1
The anti-tumor efficiency of pterostilbene is promoted with a combined
2
treatment of Fas signaling or autophagy inhibitors in triple negative breast
3
cancer cells
4
Wei-Chih Chen1, Kuei-Yang Hsu2, Chao-Ming Hung3, Ying-Chao Lin4, Ning-Sun
5
Yang5, Chi-Tang Ho6, Sheng-Chu Kuo7, and Tzong-Der Way2,8,9*
6
1
7
8
China Medical University, Taichung, Taiwan
2
9
10
Department of Biological Science and Technology, College of Life Sciences, China
Medical University, Taichung, Taiwan
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11
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The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medicine,
Department of General Surgery, E-Da Hospital, I-Shou University, Kaohsiung,
Taiwan
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Division of Neurosurgery, Buddhist Tzu Chi General Hospital, Taichung Branch,
Taiwan
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5
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
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6
Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA
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7
Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China
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Medical University, Taichung, Taiwan
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8
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University, Taichung, Taiwan
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21
22
9
Institute of Biochemistry, College of Life Science, National Chung Hsing
Department of Health and Nutrition Biotechnology, College of Health Science, Asia
University, Taichung, Taiwan
*Correspondence author:
1
23
Tzong-Der Way, Ph.D.
24
Department of Biological Science and Technology, College of Life Sciences, China
25
Medical University, Taichung, Taiwan
26
No.91 Hsueh-Shih Road, Taichung, Taiwan 40402
27
Tel: +886-4-2205-3366 ext: 2509
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Fax: +886-4-2203-1075
29
E-mail: tdway@mail.cmu.edu.tw
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38
Abstract
39
High expression of vimentin (canonical mesenchymal marker) links with poor
40
prognosis in triple negative breast cancer (TNBC), implying that vimentin may be a
41
potential biomarker in the application of TNBC therapy. Pterostilbene (PTE) shows
42
anti-invasion
43
epithelial-mesenchymal transition (EMT) in TNBC. Here, we showed that PTE
44
decreased vimentin level, but the effect was transient. PTE stimulated Fas signaling
45
which drove EMT by ERK1/2 and GSK3β/β-catenin pathways, supporting Fas
46
signaling induction involved in EMT regulation. PTE also triggered autophagy in
47
TNBC. The treatment of TNBC with 3-MA (autophagy inhibitor) not only sustained
48
PTE-inhibited EMT but also significantly promoted anti-proliferation, supporting that
49
the autophagy played a cyto-protective role and was associated with EMT. Taken
50
together, these data showed that Fas signaling and autophagy accelerated the
51
aggressiveness of TNBC. Inhibition of autophagy or Fas signaling may provide novel
52
targets for TNBC therapy.
activity,
hence,
we
investigated
whether
PTE
inhibited
53
54
Keywords: Triple-negative breast cancer; Pterostilbene; Vimentin; Fas signaling;
55
Autophagy
56
3
57
1 Introduction
58
Triple negative breast cancer (TNBC) is approximately 15-20% of breast cancers
59
which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal
60
growth factor receptor 2 (HER2). Due to high propensity for metastasis and poor
61
prognosis, TNBC is considered the most aggressive of all breast cancers. 1-3 Current
62
treatment of patients with TNBC limited to chemotherapy because of the absence of
63
specific molecular targets.4 However, TNBC standard therapy has been challenging
64
due to the data on TNBC treatment is insufficient. Hence, there is important to
65
establish new approaches to improve the poor prognosis of TNBC therapy.
66
Epithelial-mesenchymal transition (EMT), so called the loss of epithelial
67
phenotype and the acquisition of a mesenchymal characteristic, is associated with
68
tumor invasion and metastasis.5-7 Fas signaling, transforming growth factor (TGF-β),
69
hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast
70
growth factor (FGF) are demonstrated to induce EMT.8-10 A recent study showed that
71
high expression of vimentin (canonical mesenchymal marker) linked with a high
72
histological grade and poor prognosis in TNBC. Vimentin may be a potential
73
biomarker in the application of TNBC prognosis.11,12
74
Fas (APO-1/CD95) belongs to the death receptor (DR) superfamily and is well
75
known as an apoptosis-inducing receptor. Once Fas-ligand (FasL) binding with Fas
4
76
receptor, Fas activates caspase-dependent apoptosis by recruiting the Fas-associated
77
death domain (FADD), caspase 8 and caspase 10. In addition, Fas induces
78
non-apoptotic events in various cell types, such as the proliferation of human T
79
lymphocytes and human fibroblasts, the regulation of proinflammatory cytokines and
80
chemokines, tumor growth, and motility.13-15
81
Autophagy has been recognized to provide cells in adapting to the surroundings
82
via recycling cellular constituents in various physiopathology conditions, including
83
nutrient and growth factor deprivation, stress, microbial infection and diseases.16
84
Using pharmacological or genetic approaches to inhibit autophagy would enhance the
85
shift of cancer cells in apoptosis.17-19 It means that autophagy promoted both cell
86
survival and cell death. Autophagy has also been indicated to mediate tumor
87
progression including the aggressive characteristics of cancer cells, invasion and
88
metastasis.
89
Pterostilbene (trans-3,5-dimethoxy-4 ʹ -hydroxystilbene, PTE) is a natural
90
dimethylated analog of resveratrol from blueberries. The pharmacological properties
91
of PTE are similar to resveratrol such as anti-cancer, anti-inflammation, anti-oxidant,
92
anti-proliferative and analgesic properties in several cancer cells including breast,
93
melanoma, colon, liver, gastric and bladder.20-22 Compared to resveratrol,
94
pterostilbene has higher oral bioavailability (80% versus 20%), higher potential for
5
95
cellular uptake, longer half-life (105 minutes versus 14 minutes).23 In addition, dietary
96
administration of high doses of PTE (up to 3 g/Kg bw/day for 28 days) to mice has
97
been shown to be nontoxic.24,
98
HepG2 cells.26 However, the effects of PTE on TNBC and the underlying mechanisms
99
of EMT remain unclear. In the present study we investigated whether PTE could
100
inhibit EMT in TNBC and the possible regulatory mechanisms. Interestingly, our
101
results demonstrated that EMT inhibition by PTE is associated with decreased in
102
vimentin protein level, but the effect is transient. The novelty of this study is that Fas
103
signaling and autophagy contributed to EMT promotion in TNBC. Hence, a combined
104
treatment of PTE with inhibitors of Fas signaling or autophagy may be a beneficial
105
therapy for TNBC patients.
25
PTE reduced tumor invasion and metastasis of
106
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2 Materials and methods
108
2.1 Cell culture
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The human breast cancer cell lines used in this study were MDA-MB-231 and
110
BT-549. MDA-MB-231 and BT-549 cells were mesenchymal phenotype with high
111
vimentin expression but undetectable E-cadherin expression. BT-549 cells were
112
grown in RPMI 1640 (Invitrogen Corporation, Carlsbad, CA, USA); MDA-MB-231
113
cells were grown in DMEM/F12 (Invitrogen Corporation, Carlsbad, CA, USA).
6
114
Medium was supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine,
115
100 U penicillin and 100 g streptomycin (Invitrogen Corporation, Carlsbad, CA,
116
USA). All cell lines were grown in a humidified incubator at 37 °C under 5% CO2 in
117
air.
118
2.2 Reagents and Antibodies
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PTE was provided by Chi-Tang Ho (Department of Food Science, Rutgers
120
University). The purity of PTE is 96%. PTE was resuspended in DMSO.
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
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3-methyladenine [3-MA (autophagy inhibitor)] and primary antibody -actin were
123
purchased from Sigma Chemical Co. (St. Louis, Mo, USA). PD98059 [an
124
extracellular signal-regulated kinase1/2 (ERK1/2) inhibitor] was purchased from
125
Calbiochem (EMD Chemical, San Diego, CA, USA). NOK-1 (FasL inhibitor) and
126
primary antibody Fas were purchased from Santa Cruz Biotechnology (Santa Cruz,
127
CA, USA). All inhibitors were added into the culture medium 2 h before treatment.
128
Primary antibodies E-cadherin, Zeb1, FasL, Twist, MMP2, MMP9, phospho-GSK3β
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(S2448), β-catenin, Beclin-1, LC3-Ⅰ/Ⅱ and -actin were purchased from Cell
130
Signaling Technology (Beverly, MA, USA). Primary vimentin was purchased from
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Abcam Inc. (Cambridge, MA, USA). Secondary antibodies, HRP-conjugated Goat
tetrazolium
7
bromide
(MTT),
132
anti-Mouse IgG and Goat anti-Rabbit IgG, were obtained from Millipore (Billerica,
133
MA, USA).
134
2.3 Cell viability assay
135
MDA-MB-231 and BT-549 cells were seeded in a 24-well plate (2104 cells/well)
136
overnight, and then were treated with indicated times of PTE with or without
137
NOK-1or NOK-1 only. Cell viability was examined by the MTT assay. Briefly, 80 L
138
MTT solution (2 mg/mL) was added to each well to make a final volume of 500 L
139
and incubated for 1.5 h at 37 °C. The supernatant was aspirated, and the
140
MTT-formazan crystals formed by metabolically viable cells were dissolved in 500
141
L of DMSO. Finally, the absorbance at O.D. 570 nm was detected by enzyme-linked
142
immunosorbent assay (ELISA) reader.
143
2.4 Morphology observation
144
MDA-MB-231 and BT-549 cells (2104) were seeded in each well of a 24-well plate
145
and incubated in a 37°C incubator with 5% CO2 overnight. MDA-MB-231 and
146
BT-549 cells were treated with indicated times of PTE with or without NOK-1or
147
NOK-1 only and then cells incubated at 37°C for 48 h. Representative photographs
148
were taken at 200x magnification using a Nikon TE2000-U inverted microscope.
149
2.5 Quantitative real-time PCR
150
Total RNA was isolated from cultured cells using the TRIzol reagent (Life
8
151
Technologies, Inc., Grand Island, NY, USA) according to the manufacturer’s
152
instructions. Real-time quantitative PCR was performed using the Real-time PCR
153
system 7900 (Applied Biosystems). The primer sequences for E-cadherin, Vimentin
154
and glyceraldehyde-6-phosphoate dehydrogenase (GAPDH; used as internal control)
155
are as follows: GAPDH (5’-AAGGTCGGAGTCAACGGATTTG-3’, 5’-CCATGGGT
156
GGAATCATATTGGAA-3’), E-cadherin (5’-GACCGGTGCAATCTTCAAA-3’, 5′-T
157
TGACGCCGAGAGCTACAC-3’), Vimentin (5’-GACAATGCGTCTCTGGCACGT
158
CTT-3’, 5’-TCCTCCGCCTCCTGCAGGTTCTT-3’). The thermal cycle condition
159
included maintaining the reactions at 50℃for 2min and at 95℃ for 10 min, and then
160
alternating for 40 cycles between 95℃ for 15 s and 60℃ for 1 min. The relative
161
gene expression for each sample was determined using the formula 2 (
162
(GAPDH)- Ct (target))
163
levels.
164
2.6 Western blot analysis
165
Cells were seeded onto a 100-mm culture dishes (1106/dish) containing 10% FBS.
166
Cells were than treated with various agents as indicated in the figure captions. After
167
treatment, the total proteins were extracted by adding 50 L of gold lysis buffer (50
168
mM Tris–HCl, pH 7.4; 1 mM NaF; 150 mM NaCl; 1 mM EGTA; 1 mM
169
phenylmethylsulfonyl floride; 1% NP-40; and 10 mg/ml leupeptin) to the cell pellets.
Ct
) = 2(Ct
, which relected the target gene expression normalized to GAPDH
9
170
Lysate proteins were determined by the Lowry protein assay (Bio-Rad Laboratories).
171
The samples (50 g of proteins) of total cell lysates were resolved by sodium dodecyl
172
sulfate-polyacrylamide gel electrophoresis and (SDS-PAGE), transferred to
173
nitrocellulose membranes. Membranes were blocked with 5% BSA (Sigma, St. Louis,
174
MO, USA) for 1 h at room temperature, and probed with primary antibody for 1.5 h at
175
room temperature or overnight at 4 °C followed by HRP-conjugated appropriated
176
secondary antibodies.
177
2.7 Statistical analysis
178
One-way analysis of variance (ANOVA) was used for the comparison of more than
179
two mean values. Results represent at least two to three independent experiments.
180
Results with a P value less than 0.05 were considered statistically significant. *, p 
181
0.05.
182
183
3 Results
184
3.1 Proliferation-inhibitory effect of PTE on TNBC cells
185
To evaluate the cell proliferation of PTE against mesenchymal phenotype TNBC cells,
186
MDA-MB-231 and BT-549 cells were treated with various concentrations for 48 h
187
and determined using the MTT assay. PTE exhibited the cell growth inhibition in a
188
dose-dependent manner (Figure 1B). This data showed that PTE decreased cell
10
189
proliferation in TNBC cells.
190
3.2 PTE transiently regulated EMT in TNBC cells
191
Previously, PTE has been reported to exert an inhibition of tumor invasion,26
192
suggesting that PTE may inhibit EMT in TNBC cells. To test this hypothesis, we
193
examined PTE effects on morphological changes in MDA-MB-231 and BT-549 cells
194
and representative photographs were taken at 200x magnification using a Nikon
195
TE2000-U inverted microscope. MDA-MB-231 and BT-549 cells exposed to PTE for
196
6 and 12 h displayed morphology changes for towards a classic ‘cobblestone’
197
epithelial morphology. However, both cells reversed to form a more fibroblast-like
198
morphology, a critical marker of EMT (Figure 2A and 2B). MDA-MB-231 and
199
BT-549 cells are mesenchymal phenotype TNBC which lack detectable E-cadherin
200
expression.27, 28 We next examined the effect of PTE on E-cadherin and vimentin
201
protein expression. After treatment with PTE for 6 and 12 h, the E-cadherin protein
202
level was temporary upregulated, but did not detect after 24 h treatment in
203
MDA-MB-231 cells. After treatment with PTE for 6 h, E-cadherin was slight detected
204
and undetected after 12 h and 24 h treatment in BT-549 cells. Inversely, the vimentin
205
protein level was temporary downregulated, however, after 24 h the downregulation
206
of vimentin was restored (Figure 2C and 2D). We also examined the effect of PTE on
207
E-cadherin and Vimentin transcription levels. After treatment with PTE, E-cadherin
11
208
mRNA was detected in MDA-MB-231 and BT-549 cells, respectively. Vimentin
209
mRNA was down-regulated after PTE-treatment in MDA-MB-231 and BT-549 cells,
210
respectively (Figure 2E and 2F). These data showed that PTE regulated transiently
211
E-cadherin and vimentin via transcription level in TNBC cells. We next determined
212
whether PTE regulated EMT through EMT-inducing transcription factors. In
213
MDA-MB-231 cells, Zeb1 protein level was slightly downregulated and recovered
214
after 6 h treatment, however, twist protein level did not alter after PTE treatment. In
215
BT-549 cells, PTE decreased Zeb1 and twist protein levels during 6 and 12 h
216
treatment, however, after 24 h PTE treatment, the expression of Zeb1 and twist were
217
recovered. We also determined whether PTE regulated MMP2 and MMP9 expressions
218
which are related to the invasion and metastasis. The results were similar to Figure 2C
219
and 2D, the expressions of MMP2 and MMP9 were decreased in 6-12 h post-PTE
220
incubation interval but the expressions were reversed after 24 h treatment in
221
MDA-MB-231 cells (Figure 2G) and BT-549 cells (Figure 2G and 2H). These results
222
showed that the inhibition of EMT by PTE is transient in TNBC cells.
223
3.3 Fas signaling contributed to PTE transiently regulated EMT
224
We next explored the mechanism of PTE transiently regulated EMT in TNBC cells.
225
PTE induced apoptosis through Fas/FasL-mediated pathway29 which related with
226
EMT induction,8 suggesting that PTE may regulate EMT through Fas signaling in
12
227
TNBC cells. To test this hypothesis, MDA-MB-231 and BT-549 cells were incubated
228
with PTE for 6, 12, and 24 h and determined Fas protein expression by western blot
229
assay. PTE stimulated the expression of Fas and FasL and the maximum expression
230
were detected at 24 h (Figure 3A) and 12 h (Figure 3B), respectively. To further
231
explore whether Fas signaling mediated in transient regulation of EMT by PTE, we
232
used NOK-1 (FasL inhibitor) to inhibit Fas signaling in TNBC cells. To examine
233
whether NOK-1 is able to block PTE-induced morphological alternation in
234
MDA-MB-231 and BT-549 cells, representative photographs were taken. The
235
morphology of NOK-1 treatment only was triggered from ‘fibroblast-like’ to
236
‘epithelial-like’ in MDA-Mb-231 and BT-549 cells, respectively. Addition of NOK-1
237
to PTE treatment maintained PTE-induced ‘epithelial-like’ morphology (Figure 3C
238
and 3D). It is also interesting to note that cotreatment with NOK-1 and PTE displayed
239
an irregulated appearance suggesting another cellular transformation, such as cell
240
death program, it may be undergoing. We also determined the vimentin expression.
241
After 24 h PTE treatment, the downregulation of vimentin was restored, however,
242
cotreatment with NOK-1 and PTE repressed the restored expression of vimentin in
243
MDA-MB-231 and BT-549 cells (Figure 3E and 3F). Moreover, we investigated
244
whether NOK-1 affect PTE-induced growth inhibition. Pretreated with NOK-1 for 2 h
245
in the absence or presence of PTE, NOK-1 alone had no significant effect on growth
13
246
inhibition but the cell proliferation was significantly inhibited after combined
247
treatment with NOK-1 and PTE (Figure 3G). NOK-1 enhanced PTE-induced growth
248
inhibition in both MDA-MB-231 and BT-549 cells, respectively. These results showed
249
that Fas signaling might contribute to the transient regulation of EMT by PTE and Fas
250
signaling inhibition increased the effect of PTE-induced growth inhibition.
251
3.4 Activation of ERK1/2 related with PTE transiently regulated EMT
252
ERK1/2 was activated during Fas-induced EMT in gastrointestinal cancer,8 suggesting
253
that the transient regulation of EMT by PTE might include ERK1/2 activation. To test
254
this hypothesis, MDA-MB-231 and BT-549 cells were incubated with PTE for 15, 30,
255
60 and 90 min and determined p-ERK1/2 and ERK1/2 protein expressions by western
256
blot assay. After PTE treatment, p-ERK1/2 was activated in MDA-MB-231 and
257
BT-549 cells (Figure 4A and 4B). Moreover, when both cells were pretreated with
258
PD98059 (10 M) for 2 h before PTE treatment, the cotreatment repressed the
259
restored expression of vimentin in MDA-MB-231 and BT-549 cells (Figure 4C and
260
4D). These results showed that ERK1/2 activation was acquired in PTE transiently
261
regulated EMT in TNBC cells.
262
3.5 Glycogen synthase kinase-3 beta (GSK3β) and -catenin involved in PTE
263
transiently regulated EMT
264
GSK3β inhibition was phosphorylated by ERK1/2.30 ERK1/2 activation was acquired
14
265
in PTE transiently regulated EMT, suggesting that GSK3β and β-catenin might be
266
included in PTE transiently regulated EMT. To test this hypothesis, MDA-MB-231
267
and BT-549 cells were incubated with PTE for 6, 12 and 24 h and determined the
268
protein levels of p-GSK3β and β-catenin. The protein levels of p-GSK3β and
269
β-catenin were upregulation in MDA-MB-231 and BT-549 cells (Figure 5A and 5B).
270
We pretreated with NOK-1 for 2 h in the absence or presence of PTE to confirm
271
whether GSK3β inactivation was regulated by Fas signaling. The accumulation of
272
pGSK3β and β-catenin were suppressed by NOK-1 in the presence of PTE for 24 h
273
(Figure 5C and 5D). These results showed that PTE transiently regulated EMT might
274
be through the GSK3β/β-catenin pathway in TNBC cells.
275
3.6 Autophagy induction involved in PTE transiently regulated EMT
276
PTE treatment induced autophagy at an earlier stage and then apoptosis at a later stage
277
in human bladder cancer cells.31 We next determined whether PTE treatment induced
278
autophagy in TNBC cells. The autophagy markers (LC3-Ⅰ/Ⅱ) and autophagy-related
279
protein (Beclin-1) were determined by western blotting. PTE caused the conversion of
280
LC3 from LC3-Ⅰ to LC3-Ⅱ and increased Beclin-1 protein level in MDA-MB-231
281
and BT-549 cells (Figure 6A and Figure 6B). These results indicated that PTE induced
282
growth inhibition through autophagy. Previous studies suggested that autophagy
283
induced cancer cell EMT and invasion,32,33 suggesting that autophagy induction might
15
284
be involved in PTE transiently regulated EMT. MDA-MB-231 and BT-549 cells were
285
pretreated with 3-MA (autophagy inhibitor) for 2 h in the absence or presence of PTE.
286
After 24 h PTE treatment, the downregulation of vimentin was restored, however,
287
cotreatment with 3-MA and PTE repressed the restored expression of vimentin in
288
MDA-MB-231 and BT-549 cells (Figure 6C and 6D). We further examined whether
289
NOK-1 affected PTE-induced autophagy. MDA-MB-231 and BT-549 cells were
290
pretreated with NOK-1 for 2 h in the absence or presence of PTE. NOK-1 repressed
291
PTE-induced the conversion of LC3 from LC3-Ⅰ to LC3-Ⅱ(Figure 6E and 6F).
292
Moreover, we investigated the role of autophagy in PTE caused growth inhibition.
293
Pretreated with 3-MA for 2 h in the absence or presence of PTE, 3-MA alone had no
294
significant effect on growth inhibition but the cell proliferation was significantly
295
enhanced after combined treatment with 3-MA and PTE (Figure 6G). These results
296
showed that autophagy induction by PTE played a cyto-protective and an EMT
297
regulative role in TNBC cells. Additionally, NOK-1 decreased the induction of a
298
protective form of autophagy by PTE.
299
300
4 Discussion
301
PTE is considered a chemopreventive agent and exhibits antiproliferative effects on
302
several types of cancer cells through various pathways including PI3K, MAPK,
16
303
adenosine monophosphate activated protein kinase (AMPK), apoptosis, autophagy,
304
metastatic activity inhibition, ROS, and cytosolic Ca2+ overload.34,35 PTE regulated
305
NO accumulation which related to intracellular adhesion molecules expression,36
306
leading to the inhibition of the potential metastasis in mouse B16 melanoma F10
307
cells.37,38 PTE significantly inhibited MMP-9 gene expression through suppression of
308
the MAPK, PI3K/NF-κB and AP-1-signaling pathways and resulted in blocking
309
invasion and metastasis of human hepatocellular carcinoma cells.26
310
EMT was related to cancer stem cells (CSCs) which contributed to poor
311
prognosis, drug-resistance, and metastasis of cancers. CD44 and CD24 are two of
312
widely identified and isolated breast cancer stem cell markers.39 PTE effectively
313
inhibited tumor-associated macrophages (TAMs)-enriched CD44+/CD24- percentage
314
via modulating NF-B/microRNA 448 circuit.40 Our data showed that PTE transiently
315
inhibited EMT during 6 and 12 h (Figure 2). In addition to apoptosis induction, Fas
316
signaling activated multiple signaling pathways including NF-κB, MAPK and
317
PI3K/AKT to induce non-apoptotic events such as growth regulation, EMT, migration
318
and invasion in cancer cells.41-45 PTE has been demonstrated to induce apoptosis
319
through mitochondrial and Fas/FasL pathway in human gastric adenocarcinoma
320
cells.29 We question whether PTE regulated EMT by modulating Fas signaling. Our
321
results showed that PTE activated Fas/FasL pathway (Figure 3A and 3B) and induced
17
322
cell death by apoptosis and autophagy in TNBC cells (Figure 1B, Figure 6A and 6B).
323
Furthermore, preventing Fas signaling held the ability of PTE-decreased EMT (Figure
324
3E and 3F), suggesting that Fas signaling contributed to the promotion of EMT.
325
ERK1/2 inhibition and loss of Snail or twist expression suppressed Fas
326
signaling-induced EMT and motility, suggesting ERK1/2 activation, Snail and twist
327
were essential for Fas signaling-induced EMT and motility promotion.8 Similarly, our
328
data showed that ERK1/2 inhibition inhibited Fas-induced EMT during PTE treatment
329
(Figure 4).
330
The serine/threonine kinase, GSK3β is inactivated through various signal
331
mechanisms such as PI3K/AKT and ERK1/2 pathways and regulates cell cycle
332
progression, anti-apoptosis, invasion and various oncogenic transcriptional factors
333
such as vimentin, uPAR and metalloproteinases. Inactivation of GSK3β results in
334
stabilization of β-catenin which translocates from cytosol into nucleus and then binds
335
to the T cell-specific transcription factor (TCF)/ lymphoid enhancer-binding factor
336
(LEF) to alter the target genes and then to induce EMT and cancer metastasis.46-48
337
FasL treatment inactivated GSK3β which leaded to accumulated Snail and β-catenin
338
in nuclear and, thus, E-cadherin was downregulated and vimentin and MMP9 were
339
upregulated.49 Similarly, our data showed that PTE regulated EMT by Fas signaling
340
through modulating GSK3β and β-catenin (Figure 5).
18
341
Autophagy induces cyto-protective role in breast cancer cells50 and promotes invasion
342
by inducing EMT through TGF-β/Smad3 signaling modulation in HCC cells,
343
indicating that autophagy accelerates the invasion of cancer cells.33,51 Autophagy
344
induction is known to relate with T-cell-mediated immune surveillance and EMT.52
345
Our data showed that autophagy induction by PTE played a cyto-protective role
346
(Figure 6G) and autophagy inhibition by 3-MA maintained PTE-inhibited EMT
347
(Figure 6C and 6D), suggesting the regulation of EMT by PTE related with autophagy.
348
Similarly, NOK-1 increased the effect of PTE-inhibited anti-proliferation may relate
349
with protective autophagy inhibition (Figure 3G). It may suggest that Fas signaling
350
inhibition increased PTE-triggered growth inhibition via switching autophagy to
351
apoptosis. Our results suggest that PTE-induced a protective form of autophagy
352
against cell death and the inhibition of autophagy could enhance PTE-caused cell
353
death through Fas signaling inhibition.
354
In summary, we demonstrated that PTE transiently inhibited EMT due to Fas
355
signaling stimulation and autophagy induction in TNBC cells. The broader
356
implication of this study supports that Fas signaling and autophagy enhance
357
aggressiveness in TNBC and could lead to the development of target therapy. Further
358
studies will focus on whether EMT regulation by PTE was similar to other cancer cell
359
types.
19
360
361
Abbreviations
362
TNBC, triple negative breast cancer; EMT, epithelial-mesenchymal transition; PTE,
363
pterostilbene; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
364
diphenyl tetrazolium bromide; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide;
365
ECL; enhanced chemiluminescence; ER, estrogen receptor; HER2, epidermal growth
366
factor receptor-2; PR, progesterone receptor; 3-MA, 3-Methyladenine; DMSO,
367
dimethyl sulfoxide; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient
368
Mixture F12; RPMI 1640, Roswell Park Memorial Institute (RPMI) 1640.
369
370
371
Acknowledgement
372
This study is supported in part by Taiwan Ministry of Health and Welfare Clinical
373
Trial and Research Center of Excellence (DOH102-TD-B-111-004)
374
375
376
377
378
20
379
References
380
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Figure legends
487
Figure 1. The anti-proliferation activities of PTE against TNBC cells. (A)
488
Structure of PTE. (B) MDA-MB-231 and BT-549 cells were treated with various
26
489
concentrations (5, 10, 20, 40 and 80 M) of PTE for 48 h. Cell proliferation was
490
assessed using the MTT assay. The percentage of cell growth inhibition was
491
calculated by the absorption of control cells as 100%. *p  0.05 compared with
492
control group. a and c, MDA-MB-231; b and d, BT-549.
493
Figure 2. PTE transiently regulated EMT in TNBC cells. MDA-MB-231 and
494
BT-549 cells were treated with 80 M PTE for 6, 12 and 24 h. (A), (B) Phase-contrast
495
images of MDA-MB-231 and BT-549 cells. The sub-confluent cultures were shown
496
the morphological differences. (C), (D) Cells were then harvested and lysed for the
497
detection of E-cadherin and vimentin and β-actin. (E), (F) Cells were then harvested
498
and RNA was isolated. Real-time PCR was performed using primers directed against
499
E-cadherin, vimentin and GAPDH. (G), (H) Cells were then harvested and lysed for
500
the detection of Zeb1, twist, MMP2, MMP9 and β-actin. Western blot data presented
501
are representative of those obtained in at least three separate experiments. The values
502
below the figures represent change in protein expression of the bands normalized to
503
β-actin.
504
Figure 3. PTE transiently regulated EMT through Fas-signaling in TNBC cells.
505
(A) MDA-MB-231 and (B) BT-549 cells were incubated with PTE (80 M) for 6, 12
506
and 24 h. Cells were then harvested and lysed for the detection of Fas and FasL and
27
507
β-actin. MDA-MB-231 and BT-549 cells were incubated PTE (80 M) for 24 h or
508
pretreated with NOK-1 (10 g/mL) before PTE treatment. Phase-contrast images of
509
(C) MDA-MB-231 and (D) BT-549 cells. The sub-confluent cultures were shown the
510
morphological differences. (E), (F) Cells were then harvested and lysed for the
511
detection of vimentin and β-actin. Western blot data presented are representative of
512
those obtained in at least three separate experiments. The values below the figures
513
represent change in protein expression of the bands normalized to β-actin. (G) Cell
514
proliferation was assessed using the MTT assay. The percentage of cell growth
515
inhibition was calculated by the absorption of control cells as 100%. *p  0.05
516
compared with control group. a, MDA-MB-231; b, BT-549.
517
518
Figure 4. Fas-mediated activation of ERK1/2 corrected with PTE transiently
519
regulated EMT. (A) MDA-MB-231 and (B) BT-549 cells were incubated with PTE
520
(80 M) for 15, 30, 60 and 90 min. Cells were then harvested and lysed for the
521
detection of ERK1/2, p-ERK1/2 and β-actin. (C) MDA-MB-231 and (D) BT-549 cells
522
were incubated with PTE (80 M) for 24 h or pretreated with PD98059 (10 M).
523
Cells were then harvested and lysed for the detection of vimentin and β-actin. Western
524
blot data presented are representative of those obtained in at least three separate
525
experiments. The values below the figures represent change in protein expression of
28
526
the bands normalized to β-actin.
527
Figure 5. Fas-induced glycogen synthase kinase-3 beta (GSK3) inhibition
528
involved in PTE transiently regulated EMT. (A) MDA-MB-231 and (B) BT-549
529
cells were incubated with PTE (80 M) for 6, 12 and 24 h. Cells were then harvested
530
and lysed for the detection of p-GSK3, -catenin and β-actin. (C) MDA-MB-231
531
and (D) BT-549 cells were incubated with PTE (80 M) for 24 h or pretreated with
532
NOK-1 (10 g/mL). Cells were then harvested and lysed for the detection of
533
p-GSK3, -catenin and β-actin. Western blot data presented are representative of
534
those obtained in at least three separate experiments. The values below the figures
535
represent change in protein expression of the bands normalized to β-actin.
536
Figure 6. PTE-induced autophagy participated in PTE transiently regulated
537
EMT. (A) MDA-MB-231 and (B) BT-549 cells were incubated with PTE (80 M) for
538
6, 12 and 24 h. Cells were then harvested and lysed for the detection of LC3Ⅰ/Ⅱ,
539
Beclin-1 and β-actin. (C) MDA-MB-231 and (D) BT-549 cells were incubated with
540
PTE (80 M) for 24 h or pretreated with 3-MA (5 mM). Cells were then harvested
541
and lysed for the detection of vimentin and β-actin. MDA-MB-231(E) and BT-549 (F)
542
cells were incubated PTE (80 M) for 24 h or pretreated with NOK-1 (10 g/mL)
543
before PTE treatment. Cells were then harvested and lysed for the detection of LC3Ⅰ
544
/Ⅱ and β-actin. Western blot data presented are representative of those obtained in at
29
545
least three separate experiments. The values below the figures represent change in
546
protein expression of the bands normalized to β-actin. (G) MDA-MB-231 and BT-549
547
cells were incubated with PTE (80 M) for 24 h or pretreated with 3-MA (5 mM).
548
Cell proliferation was assessed using the MTT assay. The percentage of cell growth
549
inhibition was calculated by the absorption of control cells as 100%. *p  0.05
550
compared with control group. a, MDA-MB-231; b, BT-549.
30
551
552
31
553
32
554
33
555
34
556
35
557
36