1
Brain-derived neurotrophic factor increases vascular endothelial growth factor
2
expression and enhances angiogenesis in human chondrosarcoma cells
3
4
Chih-Yang Lin1#, Shih-Ya Hung2,3#, Hsien-Te Chen4,5,6, Hsi-Kai Tsou6,7,8,
5
Yi-Chin Fong4,5, Shih-Wei Wang9, Chih-Hsin Tang1,10,11*
6
1
7
Graduate Institute of Basic Medical Science, China Medical University, Taichung,
Taiwan
8
2
9
Epigenome Research Center, China Medical University Hospital, Taichung Taiwan
3
10
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China
Medical University, Taichung, Taiwan
11
4
12
School of Chinese Medicine, College of Chinese Medicine, China Medical
University, Taichung, Taiwan
13
5
14
Department of Orthopedic Surgery, China Medical University Hospital, Taichung,
Taiwan
15
6
16
Department of Materials Science and Engineering, Feng Chia University,Taichung,
Taiwan
17
7
18
Department of Neurosurgery, Taichung Veterans General Hospital, Taichung,
Taiwan
19
8
20
Department of Early Childhood Care and Education, Jen-Teh Junior College of
Medicine, Nursing and Management, Miaoli County, Taiwan
21
9
22
Department of Medicine, Mackay Medical College, New Taipei City, Taiwan
10
23
Department of Pharmacology, School of Medicine, China Medical University,
Taichung, Taiwan
24
11
25
Department of Biotechnology, College of Health Science, Asia University,
Taichung, Taiwan
26
27
28
#
These authors contributed equally to this work.
29
1
30
*Corresponding author
31
Chih-Hsin, Tang PhD
32
Graduate Institute of Basic Medical Science, China Medical University
33
No. 91, Hsueh-Shih Road, Taichung, Taiwan
34
Tel: (886) 4-22052121 Ext. 7726. Fax: (886) 4-22333641.
35
E-mail: chtang@mail.cmu.edu.tw
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2
37
Abstract
38
Chondrosarcomas are a type of primary malignant bone cancer, with a potent
39
capacity for local invasion and distant metastasis. Brain-derived neurotrophic factor
40
(BDNF) is commonly upregulated during neurogenesis. The aim of the present study
41
was to examine the mechanism involved in BDNF-mediated vascular endothelial
42
growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells.
43
Here, we knocked down BDNF expression in chondrosarcoma cells and assessed their
44
capacity to control VEGF expression and angiogenesis in vitro and in vivo. We found
45
knockdown of BDNF decreased VEGF expression and abolished chondrosarcoma
46
conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in
47
vivo in the chick chorioallantoic membrane and Matrigel plug nude mouse models. In
48
addition, in the xenograft tumor angiogenesis model, the knockdown of BDNF
49
significantly reduced tumor growth and tumor-associated angiogenesis. BDNF
50
increased VEGF expression and angiogenesis through the TrkB receptor, PLCγ,
51
PKCα, and the HIF-1α signaling pathway. Finally, we analyzed samples from
52
chondrosarcoma patients by immunohistochemical staining. The expression of BDNF
53
and VEGF protein in 56 chondrosarcoma patients was significantly higher than in
54
normal cartilage. In addition, the high level of BDNF expression correlated strongly
55
with VEGF expression and tumor stage. Taken together, our results indicate that
56
BDNF increases VEGF expression and enhances angiogenesis through a signal
57
transduction pathway that involves the TrkB receptor, PLCγ, PKCα, and the HIF-1α.
58
Therefore, BDNF may represent a novel target for anti-angiogenic therapy for human
59
chondrosarcoma.
60
61
62
Keywords: BDNF; TrkB; Chondrosarcoma; VEGF; Angiogenesis.
63
Running title: BDNF induces angiogenesis in chondrosarcoma.
64
3
65
1. Introduction
66
Chondrosarcomas are formed by the abnormal proliferation of cartilage. It
67
accounts for about 26% of bone cancer cases worldwide. Usually occurring in males
68
between 10 and 80 years of age, the tumors generally appear on the scapula, sternum,
69
ribs, or pelvis [1]. Clinically, surgical resection remains the primary mode of therapy
70
for chondrosarcoma. Chondrosarcoma can easily metastasize to other organs, such as
71
the lung and liver [2-4]. When distant metastasis occurs, the patient has a poor
72
prognosis. Because there is currently no effective adjuvant therapy, the development
73
of novel molecular mechanisms is very important for the treatment of
74
chondrosarcoma metastasis [5].
75
Tumor metastasis includes many functions, such as proliferation [6], migration
76
[7], invasion [8], and angiogenesis [9]. Among these, the most important step is
77
angiogenesis because when the tumor reaches a certain size, it requires more nutrients
78
and oxygen to support its growth. Unfortunately, the tumor can metastasize to remote
79
organs through these new blood vessels. Vascular endothelial growth factor (VEGF),
80
a protein that plays important roles in embryonic development, wound healing, and
81
tumor angiogenesis [10]. In addition, VEGF has been associated with angiogenesis in
82
breast [11], lung [12], pancreatic [13], and cervical carcinomas [14]. Many drugs
83
reduce cancer growth and metastasis by inhibiting VEGF expression [15, 16].
84
Therefore, VEGF is a critical target in cancer therapy and metastasis.
85
Brain-derived neurotrophic factor (BDNF) is a small basic protein. It is a
86
member of the neurotrophin family of growth factors, and it is a physiologically
87
important nerve growth factor [17]. BDNF and its specific receptor, tropomyosin
88
related kinase B (TrkB), play key roles in neural development, and some studies have
89
suggested a role for BDNF in cancer cell proliferation [18], survival [19], metastasis
90
[2], and angiogenesis [20]. BDNF promotes metastasis and angiogenesis through
91
VEGF-dependent activation in several tumor types, including ovarian cancers [21],
92
hepatocellular carcinoma [22], oral squamous cell carcinoma [23], multiple myeloma
4
93
[24], and neuroblastoma [25]. Therefore, BDNF could be a novel target for tumor
94
metastasis and angiogenesis.
95
Because the prognosis of patients with distant metastasis of chondrosarcoma is
96
very poor [26], the development of an anti-angiogenic and anti-metastatic therapy
97
could be useful for these patients. BDNF promotes chondrosarcoma metastasis
98
through the upregulation of integrin 5 and matrix metalloproteinase-1 [2, 27].
99
However, the role of BDNF in VEGF expression and angiogenesis in human
100
chondrosarcoma is largely unknown. Here, we report that BDNF promotes VEGF
101
expression in human chondrosarcoma via the activation of the TrkB receptor, PLCγ,
102
PKCα, and HIF-1α signaling pathways. In addition, the knockdown of BDNF
103
significantly reduced VEGF expression and angiogenesis in vivo. Our results indicate
104
that BDNF plays an important role in the metastasis and angiogenesis of human
105
chondrosarcoma. Therefore, BDNF might be a new therapeutic target for the
106
treatment of chondrosarcoma metastasis.
107
108
109
2. Experimental Section
110
2.1. Materials
111
Rabbit polyclonal antibodies specific for p-PLCγ and p-PKCα were purchased
112
from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies
113
specific for BDNF, TrkB, PLCγ, PKCα, β-actin, and PCNA were purchased from
114
Santa Cruz Biotechnology (Santa Cruz, CA, USA). HIF-1α, VEGF, and CD31
115
antibodies were purchased from Abcam (Cambridge, MA, USA). GF109203X and
116
Gö6976 were purchased from Enzo Biochem Inc (New York, NY, USA).
117
Recombinant human BDNF and VEGF were purchased from R&D Systems
118
(Minneapolis, MN, USA). DMEM, α-MEM, fetal bovine serum (FBS), and all the
119
other cell culture reagents were purchased from Gibco-BRL life technologies (Grand
120
Island, NY, USA). The PKCα dominant negative mutant was provided by Dr. V.
121
Martin (Louis Pasteur de Strasbourg University, France). The HIF-1 dominant
5
122
negative mutant and pHRE-luciferase construct were provided by Dr. W.M. Fu
123
(National Taiwan University, Taiwan). The pSV-β-galactosidase vector and luciferase
124
assay kit were purchased from Promega (Madison, WI, USA). All other chemicals
125
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
126
127
2.2. Cell culture
128
The human chondrosarcoma cell line (JJ012) was kindly provided by Dr. Sean P.
129
Scully (University of Miami School of Medicine, Miami, FL, USA) [28]. We used
130
lentiviral transfection to transfect BDNF-shRNA or control-shRNA plasmids into
131
JJ012 cells. After 48 h, the medium was replaced. Subsequently, 10 µg/mL
132
puromycin (Life Technologies) was used to select stable transfectants. Thereafter, the
133
selection medium was replaced every 3 days. After 2 weeks of selection in
134
puromycin, clones of resistant cells were isolated. All JJ012 cells were cultured in
135
Dulbecco’s modified Eagle’s medium (DMEM)/-MEM supplemented with 10%
136
FBS. These cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
137
The human endothelial progenitor cells (EPCs) used in this study were approved
138
by the Institutional Review Board of Mackay Medical College, New Taipei City,
139
Taiwan (reference number: P1000002), and all subjects gave informed written
140
consent before enrollment in this study. After collected peripheral blood (80 ml) from
141
healthy donors, the peripheral blood mononuclear cells (PBMCs) were fractionated
142
from other blood components by centrifugation on Ficoll-Paque plus (Amersham
143
Biosciences, Uppala, Sweden) according to the manufacturer’s instructions.
144
CD34-positive progenitor cells were obtained from the isolated PBMCs using CD34
145
MicroBead kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch
146
Gladbach, Germany). The maintenance and characterization of CD34-positive EPCs
147
were performed as described previously [29, 30]. Briefly, human CD34-positive EPCs
148
were maintained and propagated in MV2 complete medium consisting of MV2 basal
149
medium and growth supplement (PromoCell, Heidelberg, Germany) supplemented
150
with 20% FBS (HyClone, Logan, UT, USA). The cultures were seeded onto 1%
6
151
gelatin-coated plastic ware and maintained at 37°C in a humidified atmosphere of 5%
152
CO2 [30].
153
154
2.3. Western blot analysis
155
Cellular lysates were prepared as described previously [31]. Proteins were
156
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
157
transferred to Immobilon polyvinyl difluoride membranes (Immobilon P, Millipore).
158
The blots were blocked with 4% non-fat milk for 1 h at room temperature and then
159
probed with rabbit anti-human antibodies against p-PLCγ, PLCγ, p-PKCα, PKCα,
160
HIF-1α, VEGF, β-actin, PCNA, BDNF, or TrkB (1:1,000) for 1 h at room
161
temperature. After 3 washes, the blots were subsequently incubated with a goat
162
anti-rabbit or goat anti-mouse peroxidase-conjugated secondary antibody (1:1,000)
163
for 1 h at room temperature. The blots were visualized by enhanced
164
chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY,
165
USA).
166
167
2.4. Quantitative real-time polymerase chain reaction
168
Total RNA was extracted from chondrosarcoma cells by using a TRIzol kit
169
(MDBio Inc., Taipei, Taiwan). The reverse transcription reaction was performed
170
using 1 g of total RNA that was reverse transcribed into cDNA using oligo(dT)
171
primers [32]. Quantitative real-time PCR (qPCR) analysis was performed using
172
Taqman® One-Step RT-PCR Master Mix (Applied Biosystems, CA). Two microliters
173
of cDNA template was added to each 25-μL reaction with sequence-specific primers
174
and Taqman® probes. The sequences for all target gene primers and probes were
175
purchased commercially. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was
176
used as an endogenous control to normalize expression data (Applied Biosystems).
177
qPCR assays were carried out in triplicate in a StepOnePlus sequence detection
178
system. The cycling conditions were as follows: initial 10-min polymerase activation
179
at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. To calculate the
7
180
cycle number at which the transcript was detected (CT), the threshold was set above
181
the non-template control background and within the linear phase of target gene
182
amplification.
183
184
2.5. Transient transfection and reporter gene assay
185
Human chondrosarcoma cells were plated in 12-well dishes. DNA and
186
Lipofectamine 2000 (LF2000; Invitrogen) were premixed for 20 min and then applied
187
to the cells. Twenty-four hours after transfection, the cells were incubated with the
188
indicated agents. Cell extracts were prepared and used for measuring the luciferase
189
and β-galactosidase activities.
190
ON-TARGETplus siRNAs of PLCγ, PKCα, HIF-1α, and control were purchased
191
from Dharmacon Research (Lafayette, CO, USA). Transient transfection of siRNAs
192
was carried out using DharmaFECT1 transfection reagent. The siRNA (100 nM) was
193
formulated with DharmaFECT1 transfection reagent according to the manufacturer's
194
instruction.
195
196
2.6. Preparation of conditioned medium (CM)
197
Human chondrosarcoma cell line (JJ012 cells) was plated in 6-well dishes and
198
grown to confluence. The culture medium was exchanged with serum-free
199
DMEM/α-MEM medium. CM were collected 2 days after the change of media and
200
stored at -20°C until use. In the series of experiments, JJ012 cells were pretreated for
201
30 min with inhibitors, including K252a, U73122, U73343, GF109203X, Gö6976,
202
HIF-1 inhibitor, or vehicle control (0.1% DMSO), followed by treatment with BDNF
203
for 24 h to prevent signaling via the BDNF pathway.
204
205
2.7. Migration assay
206
Migration activity was measured using a Transwell assay (Costar, NY; pore size,
207
8-μm). Approximately 1  104 cells were added to the upper chamber in 200 L of
208
10% FBS MV2 complete medium. The lower chamber contained 150 l MV2
8
209
complete medium and 150 l CM. The plates were incubated for 24 h at 37°C in 5%
210
CO2, and cells were fixed in 3.7% formaldehyde solution for 15 min and stained with
211
0.05% crystal violet in PBS for 15 min. Cells on the upper side of the filters were
212
removed with cotton-tipped swabs, and the filters were washed with PBS. Cells on the
213
underside of the filters were examined and counted under a microscope. Each clone
214
was plated in triplicate for each experiment, and each experiment was repeated at least
215
3 times.
216
217
2.8. ELISA assay
218
Human chondrosarcoma cells were cultured in 24-well plates. Cells were
219
incubated in a humidified incubator at 37°C for 24 h. To examine the downstream
220
signaling pathways involved in BDNF treatment, cells were pretreated with various
221
inhibitors for 30 min before the addition of BDNF (50 ng/mL). After incubation, the
222
medium was removed and stored at -80°C until the assay was performed. VEGF in
223
the medium was assayed using a VEGF enzyme immunoassay kit (Peprotech,
224
Offenbach, Germany), according to the procedure described by the manufacturer.
225
226
2.9. Chromatin immunoprecipitation assay
227
Chromatin immunoprecipitation analysis was performed as described previously
228
[33]. DNA immunoprecipitated with an anti-HIF-1α Ab was purified and extracted
229
with phenol-chloroform. The purified DNA pellet was subjected to PCR. PCR
230
products were resolved by 2% agarose gel electrophoresis and visualized under UV
231
light.
232
5′-CCAAGTTTGTGGAGCTGA-3′ were utilized for amplification across the VEGF
233
promoter region [34].
The
primers
5′-CCTTTGGGTTTTGCCAGA-3′
and
234
235
2.10. Tube formation assay
236
Matrigel (BD Biosciences, Bedford, MA, USA) was dissolved overnight at 4°C
237
and 48-well plates were prepared with 100 μL Matrigel in each well, after coating and
9
238
incubating at 37°C overnight. EPCs (3 × 104) were cultured in 200 μL media which
239
containing 50% MV2 medium and 50% CM. After 6 h of incubation at 37°C, EPC
240
tube formation was assessed with a photomicroscope, and each well was
241
photographed at 200× magnification. Tube branches and total tube length were
242
calculated using MacBiophotonics Image J software.
243
244
2.11. Chick chorioallantoic membrane (CAM) assay
245
Fertilized chicken eggs were incubated at 38°C with 80% humidity. A small
246
window was made in the shell on day 3 of chick embryo development under aseptic
247
conditions. The window was resealed with adhesive tape and eggs were returned to
248
the incubator until day 3 of chick embryo development. On day 7, CM from
249
JJ012/control-shRNA or JJ012/BDNF-shRNA cells (2 × 104 cells) deposited in the
250
center of the CAM. At 11 days, CAMs were collected for microscopy and
251
photographic documentation. Angiogenesis was quantified by counting the number of
252
blood vessel branches; at least 10 viable embryos were tested for each treatment. All
253
animal work was done in accordance with a protocol approved by the China Medical
254
University (Taichung, Taiwan) Institutional Animal Care and Use Committees.
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256
2.12. In vivo tumor xenograft study
257
Four-week-old male CB17-SCID mice were randomized into 2 groups.
258
Experimental cells growing exponentially were implanted into 10 SCID mice by
259
subcutaneous (SC) injection of 2 × 106 cells (JJ012/control-shRNA or
260
JJ012/BDNF-shRNA resuspended in 200 μL of containing 50% serum-free
261
DMEN/α-MEM and 50% Matrigel) injected into the right flank. Mice were observed
262
at 5 weeks by the Xenogen IVIS system and images were captured 10 min after
263
D-luciferin injection with a 60-s exposure using a CCD camera. Mice were
264
euthanized by subjecting them to CO2 inhalation and the tumors were removed and
265
fixed in 10% formalin, and their volume and weight were measured. The hemoglobin
266
content was assayed using the Drabkin’s reagent kit (Sigma).
10
267
268
2.13. Matrigel plug assay
269
Matrigel plug angiogenesis assay was adapted from a previously described assay
270
[35]. Four-week-old male nude mice were randomized into 3 groups: serum-free
271
medium, JJ012/control-shRNA CM, or JJ012/BDNF-shRNA CM resuspended with
272
Matrigel. Mice were subcutaneously injected with 300 μL of Matrigel. After 7 days,
273
Matrigel pellets were harvested, partially fixed with 4% formalin, embedded in
274
paraffin, and subsequently processed for immunohistochemistry staining for vessel
275
marker CD31.
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2.14. Immunohistochemistry (IHC)
278
The human chondrosarcoma tissue array was purchased from Cybrdi (Rockville,
279
MD, USA) and Biomax (Rockville, MD, USA). The 8 cases for normal cartilage, 26
280
cases for grade I chondrosarcoma, 12 cases for grade II chondrosarcoma, and 18 cases
281
for grade III chondrosarcoma); (Grade I chondrosarcoma grows relatively slowly, has
282
cells whose histological appearance is quite similar to cells of normal cartilage, and
283
have much less aggressive invasive. Grades 2 and 3 are increasingly faster-growing
284
cancers, with more varied and abnormal-looking cells, and are much more likely to
285
infiltrate surrounding tissues). The tissues were placed on glass slides, rehydrated, and
286
incubated in 3% hydrogen peroxide to block the endogenous peroxidase activity.
287
After trypsinization, the sections were blocked by incubation in 3% bovine serum
288
albumin in PBS. The primary polyclonal antibodies, rabbit anti-human BDNF and
289
VEGF, were applied to the slides at a dilution of 1:200 and incubated at 4°C
290
overnight. After being washed 3 times in PBST, the samples were treated with goat
291
anti-rabbit IgG biotin-labeled secondary antibodies at a dilution of 1:50. Bound
292
antibodies were detected with an ABC kit (Vector Laboratories). The slides were
293
stained with chromogen diaminobenzidine, washed, counterstained with Delafield's
294
hematoxylin, dehydrated, treated with xylene, and mounted.
295
11
296
2.15. Statistical analysis
297
Data were presented as mean ± standard error of the mean. Statistical analysis of
298
comparisons between 2 samples was performed using Student’s t test. Statistical
299
comparisons of more than 2 groups were performed using 1-way analysis of variance
300
with Bonferroni’s post-hoc test. In all cases, p < 0.05 was considered significant.
301
302
303
3. Results
304
3.1. Knockdown of BDNF decreases VEGF expression in vitro and angiogenesis
305
in vivo.
306
BDNF has cited to promote the metastasis of human chondrosarcoma cells [2,
307
27]. However, the effects of BDNF on VEGF expression and angiogenesis in
308
chondrosarcoma
309
BDNF-shRNA-expressing chondrosarcoma cells (JJ012/BDNF-shRNA) to examine
310
the role of BDNF in VEGF expression and angiogenesis. The overexpression of
311
BDNF-shRNA reduced BDNF and VEGF expression, as shown in Figures 1A-C, by
312
using qPCR, Western blotting, and ELISA assays (~35%, ~55%, and ~40%,
313
respectively). However, knockdown of BDNF did not affect the cell proliferation of
314
chondrosarcoma cells (Fig. 1D). In angiogenic processes, endothelial cells must
315
undergo migration, proliferation, and tube formation to form tumor neovessel [36].
316
Subsequent study indicates that tumor angiogenesis is also supported by the
317
mobilization and functional incorporation of EPCs [37]. Therefore, we next
318
determined whether knockdown BDNF reduced angiogenesis in EPCs. Our results
319
showed in Figures 1E and F that knockdown of BDNF reduced chondrosarcoma
320
CM-mediated EPC migration and tube formation (~60% and ~45%, respectively). On
321
the other hand, the effect of BDNF on angiogenesis in vivo was evaluated using the in
322
vivo model of the CAM assay. CM from JJ012/control-shRNA increased angiogenesis
323
in CAM (Fig. 1G). However, BDNF shRNA completely reduced angiogenesis in CAM
324
(Fig. 1G). We also analyzed Matrigel plug formation following subcutaneous
are
largely
unknown.
12
Therefore,
we
established
325
implantation in mice. Knockdown of BDNF reduced vascular formation in the
326
Matrigel and the expression of vessel marker CD31 (Fig. 1H & I). These results
327
indicated that knockdown of BDNF decreases VEGF expression and angiogenesis in
328
vitro and in vivo.
329
330
3.2. Knockdown of BDNF decreases tumor-associated angiogenesis in vivo
331
In order to investigate whether the knockdown of BDNF was able to inhibit
332
tumor-induced angiogenesis in vivo, a xenograft tumor-induced angiogenesis model
333
was used. We used two chondrosarcoma cell lines (JJ012/control-shRNA and
334
JJ012/BDNF-shRNA) mixed with Matrigel and injected them into the right flanks of
335
SCID mice. We observed the mice 5 weeks later by using the Xenogen IVIS system
336
and found that the knockdown of BDNF reduced tumor growth (Fig. 2A-D). We
337
quantified the level of angiogenesis by determining the hemoglobin content of the
338
tumor
339
chondrosarcoma-induced angiogenesis in vivo (Fig. 2E). The hemoglobin content also
340
correlated with the tumor volume in these tumors (Fig. 2F). Finally, ex vivo analysis
341
of tumors excised from the mice showed decreased BDNF and VEGF protein level
342
(~55% and ~60%, respectively), by using qPCR, Western blotting, and ELISA assays,
343
and CD31 expression in the JJ012/BDNF-shRNA group (Fig. 2G-I). Overall, these
344
results suggest that BDNF promotes angiogenesis and tumor growth in vivo.
and
found
that
reducing
BDNF
expression
diminished
345
346
3.3. BDNF induces VEGF expression and angiogenesis through the TrkB
347
receptor
348
Next, we directly applied BDNF to chondrosarcoma cells and examined VEGF
349
expression. Stimulation of human chondrosarcoma cells with BDNF increased VEGF
350
expression in a dose- and time-dependent manner (Fig. 3A-E) (~2.5 to 4 fold). We
351
then determined whether the BDNF-dependent VEGF expression induced
352
angiogenesis in EPCs. We found that CM promoted EPCs cells migration and tube
353
formation (Fig. 3F & G) (~2.5 fold). To elucidate whether BDNF-dependent VEGF
13
354
plays an important role in angiogenesis, the VEGF antibody was used. As shown in
355
Figures 3F and G, incubation of CM with VEGF antibody reduced BDNF-induced
356
migration and tube formation in EPC. These data indicated that BDNF increases
357
VEGF expression promotes angiogenesis in human chondrosarcoma cells.
358
Previous studies have shown that BDNF exerts its effects by interacting with a
359
specific TrkB receptor [38, 39]. We therefore determined whether the TrkB receptor
360
is involved in BDNF-induced VEGF expression and angiogenesis in chondrosarcoma.
361
Pretreatment of cells with the TrkB receptor inhibitor K252a for 30 min or
362
transfection of cells with TrkB shRNA for 24 h reduced BDNF-induced increases in
363
VEGF expression (Fig. 3H-J). In addition, CM from chondrosarcoma demonstrated
364
that the TrkB inhibitor or shRNA reduced BDNF-mediated migration and tube
365
formation of EPCs (Fig. 3K & L). These data suggested that BDNF enhanced VEGF
366
expression and promoted angiogenesis through the TrkB receptor.
367
368
3.4. PLCγ and PKCα activation are involved in BDNF-induced VEGF expression
369
and angiogenesis
370
The PLCγ/PKCα pathway is a common downstream molecule of the TrkB
371
receptor [40, 41]. We next examined whether the PLCγ/PKCα pathway is involved in
372
BDNF-mediated VEGF expression and angiogenesis. The results show that
373
pretreatment of cells for 30 min with PLCγ inhibitor U7122 or PKCα inhibitors
374
GF109203X and Gö6976, or transfection of cells for 24 h with PLCγ and PKCα
375
siRNAs, or with a PKCα mutant abolished BDNF-induced VEGF expression, but this
376
was not observed with PLCβ inhibitor U73343 (Fig. 4A-F). In addition,
377
BDNF-mediated EPC migration and tube formation were diminished by pre-treatment
378
with PLCγ and PKCα inhibitors or siRNAs (Fig. 4G-J). Moreover, incubation of
379
JJ012 cells with BDNF increased PLCγ and PKCα phosphorylation in a
380
time-dependent manner (Fig. 4K). Pretreatment of cells with K252a or U73122
381
markedly inhibited BDNF-induced PLCγ and PKCα phosphorylation (Fig. 4L). Based
382
on these results, it appears that BDNF acted through the TrkB, PLCγ, and PKCα
14
383
pathways to enhance VEGF expression and angiogenesis in human chondrosarcoma
384
cells.
385
386
3.5. Involvement of HIF-1α in BDNF-induced angiogenesis and VEGF expression
387
Recent studies have shown that HIF-1α activation is necessary for VEGF
388
expression and angiogenesis of human chondrosarcoma [42, 43]. To examine whether
389
HIF-1α activation is involved in the signal transduction pathway leading to VEGF
390
expression caused by BDNF, we pretreated cells for 30 min with an HIF-1 inhibitor or
391
transfected cells for 24 h with HIF-1α siRNA or a mutant, all of which markedly
392
reduced VEGF expression (Fig. 5A & B). In addition, as shown in Figures 5C & D,
393
the HIF-1α inhibitor, siRNA, or mutant also reduced BDNF-mediated EPC migration
394
and tube formation. Results from western blotting indicated that BDNF increased the
395
protein level of HIF-1α (~ 4 fold), but not the HIF-1β in a time-dependent manner
396
(Fig. 5E). We then examined whether BDNF could up-regulate HIF-1 protein in
397
chondrosarcoma via an increase in mRNA level. We found that BDNF did not affect
398
the mRNA level of HIF-1 (Fig. 5F). Based on the above findings, we suggest that
399
BDNF increases the stability of HIF-1α protein. Regulation of HIF-1α seems to occur
400
principally at the protein level by HIF-1α stabilization and activation [44]. Yen et al.,
401
has been reported that diosgenin increased HIF-1α protein through enhancing HIF-1α
402
protein stability. However, the detail role of BDNF in stability of HIF-1α protein is
403
needs further examination.
404
To examine whether the TrkB/PLCγ/PKCα signaling pathway is involved in
405
BDNF-induced HIF-1α activation, cells were pretreated with K252a, U73122,
406
GF109203X, and Gö6976. Reduced BDNF-induced HIF-1 accumulation in the
407
nucleus and HIF-1α binding to the HRE element on the VEGF promoter (Fig. 5G & H)
408
was observed. HIF-1α activation was also evaluated by analyzing the HRE luciferase
409
activity. BDNF-mediated HRE luciferase activity was reduced by pre-treatment with
410
K252a, U73122, GF109203X, Gö6976, and HIF inhibitor or by co-transfection with
411
TrkB, PLCγ, PKCα, and HIF-1α siRNA (Fig. 5I & J). Therefore, TrkB receptor,
15
412
PLCγ, and PKCα signaling pathways are involved in BDNF-mediated HIF-1α
413
activation.
414
415
3.6. BDNF and VEGF expression correlates with the tumor stage of
416
chondrosarcoma patients
417
To determine the clinical significance of BDNF and VEGF expression in patients
418
with chondrosarcoma, we analyzed samples from chondrosarcoma patients by
419
immunohistochemical staining. The expression of BDNF and VEGF was significantly
420
higher in chondrosarcoma patients than in healthy individuals (Fig. 6A–C). In
421
addition, the high level of BDNF expression correlated strongly with VEGF
422
expression and tumor stage (Fig. 6A–C). Quantitative data also indicated that the
423
expression of BDNF correlated with the expression of VEGF in human
424
chondrosarcoma patients (Fig. 6D). Taken together, these results indicate that BDNF
425
and VEGF expression correlates with tumor stage in patients with chondrosarcoma.
426
427
428
4. Discussion
429
Chondrosarcoma is a rare but deadly bone cancer [45]. It is the second most
430
common type, accounting for nearly 26% of all bone cancers. Unlike other
431
mesenchymal malignancies such as osteosarcoma and Ewing’s sarcoma, which cause
432
dramatic increases in long-term patient survival with the advent of systemic
433
chemotherapy, chondrosarcoma shows a predilection for metastasis to the lungs,
434
resulting in a poor prognosis for the patient because of the lack of an effective
435
adjuvant therapy. Tumor metastasis is a result of many processes. Among these, the
436
most important is angiogenesis. Therefore, it is important to explore potential targets
437
for preventing chondrosarcoma angiogenesis and metastasis. In the current study, we
438
using immunohistochemistry analysis and found that the expression of BDNF and
439
VEGF in chondrosarcoma patients correlated with tumor grade and significantly
440
higher than normal cartilage. Chondrosarcoma CM-mediated tube formation and
16
441
angiogenesis was abolished by BDNF shRNA. In addition, BDNF knockdown
442
reduced angiogenesis and tumor growth in vivo. Our study suggests that BDNF
443
increased VEGF expression and subsequently promoted angiogenesis in human
444
chondrosarcoma cells. One of the mechanisms underlying the BDNF-induced VEGF
445
production and angiogenesis appeared to be activation of the TrkB receptor, as well as
446
the PLCγ, PKCα, and HIF-1α pathways. Therefore, BDNF may a novel target for
447
chondrosarcoma angiogenesis and metastasis. BDNF has been report to increase tube
448
formation in endothelial cells [46]. Direct application of BDNF (50 ng/ml) also
449
increased ~ 1.5 fold tube formation in EPCs (data not shown). In this study, we didn’t
450
exchange the medium after BDNF stimulation (that contained the BDNF). However,
451
VEGF antibody abolished CM-mediated tube formation in EPCs. Therefore,
452
BDNF-induced VEGF plays key role in BDNF-increased tube formation.
453
Nevertheless, in the pathological condition, BDNF may direct or indirect (through
454
increasing VEGF) promotes angiogenesis in EPCs.
455
It have been reported that BDNF induces neuronal survival, differentiation, and
456
proliferation via the specific receptor, TrkB [18, 19, 47]. The TrkB receptor has been
457
reported to mediate BDNF-induced metastasis in human chondrosarcoma cells [2, 27].
458
In this study, we found that pretreatment of chondrosarcoma cells with a TrkB
459
inhibitor or with shRNA blocked BDNF-induced VEGF expression. Furthermore,
460
TrkB inhibitor or shRNA pretreatment also diminished BDNF-mediated migration
461
and tube formation of EPCs. Therefore, the BDNF-TrkB interaction mediated VEGF
462
expression and angiogenesis in human chondrosarcoma cells.
463
The key oncogenic signaling pathway has been linked to TrkB receptor
464
activation, including the regulation of downstream signaling pathways, such as the
465
PLC/PKC cascade [48]. PLCγ is a member of the PLC serine/threonine family;
466
phosphorylation of tyrosine783 activates its enzyme activity, transmits signals, and
467
regulates corresponding cellular effects. The PKCα protein is a common downstream
468
molecule of PLCγ. PKCα has been characterized at the molecular level and has been
469
found to mediate several cellular molecular responses, such as induction of VEGF
17
470
expression [49, 50]. Here, we demonstrated that PLCγ and PKCα inhibitors or
471
siRNAs antagonized BDNF-mediated VEGF expression and angiogenesis, suggesting
472
that PLCγ and PKCα activation is an obligatory event in BDNF-induced VEGF
473
expression and angiogenesis in human chondrosarcoma cells. In addition, stimulating
474
cells with BDNF also increased PLCγ and PKCα phosphorylation. Furthermore,
475
pretreatment of cells with K252a or U73122 reduced BDNF-promoted PLCγ and
476
PKCα phosphorylation. Thus, our results provide evidence that BDNF upregulates
477
VEGF expression and angiogenesis in human chondrosarcoma cells via the TrkB
478
receptor, and the PLCγ and PKCα signaling pathways.
479
HIF-1 is thought to play a major role in VEGF expression [51]. HIF-1α activates
480
VEGF expression by binding to the HRE site within the VEGF promoter in response
481
to hypoxia [52]. Similarly, the activation of HIF-1α by BDNF also resulted in the
482
induction of VEGF transcription activity through the HRE site, although many other
483
possible response elements, including activator protein-2, nuclear factor-κB, and
484
simian virus 40 promoter factor 1, are located within the VEGF promoter. In the
485
current study, a HIF-1α inhibitor, siRNA, or mutant reduced BDNF-induced VEGF
486
expression and angiogenesis. Incubation of cells with BDNF promoted HIF-1, but
487
not HIF-1, expression in a time-dependent manner. However, BDNF did not affect
488
the mRNA expression of HIF-1. It is apparent that BDNF-induced VEGF expression
489
is substantially mediated by the HRE. In addition, TrkB receptor inhibitor reduced
490
BDNF-induced HIF-1 accumulation in the nucleus and HIF-1 binding to HER
491
element. Therefore, BDNF/TrkB/HIF-1 axis plays key role in BDNF-induced
492
angiogenesis. In addition to cancer metastasis, a similar signal pathway has also been
493
reported in the BDNF induced neuroblastoma migration, which through
494
BDNF/TrkB/HIF-1 axis [25]. Therefore, BDNF/TrkB/HIF-1 axis may have a role
495
in treatment of cancer angiogenesis and metastasis.
496
The rate-limiting step in metastasis and a critical stage in cancer progression is
497
the acquisition of motility by a tumor cell. Lung metastasis is the major cause of
498
mortality for patients with chondrosarcoma [53, 54]. In addition, angiogenesis plays
18
499
an important step in tumor metastasis. Thus, development of an anti-angiogenic and
500
anti-metastatic therapy could conceivably be useful in these patients. In the current
501
study, using immunohistochemistry analysis, we found that BDNF and VEGF
502
expression in specimens from patients with chondrosarcoma correlated with tumor
503
grade and was significantly higher than that of normal cartilage specimens. To the
504
best our knowledge, the present study is the first to demonstrate a correlation between
505
BDNF and VEGF expression in patients with chondrosarcoma. Further studies are
506
required to determine whether increased BDNF and VEGF expression predicts
507
relapse-free and progression-free survival. We also found that BDNF induced VEGF
508
expression and subsequently promoted angiogenesis and tumor growth in human
509
chondrosarcoma cells through activation of the TrkB receptor and the PLCγ, PKCα,
510
and HIF-1 signaling pathways (Fig. 6E). These findings may provide a better
511
understanding of the mechanisms of metastasis and may lead to the development of
512
effective therapies of chondrosarcoma.
513
514
515
516
Acknowledgments
517
We thank Dr. W.M. Fu for providing HIF-1 mutant and HRE promoter
518
construct. This work was supported by grants from the Ministry of Science and
519
Technology of Taiwan (NSC102-2632-B-039-001-MY3; 102-2320-B-039-030-MY3;
520
103-2628-B-039-002-MY3) and China Medical University Hospital (DMR-103-047).
521
522
523
524
525
Conflict of Interest statement
All authors have no financial or personal relationships with other people or
organizations that could inappropriately influence our work.
526
19
536
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712
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714
Figure legends
715
Fig. 1 Knockdown of BDNF decreases VEGF expression in vitro and
716
angiogenesis in vivo. (A-C) The protein and mRNA levels of BDNF and
717
VEGF in JJ012/control-shRNA and JJ012/BDNF-shRNA cells were
718
measured using Western blotting, qPCR, and ELISA (n=5). (D) The cell
719
viability of BDNF-shRNA and control-shRNA cells was examined by MTT
720
assay.
721
JJ012/control-shRNA or JJ012/BDNF-shRNA for 24 h was examined using
722
a Transwell assay (F) and tube formation was photographed under a
723
microscope (n=4). (G) Chick embryos were incubated for 4 days with
724
serum-free medium, JJ012/control-shRNA CM, or JJ012/BDNF-shRNA CM
725
and then resected, fixed, and photographed with a stereomicroscope (n=5).
726
(H & I) Mice were injected subcutaneously with Matrigel mixed with
727
serum-free medium, JJ012/control-shRNA CM, or JJ012/BDNF-shRNA CM
728
for 7 days. The plugs were excised from mice and photographed or stained
729
with CD31 (n=6). Scale bar = 20 m. Results are expressed as the mean ±
730
S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with
731
BDNF-treated group.
(E)
Cell
migration
of
EPCs
incubated
in
CM
with
732
733
Fig. 2 Knockdown of BDNF reduces tumor-associated angiogenesis in vivo. (A-E)
734
JJ012/control-shRNA or JJ012/BDNF-shRNA cells were mixed with
735
Matrigel and injected into flank sites of mice, which were observed for 5
736
weeks and then euthanized prior to tumor resection. The tumors were
737
photographed with a microscope, weight and volume were measured, and
738
hemoglobin levels were quantified (n=6). (F) Correlation between tumor
739
volume and hemoglobin levels. (G-I) Immunofluorescence staining of BDNF
740
and VEGF, Western blotting, or immunohistochemical detection of CD31 in
741
tissues from chondrosarcoma cell xenografts (n=6). Scale bar = 20 m.
742
Results are expressed as the mean ± S.E. *, p < 0.05 compared with control.
25
743
744
Fig. 3 BDNF increases VEGF expression and angiogenesis via the TrkB receptor
745
(A - C) JJ012 cells were incubated with various concentrations of BDNF
746
(30-100 ng/mL) for 24 h, and VEGF expression was examined using qPCR,
747
western blotting, and ELISA (n=5). (D & E) JJ012 cells were incubated with
748
BDNF (50 ng/ml) for indicated time intervals, the protein and mRNA
749
expression of VEGF were examined by using qPCR and Western blot (n=4).
750
(F & G) JJ012 cells were incubated with various concentrations of BDNF for
751
24 h or incubated with BDNF (50 ng/mL) for 24 h followed by stimulation
752
with VEGF antibody (5 μg/mL) for 30 min. The medium was collected as
753
CM and applied to EPCs cells for 24 h. Cell migration and capillary-like
754
structure formation in EPCs were examined by Transwell and tube formation
755
assays (n=4). (H-J) Cells were pretreated with K252a (100 nM) for 30 min or
756
transfected with TrkB shRNA for 24 h, followed by stimulation with BDNF
757
(50 ng/mL), and VEGF expression was measured by qPCR, ELISA, and
758
western blotting (n=4). (K & L) JJ012 cells were pretreated with K252a (100
759
nM) for 30 min or transfected with TrkB shRNA for 24 h followed by
760
stimulation with BDNF (50 ng/mL). The medium was collected as CM and
761
then applied to EPCs cells for 24 h. The cell migration and capillary-like
762
structure formation in EPCs were examined by Transwell and tube formation
763
assays (n=4). Results are expressed as the mean ± S.E. *, p < 0.05 compared
764
with control; #, p < 0.05 compared with BDNF-treated group.
765
766
Fig. 4 The PLCγ/PKCα pathway is involved in BDNF-mediated VEGF
767
expression and angiogenesis. Cells were pretreated for 30 min with U7322
768
(10 μM), U73343 (10 μM), GF109203X (3 μM), and Gö6976 (3 μM) or
769
transfected with PLCγ and PKCα siRNAs or a dominant negative (DN)
770
mutant of PKCα for 24 h, followed by stimulation with BDNF for 24 h. (A-F)
771
The VEGF mRNA and protein expression was examined using qPCR,
26
772
ELISA, and western blotting (n=5). (G-J) The medium was collected as CM
773
and then applied to EPCs for 24 h. Cell migration and capillary-like structure
774
formation in EPCs were examined by Transwell and tube formation assays
775
(n=4). (K) JJ012 cells were incubated with BDNF (50 ng/mL) for the
776
indicated time intervals, and PLCγ or PKCα phosphorylation was examined
777
by western blotting (n=4). (L) JJ012 cells were pretreated for 30 min with
778
K252a or U73122 for 30 min, followed by stimulation with BDNF (50
779
ng/mL) for 15 min, and PLCγ or PKCα phosphorylation was determined by
780
western blotting (n=4). Results are expressed as the mean ± S.E. *, p < 0.05
781
compared with the control; #, p < 0.05 compared with the BDNF-treated
782
group.
783
784
Fig. 5 HIF-1α activation is involved in BDNF-promoted VEGF expression and
785
angiogenesis. Cells were pretreated for 30 min with HIF-1 inhibitor (10 μM)
786
or transfected with a dominant negative (DN) mutant and siRNA of HIF-1α
787
for 24 h, followed by stimulation with BDNF for 24 h. (A & B) The VEGF
788
mRNA and protein expression was examined using qPCR, ELISA, and
789
western blotting (n=5). (C & D) The medium was collected as CM and then
790
applied to EPCs for 24 h. The cell migration and capillary-like structure
791
formation in EPCs were examined by Transwell and tube formation assays
792
(n=4). (E & F) Cells were incubated with BDNF for the indicated time
793
intervals; HIF-1α protein and mRNA expression were examined by western
794
blotting and qPCR, respectively. JJ012 cells were pretreated with K252a,
795
U73122, GF109203X, or Gö6976 for 30 min, and then stimulated with
796
BDNF for 240 min. (G) HIF-1α expression in the nucleus was determined by
797
western blotting (n=4). (H) HIF-1α binding to HRE was determined by the
798
chromatin immunoprecipitation assay (n=4). (I & J) Cells were pretreated
799
with K52a, U73122, GF109203X, Gö6976, and HIF-1 inhibitor for 30 min or
800
transfected with mutants or siRNAs against TrkB, PLCγ, PKCα, and HIF-1α
27
801
before exposure to BDNF. HRE luciferase activity was measured, and the
802
results were normalized to β-galactosidase activity (n=5). Results are
803
expressed as the mean ± S.E. *, p < 0.05 compared with the control; #, p <
804
0.05 compared with the BDNF-treated group.
805
806
Fig. 6 BDNF and VEGF expression correlates with tumor stage of patients with
807
chondrosarcoma (A-C) Immunohistochemistry of BDNF and VEGF
808
expressions in normal cartilage and chondrosarcoma tissue. The correlation
809
and quantitative data are shown in (D). (E) Schematic diagram of the
810
signaling pathways involved in BDNF, which regulates VEGF production
811
and subsequently promotes angiogenesis in human chondrosarcoma cells
812
through activation of the TrkB, PLCγ, PKCα, and HIF-1α pathways. Scale
813
bar = 20 m.
28