation are critical for PTER/TRAIL-induced

303

apoptosis.

304

Upregulation of DR4 and DR5 by PTER are ERK1/2 and p38 dependent.

305

Several studies have shown that the signals of MAPKs are very likely related to the

306

induction of TRAIL receptor,23,24 we examined whether ERK1/2, p38, or JNK 18


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activation exerted their role in PTER-induced DR4 and DR5 up-regulation. Our data

308

indicated that treatment with PTER resulted in an activation of ERK1/2, p38 and

309

JNK1/2 in a time-dependent manner (Figure 6A). Next, to investigate whether PTER

310

upregulated DR4 and DR5 expression by modulating the activation of ERK1/2, p38

311

and JNK1/2, the inhibitors for p38 (SB203580), JNK1/2 (SP600125), and ERK1/2

312

(PD98059) were used. We found that treatment with SP600125 (JNK1/2 inhibitor)

313

cannot inhibit PTER-induced DR4 and DR5 upregulation (Figure 6B). However, the

314

SB203580 (p38 inhibitor) (Figure 6C) and PD98059 (ERK1/2 inhibitor) (Figure 6D)

315

suppressed PTER-induced DR4 and DR5 upregulation, which suggested that ERK1/2

316

and p38 were needed for DR4 and DR5 upregulation. Next, we examined whether

317

ROS regulated PTER-induced the activation of ERK1/2 and p38. We found that

318

pretreating cells with NAC suppressed PTER-induced the phosphorylation of ERK1/2

319

and p38 (Figure 6E). Overall, our findings indicate that PTER-induced ROS

320

production is important for the activation of ERK1/2 and p38, which in turn led to

321

induction of the DR4 and DR5.

322

PTER induces ER stress response and leads to DR4 and DR5-dependent

323

apoptosis. Recent study demonstrated that ER stress are critical for TRAIL-induced

324

cell death by inducing the expression of DR5.25,26 We next investigated whether PTER

325

affected the ER stress-induced pathways. We observed PTER increased the protein 19



326

expression or phosphorylation of ER stress marker (e.g. p-eIF2 and GRP78) in a

327

dose- (Figure 7A) and time-dependent manner (Figure 7B). Since oxidative stress was

328

involved in ER stress-induced ischemic neuronal cell death,27 we further demonstrated

329

whether ROS production could involve in PTER-induced ER stress. As shown in

330

Figure 7C, NAC pretreatment inhibited PTER/TRAIL-induced phosphorylation of

331

eIF2 and GRP78 upregulation. These data support our hypothesis that ROS

332

generation is critical for PTER/TRAIL-induced ER stress.

333

PTER induces DR4 and DR5 expression through CHOP activation. Recent

334

study showed that CHOP is ER stress-induced transcription factor and important

335

transcription factor of DR5,13 we next to examine whether PTER induced CHOP

336

expression. We found that PTER treatment could result in a greatly increased amount

337

of CHOP protein levels in a dose- (Figure 8A) and time-dependent manner (Figure

338

8B). We further demonstrated whether ROS generation is critical for PTER-induced

339

upregulation of CHOP and DRs. We observed NAC pretreatment significantly

340

attenuated PTER-induced upregulation of DR4, DR5 and CHOP expression (Figure

341

8C). We used CHOP siRNA to examine whether CHOP played a critical role in

342

PTER-induced upregulation of DR4 and DR5. The results revealed that transfection

343

with CHOP siRNA significantly attenuated PTER-induced upregulation of DR4 and

344

DR5 (Figure 8D), while control-transfected (scrambled RNA) had no effect. A 20


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345

combination of 40 M PTER and 100 ng/mL TRAIL significantly induced apoptosis

346

(Figure 8E), while CHOP siRNA significantly attenuated PTER/TRAIL-induced

347

apoptosis. This result suggests that CHOP seems to play an important role in

348

PTER-induced upregulation of DR4 and DR5 and PTER/TRAIL-induced apoptosis.

349

Upregulation of CHOP by PTER is mediated through ERK1/2 and p38. We

350

next investigated whether PTER-induced CHOP up-regulation through ERK1/2 and

351

p38 activation. Our study found that the SB203580 (p38 inhibitor) (Figure 8F) and

352

PD98059 (ERK1/2 inhibitor) (Figure 8G) suppressed PTER-induced CHOP

353

up-regulation. We future determined whether PTER/TRAIL-induced apoptosis through

354

ERK1/2 and p38 activation. The results revealed that PTER/TRAIL significantly

355

increased apoptosis, however, SB203580 (p38 inhibitor) and PD98059 (ERK1/2

356

inhibitor) significantly attenuated PTER/TRAIL-induced apoptosis (Figure 8H). These

357

studies indicate that PTER-induced CHOP upregulation and PTER/TRAIL-induced

358

apoptosis are mediated through ERK1/2 and p38 activation.

359 360 361 362 363 21



364

DISCUSSION

365

Although amount of tumors remain sensitive to TRAIL-induced apoptosis, while

366

some mismatch-repair-deficient tumors evade the proapoptotic effects of TRAIL.

367

Therefore, it is important to find improved and optimized methods that sensitize tumor

368

cells to TRAIL. In our current study, we demonstrated that PTER potentiated

369

TRAIL-resistant TNBC cells induced apoptosis by up-regulating the expression of

370

DR4 and DR5 and by down-regulating the expression of survival proteins. Moreover,

371

PTER-induced DR4 and DR5 upregulation are mediated through ROS/ ERK1/2 and

372

p38/CHOP pathway.

373

Previous study indicated that most human breast cancer cell lines are resistant to

374

TRAIL-mediated apoptosis.28 The present study found that BT-20 cells were

375

TRAIL-resistant cells and MDA-MB-468 cells were less sensitive to TRAIL. Recent

376

study found that the mesenchymal phenotype TNBC cells are TRAIL-sensitive, and the

377

TRAIL-resistant TNBC cells have an epithelial phenotype.29 Our results were

378

consistent with previous studies, the TRAIL-resistant TNBC cells (BT-20 and

379

MDA-MB-468 cells) had an epithelial phenotype. Specifically, our studies found that

380

PTER greatly improved the cytotoxic activity and the antitumor efficacy of TRAIL in

381

TRAIL-resistant TNBC cells. Moreover, our results demonstrated for the first time that

382

PTER enhanced TRAIL-induced caspase-8, caspase-3 activation and PARP cleavage in 22


Page 22 of 53

Page 23 of 53

383


TNBC cells.

384

Several TRAIL resistance mechanisms have been proposed, including

385

overexpression of antiapoptotic Bcl-2 family members and decoy receptors.7-10 In our

386

study, we found that PTER exerted its effects via several mechanisms. First, PTER

387

decreased the expression of DcR-1 and DcR-2. Moreover, PTER significantly

388

down-regulated the expression of anti-apoptotic Bcl-2 family members, including

389

Bcl-2, Bcl-xL, cFLIP-L, cFLIP-S, Survivin, and XIAP.30, 31 Recent study found that

390

downregulation of c-FLIP expression sensitizes TRAIL-induced apoptosis in

391

chemotherapy.32 The proapoptotic Bcl-2 family members have been shown to block

392

apoptosis by keeping mitochondrial function.33 Kaempferol enhances TRAIL-mediated

393

apoptosis

394

proteasome-mediated degradation.34 In agreement with the study, our results indicated

395

that

396

downregulating Survivin. Overall, our results indicate that Bcl-2, Bcl-xL, cFLIP-L,

397

cFLIP-S, Survivin, and XIAP down-regulation contributed to PTER-facilitated

398

TRAIL-mediated apoptosis.

in

PTER

human

enhanced

glioma

cells

through

TRAIL-mediated

downregulating

apoptosis

in

TNBC

Survivin

cells

via

through

399

Studies have shown that DR4 or/and DR5 upregulation can lead to

400

TRAIL-resistant cells to TRAIL-responsive apoptosis.35,36 Here, we have shown that

401

PTER-induced DR4 and DR5 upregulation can convert TRAIL-resistant TNBC cells to 23



402

TRAIL-responsive apoptosis, as gene silencing of DR5 by RNAi attenuated the

403

PTER/TRAIL-induced apoptosis. Moreover, we reported that PTER enhanced

404

TRAIL-induced caspase activation in TNBC cells through up-regulating DR4 and DR5

405

expression. To our best knowledge, our results highlight the combined PTER and

406

TRAIL treatment induced apoptosis through a novel DRs-mediated mechanism in

407

TNBC cells.

408

Recent studies reported that ROS generation could trigger TRAIL-dependent

409

apoptosis through the up-regulation of DRs.37,38 Numerous studies have been described

410

that some flavonoids generate ROS in cancer cells. Yang et al. found that

411

8-bromo-7-methoxychrysin (BrMC) induces apoptosis through the ROS generation in

412

human hepatocellular carcinoma cells.39 Moreover, PTER represses the proliferation of

413

human esophageal cancer cells by upregulating ROS generation and ER stress.40 In

414

agreement with the previous studies, our results indicated that PTER upregulated ROS

415

generation, and NAC, ROS scavengers, attenuated PTER-induced upregulation of DR4

416

and DR5. Alosi et al. indicated that PTER increased mitochondrial ROS production to

417

trigger human breast cancer cell apoptosis.41 Mannal et al. found that treatment with

418

PTER increased O2− production and caspase-3 activity in breast cancer cells.42 Studies

419

performed by Chakraborty et al. indicated that treatment with PTER induced apoptosis

420

through the production of H2O2 and singlet oxygen in breast cancer cells.43 24


Page 24 of 53

Page 25 of 53


421

Collectively, our study consistent with these findings, imply that PTER increases

422

cellular oxidation to induce TNBC apoptosis.

423

MAPKs have been shown to play a key role in ROS downstream signaling

424

pathways. Moreover, various studies have suggested that MAPKs, including ERK1/2,

425

p38, and JNK, activation involves in DRs upregulation.44,45 In our study, we found that

426

PTER-induced DR4 and DR5 induction was ERK1/2 and p38 dependent, but not JNK.

427

Our study consistent with a previous study showing that ERK1/2 and p38 activation

428

mediated emodin-induced apoptosis through upregulating DRs expression.46 Moreover,

429

ampelopsin also upregulates DR4 and DR5 expression through the ERK1/2 and p38

430

activation.47

431

Several ER stress inducers, such as thapsigargin,12 tunicamycin11 and MG132,48

432

have been implicated in the up-regulation of DR5. Here, we found that PTER treatment

433

increased the phosphorylation of eIF2a and upregulation of GRP78, suggesting that

434

PTER induced ER stress. CHOP is an inducible ER stress transcription factor involved

435

in DR5 gene transcription, and provided evidence that this link between DR5 and ER

436

stress.13 Here, our findings suggest that CHOP may still be an important mediator for

437

PTER-induced DR4 and DR5 upregulation. Using CHOP siRNA in BT-20 cells

438

significantly blocked PTER-induced DR4 and DR5 upregulation. Therefore, this

439

suggests that PTER upregulated DR4 and DR5 through the ER stress-induced CHOP 25



440

expression in TNBC cells. Recent study found that berberine induced apoptosis via ER

441

stress was mediated by ROS in human glioblastoma cells.49 Here, our data revealed

442

that PTER induced CHOP and GRP78 expression via ER stress, while NAC

443

pretreatment attenuated these effects. Overall, our study consistent with these findings,

444

imply that generation of ROS activates ER stress and CHOP protein expression in

445

TNBC cells.

446

These above results indicated that PTER induced ER stress, CHOP, and DR4/DR5

447

expression via ROS generated. Moreover, PTER upregulated ROS generation plays

448

important role in PTER/TRAIL-induced apoptosis in TNBC cells. Recent study found

449

that piperlongumine selectively induced apoptosis and preferentially inhibited

450

migration/invasion via ROS-ER-MAPKs-CHOP axis in HCC cells.50 The possibility is

451

that ER stress-induced ERK1/2 and p38 activation and then inducing the expression of

452

CHOP. Further study is necessary to elucidate whether ERK1/2 and p38 could provide

453

a key mechanistic link between ROS and ER stress.

454

Recent study found that phase II metabolism, including sulfation and

455

glucuronidation represents the main clearance pathways for PTER.51 Moreover, the

456

levels of the PTER metabolites (both glucuronide and sulfate conjugates) in plasma

457

were significantly higher than the parent compound.51 Recent study also found that

458

PTER exhibits capacity limited elimination as the conjugating enzymes may be 26


Page 26 of 53

Page 27 of 53


459

saturated at higher doses.52 Interestingly, PTER exhibited superior metabolic stability

460

than resveratrol (ten-fold longer transit time and five-fold lower clearance than

461

resveratrol). This result can be justified by resveratrol has three hydroxyl groups, while

462

PTER contains one hydroxyl group and two methoxy groups. The two methoxy groups

463

cause PTER to be more lipophilic and increase oral absorption. Recent studies found

464

that given oral intake of PTER, no deaths or abnormal changes were observed in

465

mice.53 In this study, we found that PTER exhibited anticancer activation at the

466

concentrations of 40 µM. It is interesting to know how many dietary intakes are needed

467

to reach systemic concentrations of 40 µM in human. After 50 and 250 mg/kg PTER

468

intraperitoneal administration, Pan et al. found that the plasma levels of PTER were

469

2.24 and 26.85 μg/mL at 30 min, respectively. Moreover, the plasma levels of PTER

470

were 0.05 and 0.39 μg/mL at 24 h, respectively.54 According to this previous study, we

471

suggested that PTER at 40 μM is corresponding to ∼568 mg of daily administration in

472

70 kg body weight adult human. In future, the focus of a Phase I study will be needed

473

to evaluate the safety of PTER and dosing in humans.

474

Overall, we demonstrate that PTER enhances TRAIL-induced apoptosis via the

475

upregulation of DR4 and DR5 in TNBC cells. Moreover, PTER induces TRAIL

476

sensitivity via the ROS/ER stress/ERK1/2 and p38/CHOP signaling pathways. PTER

477

also down-regulated anti-apoptotic Bcl-2 family members, including Bcl-2, Bcl-xL, 27



478

cFLIP-L, cFLIP-S, Survivin, and XIAP. All of these data indicate that PTER could be

479

developed as a novel chemopreventive agent for TNBC.

480 481 482 483 484

485

ACKNOWLEDGMENT

486

We would like to thank Ming-Ching Kao (China Medical University) for his assistance

487

in editing the manuscript.

488 489 490 491 492

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1234-1242. 37



667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682

Figure legends

683

Figure 1. PTER induced apoptosis in TRAIL-resistant cells. (A) Chemical structure

684

of PTER. (B) BT-20 and MDA-MB-468 cells were treated with TRAIL (0, 25, 50, 100,

685

200 ng/mL) for 48 h and cell viability was quantitated by MTT assay. (C) BT-20 and 38


Page 38 of 53

Page 39 of 53


686

MDA-MB-468 cells were treated with various concentrations of PTER (0, 10, 20, 40

687

or 80 M) for 48 h and cell viability was quantitated by MTT assay. (D) In the colony

688

formation assay, BT-20 cells were treated with 10, 20, 40 μM PTER and stained with

689

Giemsa. (E) BT-20 cells were treated with 40 μM PTER for 12, 24, 48 h. (F) BT-20

690

cells were treated with various concentrations of PTER (0, 10, 20, 40 or 80 M) for 48

691

h. Cells were stained with PI, and the sub-G1 fraction was analyzed using flow

692

cytometry. (G) BT-20 cells were treated with various concentrations of PTER (0, 10,

693

20, 40 or 80 M) for 48 h. (H) BT-20 cells were treated with 40 μM for 12, 24, 48 h.

694

Whole-cell extracts were prepared and analyzed by Western blotting using antibodies

695

against PARP and cleaved-PARP. Western blot data presented are representative of

696

those obtained in at least three separate experiments. Data are expressed as mean ± SD.

697

*, P < 0.05, **, P < 0.01.

698

Figure 2. PTER-potentiated TRAIL induced apoptosis of TNBC cells. (A) BT-20

699

and MDA-MB-468 cells were treated with TRAIL (100 ng/mL) and with or without 40

700

M PTER for 48 h. Cell viability was determined by MTT assay, as described in

701

Materials and Methods. #,

702

colony formation assay, BT-20 and MDA-MB-468 cells were treated with TRAIL (100

703

ng/mL) and with or without 40 M PTER and stained with Giemsa. (C) BT-20 and

704

MDA-MB-468 cells were treated with TRAIL (100 ng/mL) and with or without 40 M

##

P < 0.05 significant when compared to PTER. (B) In the

39



705

PTER for 48 h. Cells were stained with PI, and the sub-G1 fraction was analyzed using

706

flow cytometry. Data are expressed as mean ± SD. #, P < 0.001 significant when

707

compared to PTER. (D) BT-20 and MDA-MB-468 cells were treated with TRAIL (100

708

ng/mL) and with or without 40 M PTER for 48 h. Whole-cell extracts were prepared

709

and analyzed by Western blotting using antibodies against pro-caspase-3,

710

pro-caspase-8, pro-caspase-9, and cleaved-PARP. Western blot data presented are

711

representative of those obtained in at least three separate experiments.

712

Figure 3. Effects of PTER on antiapoptotic and proapoptotic proteins expression.

713

(A) BT-20 cells were treated with various concentrations of PTER (0, 10, 20, 40 or 80

714

M) for 48 h. (B) BT-20 cells were treated with 40 μM for 12, 24, 48 h. Whole-cell

715

extracts were prepared and analyzed by Western blotting using antibodies against

716

cFLIP-L, cFLIP-S, Bcl-2, Bcl-xL, Surviving and XIAP. (C) BT-20 cells were treated

717

with various concentrations of PTER (0, 10, 20, 40 or 80 M) for 48 h. (D) BT-20 cells

718

were treated with 40 μM for 12, 24, 48 h. Whole-cell extracts were prepared and

719

analyzed by Western blotting using antibodies against Bax and Bid. Western blot data

720

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

721

Figure 4. PTER induced DR4 and DR5 expression and suppressed decoy

722

receptors. (A) BT-20 cells were treated with various concentrations of PTER (0, 10,

723

20, 40 or 80 M) for 48 h. (B) BT-20 cells were treated with 40 μM for 12, 24, 48 h. 40


Page 40 of 53

Page 41 of 53


724

Whole-cell extracts were prepared and analyzed for DR4 and DR5 expression by

725

Western blotting. (C) BT-20 cells were treated with 40 μM for 48 h, and flow

726

cytometry were used to analyze DR5 surface expression. (D) BT-20 cells were

727

transfected with DR5 shRNA. Twenty-four hours after the transfection, the cells were

728

treated with TRAIL (100 ng/mL) and 40 M PTER for 48 h. Cell viability was

729

determined by MTT assay, as described in Materials and Methods. Data are expressed

730

as mean ± SD. **, P < 0.01. (E) Several cancer cells were treated with 40 μM for 48 h.

731

Whole-cell extracts were prepared and analyzed for DR4 and DR5 expression by

732

Western blotting. (F) BT-20 cells were treated with various concentrations of PTER (0,

733

10, 20, 40 or 80 M) for 48 h. (G) BT-20 cells were treated with 40 μM for 12, 24, 48

734

h. Whole-cell extracts were prepared and analyzed for DcR1 and DcR2 expression by

735

Western blotting. Western blot data presented are representative of those obtained in at

736

least three separate experiments.

737

Figure 5. Upregulation of DR4 and DR5 by PTER was mediated by ROS. (A)

738

BT-20 cells were treated with 40 μM PTER for indicated times and then stained with

739

DCFDA. (B) BT-20 cells were pretreated with 10 mM NAC for 1 h, and then treated

740

with or without PTER (0, 10, 20, or 40 M) for 6 h and then stained with DCFDA. The

741

flow cytometry was used to determine the fluorescence intensity of DCFDA in the cells.

742

(C) BT-20 cells were pretreated with 1, 5, or 10 mM NAC for 1 h, and then treated 41



743

with 40 M PTER for 48 h. Whole-cell extracts were prepared and analyzed for DR4

744

and DR5 expression by Western blotting. (D) BT-20 cells were pretreated with 10 mM

745

NAC for 1 h, and then treated with 40 M PTER or 100 ng/mL TRAIL for 48 h.

746

Whole-cell extracts were prepared and analyzed for PARP and Cleaved-PARP

747

expression by Western blotting. Western blot data presented are representative of those

748

obtained in at least three separate experiments. (E) BT-20 cells were pretreated with 10

749

mM NAC for 1 h, and then treated with 40 M PTER or 100 ng/mL TRAIL for 48 h.

750

Cells were stained with PI, and the sub-G1 fraction was analyzed using flow cytometry.

751

Data are expressed as mean ± SD. **, P < 0.01.

752

Figure 6. Upregulation of DR4 and DR5 were ERK1/2, and p38 dependent. (A)

753

BT-20 cells were treated with 40 μM PTER for 12, 24, 48 h and whole-cell extracts

754

were subjected to Western blotting for phosphorylated ERK1/2, p38, JNK and ERK1/2,

755

p38, and JNK. BT-20 cells were pretreated with 20 μM JNK inhibitor, SP600125 (B);

756

and 10 μM p38 inhibitor, SB203580 (C) 20 μM ERK1/2 inhibitor, PD98059 (D) for 1 h

757

and then treated with 40 μM PTER for 48 h. Whole-cell extracts were prepared and

758

analyzed by Western blotting using DR4 and DR5 antibodies. (E) BT-20 cells were

759

pretreated with 10 mM NAC for 1 h, and then treated with 40 M PTER for indicated

760

times. Whole-cell extracts were prepared and analyzed for phosphorylated ERK1/2,

761

p38, JNK and ERK1/2, p38, and JNK. Western blot data presented are representative 42


Page 42 of 53

Page 43 of 53


762

of those obtained in at least three separate experiments.

763

Figure 7. PTER induced ER stress. (A) BT-20 cells were treated with various

764

concentrations of PTER (0, 10, 20, 40 or 80 M) for 48 h. (B) BT-20 cells were treated

765

with 40 μM for 12, 24, 48 h. Whole-cell extracts were prepared and analyzed for

766

p-eIF2 and GRP78 expression by Western blotting. (C) BT-20 cells were pretreated

767

with 10 mM NAC for 1 h, and then treated with 40 M PTER or 100 ng/mL TRAIL

768

for 48 h. Whole-cell extracts were prepared and analyzed for p-eIF2 and GRP78

769

expression by Western blotting. Western blot data presented are representative of those

770

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

771

change in protein expression of the bands normalized to β-actin.

772

Figure 8. Induction of DR4 and DR5 by PTER was mediated through of CHOP

773

activation. (A) BT-20 cells were treated with various concentrations of PTER (0, 10,

774

20, 40 or 80 M) for 48 h. (B) BT-20 cells were treated with 40 μM for 12, 24, 48 h.

775

Whole-cell extracts were prepared and analyzed for CHOP expression by Western

776

blotting. (C) BT-20 cells were pretreated with 10 mM NAC for 1 h, and then treated

777

with 40 M PTER or 100 ng/mL TRAIL for 48 h. BT-20 cells were transfected with

778

CHOP siRNA. (D) Twenty-four hours after the transfection, the cells were treated with

779

40 M PTER for 48 h. Whole-cell extracts were prepared and analyzed for CHOP,

780

DR4, and DR5 expression by Western blotting. (E) Twenty-four hours after the 43



781

transfection, the cells were treated with 40 M PTER or 100 ng/mL TRAIL for 48 h.

782

Cells were stained with PI, and the sub-G1 fraction was analyzed using flow cytometry.

783

BT-20 cells were pretreated with (F) 10 μM p38 inhibitor, SB203580 and (G) 20 μM

784

ERK1/2 inhibitor, PD98059 for 1 h and then treated with 40 μM PTER for 48 h.

785

Whole-cell extracts were prepared and analyzed by Western blotting using CHOP

786

antibody. (H) BT-20 cells were pretreated with 10 μM p38 inhibitor, SB203580 or 20

787

μM ERK1/2 inhibitor, PD98059 for 1 h and then treated with 40 M PTER or 100

788

ng/mL TRAIL for 48 h. Cells were stained with PI, and the sub-G1 fraction was

789

analyzed using flow cytometry. Western blot data presented are representative of those

790

obtained in at least three separate experiments. Data are expressed as mean ± SD. **, P

791

< 0.01, ***, P < 0.001.

792

44


Page 44 of 53

Page 45 of 53


Figure 1 (A)

(B)

(C)

(D)

﹡

﹡

﹡

﹡ ﹡ ﹡ ﹡

﹡ ﹡

10 μM

CTL

20 μM

40 μM PTER

(F)

(E)

﹡ sub-G1 (%)

﹡ sub-G1 (%)

﹡ ﹡ ﹡

CTL

12 h

24 h

﹡ ﹡

10 M

CTL

48 h

20 M

40 M

PTER (48 h)

PTER (40 M)

(H)

(G) PTER

( M ) PARP

▼

Cleaved-PARP

▼

C

-actin

▼

-actin

40

▼

Cleaved-PARP

20

▼

PARP

10

▼

C

PTER


12

24

48

(h)


Page 46 of 53

Figure 2 (A)

(B) CTL

TRAIL

PTER

Combine

BT-20

MDA-MB-468

(D)

MDA-MB-468

BT-20

TRAIL (100 ng/mL)

Pro-Caspase-3

▼

Pro-Caspase-9

▼

Pro-Caspase-8

▼

Cleaved-PARP

▼

PARP

▼

PTER (40 M)

-actin

▼

(C)


-

+ -

+

+ +

-

+ -

+

+ +

Page 47 of 53


Figure 3 (A)

(B) PTER

Bcl-2 Bcl-xL

Survivin

▼

XIAP

▼

-actin

(C)

24

48

( h)

▼

▼

▼

▼

cFLIP-S

12

▼

-actin

▼

XIAP

cFLIP-L

▼

Survivin

C

▼

Bcl-xL

( M )

▼

Bcl-2

40

▼

cFLIP-S

20

▼

cFLIP-L

10

▼

C

PTER

(D) PTER

Bid

t-Bid -actin

▼

▼

Bax

▼

C

▼

-actin

( M )

▼

t-Bid

40

▼

Bid

20

▼

Bax

10

▼

C

PTER


12

24

48

(h)

Figure 4


(A)

Page 48 of 53

(B) PTER ( M )

DR4

▼

-actin

(C)

(D) PTER Control Control

12

(h)

24

48

+ 4 +

PTER 40 M

▼

DR5

▼

C

Cell viability (% of control)

-actin

40

▼

DR4

20

▼

DR5

10

▼

C

PTER

**

120 100 80 60 40 20 0

– 1 –

+ 2 + Vector

DR5

– 3 –

TRAIL 100 ng/mL

DR5 shRNA

(E) HL60

-

DR4

▼

-actin

▼

DR5

+

MCF-7

-

-

PC3

-

+

-

+

DU145

+

-

+

A549

-

+

H1299

-

+

DR4

▼

-actin

▼

▼

PTER DR5

+

MDA-MB-231

▼

PTER

SKOV3

(F)

(G) PTER (M)

C DcR1

DcR2

DcR2

ACS Paragon Plus Environment -actin ▼

-actin

▼

DcR1

▼

40

▼

20

▼

10

▼

C

PTER 12

24

48

(h)

Page 49 of 53


Figure 5 (B) ROS production (MFI)

(A)

(C)

PTER

0

10

20

40

40

0

NAC

0

0

0

0

10

10

(D) 0

1

5

10

10

TRAIL (100 ng/mL)

－

＋

－

＋

＋

－

＋

＋

＋

＋

－

PTER (40 μM)

－

－

＋

＋

＋

－

NAC (10 mM)

－

－

－

－

＋

＋

DR5

Cleaved-PARP

▼

-actin

▼

-actin

▼

DR4

▼

PARP

▼

0 －

▼

NAC (mM) PTER (40 μM)

(E)

Sub-G1 (%)

﹡ ﹡

TRAIL (100 ng/mL)

－

＋

－

＋

＋

－

PTER (40 μM)

－

－

＋

＋

＋

－

NAC (10 mM)

－

－

－

－

＋

＋


Figure 6


(A)

(B)

PTER

p38

▼

p-p38

▼

β-actin

▼

p-JNK 1/2

48

(h) SP600125 (20 μmol/L)

DR5

－

－

＋

＋

－

＋

＋

－

▼

PTER (40 μM)

DR4

▼

▼▼

JNK 1/2

24

-actin

▼

▼▼

p-ERK 1/2

12

▼ ▼

ERK 1/2

▼▼

C

Page 50 of 53

(C)

(D)

SB203580 (10 μmol/L)

－

＋

＋

－

＋

＋

－

PD98059 (20 μmol/L) PTER (40 μM)

DR5

(E) NAC

Medium PTER (40 μM)

C

12

24

48

C

12

24

48 (h) ▼ ▼

p-ERK 1/2

▼▼

ERK 1/2

▼

p-p38

▼

p38

▼▼

p-JNK 1/2

▼▼

JNK 1/2

▼

ACS Paragon Plus Environment -actin

▼

▼

-actin

DR4

-actin

▼

DR4

▼

DR5

▼

▼

PTER (40 μM)

－

－

－

＋

＋

－

＋

＋

－

Page 51 of 53


Figure 7 (A)

(B) PTER ( M )

3.58

1

5.89

▼

2.65

4.85

6.75

1

▼

-actin

(C) TRAIL (100 ng/mL)

＋

－

＋

＋

－

PTER (40 μM)

－

－

＋

＋

＋

－

NAC (10 mM)

－

－

－

－

＋

＋

0.95

2.58

2.82

p-eIF2

▼

－

GRP78

1.25

0.85

2.45

2.72

1.35

1.14

▼

1

-actin

1.56

▼

1


24

48

0.84

2.85

3.78

▼

1.85

12

▼

C

GRP78 1

-actin

40

p-eIF2 1

GRP78

20

▼

p-eIF2

10

▼

C

PTER

1.35

3.87

5.78

(h)

Figure 8


(A)

Page 52 of 53

(B) PTER ( M )

C CHOP

▼

40

-actin

▼

-actin

20

▼

CHOP

10

▼

C

PTER

(C)

12

24

(h)

48

(D) －

＋

－

＋

＋

－

PTER (40 μM)

－

－

＋

＋

＋

－

NAC (10 mM)

－

－

－

－

＋

＋

▼

DR5

▼

▼

DR4

-actin

(E)

-

siCHOP

scramble

+

-

+

-

+

－

－

＋

＋

－

＋

＋

－

▼

-actin

▼

DR5

CHOP

▼

DR4

PTER (40 M)

▼

CHOP

Control

▼

TRAIL (100 ng/mL)

(F) Sub-G1 (%)

SB203580 (10 μmol/L)

－

＋

＋

＋

－

－

PTER (40 μM)

－

＋

＋

＋

－

－

siCHOP

－

－

＋

－

＋

－

scramble

－

－

－

＋

－

＋

▼

TRAIL (100 ng/mL)

CHOP

-actin

▼

PTER (40 μM)

PD98059 (20 μmol/L)

CHOP

▼

-actin

▼

PTER (40 μM)

－

－

＋

＋

－

＋

＋

－

Apoptosis (%)

(H)

(G)

TRAIL (100 ng/mL)

－

＋

＋

＋

－

PTER (40 μM)

－

＋

＋

＋

－

－－

SB203580 (10 μmol/L)

－

－

＋

－

＋

－

PD98059 (20 μmol/L) ACS Paragon Plus Environment

－

－

－

＋

－

＋

Page 53 of 53


Phase I and II metabolism

Pterostilbene sulfate


Pterostilbene glucuronide

Pterostilbene Enhances TRAIL-Induced Apoptosis through the

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