- Email: [email protected]
A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells
E XP ER I ME NTAL C E LL RE S E ARCH
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells Q1
Talia Hatkevich, Joseph Ramos, Idalys Santos-Sanchez, Yashomati M. Pateln
Q2 Department of Biology, University of North Carolina at Greensboro, Greensboro, NC, 27412, United States
article information
abstract
Article Chronology:
Since over 60% of breast cancers are estrogen receptor positive (ERþ), many therapies have
Received 25 March 2014
targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-
Received in revised form
estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth
9 May 2014
factor promoting phosphorylation of the ER. Other proliferation pathways such as the
Accepted 20 May 2014
phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting
Keywords:
multiple cellular proliferation and survival pathways at the onset of treatment is critical for the
Naringenin
development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an
Tamoxifen
inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam
Breast cancer
used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use
Apoptosis
serum containing medium in order to retain all the potential inducers of cell proliferation so as
Cell proliferation
not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells. & 2014 Published by Elsevier Inc.
Breast cancer is one of the most common forms of cancer in women. Approximately 60% of breast cancers are estrogen receptor positive (ERþ) and utilize the ER to promote cell proliferation [1]. Activation of the ER is regulated by both estrogen binding and phosphorylation. Thus the ER is activated
in both a ligand-dependent and ligand-independent manner to promote cell proliferation and survival. The ER is localized throughout the cell and at the plasma membrane [2–4]. Liganddependent activation of the ER involves estrogen binding to the ER, homodimerization and then translocation to the nucleus to
n
Corresponding author. Fax: þ1 336 334 5839. E-mail address: [email protected] (Y.M. Patel).
http://dx.doi.org/10.1016/j.yexcr.2014.05.017 0014-4827/& 2014 Published by Elsevier Inc.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
94 95 96 97 98 99 100 101 102 103 104 105
2
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
E XP E RI ME N TAL CE L L R ES E ARC H
bind estrogen-responsive elements (EREs) of target genes [2–5]. Approximately 180 estrogen responsive genes regulating cell proliferation, cell cycle control, transcriptional regulation, and metabolic processes have been identified in ERþ mammary cells [2–4]. In a ligand-independent manner the ER is regulated by phosphorylation from activated growth factor receptors such as, epidermal growth factor (EGF), insulin-like growth factor (IGF), insulin receptor and Her2/neu [6,7]. Growth factor receptors can activate the ER through several signaling pathways including the MAPK (Ras–Raf–MEK–ERK1/2) and PI3K (AKT) pathways [6–8]. Both extracellular signal-regulated kinases (ERK1/2) and AKT activate the ER [6–8]. Thus many therapies have targeted the ER. Most ERþ breast cancers are responsive to anti-estrogen therapies such as tamoxifen (Tam) [9]. Tam is a selective estrogen receptor modulator (SERM) that functions as an antagonist to the ER in breast tissue. Tam binds the ER and impairs transcription of estrogenresponsive genes which arrest cells in the G0/G1 phase of the cell cycle [9]. Furthermore, ligand-independent activation of the ER has increased the need for treatments that target not only the ER but also these signaling pathways to impair cell proliferation. In order to target multiple growth promoting pathways, combination therapies using Tam and kinase inhibitors for breast cancer are being investigated [10,11]. However, single kinase inhibitors are rarely clinically successful, so now therapies are targeting multiple kinases [10,11]. Since many kinase inhibitors have numerous side effects, this approach may not be feasible. An alternative approach is the use of bioactive food components. Over the past few decades the use of dietary components (such as vitamins and flavonoids) has shown promise in the prevention and treatment of various diseases including cancer. We have identified the grapefruit flavanone naringenin (Nar) as an inhibitor of both the PI3K and MAPK pathways [12,13]. The grapefruit flavanone, Nar has been shown to inhibit both cell proliferation and motility by interfering with the PI3K and MAPK pathways [14–17]. Short-term exposure to Nar has been shown to inhibit the phosphorylation of ERK1/2 and AKT in MCF7 breast cancer cells [13–17]. Furthermore, Nar has been reported to induce apoptosis in colon, breast, and uterine cancer cell lines expressing ERα or ERβ [14,16,17]. In addition Nar has been identified as a xenoestrogen. Many xenoestrogen have structures that mimic natural steroid hormones like estrogens that enable them to interact with ERs as agonists or antagonists [17,18]. Bulzomi et al. demonstrated that Nar can bind directly to the estrogen receptor as an antagonist [14]. Collectively, these findings may explain why individuals consuming a diet rich in flavanones such as Nar show increased protection against the development of endocrine tumors [18]. The ability of Nar to both impair the MAPK and PI3K pathways and to function as an antagonist to the estrogen receptor make it ideal as a cotherapy with Tam in ERþ breast cancer cells. Since Tam and Nar inhibit ligand-dependent activation of the ER by acting as estrogen antagonists and Nar has been shown to inhibit ERK1/2 and AKT phosphorylation, which would block phosphorylation and thus activation of the ER, a Nar–Tam combination would inhibit multiple proliferation and survival pathways in ERþ breast cancer cells. Collectively, these findings suggest that a Nar–Tam combination would be effective in impairing the proliferation of ERþ breast cancer cells. In this study we show that a combination Nar–Tam treatment inhibits MCF-7 cell
] (]]]]) ]]]–]]]
proliferation and viability to a greater extent than either treatment alone in both MCF-7 breast cancer cells. Furthermore we demonstrate that a Nar–Tam treatment requires lower concentrations of both compounds when used in combination.
Materials and methods Materials The MCF-7 cell line (HTB-22) was purchased from ATCC. Dulbecco's Modified Eagle Medium (DMEM) was obtained from Gibco (Grand Island, NY). Phenol red-free media, L-glutamine, charcoal-stripped fetal bovine serum, bovine Insulin, anti-rabbit secondary antibody, naringenin and 4-OH-tamoxifen were from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was purchased from Thermo Scientific HyClone (Logan, UT). Guava Via-Count Reagent was from Millipore (Billerica, MA). Phospho-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, phospho-AKT (Ser473), cleaved PARP and Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA). ERα antibody (HC-20) was from Santa Cruz Biotechnology (Dallas, TX). Actin antibody was obtained from Abcam (Cambridge, UK). AlexaFluor 488 conjugated goat anti-rabbit secondary antibody was from Jackson ImmunoResearch (West Grove, PA). The enhanced chemiluminescence (ECL) detection kit was from ThermoScientific (Waltham, MA).
Cell culture MCF-7 breast cancer cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 0.01 mg/mL bovine insulin, and 100 U/mL penicillin/streptomycin). This medium will be referred to as Full Medium. Cells were maintained at 37 1C with 5% CO2. Media was changed every two to three days and cells were passed once they attained 80% confluence.
Via-Count cell density and viability assays Treated cells were washed twice with PBS and subjected to trypsin. Cells were centrifuged at 5000 g for 5 min, supernatants were discarded and the pelleted cells were resuspended in PBS. A 1:20 dilution of cells was incubated in ViaCount Reagent for 5 min in the dark. Viability was analyzed by Guava easy-CyteTM Flow Cytometry (Millore) using the ViaCount software.
Immunoblot analysis Cell lysates were prepared as previously described [13]. Proteins were separated by 10% SDS-PAGE, and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with the indicated antibody and visualized by enhanced chemiluminescent (ECL) and a Bio-Rad ChemiDoc XRS system. The resulting bands were quantified using Quantity One analysis software.
Immunofluorescence Treated cells were grown on coverslips. Cells were washed with 1XPBS, fixed with 3.7% paraformaldehyde, and permeabilized in
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
E X PE R IM EN TA L C ELL R E S EA RC H
0.25% Triton. Cells were incubated with an anti-estrogen receptor (ERα) antibody (1:100) dilution for 1 h. Following incubation with the secondary antibody, cells were washed and treated with a 1:1000 DAPI solution for 5 min. Coverslips were mounted and then visualized using an Olympus iX81 Motorized Inverted Confocal Microscope equipped with Fluoview FV500 software.
Quantification of ERα distribution within the cell ERα levels were quantified via intensity of fluorescence in both the cytoplasm and the nucleus. The relative intensity of immunofluroescence was quantified using Image-Pro Plus software (Silver Spring, MD). Briefly, Intensity of fluorescence signal at the nucleus and the cytoplasm were measured and the ratio of the nuclear/cytoplasmic signal was averaged for individual cells (n¼ 6) under various conditions. A percent change formula, [(TreatmentBasal)/(Basal) 100%], was used to determine the change in protein localization.
Statistical analysis Data are stated as means7SEM. The significance of comparing means was assessed by two-way analysis of Student's t-test (StatPlus, AnalystSoft).
Results A Nar–Tam combination impairs cell proliferation and survival in MCF-7 cells Previous studies examined the role of Nar on cell proliferation, ER regulation and growth factor signaling pathways in charcoal stripped serum in the presence or absence of exogenously added 17β-estradiol (E2) for short time periods (15 min–24 h) [14–16,19]. While these studies aimed to establish a relationship between Nar, E2 and ER, they do not account for possible other targets of Nar that
Doubling Time (Normalized to untreated)
150 125 100 75
* *
50 25 uM Nar 0
may be absent in charcoal-stripped serum. While charcoal stripping significantly lowers the concentrations of lipophilic hormones it also removes growth factors from the serum. Both E2 (a lipophilic hormone) and growth factors are cellular targets of Nar that are involved in cell proliferation. In order to determine the role of Nar, we examined the effects of Nar in the presence of serum containing medium (DMEM supplemented with 10% FBS) which will be referred to as “full medium” to retain all the potential inducers of cell proliferation present in the serum. First we determined the concentration of Nar required to impair proliferation of MCF-7 cells in the presence of full medium. We cultured cells in full medium in the presence of increasing concentrations of Nar and assayed cell doubling times. As seen in Fig. 1A Nar has a concentration dependent inhibition of cell doubling times. As seen previously Nar was able to impair proliferation at a concentration of 50 and 100 mM but maximal inhibition was observed at 250 mM. In previous studies Nar concentrations as high as 100 mM show the greatest inhibition of cell proliferation. Our findings show that a slightly greater Nar concentration is required in the presence of serum-containing medium to achieve maximal inhibition of cell proliferation. Next we examined the effect of a Nar–Tam combination on doubling time of MCF-7 cells cultured in full medium. We used the established concentration of Tam (100 nM). Since both Nar and Tam impair cell proliferation we wanted to determine if a Nar– Tam treatment would be more effective and either treatment alone. MCF-7 cells were treated with either Tam (100 nM), Nar (250 mM), or both in full medium. Control cells were treated with the vehicles for Tam (ethanol) and Nar (DMSO). Cells were plated and cultured for 7 days in the presence of each treatment. Our results show that treatment with either Nar alone or Tam alone resulted in a 2-fold increase in doubling time when compared to untreated MCF-7 cells. However, the combination treatment demonstrated a synergistic 13fold increase in doubling time (Fig. 1B). These studies also show that slightly higher concentrations of Nar (250 mM instead of 100 mM) are required to impair cell proliferation in the presence of serumcontaining medium.
*
175 Doubling Time (h)
226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
3
] (]]]]) ]]]–]]]
50
100
250
20 15 10 5
Tam Nar -
+ -
+
+ +
Fig. 1 – Effect of Nar and Tam on doubling times of MCF-7 cells cultured in full medium. (A) MCF-7 cells (2.45 104 cells/ml) are cultured in full media (DMEMþFBS) in the absence or presence of varying concentrations of Nar as indicated. Media is replaced every 2 days. Cells are collected and counted after 6 days. (B) MCF-7 cells are grown for 7 days in full media (DMEMþFBS) in the presence or absence of (Tam) (100 nM), Nar (250 lM) or a combination. After 7 days, the cells are replated for an additional 7 days. The media is changed and the treatments are reapplied every 2 days. Following treatment cell density is quantified and doubling times are calculated using the formula, Td ¼ (t2 t1) log 2/log(q2/q1) where “t” is the time in hours and “q” is the number of cells counted at each time point. The values from each treatment group are presented as a percentage of an untreated control. The results are the means7SEM of three independent experiments. Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
4
Nar–Tam impairs MCF-7 cell viability Next we wanted to determine the mechanism of action of Nar–Tam on decreasing cell density. To determine whether a Nar–Tam combination would impair cell viability to a greater extent than either treatment alone, we treated MCF-7 cells with Tam, Nar, or a combination of the two (Fig. 2A). As expected, Tam treatment showed a decrease in cell viability. Nar treatment alone significantly reduced (40%) cell viability by day 5, confirming the cytotoxic behavior of Nar (Fig. 2A). The combination treatment had even a greater reduction in cell viability on days 4 and 5 (65%). We also assayed cell death on the various days of treatment. As shown previously, Tam had an effect on cell death. As expected there was no effect of Nar–Tam on days 2 and 3 but a significant increase in cell death on days 4 and 5 after treatment. Nar treatment alone also increased cell death although to a lesser extent than the Nar–Tam treatment. Collectively, our results show that a Nar–Tam treatment elicited a greater cytostatic and cytotoxic effect than either treatment alone. These findings are critical since they examine the long term effects of a Nar–Tam combination in serum-containing medium.
Lower concentrations of Nar and Tam are required in the Nar–Tam combination to impair cell proliferation and viability Next, we investigated whether a lower concentration of either compound would elicit similar effects as the initial treatment used (250 μM Nar and 100 nM Tam on MCF-7 cells on day 4) on cell density and viability (Fig. 3). To do this, we treated MCF-7 cells with Nar (250 μM) and decreasing concentrations of Tam, Tam (100 nM) and decreasing concentrations of Nar or varying concentrations of both Nar and Tam for 4 days in full medium and then determined cell density and viability. Cell density did not change when Tam was reduced during the dual treatment (Fig. 3A). In contrast, however, lowering the Nar concentration (o200 μM) resulted in a significant increase in cell density (Fig. 3B). Next, we wanted to determine the lowest concentrations of both Nar and Tam. A similar level of impaired cell proliferation to that seen in the initial treatment was seen at 200 μM Nar and 25 nM Tam (Fig. 3C). The other two treatments were not able to significantly impair cell density.
] (]]]]) ]]]–]]]
Next we performed similar studies to determine the lowest concentration of Nar–Tam on cell viability (Fig. 3 panels D–F). Reduction of Tam in the presence of 250 μM Nar did not affect cell viability (Fig. 3D). However lowering the concentration of Nar below 200 μM significantly increased cell viability compared to the initial treatment (Fig. 3E). Finally, we treated cells with varying concentrations of both agents for 4 days and assessed cell viability. There was a significant increase in cell viability in samples treated with Nar concentrations less than 200 μM when compared to the initial treatment (Fig. 3F). These studies indicate that 25 nM Tam and 200 μM Nar is the lowest concentration of both compounds that will achieve the same response as the initial treatment on cell viability. Collectively, our results indicate that Nar has a dose dependent effect on cell viability and density below 200 μM. In contrast, Tam does not alter either cell density or viability in the presence of Nar. Furthermore, our studies show that the concentrations of both Nar and Tam can be reduced to achieve maximal effects on cell density and viability when used in combination in the presence of serum containing medium.
Nar–Tam reduces the total protein levels of ERK1/2 and AKT To determine the mechanism by which Nar–Tam elicits effects on cell density and viability, we investigated the known targets of Nar. Nar has been shown to inhibit the phosphorylation but not the expression levels of ERK1/2 and AKT in MCF-7 cells cultured in stripped serum for 24 h [15]. To investigate the effect of Nar and Nar–Tam on ERK1/2 and AKT, we treated MCF-7 cells with Tam, Nar, and Nar–Tam for 4 days. We examined the expression levels as well as the phosphorylation status of ERK1/2 and AKT (Fig. 4A–C). Tam treatment did not affect the expression of ERK1/2 or AKT (Fig. 4A and B). In contrast, Nar and to a greater extent Nar–Tam significantly reduced the expression of ERK1/2 and AKT when compared to the control (Fig. 4A and B). The levels of actin were unaffected indicating that the effect of Nar was specific. Next we examined the effect of the treatments on the phosphorylation status of ERK1/2 and AKT. Surprisingly, none of the treatments significantly altered the phosphorylation status of ERK 1/2 or AKT relative to total protein levels (Fig. 4A and C). These results demonstrate that Nar has effects on ERK1/2 and AKT protein levels in MCF-7 cells.
120 % Cell Death (Normalized to Untreated)
% Viable Cells (Normalized to untreated)
346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405
E XP E RI ME N TAL CE L L R ES E ARC H
100 80
*
60 40
* *
20
0 2
3
4 Days
5
80
Tam Nar Nar-Tam
*
60
* 40
*
20
2
3
4
5
Days
Fig. 2 – Effect of Nar on cell viability. MCF-7 cells are grown in full medium in the presence of Tam (100 nM) (circle), Nar, (250 μM) (square) or a combination of the two (triangle) for the indicated times. (A) Cell viability and (B) cell death are determined by flow cytometry. Results are the means7SEM of three independent experiments. *po0.05. Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
E X PE R IM EN TA L C ELL R E S EA RC H
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525
] (]]]]) ]]]–]]]
5
Fig. 3 – A Nar–Tam combination requires lower concentration of both compounds to achieve optimal reduction in cell density and viability. MCF-7 cells are grown in full medium in the presence of either varying concentrations of Nar or Tam or both for four days. (A–C) Cell density and (D–F) cell viability are determined by flow cytometry. Results are the means7SEM of three independent experiments. Significance at Po0.05 is compared to the initial treatment (*).
Nar–Tam promotes apoptosis via activation of caspases 7 and 9
in Nar and Nar–Tam treated cells compared to levels present in untreated cells.
Our studies show that Nar and Nar–Tam inhibit cell density in part by increasing cell death. To investigate the mechanism of the effect of Nar and Nar–Tam on viability, we assayed for the presence of apoptotic cells by flow cytometry (Fig. 5A). Our results show that both Nar–Tam and Nar increased the number of apoptotic cells starting on day 4 of treatment when compared to untreated and Tam treated cells cultured in full medium. To determine how Nar induced apoptosis we examined members of the caspase family. Studies have shown that Nar induces apoptosis via caspase 3 activation in HeLa cells transfected with ERα [14,16]. Since MCF-7 cells do not express caspase 3 but do express caspases 7 and 9, we wanted to investigate whether these caspases were involved in Nar induced apoptosis (Fig. 5B and C) [20]. Caspase 7 was significantly reduced in Nar and Nar–Tam treated cells (Fig. 5B and C). Similarly, caspase 9 was undetectable in Nar and Nar–Tam treated cells (Fig. 5B). These results show that caspases 7 and 9 are cleaved and activated, in Nar–Tam treated cells. We also examined the downstream target of caspase 7, PARP. As shown in Fig.5B, we detected increased levels of cleaved PARP
Nar–Tam influences ER localization Since both Nar and Tam interact with ERα [11,17,21], we wanted to investigate the localization of ERα in the various treatments. To investigate the localization pattern of ERα, we treated MCF-7 cells with Nar, Tam or Nar–Tam for 4 days in serum-containing medium and then visualized ERα by confocal microscopy (Fig. 6A and B). The ER is localized throughout the cell. Upon estrogen binding, the ER-estrogen complex translocates to the nucleus. As shown in Fig. 6A (panels a–c) the ER is localized throughout the cell with more intense staining in the nucleus. As described previously, our studies also show that Tam induces the translocation of the ERα from the cytosol to the nucleus in full medium (Fig. 6A and B) [2]. In contrast, Nar treated cells showed an accumulation of ERα in the cytoplasm (Fig. 6A and B). Nar–Tam treated cells exhibited an even distribution of ERα in the nucleus and the cytoplasm, suggesting that both Nar and Tam independently influence the localization of ERα in the combination treatment.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585
6
Vehicle
Tam
Nar
Nar-Tam
] (]]]]) ]]]–]]]
Vehicle Tam Nar Nar-Tam
150
Percent of Vehicle
pERK 1/2
ERK 1/2
pAKT AKT
100
* *
50
* *
* *
Actin ERK1
ERK2
AKT
Vehicle Tam Nar Nar-Tam
350
Percent of Vehicle
586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645
E XP E RI ME N TAL CE L L R ES E ARC H
300 250 200 150 100 50
pERK1/ pERK2 pAKT/ ERK1 /ERK2 AKT
Fig. 4 – Nar–Tam reduces the levels of proteins involved in proliferation and survival. MCF-7 cells are grown in full medium either untreated (Vehicle) or in the presence of Tam (25 nM), Nar (200 μM), or a combination of the two for 4 days. Protein lysates are prepared. (A) Lysates are subjected to SDS-PAGE and immunoblotted using antibodies against pERK, ERK, pAKT, AKT, and actin. Immunoblot results are quantified by densitometry for (B) total protein levels and (C) phospho proteins. ERK results are the means7SEM of four independent experiments. AKT results are the means7SEM of three independent experiments. Significance at Po0.05 is compared to vehicle (*).
Discussion Since over half of breast cancers are ERþ and depend on estrogen to promote proliferation, the focus of pharmaceutical treatments has been to target the ER. The ER is regulated not only by estrogen but also by phosphorylation mediated by the activation of kinase cascades. Currently, the use of Tam in conjunction with multiple kinase inhibitors is being investigated for the treatment of breast cancers [22]. Natural products such as Nar have also been shown to have anti-proliferative effects. In this study we investigated the effect of Nar in conjunction with Tam to impair cell proliferation and viability in MCF-7 breast cancer cells. Our study demonstrates that a Nar–Tam combination elicits its effects by targeting multiple proliferative and apoptotic pathways resulting in greater inhibition of proliferation and cell viability than either treatment alone. We first focused our studies on the effects of Nar–Tam on MCF7 cells in serum-containing medium to retain all the potential inducers of cell proliferation present in the serum. Initially we determined the optimal concentration required to achieve maximal inhibition of proliferation and viability of each compound alone, and then we used these concentrations in combination. Overall, our studies indicate that Nar–Tam inhibited MCF-7 cell proliferation and viability better than either component alone.
Next we determined the optimal concentration of the Nar–Tam treatment on cell proliferation and viability. The reduction of Tam concentration showed no effect on cellular proliferation or viability when compared to the initial concentration when used in conjunction with Nar. Conversely there was a dose-dependent effect of Nar on both cell proliferation and viability. Taken together, our results show that when used in combination lower concentration of both Nar and Tam can be used to achieve optimal inhibition of cell proliferation and viability. In order to determine the mechanism of action of the Nar–Tam combination on cell proliferation and viability, we examined the known targets of Nar and Tam. Our previous studies demonstrated that Nar impairs the phosphorylation of ERK1/2 and AKT when cells were exposed for short time periods (4 h) [13]. In this study we exposed MCF-7 cells to Nar for several days and then examined the expression levels and phosphorylation status of ERK1/2 and AKT. Surprisingly Nar treatment reduced the overall levels of ERK1/2 and AKT which was not observed at shorter time periods. Currently inhibitors of both the ERK and AKT pathways are targets in breast cancer clinical trials. Nar reduced cell density by not only impairing cell proliferation but also by inducing apoptosis via activating caspase 9 and caspase 7. Although we were unable to detect the cleaved forms of the caspases, others
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705
E X PE R IM EN TA L C ELL R E S EA RC H
20
% Mid Apoptotic
Control
Control Tam Nar Nar-Tam
15
*
10
2
3
Nar
Nar-Tam
Caspase 9
* *
5
Tam
Caspase 7
*
Cleaved PARP
4
Actin
5
Days
140 120
Caspase 7 % of Control
100 80
* *
60 40 20
Fig. 5 – Role of Nar–Tam on apoptosis. MCF-7 cells are grown in full medium alone “Control” or in the presence of Tam (25 nM), Nar (200 μM), or a combination of the two. (A) Apoptosis is determined at the indicated time points by flow cytometry. *po0.05. (B) On day 4 protein lysates are prepared and subjected to SDS-PAGE and immunoblotted using antibodies against caspase 7, caspase 9, cleaved PARP and actin. (C) Caspase 7 levels are quantified using densitometry. Results are the means7SEM of three independent experiments. Significance at Po0.05 is compared to vehicle (*).
ER
DAPI
Merge 1.0
Vehicle 0.8
Tam
Nuclear/Total
706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765
7
] (]]]]) ]]]–]]]
0.6 0.4 0.2
Nar
Nar-Tam
Fig. 6 – Nar localizes ER to the cytoplasm. MCF-7 cells are grown in full medium alone “Vehicle” or in the presence of Tam (25 nM), Nar (200 μM), or a combination of the two for 4 days on coverslips. (A) On day 4, cells are fixed and stained with anti-ERα antibody and DAPI and then subjected to confocal microscopy. (B) ERα nuclear localization is quantified. Results are the means7SEM of three independent experiments. Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825
8
826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885
E XP E RI ME N TAL CE L L R ES E ARC H
have shown that a reduction in the full length caspases indicates their cleavage and thus activation [20,23]. To support the idea of caspase-mediated apoptosis we examined the downstream target of caspase 7, PARP. Our findings show that Nar treatment increased the levels of cleaved PARP in MCF-7 cells. Since the ER is also a target of both Nar and Tam we examined the effect of these treatments on ER localization. Normally, estrogen binding to ERα leads to translocation of the complex to the nucleus and recruitment of co-activators to regulate transcription of estrogen-responsive genes [2]. However, when Tam binds to ERα it causes a conformational change in the receptor leading to a reduction in binding of co-activators and/or promoters of estrogen-responsive genes. In the absence of DNA binding the Tam/ERα complex is localized to the cytosol allowing it to interact with kinase signaling pathways such as the PI3K and MAPK pathways [24–29]. Fan et al. also noted a change in ERα localization from the nucleus to the cytoplasm following Tam treatment [30]. Our results show that Nar treatment also causes a relocalization of ERα to the cytoplasm. We are the first to specifically note a perinuclear localization pattern of ERα in Nar treated cells. Thus future studies are required to determine if Nar relocalization of ERα outside the nucleus results in reduced expression of estrogen-responsive genes. In summary, our studies demonstrate that a Nar–Tam combination results in greater inhibition of cellular proliferation and viability then either component alone in MCF-7 cells cultured in serum containing medium for longer time points. We also show that use of the combination treatment requires lower concentrations of both components to achieve optimal results. Furthermore we show that Nar targets ERK, AKT and the caspase pathway to exert its effects on proliferation and viability. Additionally, our studies show that Nar localizes ERα to the cytoplasm. Collectively our results enhance the understanding of the mechanism of action of Nar in conjunction with Tam in MCF-7 cells.
references [1] C. DeSantis, J. Ma, L. Bryan, A. Jemal, Breast cancer statistics, 2013, CA Cancer J. Clin. 64 (2014) 52–62. [2] B.J. Deroo, K.S. Korach, Estrogen receptors and human disease, J. Clin. Investig. 116 (2006) 561–570. [3] S. Safe, K. Kim, Non-classical genomic estrogen receptor (ER)/ specificity protein and ER/activating protein-1 signaling pathways, J. Mol. Endocrinol. 41 (2008) 263–275. [4] S. Sommer, S.A. Fuqua, Estrogen receptor and breast cancer, Semin. Cancer Biol. 11 (2001) 339–352. [5] V. Kumar, S. Green, G. Stack, M. Berry, J.R. Jin, et al., Functional domains of the human estrogen receptor, Cell 51 (1987) 941–951. [6] G. Bunone, P.A. Briand, R.J. Miksicek, D. Picard, Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation, EMBO J. 15 (1996) 2174–2183. [7] P.H. Driggers, J.H. Segars, Estrogen action and cytoplasmic signaling pathways. Part II: the role of growth factors and phosphorylation in estrogen signaling, Trends Endocrinol. Metab. 13 (2002) 422–427. [8] S.E. Ghayad, J.A. Vendrell, S. Ben Larbi, C. Dumontet, I. Bieche, et al., Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways, Int. J. Cancer 126 (2010) 545–562.
] (]]]]) ]]]–]]]
[9] M. Baum, Tamoxifen–the treatment of choice, Why look for alternatives? Br J Cancer 78 (Suppl 4) (1998) S1–S4. [10] S. Ali, R.C. Coombes, Endocrine-responsive breast cancer and strategies for combating resistance, Nat. Rev. Cancer 2 (2002) 101–112. [11] C.G. Sanchez, C.X. Ma, R.J. Crowder, T. Guintoli, C. Phommaly, et al., Preclinical modeling of combined phosphatidylinositol-3kinase inhibition with endocrine therapy for estrogen receptorpositive breast cancer, Breast Cancer Res. 13 (2011) R21. [12] A.W. Harmon, Y.M. Patel, Naringenin inhibits phosphoinositide 3kinase activity and glucose uptake in 3T3-L1 adipocytes, Biochem. Biophys. Res. Commun. 305 (2003) 229–234. [13] A.W. Harmon, Y.M. Patel, Naringenin inhibits glucose uptake in MCF-7 breast cancer cells: a mechanism for impaired cellular proliferation, Breast Cancer Res. Treat. 85 (2004) 103–110. [14] P. Bulzomi, A. Bolli, P. Galluzzo, F. Acconcia, P. Ascenzi, et al., The naringenin-induced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background, IUBMB Life 64 (2012) 690–696. [15] P. Galluzzo, P. Ascenzi, P. Bulzomi, M. Marino, The nutritional flavanone naringenin triggers antiestrogenic effects by regulating estrogen receptor alpha-palmitoylation, Endocrinology 149 (2008) 2567–2575. [16] P. Totta, F. Acconcia, S. Leone, I. Cardillo, M. Marino, Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involvement of estrogen receptor alpha and beta signalling, IUBMB Life 56 (2004) 491–499. [17] F. Virgili, F. Acconcia, R. Ambra, A. Rinna, P. Totta, et al., Nutritional flavonoids modulate estrogen receptor alpha signaling, IUBMB Life 56 (2004) 145–151. [18] M. Marino, M. Pellegrini, P. La Rosa, F. Acconcia, Susceptibility of estrogen receptor rapid responses to xenoestrogens: physiological outcomes, Steroids 77 (2012) 910–917. [19] P. Bulzomi, A. Bolli, P. Galluzzo, S. Leone, F. Acconcia, et al., Naringenin and 17beta-estradiol coadministration prevents hormone-induced human cancer cell growth, IUBMB Life 62 (2010) 51–60. [20] H. Kurokawa, K. Nishio, H. Fukumoto, A. Tomonari, T. Suzuki, et al., Alteration of caspase-3 (CPP32/Yama/apopain) in wild-type MCF-7, breast cancer cells, Oncol. Rep. 6 (1999) 33–37. [21] M. Clemons, S. Danson, A. Howell, Tamoxifen (Nolvadex): a review, Cancer Treat. Rev. 28 (2002) 165–180. [22] E.M. Fox, C.L. Arteaga, T.W. Miller, Abrogating endocrine resistance by targeting ERalpha and PI3K in breast cancer, Front. Oncol. 2 (2012) 145. [23] J. Li, P. Chen, N. Sinogeeva, M. Gorospe, R.P. Wersto, et al., Arsenic trioxide promotes histone H3 phosphoacetylation at the chromatin of CASPASE-10 in acute promyelocytic leukemia cells, J. Biol. Chem. 277 (2002) 49504–49510. [24] S. Kato, H. Endoh, Y. Masuhiro, T. Kitamoto, S. Uchiyama, et al., Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase, Science 270 (1995) 1491–1494. [25] H. Kurokawa, A.E. Lenferink, J.F. Simpson, P.I. Pisacane, M.X. Sliwkowski, et al., Inhibition of HER2/neu (erbB-2) and mitogenactivated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells, Cancer Res. 60 (2000) 5887–5894. [26] K. Liang, W. Jin, C. Knuefermann, M. Schmidt, G.B. Mills, et al., Targeting the phosphatidylinositol 3-kinase/Akt pathway for enhancing breast cancer cells to radiotherapy, Mol. Cancer Ther. 2 (2003) 353–360. [27] A. Migliaccio, M. Di Domenico, G. Castoria, A. de Falco, P. Bontempo, et al., Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells, EMBO J. 15 (1996) 1292–1300.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945
E X PE R IM EN TA L C ELL R E S EA RC H
946 947 948 949 950 951 952 953
[28] M. Sun, J.E. Paciga, R.I. Feldman, Z. Yuan, D. Coppola, et al., Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K, Cancer Res. 61 (2001) 5985–5991. [29] E. Tokunaga, A. Kataoka, Y. Kimura, E. Oki, K. Mashino, et al., The association between Akt activation and resistance to hormone
] (]]]]) ]]]–]]]
9
therapy in metastatic breast cancer, Eur. J. Cancer 42 (2006) 629–635. [30] P. Fan, J. Wang, R.J. Santen, W. Yue, Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells, Cancer Res. 67 (2007) 1352–1360.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
954 955 956 957 958 959 960
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells Q1
Talia Hatkevich, Joseph Ramos, Idalys Santos-Sanchez, Yashomati M. Pateln
Q2 Department of Biology, University of North Carolina at Greensboro, Greensboro, NC, 27412, United States
article information
abstract
Article Chronology:
Since over 60% of breast cancers are estrogen receptor positive (ERþ), many therapies have
Received 25 March 2014
targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-
Received in revised form
estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth
9 May 2014
factor promoting phosphorylation of the ER. Other proliferation pathways such as the
Accepted 20 May 2014
phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting
Keywords:
multiple cellular proliferation and survival pathways at the onset of treatment is critical for the
Naringenin
development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an
Tamoxifen
inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam
Breast cancer
used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use
Apoptosis
serum containing medium in order to retain all the potential inducers of cell proliferation so as
Cell proliferation
not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells. & 2014 Published by Elsevier Inc.
Breast cancer is one of the most common forms of cancer in women. Approximately 60% of breast cancers are estrogen receptor positive (ERþ) and utilize the ER to promote cell proliferation [1]. Activation of the ER is regulated by both estrogen binding and phosphorylation. Thus the ER is activated
in both a ligand-dependent and ligand-independent manner to promote cell proliferation and survival. The ER is localized throughout the cell and at the plasma membrane [2–4]. Liganddependent activation of the ER involves estrogen binding to the ER, homodimerization and then translocation to the nucleus to
n
Corresponding author. Fax: þ1 336 334 5839. E-mail address: [email protected] (Y.M. Patel).
http://dx.doi.org/10.1016/j.yexcr.2014.05.017 0014-4827/& 2014 Published by Elsevier Inc.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
94 95 96 97 98 99 100 101 102 103 104 105
2
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
E XP E RI ME N TAL CE L L R ES E ARC H
bind estrogen-responsive elements (EREs) of target genes [2–5]. Approximately 180 estrogen responsive genes regulating cell proliferation, cell cycle control, transcriptional regulation, and metabolic processes have been identified in ERþ mammary cells [2–4]. In a ligand-independent manner the ER is regulated by phosphorylation from activated growth factor receptors such as, epidermal growth factor (EGF), insulin-like growth factor (IGF), insulin receptor and Her2/neu [6,7]. Growth factor receptors can activate the ER through several signaling pathways including the MAPK (Ras–Raf–MEK–ERK1/2) and PI3K (AKT) pathways [6–8]. Both extracellular signal-regulated kinases (ERK1/2) and AKT activate the ER [6–8]. Thus many therapies have targeted the ER. Most ERþ breast cancers are responsive to anti-estrogen therapies such as tamoxifen (Tam) [9]. Tam is a selective estrogen receptor modulator (SERM) that functions as an antagonist to the ER in breast tissue. Tam binds the ER and impairs transcription of estrogenresponsive genes which arrest cells in the G0/G1 phase of the cell cycle [9]. Furthermore, ligand-independent activation of the ER has increased the need for treatments that target not only the ER but also these signaling pathways to impair cell proliferation. In order to target multiple growth promoting pathways, combination therapies using Tam and kinase inhibitors for breast cancer are being investigated [10,11]. However, single kinase inhibitors are rarely clinically successful, so now therapies are targeting multiple kinases [10,11]. Since many kinase inhibitors have numerous side effects, this approach may not be feasible. An alternative approach is the use of bioactive food components. Over the past few decades the use of dietary components (such as vitamins and flavonoids) has shown promise in the prevention and treatment of various diseases including cancer. We have identified the grapefruit flavanone naringenin (Nar) as an inhibitor of both the PI3K and MAPK pathways [12,13]. The grapefruit flavanone, Nar has been shown to inhibit both cell proliferation and motility by interfering with the PI3K and MAPK pathways [14–17]. Short-term exposure to Nar has been shown to inhibit the phosphorylation of ERK1/2 and AKT in MCF7 breast cancer cells [13–17]. Furthermore, Nar has been reported to induce apoptosis in colon, breast, and uterine cancer cell lines expressing ERα or ERβ [14,16,17]. In addition Nar has been identified as a xenoestrogen. Many xenoestrogen have structures that mimic natural steroid hormones like estrogens that enable them to interact with ERs as agonists or antagonists [17,18]. Bulzomi et al. demonstrated that Nar can bind directly to the estrogen receptor as an antagonist [14]. Collectively, these findings may explain why individuals consuming a diet rich in flavanones such as Nar show increased protection against the development of endocrine tumors [18]. The ability of Nar to both impair the MAPK and PI3K pathways and to function as an antagonist to the estrogen receptor make it ideal as a cotherapy with Tam in ERþ breast cancer cells. Since Tam and Nar inhibit ligand-dependent activation of the ER by acting as estrogen antagonists and Nar has been shown to inhibit ERK1/2 and AKT phosphorylation, which would block phosphorylation and thus activation of the ER, a Nar–Tam combination would inhibit multiple proliferation and survival pathways in ERþ breast cancer cells. Collectively, these findings suggest that a Nar–Tam combination would be effective in impairing the proliferation of ERþ breast cancer cells. In this study we show that a combination Nar–Tam treatment inhibits MCF-7 cell
] (]]]]) ]]]–]]]
proliferation and viability to a greater extent than either treatment alone in both MCF-7 breast cancer cells. Furthermore we demonstrate that a Nar–Tam treatment requires lower concentrations of both compounds when used in combination.
Materials and methods Materials The MCF-7 cell line (HTB-22) was purchased from ATCC. Dulbecco's Modified Eagle Medium (DMEM) was obtained from Gibco (Grand Island, NY). Phenol red-free media, L-glutamine, charcoal-stripped fetal bovine serum, bovine Insulin, anti-rabbit secondary antibody, naringenin and 4-OH-tamoxifen were from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was purchased from Thermo Scientific HyClone (Logan, UT). Guava Via-Count Reagent was from Millipore (Billerica, MA). Phospho-p44/42 MAP kinase (Thr202/Tyr204), p44/42 MAP kinase, phospho-AKT (Ser473), cleaved PARP and Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA). ERα antibody (HC-20) was from Santa Cruz Biotechnology (Dallas, TX). Actin antibody was obtained from Abcam (Cambridge, UK). AlexaFluor 488 conjugated goat anti-rabbit secondary antibody was from Jackson ImmunoResearch (West Grove, PA). The enhanced chemiluminescence (ECL) detection kit was from ThermoScientific (Waltham, MA).
Cell culture MCF-7 breast cancer cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 0.01 mg/mL bovine insulin, and 100 U/mL penicillin/streptomycin). This medium will be referred to as Full Medium. Cells were maintained at 37 1C with 5% CO2. Media was changed every two to three days and cells were passed once they attained 80% confluence.
Via-Count cell density and viability assays Treated cells were washed twice with PBS and subjected to trypsin. Cells were centrifuged at 5000 g for 5 min, supernatants were discarded and the pelleted cells were resuspended in PBS. A 1:20 dilution of cells was incubated in ViaCount Reagent for 5 min in the dark. Viability was analyzed by Guava easy-CyteTM Flow Cytometry (Millore) using the ViaCount software.
Immunoblot analysis Cell lysates were prepared as previously described [13]. Proteins were separated by 10% SDS-PAGE, and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated with the indicated antibody and visualized by enhanced chemiluminescent (ECL) and a Bio-Rad ChemiDoc XRS system. The resulting bands were quantified using Quantity One analysis software.
Immunofluorescence Treated cells were grown on coverslips. Cells were washed with 1XPBS, fixed with 3.7% paraformaldehyde, and permeabilized in
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
E X PE R IM EN TA L C ELL R E S EA RC H
0.25% Triton. Cells were incubated with an anti-estrogen receptor (ERα) antibody (1:100) dilution for 1 h. Following incubation with the secondary antibody, cells were washed and treated with a 1:1000 DAPI solution for 5 min. Coverslips were mounted and then visualized using an Olympus iX81 Motorized Inverted Confocal Microscope equipped with Fluoview FV500 software.
Quantification of ERα distribution within the cell ERα levels were quantified via intensity of fluorescence in both the cytoplasm and the nucleus. The relative intensity of immunofluroescence was quantified using Image-Pro Plus software (Silver Spring, MD). Briefly, Intensity of fluorescence signal at the nucleus and the cytoplasm were measured and the ratio of the nuclear/cytoplasmic signal was averaged for individual cells (n¼ 6) under various conditions. A percent change formula, [(TreatmentBasal)/(Basal) 100%], was used to determine the change in protein localization.
Statistical analysis Data are stated as means7SEM. The significance of comparing means was assessed by two-way analysis of Student's t-test (StatPlus, AnalystSoft).
Results A Nar–Tam combination impairs cell proliferation and survival in MCF-7 cells Previous studies examined the role of Nar on cell proliferation, ER regulation and growth factor signaling pathways in charcoal stripped serum in the presence or absence of exogenously added 17β-estradiol (E2) for short time periods (15 min–24 h) [14–16,19]. While these studies aimed to establish a relationship between Nar, E2 and ER, they do not account for possible other targets of Nar that
Doubling Time (Normalized to untreated)
150 125 100 75
* *
50 25 uM Nar 0
may be absent in charcoal-stripped serum. While charcoal stripping significantly lowers the concentrations of lipophilic hormones it also removes growth factors from the serum. Both E2 (a lipophilic hormone) and growth factors are cellular targets of Nar that are involved in cell proliferation. In order to determine the role of Nar, we examined the effects of Nar in the presence of serum containing medium (DMEM supplemented with 10% FBS) which will be referred to as “full medium” to retain all the potential inducers of cell proliferation present in the serum. First we determined the concentration of Nar required to impair proliferation of MCF-7 cells in the presence of full medium. We cultured cells in full medium in the presence of increasing concentrations of Nar and assayed cell doubling times. As seen in Fig. 1A Nar has a concentration dependent inhibition of cell doubling times. As seen previously Nar was able to impair proliferation at a concentration of 50 and 100 mM but maximal inhibition was observed at 250 mM. In previous studies Nar concentrations as high as 100 mM show the greatest inhibition of cell proliferation. Our findings show that a slightly greater Nar concentration is required in the presence of serum-containing medium to achieve maximal inhibition of cell proliferation. Next we examined the effect of a Nar–Tam combination on doubling time of MCF-7 cells cultured in full medium. We used the established concentration of Tam (100 nM). Since both Nar and Tam impair cell proliferation we wanted to determine if a Nar– Tam treatment would be more effective and either treatment alone. MCF-7 cells were treated with either Tam (100 nM), Nar (250 mM), or both in full medium. Control cells were treated with the vehicles for Tam (ethanol) and Nar (DMSO). Cells were plated and cultured for 7 days in the presence of each treatment. Our results show that treatment with either Nar alone or Tam alone resulted in a 2-fold increase in doubling time when compared to untreated MCF-7 cells. However, the combination treatment demonstrated a synergistic 13fold increase in doubling time (Fig. 1B). These studies also show that slightly higher concentrations of Nar (250 mM instead of 100 mM) are required to impair cell proliferation in the presence of serumcontaining medium.
*
175 Doubling Time (h)
226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
3
] (]]]]) ]]]–]]]
50
100
250
20 15 10 5
Tam Nar -
+ -
+
+ +
Fig. 1 – Effect of Nar and Tam on doubling times of MCF-7 cells cultured in full medium. (A) MCF-7 cells (2.45 104 cells/ml) are cultured in full media (DMEMþFBS) in the absence or presence of varying concentrations of Nar as indicated. Media is replaced every 2 days. Cells are collected and counted after 6 days. (B) MCF-7 cells are grown for 7 days in full media (DMEMþFBS) in the presence or absence of (Tam) (100 nM), Nar (250 lM) or a combination. After 7 days, the cells are replated for an additional 7 days. The media is changed and the treatments are reapplied every 2 days. Following treatment cell density is quantified and doubling times are calculated using the formula, Td ¼ (t2 t1) log 2/log(q2/q1) where “t” is the time in hours and “q” is the number of cells counted at each time point. The values from each treatment group are presented as a percentage of an untreated control. The results are the means7SEM of three independent experiments. Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
4
Nar–Tam impairs MCF-7 cell viability Next we wanted to determine the mechanism of action of Nar–Tam on decreasing cell density. To determine whether a Nar–Tam combination would impair cell viability to a greater extent than either treatment alone, we treated MCF-7 cells with Tam, Nar, or a combination of the two (Fig. 2A). As expected, Tam treatment showed a decrease in cell viability. Nar treatment alone significantly reduced (40%) cell viability by day 5, confirming the cytotoxic behavior of Nar (Fig. 2A). The combination treatment had even a greater reduction in cell viability on days 4 and 5 (65%). We also assayed cell death on the various days of treatment. As shown previously, Tam had an effect on cell death. As expected there was no effect of Nar–Tam on days 2 and 3 but a significant increase in cell death on days 4 and 5 after treatment. Nar treatment alone also increased cell death although to a lesser extent than the Nar–Tam treatment. Collectively, our results show that a Nar–Tam treatment elicited a greater cytostatic and cytotoxic effect than either treatment alone. These findings are critical since they examine the long term effects of a Nar–Tam combination in serum-containing medium.
Lower concentrations of Nar and Tam are required in the Nar–Tam combination to impair cell proliferation and viability Next, we investigated whether a lower concentration of either compound would elicit similar effects as the initial treatment used (250 μM Nar and 100 nM Tam on MCF-7 cells on day 4) on cell density and viability (Fig. 3). To do this, we treated MCF-7 cells with Nar (250 μM) and decreasing concentrations of Tam, Tam (100 nM) and decreasing concentrations of Nar or varying concentrations of both Nar and Tam for 4 days in full medium and then determined cell density and viability. Cell density did not change when Tam was reduced during the dual treatment (Fig. 3A). In contrast, however, lowering the Nar concentration (o200 μM) resulted in a significant increase in cell density (Fig. 3B). Next, we wanted to determine the lowest concentrations of both Nar and Tam. A similar level of impaired cell proliferation to that seen in the initial treatment was seen at 200 μM Nar and 25 nM Tam (Fig. 3C). The other two treatments were not able to significantly impair cell density.
] (]]]]) ]]]–]]]
Next we performed similar studies to determine the lowest concentration of Nar–Tam on cell viability (Fig. 3 panels D–F). Reduction of Tam in the presence of 250 μM Nar did not affect cell viability (Fig. 3D). However lowering the concentration of Nar below 200 μM significantly increased cell viability compared to the initial treatment (Fig. 3E). Finally, we treated cells with varying concentrations of both agents for 4 days and assessed cell viability. There was a significant increase in cell viability in samples treated with Nar concentrations less than 200 μM when compared to the initial treatment (Fig. 3F). These studies indicate that 25 nM Tam and 200 μM Nar is the lowest concentration of both compounds that will achieve the same response as the initial treatment on cell viability. Collectively, our results indicate that Nar has a dose dependent effect on cell viability and density below 200 μM. In contrast, Tam does not alter either cell density or viability in the presence of Nar. Furthermore, our studies show that the concentrations of both Nar and Tam can be reduced to achieve maximal effects on cell density and viability when used in combination in the presence of serum containing medium.
Nar–Tam reduces the total protein levels of ERK1/2 and AKT To determine the mechanism by which Nar–Tam elicits effects on cell density and viability, we investigated the known targets of Nar. Nar has been shown to inhibit the phosphorylation but not the expression levels of ERK1/2 and AKT in MCF-7 cells cultured in stripped serum for 24 h [15]. To investigate the effect of Nar and Nar–Tam on ERK1/2 and AKT, we treated MCF-7 cells with Tam, Nar, and Nar–Tam for 4 days. We examined the expression levels as well as the phosphorylation status of ERK1/2 and AKT (Fig. 4A–C). Tam treatment did not affect the expression of ERK1/2 or AKT (Fig. 4A and B). In contrast, Nar and to a greater extent Nar–Tam significantly reduced the expression of ERK1/2 and AKT when compared to the control (Fig. 4A and B). The levels of actin were unaffected indicating that the effect of Nar was specific. Next we examined the effect of the treatments on the phosphorylation status of ERK1/2 and AKT. Surprisingly, none of the treatments significantly altered the phosphorylation status of ERK 1/2 or AKT relative to total protein levels (Fig. 4A and C). These results demonstrate that Nar has effects on ERK1/2 and AKT protein levels in MCF-7 cells.
120 % Cell Death (Normalized to Untreated)
% Viable Cells (Normalized to untreated)
346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405
E XP E RI ME N TAL CE L L R ES E ARC H
100 80
*
60 40
* *
20
0 2
3
4 Days
5
80
Tam Nar Nar-Tam
*
60
* 40
*
20
2
3
4
5
Days
Fig. 2 – Effect of Nar on cell viability. MCF-7 cells are grown in full medium in the presence of Tam (100 nM) (circle), Nar, (250 μM) (square) or a combination of the two (triangle) for the indicated times. (A) Cell viability and (B) cell death are determined by flow cytometry. Results are the means7SEM of three independent experiments. *po0.05. Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
E X PE R IM EN TA L C ELL R E S EA RC H
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525
] (]]]]) ]]]–]]]
5
Fig. 3 – A Nar–Tam combination requires lower concentration of both compounds to achieve optimal reduction in cell density and viability. MCF-7 cells are grown in full medium in the presence of either varying concentrations of Nar or Tam or both for four days. (A–C) Cell density and (D–F) cell viability are determined by flow cytometry. Results are the means7SEM of three independent experiments. Significance at Po0.05 is compared to the initial treatment (*).
Nar–Tam promotes apoptosis via activation of caspases 7 and 9
in Nar and Nar–Tam treated cells compared to levels present in untreated cells.
Our studies show that Nar and Nar–Tam inhibit cell density in part by increasing cell death. To investigate the mechanism of the effect of Nar and Nar–Tam on viability, we assayed for the presence of apoptotic cells by flow cytometry (Fig. 5A). Our results show that both Nar–Tam and Nar increased the number of apoptotic cells starting on day 4 of treatment when compared to untreated and Tam treated cells cultured in full medium. To determine how Nar induced apoptosis we examined members of the caspase family. Studies have shown that Nar induces apoptosis via caspase 3 activation in HeLa cells transfected with ERα [14,16]. Since MCF-7 cells do not express caspase 3 but do express caspases 7 and 9, we wanted to investigate whether these caspases were involved in Nar induced apoptosis (Fig. 5B and C) [20]. Caspase 7 was significantly reduced in Nar and Nar–Tam treated cells (Fig. 5B and C). Similarly, caspase 9 was undetectable in Nar and Nar–Tam treated cells (Fig. 5B). These results show that caspases 7 and 9 are cleaved and activated, in Nar–Tam treated cells. We also examined the downstream target of caspase 7, PARP. As shown in Fig.5B, we detected increased levels of cleaved PARP
Nar–Tam influences ER localization Since both Nar and Tam interact with ERα [11,17,21], we wanted to investigate the localization of ERα in the various treatments. To investigate the localization pattern of ERα, we treated MCF-7 cells with Nar, Tam or Nar–Tam for 4 days in serum-containing medium and then visualized ERα by confocal microscopy (Fig. 6A and B). The ER is localized throughout the cell. Upon estrogen binding, the ER-estrogen complex translocates to the nucleus. As shown in Fig. 6A (panels a–c) the ER is localized throughout the cell with more intense staining in the nucleus. As described previously, our studies also show that Tam induces the translocation of the ERα from the cytosol to the nucleus in full medium (Fig. 6A and B) [2]. In contrast, Nar treated cells showed an accumulation of ERα in the cytoplasm (Fig. 6A and B). Nar–Tam treated cells exhibited an even distribution of ERα in the nucleus and the cytoplasm, suggesting that both Nar and Tam independently influence the localization of ERα in the combination treatment.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585
6
Vehicle
Tam
Nar
Nar-Tam
] (]]]]) ]]]–]]]
Vehicle Tam Nar Nar-Tam
150
Percent of Vehicle
pERK 1/2
ERK 1/2
pAKT AKT
100
* *
50
* *
* *
Actin ERK1
ERK2
AKT
Vehicle Tam Nar Nar-Tam
350
Percent of Vehicle
586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645
E XP E RI ME N TAL CE L L R ES E ARC H
300 250 200 150 100 50
pERK1/ pERK2 pAKT/ ERK1 /ERK2 AKT
Fig. 4 – Nar–Tam reduces the levels of proteins involved in proliferation and survival. MCF-7 cells are grown in full medium either untreated (Vehicle) or in the presence of Tam (25 nM), Nar (200 μM), or a combination of the two for 4 days. Protein lysates are prepared. (A) Lysates are subjected to SDS-PAGE and immunoblotted using antibodies against pERK, ERK, pAKT, AKT, and actin. Immunoblot results are quantified by densitometry for (B) total protein levels and (C) phospho proteins. ERK results are the means7SEM of four independent experiments. AKT results are the means7SEM of three independent experiments. Significance at Po0.05 is compared to vehicle (*).
Discussion Since over half of breast cancers are ERþ and depend on estrogen to promote proliferation, the focus of pharmaceutical treatments has been to target the ER. The ER is regulated not only by estrogen but also by phosphorylation mediated by the activation of kinase cascades. Currently, the use of Tam in conjunction with multiple kinase inhibitors is being investigated for the treatment of breast cancers [22]. Natural products such as Nar have also been shown to have anti-proliferative effects. In this study we investigated the effect of Nar in conjunction with Tam to impair cell proliferation and viability in MCF-7 breast cancer cells. Our study demonstrates that a Nar–Tam combination elicits its effects by targeting multiple proliferative and apoptotic pathways resulting in greater inhibition of proliferation and cell viability than either treatment alone. We first focused our studies on the effects of Nar–Tam on MCF7 cells in serum-containing medium to retain all the potential inducers of cell proliferation present in the serum. Initially we determined the optimal concentration required to achieve maximal inhibition of proliferation and viability of each compound alone, and then we used these concentrations in combination. Overall, our studies indicate that Nar–Tam inhibited MCF-7 cell proliferation and viability better than either component alone.
Next we determined the optimal concentration of the Nar–Tam treatment on cell proliferation and viability. The reduction of Tam concentration showed no effect on cellular proliferation or viability when compared to the initial concentration when used in conjunction with Nar. Conversely there was a dose-dependent effect of Nar on both cell proliferation and viability. Taken together, our results show that when used in combination lower concentration of both Nar and Tam can be used to achieve optimal inhibition of cell proliferation and viability. In order to determine the mechanism of action of the Nar–Tam combination on cell proliferation and viability, we examined the known targets of Nar and Tam. Our previous studies demonstrated that Nar impairs the phosphorylation of ERK1/2 and AKT when cells were exposed for short time periods (4 h) [13]. In this study we exposed MCF-7 cells to Nar for several days and then examined the expression levels and phosphorylation status of ERK1/2 and AKT. Surprisingly Nar treatment reduced the overall levels of ERK1/2 and AKT which was not observed at shorter time periods. Currently inhibitors of both the ERK and AKT pathways are targets in breast cancer clinical trials. Nar reduced cell density by not only impairing cell proliferation but also by inducing apoptosis via activating caspase 9 and caspase 7. Although we were unable to detect the cleaved forms of the caspases, others
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705
E X PE R IM EN TA L C ELL R E S EA RC H
20
% Mid Apoptotic
Control
Control Tam Nar Nar-Tam
15
*
10
2
3
Nar
Nar-Tam
Caspase 9
* *
5
Tam
Caspase 7
*
Cleaved PARP
4
Actin
5
Days
140 120
Caspase 7 % of Control
100 80
* *
60 40 20
Fig. 5 – Role of Nar–Tam on apoptosis. MCF-7 cells are grown in full medium alone “Control” or in the presence of Tam (25 nM), Nar (200 μM), or a combination of the two. (A) Apoptosis is determined at the indicated time points by flow cytometry. *po0.05. (B) On day 4 protein lysates are prepared and subjected to SDS-PAGE and immunoblotted using antibodies against caspase 7, caspase 9, cleaved PARP and actin. (C) Caspase 7 levels are quantified using densitometry. Results are the means7SEM of three independent experiments. Significance at Po0.05 is compared to vehicle (*).
ER
DAPI
Merge 1.0
Vehicle 0.8
Tam
Nuclear/Total
706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765
7
] (]]]]) ]]]–]]]
0.6 0.4 0.2
Nar
Nar-Tam
Fig. 6 – Nar localizes ER to the cytoplasm. MCF-7 cells are grown in full medium alone “Vehicle” or in the presence of Tam (25 nM), Nar (200 μM), or a combination of the two for 4 days on coverslips. (A) On day 4, cells are fixed and stained with anti-ERα antibody and DAPI and then subjected to confocal microscopy. (B) ERα nuclear localization is quantified. Results are the means7SEM of three independent experiments. Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825
8
826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885
E XP E RI ME N TAL CE L L R ES E ARC H
have shown that a reduction in the full length caspases indicates their cleavage and thus activation [20,23]. To support the idea of caspase-mediated apoptosis we examined the downstream target of caspase 7, PARP. Our findings show that Nar treatment increased the levels of cleaved PARP in MCF-7 cells. Since the ER is also a target of both Nar and Tam we examined the effect of these treatments on ER localization. Normally, estrogen binding to ERα leads to translocation of the complex to the nucleus and recruitment of co-activators to regulate transcription of estrogen-responsive genes [2]. However, when Tam binds to ERα it causes a conformational change in the receptor leading to a reduction in binding of co-activators and/or promoters of estrogen-responsive genes. In the absence of DNA binding the Tam/ERα complex is localized to the cytosol allowing it to interact with kinase signaling pathways such as the PI3K and MAPK pathways [24–29]. Fan et al. also noted a change in ERα localization from the nucleus to the cytoplasm following Tam treatment [30]. Our results show that Nar treatment also causes a relocalization of ERα to the cytoplasm. We are the first to specifically note a perinuclear localization pattern of ERα in Nar treated cells. Thus future studies are required to determine if Nar relocalization of ERα outside the nucleus results in reduced expression of estrogen-responsive genes. In summary, our studies demonstrate that a Nar–Tam combination results in greater inhibition of cellular proliferation and viability then either component alone in MCF-7 cells cultured in serum containing medium for longer time points. We also show that use of the combination treatment requires lower concentrations of both components to achieve optimal results. Furthermore we show that Nar targets ERK, AKT and the caspase pathway to exert its effects on proliferation and viability. Additionally, our studies show that Nar localizes ERα to the cytoplasm. Collectively our results enhance the understanding of the mechanism of action of Nar in conjunction with Tam in MCF-7 cells.
references [1] C. DeSantis, J. Ma, L. Bryan, A. Jemal, Breast cancer statistics, 2013, CA Cancer J. Clin. 64 (2014) 52–62. [2] B.J. Deroo, K.S. Korach, Estrogen receptors and human disease, J. Clin. Investig. 116 (2006) 561–570. [3] S. Safe, K. Kim, Non-classical genomic estrogen receptor (ER)/ specificity protein and ER/activating protein-1 signaling pathways, J. Mol. Endocrinol. 41 (2008) 263–275. [4] S. Sommer, S.A. Fuqua, Estrogen receptor and breast cancer, Semin. Cancer Biol. 11 (2001) 339–352. [5] V. Kumar, S. Green, G. Stack, M. Berry, J.R. Jin, et al., Functional domains of the human estrogen receptor, Cell 51 (1987) 941–951. [6] G. Bunone, P.A. Briand, R.J. Miksicek, D. Picard, Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation, EMBO J. 15 (1996) 2174–2183. [7] P.H. Driggers, J.H. Segars, Estrogen action and cytoplasmic signaling pathways. Part II: the role of growth factors and phosphorylation in estrogen signaling, Trends Endocrinol. Metab. 13 (2002) 422–427. [8] S.E. Ghayad, J.A. Vendrell, S. Ben Larbi, C. Dumontet, I. Bieche, et al., Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways, Int. J. Cancer 126 (2010) 545–562.
] (]]]]) ]]]–]]]
[9] M. Baum, Tamoxifen–the treatment of choice, Why look for alternatives? Br J Cancer 78 (Suppl 4) (1998) S1–S4. [10] S. Ali, R.C. Coombes, Endocrine-responsive breast cancer and strategies for combating resistance, Nat. Rev. Cancer 2 (2002) 101–112. [11] C.G. Sanchez, C.X. Ma, R.J. Crowder, T. Guintoli, C. Phommaly, et al., Preclinical modeling of combined phosphatidylinositol-3kinase inhibition with endocrine therapy for estrogen receptorpositive breast cancer, Breast Cancer Res. 13 (2011) R21. [12] A.W. Harmon, Y.M. Patel, Naringenin inhibits phosphoinositide 3kinase activity and glucose uptake in 3T3-L1 adipocytes, Biochem. Biophys. Res. Commun. 305 (2003) 229–234. [13] A.W. Harmon, Y.M. Patel, Naringenin inhibits glucose uptake in MCF-7 breast cancer cells: a mechanism for impaired cellular proliferation, Breast Cancer Res. Treat. 85 (2004) 103–110. [14] P. Bulzomi, A. Bolli, P. Galluzzo, F. Acconcia, P. Ascenzi, et al., The naringenin-induced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background, IUBMB Life 64 (2012) 690–696. [15] P. Galluzzo, P. Ascenzi, P. Bulzomi, M. Marino, The nutritional flavanone naringenin triggers antiestrogenic effects by regulating estrogen receptor alpha-palmitoylation, Endocrinology 149 (2008) 2567–2575. [16] P. Totta, F. Acconcia, S. Leone, I. Cardillo, M. Marino, Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involvement of estrogen receptor alpha and beta signalling, IUBMB Life 56 (2004) 491–499. [17] F. Virgili, F. Acconcia, R. Ambra, A. Rinna, P. Totta, et al., Nutritional flavonoids modulate estrogen receptor alpha signaling, IUBMB Life 56 (2004) 145–151. [18] M. Marino, M. Pellegrini, P. La Rosa, F. Acconcia, Susceptibility of estrogen receptor rapid responses to xenoestrogens: physiological outcomes, Steroids 77 (2012) 910–917. [19] P. Bulzomi, A. Bolli, P. Galluzzo, S. Leone, F. Acconcia, et al., Naringenin and 17beta-estradiol coadministration prevents hormone-induced human cancer cell growth, IUBMB Life 62 (2010) 51–60. [20] H. Kurokawa, K. Nishio, H. Fukumoto, A. Tomonari, T. Suzuki, et al., Alteration of caspase-3 (CPP32/Yama/apopain) in wild-type MCF-7, breast cancer cells, Oncol. Rep. 6 (1999) 33–37. [21] M. Clemons, S. Danson, A. Howell, Tamoxifen (Nolvadex): a review, Cancer Treat. Rev. 28 (2002) 165–180. [22] E.M. Fox, C.L. Arteaga, T.W. Miller, Abrogating endocrine resistance by targeting ERalpha and PI3K in breast cancer, Front. Oncol. 2 (2012) 145. [23] J. Li, P. Chen, N. Sinogeeva, M. Gorospe, R.P. Wersto, et al., Arsenic trioxide promotes histone H3 phosphoacetylation at the chromatin of CASPASE-10 in acute promyelocytic leukemia cells, J. Biol. Chem. 277 (2002) 49504–49510. [24] S. Kato, H. Endoh, Y. Masuhiro, T. Kitamoto, S. Uchiyama, et al., Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase, Science 270 (1995) 1491–1494. [25] H. Kurokawa, A.E. Lenferink, J.F. Simpson, P.I. Pisacane, M.X. Sliwkowski, et al., Inhibition of HER2/neu (erbB-2) and mitogenactivated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells, Cancer Res. 60 (2000) 5887–5894. [26] K. Liang, W. Jin, C. Knuefermann, M. Schmidt, G.B. Mills, et al., Targeting the phosphatidylinositol 3-kinase/Akt pathway for enhancing breast cancer cells to radiotherapy, Mol. Cancer Ther. 2 (2003) 353–360. [27] A. Migliaccio, M. Di Domenico, G. Castoria, A. de Falco, P. Bontempo, et al., Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells, EMBO J. 15 (1996) 1292–1300.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945
E X PE R IM EN TA L C ELL R E S EA RC H
946 947 948 949 950 951 952 953
[28] M. Sun, J.E. Paciga, R.I. Feldman, Z. Yuan, D. Coppola, et al., Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K, Cancer Res. 61 (2001) 5985–5991. [29] E. Tokunaga, A. Kataoka, Y. Kimura, E. Oki, K. Mashino, et al., The association between Akt activation and resistance to hormone
] (]]]]) ]]]–]]]
9
therapy in metastatic breast cancer, Eur. J. Cancer 42 (2006) 629–635. [30] P. Fan, J. Wang, R.J. Santen, W. Yue, Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells, Cancer Res. 67 (2007) 1352–1360.
Please cite this article as: T. Hatkevich, et al., A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.05.017
954 955 956 957 958 959 960