Curcumin Suppresses Doxorubicin-Induced Epithelial-Mesenchymal Transition via the Inhibition of TGF-β Signaling and PI3K/AKT Signaling Pathways in Triple Negative Breast Cancer Cells
Wei-Chih Chen1, Ying-An Lai2, Ying-Chao Lin3, Jui-Wen Ma4, Li-Fen Huang5 Ning-Sun Yang6 , Chi-Tang Ho5Ho7
, Sheng-Chu Kuo6Kuo8
, and Tzong-Der Way2,4,9
*
1The Ph.D. Program for Cancer Biology and Drug Discovery, College of Pharmacy, China Medical University, Taichung, Taiwan
2Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan
3Division of Neurosurgery, Buddhist Tzu Chi General Hospital, Taichung Branch, Taiwan
4Institute of Biochemistry, College of Life Science, National Chung Hsing University, Taichung, Taiwan
5
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan, Taiwan
6
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan 5Department 7
Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA
6Graduate 8
Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, Taichung, Taiwan
9
Department of Health and Nutrition Biotechnology, College of Health Science, Asia University, Taichung, Taiwan
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
*Correspondence author: Tzong-Der Way, Ph.D.
Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan
No.91 Hsueh-Shih Road, Taichung, Taiwan 40402 Tel: +886-4-2205-3366 ext: 2509 Fax: +886-4-2203-1075 E-mail: [email protected] 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
ABSTRACT Abstract 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
Triple negative breast cancer (TNBC) is defined by a lack of expression of the Triple negative breast cancer ( TNBC) is defined as lacking estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER 2).
Therefore, targeted therapy agents may not be used, and therapy is largely limited to chemotherapy.expressions which lack specific targets for treatment and limit to chemotherapy. Doxorubicin-treatment consequently acquires undesired malignance characteristics (i.e., epithelial-mesenchymal transition (EMT) and multidrug
resistance). Our results illustrated that doxorubicin triggered EMT and resulted in the
acquisition of a mesenchymal phenotypeIn the present study, we illustrated that doxorubicin was able to induce EMT in TNBC cells. Epithelial marker E-cadherin was down-regulated and mesenchymal marker vimentin was up-regulated simultaneously in TNBC cells. Moreover, We we found that TGF-β and PI3K/AKT signaling pathwaysp-Smad2 and β-catenin protein accumulation were acquired for doxorubicin-induced EMT. Interestingly, we found that curcumin suppressed
doxorubicin-induced EMT. Curcumin reversed doxorubicin-induced morphological changes, inhibited doxorubicin-induced downregulation of E-cadherin expressions and inhibited doxorubicin-induced up-regulation of vimentin expression. We also found that curcumin inhibited doxorubicin-induced EMT by inhibiting the by inhibiting TGF-β signaling and PI3K/AKT signaling pathways. Moreover, curcumin 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
enhanced the anti-proliferative effects of doxorubicin in TNBC cellscurcumin could reduce the dosage of doxorubicin and abolish the undesired effects during clinical treatment of TNBC. In summary, our results suggest that doxorubicin in combination with curcumin may be a potential therapy for TNBC.
Key words: Triple negative breast cancer; Doxorubicin; Epithelial and mesenchymal transition; Curcumin
ABBREVIATIONS
TNBC, triple negative breast cancer; EMT, epithelial-mesenchymal transition; TGF-β, transforming growth factor-β; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyl tetrazolium bromide; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide; ECL; enhanced chemiluminescence; ER, estrogen receptor; HER2, epidermal growth factor receptor-2; PR, progesterone receptor; DMSO, dimethyl sulfoxide; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Maxture F12; RPMI 1640, Roswell Park Memorial Institute (RPMI) 1640. 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111
INTRODUCTION 1 Introduction
Triple negative breast cancer (TNBC) is an immunohistochemically defined
subtype which without the expression of estrogen receptor (ER), progesterone
receptor (PR) and human epidermal growth factor receptor 2 (HER2) expressions and
present approximately 15%-20% of all breast cancers. . TNBC is noted by high risk of
distant recurrence, death, visceral and CNS metastases.
1
.1,2 There are no targeted
agents developed specifically for TNBC at current therapy. The effective treatment
choices are limited to chemotherapy and the data about TNBC therapy are
insufficient. Hence, it is necessary to establish a standard treatment regimen for
TNBCTNBC.2
.3-7
Doxorubicin is an anthracycline drug with anti-tumor activity. It has been
considered as an excellent chemotherapeutic agent and widely used in the treatment of 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131
patients with metastatic breast cancer. 3 5
8-11 However, doxorubicin is limited in
clinical utility because of number of troublesome side effects including
myelosuppression, immunosuppression, dose-cumulative cardiotoxicity and drug
resistance, invasive potential in breast cancer cells.12 In addition, it has been found
that doxorubicin induced induces multidrug resistance and invasive potential in breast cancer cells. 6 9 .13-16
Transforming growth factor-β (TGF-β) regulates various cell behaviors
including cell proliferation, differentiation, migration and apoptosis. It has been
considered that TGF-β plays a dual role in the cancer progression. During early
stages of carcinogenesis, TGF-β acts as a tumor suppressor by inhibiting cell growth
and promoting apoptosis; but in the advanced stages of carcinogenesis, it acts as a
tumor promoter by enriching the motility and invasiveness to promote
epithelial-mesenchymal transition (EMT) and metastasis.
1 0 ,1 1 Previous publicationstudies implied that Twist, Snail and Slug were transcriptionally induced by TGF-β during EMT in various cancer cells.
7 ,1 2-13 .17,18
EMT has been attracting increased attention in studies of tumor metastasis. The morphologic alteration from losing of epithelial characteristics and polarity to acquire a mesenchymal phenotype with increased migratory and invasive properties is the most prominent characteristic of EMT. Down-regulation of epithelial markers
132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
such as E-cadherin is a hallmark of EMT. Twist, Snail, Slug and ZEB-1 are transcriptional factors which can bind the promoter of the E-cadherin gene to repress its transcription. Previous publications implied that Twist, Snail and Slug were transcriptionally induced by TGF-β during EMT in various cancer cells .14,19,20
β-catenin is one of adherent junction components which anchored with
E-cadherin to regulate cell-cell adhesion and cell migration. β-catenin can also form a
complex with lymphoid enhancer factor-1 ( LEF-1), a T-cell factor (TCF) related with Wnt/β-catenin signaling to inhibit E-cadherin gene transcription. A destruction complex, composed of adenomatosis polyposis coli (APC), axin, glycogen synthase kinase 3β (GSK3β), resulted in degradation of β-catenin by ubiquitin proteasome pathway.21 Phosphorylation of GSK3β by activation of AKT phosphorylation
accumulated intracellular catenin. The stabilization and nuclear accumulation of
β-catenin led to stimulation of EMT, stem cell maintenance and self-renew.141
-6
.22,23 Curcumin (1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione) is
a major component of turmeric (Curcuma longa L.). Curcumin has several biological
and pharmacological activities such as anti-inflammatory, anti-oxidant and
chemotherapeutic property. It has been demonstrated that Ccurcumin exhibited
exhibits non-toxicity even at high dose in laboratory animals.
1 7 , 1 8 .24,25 Several studies 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168
indicated that curcumin decreased decreases the ability of cancer cell motility,
metastasis and stem-like characteristics.
1 9 -2 2 .26-29
Besides its therapeutic effects, doxorubicin also enhances the malignancy of
treated cancers in clinical situations. Recently, EMT has attracted attention in studies
of TNBC tumor progression. We aimed to test whether transient doxorubicin
treatment induced EMT in TNBC cells, and elucidated the role of TGF-β and
PI3K/AKT signaling pathways in this process. In this study, we showed that
doxorubicin exposure induced activation of p-Smad2 and β-catenin which led to
nuclear accumulation and consequence EMT. In addition, we found that curcumin
reversed doxorubicin-induced EMT via inhibiting both of TGF-β signaling and
PI3K/AKT signaling pathways. Curcumin also enhanced the chemosensitivity of
TNBC cells to doxorubicin. 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188
MATERIALS AND METHODS 2 Materials and methods
2.1 Cell lines and culture conditions.
The human breast cancer cell lines used in this study were MB-468, MDA-MB-231, BT-549 and BT-20 cells were purchased from the American Type Culture
Collection (Manassas, VA, USA).. BT-549 and BT-20 cells were grown in RPMI 1640 (Invitrogen Corporation, Carlsbad, CA, USA). 231 and
MDA-MB-468 were grown in DMEM/F12 (Invitrogen Corporation, Carlsbad, CA, USA).
Medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100
g streptomycin and 100 U penicillin 10% fetal bovine serum (FBS) and 100 g
streptomycin (Invitrogen Corporation, Carlsbad, CA, USA). All cell lines were grown
in a humidified incubator at 37°C under 5% CO2 in air.
2.2 Reagents and antibodies. 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209
Doxorubicin, SB431542 and LY294002 were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). Curcumin was purchased from Merck Co. (Darmstadt,
Germany)MERCK. Primary antibodies E-cadherin, Snail, Twist, phospho-AKT (S473), phospho-GSK3β (S2448Ser9), β-catenin and -actin were purchased from
Cell Signaling Technology (Beverly, MA, USA). Primary vimentin was purchased
from Abcam Inc. (Cambridge, MA, USA). Secondary antibodies, HRP-conjugated
Goat anti-Mouse IgG and Goat anti-Rabbit IgG, were obtained from Millipore
(Billerica, MA, USA).
2.3 Western blotting.
Cells on 100-mm culture dishes (5105/dish) were treated with various concentrations of doxorubicin, curcumin or in combination, thenand then incubated for 48 h. After
treatment, the total proteins were extracted by adding 50 μL of gold lysis buffer (50
mM Tris–HCl, pH 7.4; 1 mM phenylmethylsulfonyl floride; 1 mM NaF; 1% NP-40;
150 mM NaCl; 1 mM EGTA; 1 mM phenylmethylsulfonyl floride; 1% NP-40; and 10
mg/mLl leupeptin) to the cell pellets. Lysate protein was measureddetermined by the
Lowry protein assay (Bio-Rad Laboratories, Berkeley, CA, USA).
Membranes were blocked with 5% BSA (Sigma, St. Louis, MO, USA) for 1 h at room
temperature, and probed with primary antibody for 1.5 h at room temperature or
overnight at 4°C followed by HRP-conjugated appropriated secondary antibodies. 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229
2.4 Morphology observation.
BT-20 cells (1104) were seeded in each well of a 24-well plate and incubated in a 37°C incubator with 5% CO2 overnight. BT-20 cells were treated with indicated
concentrations of doxorubicin with or without curcumin or curcumin only and then
cells incubated at 37°C for 48 h. Representative photographs were taken at 200x
magnification using a Nikon TE2000-U inverted microscope.
2.5 Cellular Fractionation Analysis.
Cells were harvested with trypsinization and washed twice with ice PBS. Cells
were rapidly washed once with hypotonic buffer, and allowed to swell on ice for 10
min. After centrifugation at 4°C with 720 gG (3,000 rpm) for 15 min, the supernatant was saved for cytoplasmic fraction. The nuclear pellet was added the
same buffer. After sonication, the suspension was spun at 10,000 gG (1200 8,000
rpm) for 20 min and supernatant was saved as the nuclear fraction. Equal proteins
from cytoplasmic and nuclear fraction were used for western blotting analysis.
2.6 Growth inhibition assay.
Cells were seeded in a 24-well plate (1104 cells/well) overnight, and then treated with different concentrations of doxorubicin with or without curcumin or curcumin 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
only for 48 h. Cell growth inhibition was examined by the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Briefly, 80 μL MTT
solution (2 mg/mLl, Sigma Chemical Co., St. Louis, MO, USA) was added to each
well to make a final volume of 500 μL and incubated for 1.5 h at 37°C. The
supernatant was aspiratedremoved, and added 500 μL of DMSO to dissolve the
MTT-formazan crystals which formed by metabolically viable cells.were dissolved in 500
μL of DMSO. Finally, the absorbance at O.D. 570 nm was detected by enzyme-linked
immunosorbent assay (ELISA) reader.
2.7 Statistical analysis.
One-way analysis of variance (ANOVA) was used for the comparison of more than
two mean values. Results represent at least two to three independent experiments and
are shown as averages ± S.E.M. Results with a P value less than 0.05 were considered
statistically significant. *, p 0.05.; **, p 0.001. RESULTS 3 Results 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269
3.1 Doxorubicin treatment induces EMT in TNBC cells.
Recently, doxorubicin exposure not only caused causes apoptosis but also induced
induces multidrug resistance in breast cancer cells.7
.14 TNBC is an aggressive form of breast cancer and resistance to available treatments. Therefore, we first aimed to
examine whether doxorubicin induced EMT in TNBC cells. Following treatment with
various concentrations of doxorubicin (0.15, 0.31 and 0.62 M, respectively) for 48 h,
vimentin (mesenchymal marker) was up-regulation in mesenchymal phenotype
(MDA-MB-231 and BT-549) and epithelial phenotype (BT-20 and MDA-MB-468)
TNBC cells. However, E-cadherin (epithelial marker) was down-regulation in BT-20
and MDA-MB-468 cells (Fig.ure 1 A and 1B). MDA-MB-231 and BT-549 did not show E-cadherin expression (data not shown) (Fig. 1 A and B). BT-20 was chosen for
further experiments. The morphological change is another critical characteristic for
EMT. As compared with untreated cells, doxorubicin exposure induced a loose cell
contact and acquired of fibroblast-like appearance in epithelial phenotype BT-20 cells
(Figure. 1C). We further examined whether EMT-inducing regulators, Snail and
Twist involved in doxorubicin-induced EMT. Western blotting analysis showed that
doxorubicin increased Snail and Twist expressions (Fig. ure 1D). Our results exhibited that doxorubicin contributed TNBC to acquire EMT characteristics.
270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
3.2 Effect of TGF-β pathway on doxorubicin-induced EMT.
TGF-β has been shown to induce EMT and to attribute metastatic progression in
various cancer cells.10
.17 WeWe questioned whether doxorubicin-induced EMT
involved TGF-β pathway. Since TGF- induces Smad2 Ser465/467-phosphorylation,
we examined the effect of doxorubicin exposure on Ser465/467-phosphorylation of
Smad2 expression. Doxorubicin exposure significantly increased the
Ser465/467-phosphorylation of Smad2 in BT-20 cells (Fig. ure 2A). We used a specific inhibitor
of TGF-β receptor kinase, SB431542, to confirm doxorubicin activated TGF-β
pathway in BT-20 cells. BT-20 cells were treated with doxorubicin and SB431542
alone or in combination for 48 h. We found that SB431542 abrogated
doxorubicin-increased Ser465/467-phosphorylation of Smad2 (Fig. ure 2B). Phosphorylation of Smad2 translocates to nucleus and participates in transcriptional activation of
responsive genes for EMT. Thus, we also confirmed the subcellular location of
p-Smad2 in BT-20 cells under the doxorubicin treatment. Compared to untreated cells,
treatment with doxorubicin elevated decreased the expression of p-Smad2 in the
cytosol fraction and elevated accumulation in the nuclear fraction (Fig. ure 2C). Next, we confirmed whether doxorubicin-induced EMT via activating TGF-β
signaling. BT-20 cells were treated with doxorubicin and SB431542 alone or in
combination for 48 h. SB431542 did abrogate doxorubicin-induced vimentin up-289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307
regulation but did not abrogate doxorubicin-induced E-cadherin down-regulation
(Fig. ure 2D). We then questioned whether SB431542 affected doxorubicin-induced
EMT-inducing factors, Snail and Twist. Our data showed that SB431542 only
abrogated increased Twist protein level but did not inhibit
doxorubicin-increased Snail protein level (Figure. 2E). Taken together, doxorubicin doxorubicin-increased
p-Smad2 expression and accumulation in nuclear which led to take part in EMT
induction.
3.3 Doxorubicin induces activation of β-catenin in TNBC cells.
Aberrant activation of β-catenin in breast cancer related with poor prognosis and is
another important regulator for possessed EMT.14,15
.22,23 We questioned whether doxorubicin-induced EMT via the activation of β-catenin. Doxorubicin exposure
increased the expression of β-catenin in a temporal response observed at 1 ~ 6 h
post-doxorubicin treatment interval (Fig. ure 3A). Nuclear accumulation of β-catenin participates in transcriptional activation of responsive genes critical for maintenance
EMT. We next questioned whether doxorubicin increased β-catenin nuclear
accumulation, western blotting analysis of cytoplasmic and nuclear fractions from
doxorubicin (0.15 and 0.31 M) treated in BT-20 cells. β-catenin elevated
accumulation in the nuclear fraction of doxorubicin treated cells as compared to 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326
untreated cells (Fig. ure 3B). In our results, doxorubicin modulated β-catenin expression and activity. GSK3β is a downstream target of phosphatidylinositol 3
kinase (PI3K)/AKT and its activity is inhibited by phosphorylation leading to modulation of downstream targets. GSK3β-mediated phosphorylation of β-catenin
causes its degradation in the ubiquitin dependent proteasome pathway. We further
investigated whether GSK3β inactivation is involved in mediating doxorubicin’s
ability to stabilize β-catenin. Doxorubicin exposure increased significantly the
phosphorylation of GSK3β in a temporal manner. We also examined whether
doxorubicin-mediated GSK3β inactivation and involvement EMT properties included
AKT. Following exposure doxorubicin for 1, 3 and 6 h, the phosphorylation of AKT
was increased at 1 ~ 6 h (Fig. ure 3C). We also use LY 294002, a highly selective
inhibitor of phosphatidylinositol 3 (PI3) kinasePI3K, to determine if inhibition of
AKT activation repressed the doxorubicin-induced EMT. Treatment of LY294002,
not only reversed E-cadherin expression but alsoand reduced vimentin expression in
the presence of doxorubicin treatment (Fig. ure 3D). Taken together, β-catenin activation participated in doxorubicin-induced EMT related with PI3K/AKT pathway.
3.4 Curcumin inhibits doxorubicin-induced EMT properties.
Apart from inhibiting cell proliferation in various cancer cells, curcumin has the 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
potential to inhibit the invasion and metastasis of cancer cells.2
3
.30 We examined
whether curcumin abrogated doxorubicin-induced EMT properties. MDA-MB-231,
BT-549, BT-20 and MDA-MB-468 cells were treated with doxorubicin and curcumin
alone or in combination for 48 h. Our results revealed that doxorubicin-induced
up-regulation of vimentin expression was inhibited by treatment with curcumin in
MDA-MB-231, BT-549, MDA-MB-468 and BT-20 cells (Fig. ure 4 A and 4B). Treatment
with curcumin inhibited doxorubicin-induced downregulation of E-cadherin
expressions in MDA-MB-468 and BT-20 cells (Figure . 4B). We observed that
curcumin maintained cell contact and epithelial phenotype even exposure in
doxorubicin (Fig. ure 4C). Moreover, curcumin suppressed doxorubicin-induced the expressions of Snail and Twist (Fig. ure 4D) in BT-20 cells. Taken together, our results shown that curcumin prevented the EMT characteristics induction by
doxorubicin.
3.5 Curcumin inhibits doxorubicin-induced EMT via modulation p-Smad2 and β-catenin expressions.
Our result showed that doxorubicin increased p-Smad2 expression, we next tTo identify whether curcumin inhibiteded doxorubicin-induced EMT via the regulation
inhibition of p-Smad2 activation. Our data showed that curcumin surpressed 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364
doxorubicin-increased p-Smad2 protein level (Fig.ure 5A). We next questioned whether curcumin inhibited doxorubicin-induced β-catenin, p-AKT and p-GSK3β
protein levels. Our data showed that curcumin diminished doxorubicin-increased
β-catenin, p-AKT and p-GSK3β protein levels (Figure. 5B). Our results showed that curcumin inhibited doxorubicin-induced p-Smad2, β-catenin, p-AKT and p-GSK3β
up-regulation. Taken together, the effect of curcumin on doxorubicin-induced EMT
mediated TGF-β signaling and PI3K/AKT signaling pathways.
3.6 Curcumin enhanced the anti-proliferative effect of doxorubicin in BT-20 cells.
To test whether curcumin could enhance the anti-proliferative effect of doxorubicin,
the anti-tumor effects of doxorubicin and curcumin was assessed in BT-20 cells by
the MTT assay at 48 h. Treatment with doxorubicin and curcumin alone reduced cell
viability, in combination treatment with 20 M curcumin enhanced the anti-proliferative effect of doxorubicin in BT-20 (Figure 6A). Treatment with doxorubicin induced dose-response reduction in cell viability, in combination treatment with 20 M curcumin enhanced the anti-proliferative effect of doxorubicin in BT-20 cells (Fig. 6A). Subsequently, we also measured the cleaved forms of PARP and caspase 3
(both of apoptosis markers). We observed that curcumin enhanced the doxorubicin-365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
induced cleaved forms of PARP and Caspase 3 (Fig. ure 6B). Taken together, these
results suggested that the ability of curcumin was to sensitize BT-20 cells to
doxorubicin and it’s a potential approach.
DISCUSSION 4 Discussion
Although TNBC has high rate of metastasis and recurrence in patients that is limited to chemotherapy. Chemotherapy not only erases cancer cells but also faces undesired effects, including enhancing the aggressive ability of the treated cancer
cells, resulting in chemotherapy failure. To find out a promising approach to eliminate
cancer without opposite effects is essential. It is widely believed that combination
treatment may have a potential therapeutic benefit for improving cancer therapy.
TNBC presents with higher rates of visceral metastases, has a relatively shorter 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404
medial survival, and has limited duration of response to successive lines of chemotherapy. In this study, we found out a potential effective approach for TNBC treatment by using a combination of curcumin and doxorubicin.
EMT, a physiological process of switching from epithelial phenotype into
mesenchymal phenotype, has been indicated to increase aggressive ability of the
tumors (i.e., tumors cell migration, invasion and dissemination).12,2
3
.19,30 Our data had
shown that both epithelial and mesenchymal phenotypes of TNBC gained of EMT
characteristics after treatment with doxorubicin (Figure 1). It was consistent with
previous publications that chemotherapy agents such as doxorubicin and paclitaxel
have the undesired effects to induce drug resistant and EMT of the treated cancers.
9,2 4 -2 5 .14-16, 31-33
Accumulated evidences suggested that TGF-β plays a regulator of EMT process
and accelerates the tumor-promoting activity in various cancer progressions involving
the progression of metastatic breast cancer. Administration of doxorubicin in
MMTV/PyVmT transgenic animal model had shown to elevate TGF-β circulating
levels which were a prometastatic signal in tumor cells (Biswas et al., 2007).27
.34 Furthermore, doxorubicin treatment enhanced the properties of migration and
invasion in murine 4T1, human MCF-7 and MDA-MB-231 breast cancer cells.
8 , 30 . 13-15 Hence, we hypothesized that doxorubicin exposure stimulated TGF-β signaling and 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423
caused EMT in BT-20 cells. Our data had shown that doxorubicin treatment did
accumulate p-Smad2 in nuclear and blocked by TGF-β receptor kinase inhibitor,
SB431542. We next questioned whether doxorubicin-induced EMT was
Smad-dependent signaling. Snail and Twist are two of concomitant TGF-β
signaling-induced EMT. Our data showed that SB431542 did inhibit doxorubicin-signaling-induced
upregulation of vimentin and Twist but it is unexpected that SB431542 could not
inhibited doxorubicin-induced E-cadherin and Snail alternation (Figure. 2). Our
results showed that Smad-dependent signaling may play minor role in
doxorubicin-induced EMT. Except EMT, TGF-β regulates cell proliferation, cell cycle arrest,
extracellular matrix production and other tumorigenicity.2
6
.37 The effect of
Smad-dependent signaling induced by doxorubicin in BT-20 cells needs to be further
investigated.
TGF-β-induced EMT not only goes through Smad-dependent mechanism but
also goes through Smad-independent mechanisms.2
7 -2 8 .7,35 Activation of AKT
phosphorylated twist1 on S42 caused to enhance TGF-β signaling which kept
PI3K/AKT hyperactivation and then cancer cells acquired more aggressiveness.2
9
.36 Our results had shown that doxorubicin increased Snail and Twist expressions (Fig.
ure 1D). We questioned whether PI3K/AKT participated in doxorubicin-induced EMT. Our data had shown doxorubicin treatment induced EMT through 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442
phosphorylated AKT and GSK3β and improved β-catenin accumulation in nuclear
and inhibited by PI3K inhibitor, LY294002 ( Fig. ure 3). We considered that doxorubicin induced EMT might be through triggering PI3K/AKT pathway.
Doxorubicin is widely used in chemotherapy involving metastatic breast cancer.
However, previous studies and our data indicated that doxorubicin treatment caused
tumors more malignancy. Therefore, to search for a chemopreventive or
chemotherapeutic agent to diminish the chemoresistance to doxorubicin via inhibition
EMT will help to improve cancer therapy. Increasing evidences suggested that
curcumin reversed chemoresistance,,1
7 24 invasion,1 9 ,26and metastasis.20 .27 In this study,
we chose curcumin, an agent is known to lessen tumor motility, invasion and
metastasis, to investigate whether curcumin could prevent doxorubicin-induced EMT.
Our results showed that curcumin inhibited doxorubicin-acquired EMT properties
involving downregulation of vimentin, upregulation of E-cadherin, and to hold on the
cell-cell contact in TNBCs. Although the effect of curcumin in E-cadherin and
Viment protein level expressions was not so effective in MDA-MB-468. Curcumin may regulate other EMT-related proteins such as Zeb1, Twist1, snail, slug and N-cadherin. TNBC. We also found that curcumin inhibited doxorubicin-increased the
expressions of Snail and Twist in BT-20 (Figure. 4). To further addressed
mechanisms by curcumin inhibited doxorubicin-induced EMT. We question whether 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461
curcumin has the ability to block doxorubicin-increased p-Smad2 and β-catenin
expressions. Our results showed that curcumin inhibited doxorubicin-induced
p-Smad2, β-catenin, p-AKT and pGSK3β expressions (Figure. 5). TGFβ signaling
plays a mediator to regulate EMT through MEK/Erk, JNK/p38 MAP kinases, Rho GTPase and PI3K/AKT.31
Inhibition the signaling of PI3K by LY294002 and MEK1/2 by UO126 inhibited TGF-β-1-induced EMT, supporting that PI3K/AKT and MAPK/Erk1/2 may play a regulator role in TGF-β-1-induced EMT in A549 human lung cancer cells.32
During the past few decades, the chemopreventive efficacy of curcumin has been
extensively studied; several molecular targets by curcumin have been found.1
8
.25 For
instance, curcumin prevented paclitaxel-induced EMT through inhibiting NF-kB
signaling.20
.27 Our results showed that curcumin could repress both TGF-β signaling and PI3K/AKT signaling pathwayS. Our data also showed that curcumin enhanced
doxorubicin-triggered apoptosis (Figure. 6).
In summary, our data showed that doxorubicin-triggered apoptosis came with
EMT via TGF-β signaling and PI3K/AKTT/GSK3β signaling pathways in TNBC.
We found a chemopreventive agent, curcumin, which suppressed
doxorubicin-induced EMT and enhanced doxorubicin-triggered apoptosis. On the other hand,
curcumin could reduce the dosage of doxorubicin and abolish the undesired effects 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
during clinical treatment of TNBC. Doxorubicin in combination with curcumin may
be a potential therapy for TNBC.
Disclosure
The authors report no disclosures or conflicts of interest.
Abbreviations
TNBC, triple negative breast cancer; EMT, epithelial-mesenchymal transition; TGF-β, transforming growth factor-β; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyl tetrazolium bromide; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide; ECL; enhanced chemiluminescence; ER, estrogen receptor; HER2, epidermal growth factor receptor-2; PR, progesterone receptor; DMSO, dimethyl sulfoxide; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Maxture F12; RPMI 1640, Roswell Park Memorial Institute (RPMI) 1640. 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499
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Figure legends
Figure 1. Stimulation of EMT by doxorubicin treatment in TNBC cells. (A) 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701
MDA-MB-231 and BT-549 cells were treated with DMSO (control) or increasing
doxorubicin concentrations (0.15, 0.31 and 0.62 M) for 48 h. The cells were then
harvested and lysed for the detection of vimentin and β-actin. (B) MDA-MB-468
and BT-20 cells were treated with DMSO (control) or increasing doxorubicin
concentrations (0.15, 0.31 and 0.62 M) for 48 h. The cells were then harvested
and lysed for the detection of E-cadherin, vimentin and β-actin. (C) Phase-contrast
images of BT-20 cells. The sub-confluent cultures were shown the morphological
differences. BT-20 cells were treated with DMSO (control) or 0.15 M doxorubicin
for 48 h. (D) BT-20 cells were treated with DMSO (control) or increasing
doxorubicin concentrations (0.15, 0.31 and 0.62 M) for 48 h. The cells were then
harvested and lysed for the detection of Snail, Twist and β-actin. Western blot data
presented are representative of those obtained in at least 3 separate experiments.
The lower panel presents the average of three independent experiments. The value of the control cells was set to 1.
Figure 2. Doxorubicin increased p-Smad2 expression and nucleus accumulation in BT-20 cells. (A) BT-20 cells were treated with DMSO (control) or increasing doxorubicin concentrations (0.15, 0.31 and 0.62 M) for 48 h. The
cells were then harvested and lysed for the detection of Smad2, p-Smad2 and
β-actin. (B) BT-20 cells were treated with DMSO (control) or 0.31 M doxorubicin 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720
and 10 M SB431542 alone or in combination for 48 h. The cells were then
harvested and lysed for the detection of Smad2, p-Smad2 and β-actin. (C) BT-20
cells were treated with DMSO (control) or doxorubicin (0.15 or 0.31 M) for 36 h.
The cells were then harvested and lysed for the detection of p-Smad2, Histone 3
(H3) and tubulin. Histone 3 (H3) and tubulin served as loading controls. (D) BT-20
cells were treated with DMSO (control) or 0.31 M doxorubicin and 10 M
SB431542 alone or in combination for 48 h. The cells were then harvested and
lysed for the detection of E-cadherin, vimentin and β-actin. (E) BT-20 cells were
treated with DMSO (control) or 0.31 M doxorubicin and 10 M SB431542 alone
or in combination for 48 h. The cells were then harvested and lysed for the
detection of Snail, Twist and β-actin. Western blot data presented are representative
of those obtained in at least 3 separate experiments. The lower panel presents the
average of three independent experiments. The value of the control cells was set to 1.
Figure 3. Doxorubicin-induced EMT via the activation of β-catenin activation mediated doxorubicin-induced EMT in BT-20 cells. (A) BT-20 cells were treated with DMSO (control) or 0.31 M doxorubicin for various times (1, 3 and 6
h). The cells were then harvested and lysed for the detection of βâ-catenin and
-actin. (B) BT-20 cells were treated with DMSO (control) or doxorubicin (0.15 or 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739
0.31 M) for 36 h. The cells were then harvested and lysed for the detection of βâ
-catenin, Histone 3 (H3) and tubulin. Histone 3 (H3) and tubulin served as loading
controls. (C) BT-20 cells were treated with DMSO (control) or 0.31 M
doxorubicin for various times (1, 3 and 6 h). The cells were then harvested and
lysed for the detection of p-GSK3βâ, p-AKT, AKT and -actin. (D) BT-20 cells
were treated with DMSO (control) or 0.31 M doxorubicin and 10 M LY294002
alone or in combination for 48 h. The cells were then harvested and lysed for the
detection of E-cadherin, vimentin and -actin. Western blot data presented are
representative of those obtained in at least 3 separate experiments. The lower panel
presents the average of three independent experiments. The value of the control cells was set to 1.
Figure 4. Curcumin inhibited doxorubicin-induced EMT characteristics. (A) MDA-MB-231 and BT-549 cells were treated with DMSO (control) or 0.31 M
doxorubicin and 20 M curcumin alone or in combination for 48 h. The cells were
then harvested and lysed for the detection of vimentin and β-actin. (B)
MDA-MB-468 and BT-20 cells were treated with DMSO (control) or 0.31 M doxorubicin
and 20 M curcumin alone or in combination for 48 h. The cells were then
harvested and lysed for the detection of E-cadherin, vimentin and β-actin. (C)
Phase-contrast images of BT-20 cells. The sub-confluent cultures were shown the 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758
morphological differences. BT-20 cells were treated with DMSO (control) or 0.31
M doxorubicin and 20 M curcumin alone or in combination for 48 h. (D) BT-20
cells were treated with DMSO (control) or 0.31 M doxorubicin and 20 M
curcumin alone or in combination for 48 h. The cells were then harvested and lysed
for the detection of Snail, Twist and β-actin. Western blot data presented are
representative of those obtained in at least 3 separate experiments. The lower panel
presents the average of three independent experiments. The value of the control cells was set to 1.
Figure 5. Curcumin blocked doxorubicin-EMT via inhibiting TGF-β signaling and PI3K/AKT signaling pathways. (A) BT-20 cells were treated with DMSO (control) or 0.31 M doxorubicin and 20 M curcumin alone or in
combination for 48 h. The cells were then harvested and lysed for the detection of
p-Smad2, Smad2 and β-actin. (B) BT-20 cells were treated with DMSO (control) or
0.31 M doxorubicin and 20 M curcumin alone or in combination for 1 h. The
cells were then harvested and lysed for the detection of β-catenin, AKT, AKT,
p-GSK3β and β-actin. Western blot data presented are representative of those
obtained in at least 3 separate experiments. The lower panel presents the average of
three independent experiments. The value of the control cells was set to 1.
Figure 6. Curcumin sensitized the anti-proliferative effect of doxorubicin in 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777
BT-20 cells. (A) BT-20 cells were treated with doxorubicin (0.32 M) and curcumin (20 M) alone or combination treatment for 48 h. various concentrations of doxorubicin (0.15, 0.31, 0.62, 1.25 and 2.5 M) or combination with curcumin (20 ìM) for 48 h. Growth inhibition was determined by MTT assay. The percentage
of cell growth inhibition was calculated by the absorption of control cells as 100%.
Experiments were performed in triplicate. a. 0.15 M doxorubicin versus 0.15 M doxorubicin plus curcumin, p0.05; b. 0.31 M doxorubicin versus 0.31 M doxorubicin plus curcumin, p0.05; c. 0.62 M doxorubicin versus 0.62 M
doxorubicin plus curcumin, p0.001. (B) BT-20 cells were treated with DMSO
(control) or 0.31 M doxorubicin and 20 M curcumin alone or in combination for
48 h. The cells were then harvested and lysed for the detection of PARP, Caspase 3
and β-actin. Western blot data presented are representative of those obtained in at
least 3 separate experiments. The lower panel presents the average of three
independent experiments. The value of the control cells was set to 1. 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794
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