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Electrophoresis 2014, 35, 1719–1727 1 ´ Margarita Gonzalez-Vallinas 1 Susana Molina Gonzalo Vicente2 ´ Ruth Sanchez-Mart´ ınez1 Teodoro Vargas1 ´ Monica R. Garc´ıa-Risco2 Tiziana Fornari2 Guillermo Reglero1,2 Ana Ram´ırez de Molina1 1 IMDEA-Food
Institute, CEI UAM + CSIC, Madrid, Spain 2 Instituto de Investigacion ´ en ´ Ciencias de la Alimentacion (CIAL), CEI UAM+CSIC, Madrid, Spain
Received November 14, 2013 Revised February 6, 2014 Accepted February 7, 2014
Research Article
Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells Breast cancer is the leading cause of cancer-related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER-␣ and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER-␣ and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen-dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy. Keywords: Breast cancer / Estrogen receptor alpha / Human epidermal growth factor receptor-2 / Paclitaxel / Rosemary DOI 10.1002/elps.201400011
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Breast cancer is the most frequently diagnosed tumor among females in both economically developed and developing countries [1]. Although breast cancer death rates have been diminishing in some developed countries, mostly due to early detection and improved treatment, this type of cancer is still the leading cause of cancer-related mortality in females worldwide, thus being responsible for 14% of the total cancer deaths Correspondence: Dr. Ana Ram´ırez de Molina IMDEA-Food Institute. CEI UAM+CSIC. Ctra. de Canto Blanco 8. 28049 Madrid. Spain E-mail: [email protected] Fax: +34-91-1880756
Abbreviations: ER, estrogen receptor; ER+, estrogendependent; ER−, estrogen-independent; ER-␣, estrogen receptor alpha; HER2+, overexpression of human epidermal growth factor receptor-2; MTT, 3-(4,5-dimethyl-thyazol-2-yl)2,5-diphenyl-tetrazolium; qRT-PCR, quantitative reverse transcription PCR; SFRE, supercritical fluid rosemary extract; TNBC, triple negative breast cancer C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in 2008 [1]. Consequently, the development of novel antitumor treatments, ideally nontoxic and cost-effective, continues to be of the utmost importance. The utility of rosemary (Rosmarinus officinalis L.) extracts and their components in breast cancer prevention and treatment has been suggested by different studies. Regarding chemoprevention, a commercial extract (Herbalox type O, Kalsec Inc.), containing 40% of crude rosemary extract, showed an inhibitory effect on mammary inducedtumorigenesis in vivo, by either oral or intraperitoneal administration [2]. The effect was explained by the decrease of 7,12-dimethylbenz(a)anthracene-DNA adduct formation and the induction of glutathione S-transferase and quinone reductase activities, and mostly attributed to the carnosol content of the extract [2]. Moreover, rosemary extracts obtained by different procedures (including ethanolic, methanolic, and supercritical fluid extracts) were reported to inhibit the viability of several tumor cell types, including breast cancer [3–5], and carnosic acid was found to be the most active component regarding this activity [5, 6]. The comparison between supercritical fluid and methanolic rosemary extracts indicated that the former contain more active compounds, and are www.electrophoresis-journal.com
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therefore more effective in inhibiting cell viability [6]. Moreover, rosemary extracts obtained by supercritical fluid technology have been declared as safe for human use by the European Food Safety Authority (EFSA) [7]. Nevertheless, breast cancer is a heterogeneous disease that involves different tumor subtypes that should be differentially treated. Breast tumors are classified as ER+ subtype (estrogen-dependent tumor, which expresses estrogen receptor alpha (ER-␣) and responds to estrogens, in contrast to estrogen-independent (ER−) tumors), human epidermal growth factor receptor-2 (HER2)+ subtype (HER2 overexpressing tumor, which could be either ER+ or ER−) and triple negative breast cancer (TNBC) subtype (which lacks the expression of the three biomarkers: ER-␣, progesterone receptor, and HER2). This classification allows the prediction of tumor sensitivity to the different chemotherapeutic drugs and it is therefore used for drug selection in the clinical practice [8]. Accordingly, patients with ER+ tumors are expected to respond to hormonal therapy (e.g. tamoxifen), whereas patients with HER2+ tumors will be candidates to receive trastuzumab treatment, a mAb that targets the extracellular domain of HER2 thus inhibiting tumor cell proliferation induced by HER2 signaling pathway. In contrast, there is no targeted chemotherapy for TNBC and this tumor subtype is commonly treated with the natural antitumor agent paclitaxel in combination with anthracyclines [9]. In view of that, it is necessary to assay the activity and mechanism of rosemary extract in the different breast tumor subtypes in order to establish the potential use of rosemary in breast cancer therapy. Some investigations suggest the role of estrogen action and metabolism in the molecular mechanism of rosemary. On the one hand, dietary administration of methanolic rosemary extract increased the liver metabolism (microsomal oxidation and glucuronidation) of estradiol and estrone and inhibited their uterotropic action, thus pointing to a beneficial effect in tumors that frequently respond to estrogens such as endometrial, ovarian, and breast cancers [10]. On the other hand, the antioxidant activity of the rosemary component carnosol was reported to be at least partially mediated through the ER-␣ signaling pathway [11], and this diterpene was proved to antagonize ER-␣ with no agonist effects [12]. These results suggest a potential differential effect of rosemary against ER+ and ER− breast cancer cells. Accordingly, previous reports suggested the higher cell sensitivity to rosemary extract of ER− in comparison to ER+ cell lines [3]; however, this suggestion was based on the use of MCF-7 cells as the unique model of ER+ breast cancer [3]. In this work, we aimed to determine the sensitivity of several breast cancer cell lines from different subtypes to the antitumor activities of a supercritical fluid rosemary extract (SFRE). In addition, due to the clinical relevance of ER-␣ and HER2 signaling pathways in breast cancer cells, we used a genomic approach to explore their potential modulation by this bioactive agent. Thus, the results of this work contribute to the global Foodomics strategy for the study of the health properties attributed to rosemary, which includes the investigation of the interaction between this bioactive food component and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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gene expression [13]. Finally, we assayed the antitumor effect of SFRE in combination with some of the antitumor drugs most frequently used in each breast cancer subtype, in order to determine the patients who will most benefit from the use of SFRE within their cancer therapy.
2 Materials and methods 2.1 Rosemary extract Rosemary extract was obtained from 0.5 kg of dried and grinded rosemary leaves by supercritical fluid extraction technology in a supercritical fluid pilot plant (Thar Technology, model SF2000) as previously described [14]. The CO2 flow rate was 60 g/min and a temperature equal to 40°C was maintained in extraction cell and separators. The pressure of the extractor was 300 bar and for the first 60 min the extracted material was fractionated by setting 100 bar of pressure in the first separator (S1), and 50 bar (the pressure of the recirculation system) in the second separator (S2). Afterwards, extraction continued without fractionation for 300 min. SFRE was collected from S1, dissolved in absolute ethanol at 50 mg/mL, and stored at −20°C. The major components of SFRE were 19.56% w/w carnosic acid, 1.90% w/w carnosol, and 15.52% w/w volatile oils. Carnosic acid and carnosol were quantified by HPLC analysis, and volatile oil components were determined by GC-MS analysis, as previously described [15]. 2.2 Cell culture Breast cancer cell lines MCF-7 (ER+), T-47D (ER+), MDA-MB-231 (TNBC), UACC-812 (HER2+), and SK-BR3 (HER2+), as well as CCD 841 cells (normal epithelial cells) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured under standard conditions of temperature (37°C), humidity (95%), and carbon dioxide (5%) in DMEM (MDA-MB-231 and MCF-7), RPMI (T-47D and SK-BR-3), or EMEM (CCD 841) supplemented with 10% FBS, 2 mM glutamine, and 1% of antibioticantimycotic solution (containing 10 000 units/mL penicillin base, 10 000 g/mL streptomycin base, and 25 000 ng/mL amphotericin B; Gibco, Grand Island, NY, USA). UACC-812 cells were maintained in DMEM supplemented with 20% FBS and 20 ng/mL epidermal growth factor, as recommended by the cell supplier. The main molecular characteristics of the breast cancer cell lines are collected in Table 1. 2.3 MTT assay Cell viability was assessed by the 3-(4,5-dimethyl-thyazol-2yl)-2,5-diphenyl-tetrazolium (MTT) assay as previously described [14]. Breast cancer cells (20 000–70 000 per well, optimized according to their proliferation rate) were seeded in 24-well plates. After overnight attachment, the cells underwent the corresponding treatment with SFRE and/or the www.electrophoresis-journal.com
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Electrophoresis 2014, 35, 1719–1727 Table 1. Molecular characterization of breast cancer cell lines
Breast cancer cell line
ER (+/−)a)
HER2 (+/−)b)
PgR (+/−)c)
Tumor subtype
MCF-7 T-47D UACC-812 SK-BR-3 MDA-MB-231
+ + − − −
− − + + −
+ + − − −
ER+ ER+ HER2+ HER2+ TNBCd)
a) ER: Estrogen responsiveness. b) HER2: Her-2/ErbB2/neu overexpression. c) PgR: Progesterone receptor expression. d) TNBC: Triple Negative Breast Cancer.
antitumor drugs paclitaxel (Sigma-Aldrich, St. Louis, MO, USA), 4-hydroxy-tamoxifen (Sigma-Aldrich), or trastuzumab R , kindly provided by GenenTech, San Francisco, (Herceptin CA, USA). Following 48 h or 72 h treatment, cells were incubated with MTT solution (5 mg/mL in PBS) at 1:10 dilution for 3 h, and the MTT metabolic product (formazan) was dissolved in 200 L DMSO. The quantity of formazan obtained, which correlates with cell viability, was determined by measuring the absorbance at 560 nm. The experiments involving the use of 4-hydroxy-tamoxifen were performed in culture medium without phenol red and with 5% charcoal/dextrantreated FBS (instead of 10% FBS), in order to minimize the level of estrogens and thus avoiding possible interferences with the results. The parameters IC50 (50% cell viability inhibition), GI50 (50% growth inhibition), TGI (total growth inhibition), and LC50 (50% cell death) were calculated according to the NIH definitions using a logistic regression [16]. In order to calculate the parameters related to cell proliferation and cell death (GI50, TGI, and LC50), the cell viability before the treatment (time zero) was determined.
2.4 Anchorage-independent growth in soft agar Breast cancer MCF-7 cells (90 000 cells), previously treated with 50 g/mL (treatment A and B), or vehicle (control) during 24 h, were suspended in culture medium containing 0.4% agar and seeded on top of a solidified layer of culture medium containing 0.5% agar in p60 plates. Cell suspension was treated with 50 g/mL SFRE (treatment B) or vehicle (treatment A and control). These plates were incubated under standard conditions to allow the formation of colonies, and irrigated twice a week with 200 L of culture medium supplemented with 100 g/mL SFRE (treatment A), 50 g/mL SFRE (treatment B), or the equivalent volume of vehicle (control) for 4–5 weeks. Colonies were dyed with Crystal Violet as previously described [17]. Representative regions of 4 mm2 of each plate (16 regions per plate) were selected and the colonies they contained were counted under a microscope. The total number of colonies in each plate was calculated according to the total surface of p60 plates. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.5 Western blot analysis Cell lysates from breast cancer cells were obtained by dissolving in Laemmli buffer (60 mM Tris-HCl pH 6.8, 10% glycerol, and 2% SDS) the cell pellet previously washed with PBS, and boiling at 95°C for 5 min. Protein concentrations were determined by the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacture’s procedure. Equal amounts of protein (35–50 g) from cell lysates were fractioned by SDS-PAGE in 8% polyacrylamide gels for HER2 analysis or 10% polyacrylamide gels for ER-␣ and poly(ADP-ribose) polymerase 1 (PARP1) analyses, and transferred onto a 0.2 m nitrocellulose membrane (Bio-Rad). The membranes were incubated with 5% BSA in TTBS (TBS supplemented with 0.05% Tween-20) in order to block unspecific sites. Antibodies against PARP1 (1:125 dilution, BD Pharmingen), ER-␣ (1:200 dilution, Santa Cruz Biotechnology), HER2 (antibody 29D8, 1:1000 dilution, Cell Signaling Technology, Beverly, MA, USA), and -actin (1:2000 dilution, SigmaAldrich) were used as primary antibodies. Horseradish peroxidase conjugated antibodies anti-mouse (antibody AP130P, 1:40 000 dilution, Millipore, Billerica, MA, USA) or anti-rabbit (antibody AP106P, 1:20 000 dilution, Millipore) were used as secondary antibodies. Detection was performed using the Clarity Western ECL Substrate (Bio-Rad). Molecular weights of protein bands and densitometry were determined by the TotalLab software (TotalLab, Newcastle, UK). -actin determination was used as an endogenous control of total protein quantity.
2.6 Immunofluorescence Cells were fixed with 4% buffered paraformaldehyde for 10 min at room temperature and then permeabilized for 30 min with −20°C methanol, and stained with a 1:100 dilution of anti-HER2 (29D8, Cell Signaling) followed by incubation with Alexa 488-conjugated anti-rabbit antibody (1:1000) and/or with 4,6 diaminophenylindole (DAPI; Prolong Gold antifade, Invitrogen) to visualize nuclei. Images were captured using a Leica DM IL microscope with a 40X Plan Fluotar objective.
2.7 Gene expression analysis To analyze the expression of 84 key genes of the estrogen receptor (ER) signaling pathway, total RNA from breast cancer cells, treated with 40 g/mL SFRE or vehicle for 48 h, was extracted using the RNeasy Mini Kit with RNAse-Free DNAse kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The quantity and purity of the obtained RNA samples were determined by UV-spectroscopy (NanoDropTM 2000 Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). Samples with ratio A260:A230 above 1.7, ratio A260:A280 between 1.8 and 2.0, and concentration higher than 40 ng/L were accepted for subsequent www.electrophoresis-journal.com
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analysis. Gene expression was determined by using the Human Estrogen Receptor Signaling RT2 Profiler PCR Array (SABiosciences). For each PCR array, 500 ng were retrotranscribed with RT2 First Strand Kit, and RT2 qPCR SYBR Green/ROX Master mix was used to perform quantitative PCR (qPCR) in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer’s protocol. Results were analyzed using the RT2 ProfilerTM PCR Array Data Analysis (SABiosciences), applying the mean of B2M, HPRT1, RPL13A, GAPDH, and ACTB gene expression as endogenous control for each plate. Absolute fold change ⬎ 2 and p value ⬍ 0.05 cutoffs were used to consider a gene to be significantly modulated. Individual gene expression analysis was also performed by quantitative reverse transcription-PCR (qRT-PCR). To that end, 400 ng RNA was reverse-transcribed to cDNA using the High Capacity RNA-to-cDNA Master Mix system (Applied Biosystems, Carlsba, CA, USA), as directed by the manufacturer. qPCR was performed in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer’s protocol using the specific Taqman gene expression assays (Applied Biosystems; Hs01001580_m1 for ERBB2, Hs00174860_m1 for ESR1, Hs00170630_m1 for FOS, and Hs99999901_s1 for 18S as endogenous control). qRT-PCR data extraction was performed using the RQ Manager software (Applied Biosystems), and the 2−࢞࢞Ct method was applied to calculate the relative expression of each gene as previously described [18].
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SFRE, cell viability of normal CCD 841 cells was 39.3 ± 9.4% (mean ± SEM), whereas viability of tumor cells decreased on average until 7.8 ± 1.9% (mean ± S.E.M.) after the same treatment. This result indicates that there is a therapeutic range for the use of SFRE in the clinical practice. Regarding the differences among tumor cells, MCF-7 and UACC-812 seemed to be the most resistant cell lines at higher doses of SFRE, although the treatment at low SFRE concentrations showed the higher sensitivity of HER2+ breast cancer cells (Fig. 1A). The comparison of the parameters regarding the inhibition of 50% cell proliferation (GI50) of the different cell lines confirmed the higher sensitivity of HER2+ cell subtype to low doses of SFRE, due to GI50 values of SFRE in SK-BR-3 and UACC-812 cells are lower than those in the other breast cancer cell lines assayed (Fig. 1B). Since the concentration of phenolic compounds that can be achieved in vivo are usually low, it is probable that the sensitivity of the different cancer cell subtypes to low doses of SFRE in vitro most reliably reflects the different sensitivity of the tumor subtypes in vivo. Apoptosis induction assessed by PARP1 cleavage analysis (which is considered a prominent marker of apoptosis [19]) showed that SFRE dose-dependently induced apoptosis in T47D and SK-BR-3 cell lines, while cleaved PARP1 is almost absent in the rest of treated breast cancer cell lines (Fig. 1C). Since LC50 parameters suggest that 80 g/mL of SFRE is able to induce cell death in the breast cancer cell lines assayed, the absence of PARP1 cleavage at that concentration indicates that SFRE may also induce a different type of cell death in breast cancer cells.
2.8 Statistical analysis Results are shown as the mean ± SEM of three independent experiments, each performed in triplicate, unless otherwise indicated. Comparisons between two groups were analyzed by Student’s t-test, whereas differences among several groups were determined by one-way ANOVA with Bonferroni’s post hoc test. Statistically significant values are indicated by asterisks as follows: *p ࣘ 0.05; **p ࣘ 0.01; ***p ࣘ 0.001. IBM SPSS Statistics version 20 (SPSS, Chicago, IL) was used for statistical analysis.
3 Results 3.1 Differential effect of SFRE on cell viability inhibition and apoptosis induction of human tumor cells from distinct breast cancer subtypes Human breast cancer cells from the subtypes ER+ (MCF-7 and T-47D), HER2+ (SK-BR-3 and UACC-812), and TNBC (MDA-MB-231) were treated with increasing concentrations of SFRE, and cell viability was determined by MTT assay. SFRE decreased cell viability of the five breast cancer cell lines in a dose-dependent manner (Fig. 1A). Normal epithelial cells (CCD 841) were used to compare the effect of SFRE on tumor and normal cells. After treatment with 120 g/mL of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Inhibitory effect of SFRE on cell transformation of human breast cancer cells Since anchorage-independent proliferation is a hallmark of malignant transformation, the effect of SFRE on cell transformation was assessed by determining the capacity of the less sensitive breast cancer MCF-7 cells seeded in suspension in a semisolid medium to form colonies. Two different patterns of SFRE treatment (A and B) were compared to control (vehicletreated) cells. Both treatment A and treatment B plates involved the 24 h pretreatment with SFRE and the irrigation with SFRE twice a week, and treatment B additionally contained SFRE in the semisolid medium. As it can be observed in Fig. 1D, both SFRE treatment patterns significantly inhibited the transformation of human breast cancer cells, and the effect was markedly higher when the semisolid medium also contained SFRE, suggesting a specific dose-dependent effect.
3.3 Different modulation by SFRE of the ER signaling pathway in ER+ and ER− breast cancer cells In order to determine the different molecular mechanism of SFRE in ER+ and ER− breast cancer cells, the modulation www.electrophoresis-journal.com
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Figure 1. Differential antitumor effects of SFRE in human tumor cell lines from several breast cancer subtypes regarding cell viability, apoptosis induction, and cell transformation. (A) Cell viability inhibition (assessed by the MTT assay) of different breast cancer cell lines after 48 h treatment with increasing concentrations of SFRE. The results are expressed as the mean ± SEM of at least two independent experiments, each performed in quadruplicate. The first part of the graph was enlarged (square on the right) in order to better visualize the different sensitivity of the breast cancer cell lines to low doses of SFRE. (B) Parameters representing the SFRE concentrations (in g/mL) responsible for the 50% cell viability inhibition (IC50), 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% cell death (LC50) after 48 h treatment of the different breast cancer cell lines. The results shown are the mean ± SEM of at least four independent experiments, each performed in quadruplicate. (C) Western blot analysis of PARP1 cleavage (and -actin as endogenous control of total protein quantity) after 48 h treatment with vehicle (Cntrl) or increasing concentrations of SFRE (expressed as g/mL above each lane) of breast cancer cell lines. (D) Capacity of colony formation in soft agar of MCF-7 cells treated with SFRE compared to control (vehicle-treated) cells. Treatment A consisted of 50 g/mL SFRE pretreatment for 24 h and irrigation of plates with 200 L of culture medium containing 100 g/mL SFRE twice a week, whereas treatment B consisted of 50 g/mL SFRE pretreatment for 24 h, 50 g/mL SFRE treatment of soft agar and irrigation of plates with 200 L of culture medium containing 50 g/mL SFRE twice a week. Results are expressed as mean ± SEM of two independent experiments, each performed in triplicate. Statistically significant differences were determined by the Student’s t-test (***p < 0.001).
of the ER signaling pathway was studied by low density arrays in both ER+ (MCF-7) and ER− (MDA-MB-231) breast cancer cell lines. SFRE significantly modulated 14 genes in ER+ cells, whereas only four genes were modulated in ER− cells (Fig. 2A), suggesting a relevant role of ER signaling pathway in the antitumor action of SFRE in ER+ cells. Interestingly, SFRE downregulated the expression of ESR1, the gene encoding the receptor ER-␣, which is responsible for the proliferation of ER+ tumors. Moreover, SFRE also modulated the expression of FOS. Due to the controversial role of this gene, which has been reported to precede cell death in some cases [20, 21], we assayed its modulation in the five breast cancer cell lines (Fig. 2B). FOS gene showed to be upregulated by SFRE in T-47D and SK-BR-3, and the upregu-
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lation correlated with the induction of apoptosis assessed by PARP1 cleavage (Fig. 2C).
3.4 SFRE induces the downregulation of ER-␣ in ER+ breast cancer cells Due to the relevant role of the ER-␣ signaling pathway in breast cancer, and concretely in the ER+ tumor subtype, we validated the effect of SFRE in mRNA and protein levels of ER-␣ in MCF-7 and T-47D (ER+) breast cancer cell lines by qRT-PCR and Western blot analysis, respectively. The results showed that gene (Fig. 3A) and protein (Fig. 3B) levels of
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Figure 2. Modulation of the expression of the estrogen receptor (ER) signaling pathway genes by SFRE in ER− and ER+ human breast cancer cells. (A) Panels (left) and graphs (right) representing the modulation of the expression of genes involved in the ER signaling pathway in MDA-MB-231 (ER−) and MCF-7 (ER+) breast cancer cell lines after 48 h treatment with 40 g/mL SFRE in relation to control (vehicle-treated) cells, assessed by qRT-PCR. Results are expressed as the mean ± SD of three independent experiments. Student’s t-test was applied to assess statistically significant differences. (B and C) Modulation of FOS gene after 48 h SFRE treatment in five different breast cancer cell lines (B) and comparison with the densitometry of Western blot analysis of PARP1 cleavage after the same treatment duration (C). FOS modulation is expressed as the mean ± SEM of three independent experiments, each performed with biological triplicates, and statistically significant differences were determined by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 3. Downregulation of ER-␣ and HER2 receptors by SFRE in breast cancer cells. (A and B) Modulation of ESR1 gene (encoding ER-␣) (A) and ER-␣ protein (B) expressions in ER+ breast cancer cell lines (MCF-7 and T-47D) after 48 h treatment with increasing concentrations of SFRE in relation to control (vehicle-treated) cells. (C and D) Modulation of ERBB2 gene (encoding HER2) (C) and HER2 protein (D) expression in HER2+ breast cancer cell lines (SK-BR-3 and UACC-812) after 48 h treatment with vehicle (control) or increasing concentrations of SFRE. (A, C) Results of qRT-PCR are expressed as the mean ± SD of biological triplicates, and statistically significant differences in relation to the control (vehicle-treated cells) were determined by Student’s t-test (**p < 0.01, ***p < 0.001). (B, D) Western blot analysis of ER-␣ and HER2 was performed after treatment with increasing concentrations of SFRE (expressed in g/mL above each lane), and -actin determination was used as endogenous control of total protein quantity. Cntrl: control.
ER-␣ are inhibited by SFRE in both tumor cell lines in a dose-dependent manner.
3.5 SFRE induces the downregulation of HER2 in HER2+ breast cancer cells Since HER2-dependent signaling pathway is involved in the increased proliferative capacity of HER2+ breast cancer cells, HER2 gene and protein expression was analyzed in SK-BR3 and UACC-812 (HER2+) breast cancer cells by qRT-PCR and Western blot analysis, respectively, after 48 h treatment with increasing concentrations of SFRE. Figure 3 shows that SFRE dose-dependently decreased the HER2 gene (Fig. 3C) and protein (Fig. 3D) levels in both HER2+ breast cancer cell lines. Moreover, immunofluorescence was performed to determine the effect of SFRE on the localization of HER2 protein, due to only the receptor located at the cellular membrane is able to activate the proliferative HER2 signaling pathway. The results showed that the decrease in total HER2 levels induced by SFRE correlates with the decrease of the receptor at the plasma membrane (Supporting Information Fig. 1). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.6 SFRE enhances the antitumor effect of commonly used breast cancer chemotherapy The inhibitory effect of antitumor drugs currently used in breast cancer therapy on tumor cell viability was assessed by MTT assay and compared to the effect of the drug combined with SFRE. Each chemotherapeutic molecule was assayed in the tumor subtype in which it is applied in the clinical setting. Accordingly, tamoxifen was assayed in the ER+ subtype (T-47D cells), while trastuzumab activity was examined in the HER2+ subtype (SK-BR-3 cells) and the effect of paclitaxel was analyzed in the TNBC subtype (MDA-MB-231 cells). The results showed that every combination assayed inhibited tumor cell viability to a significantly higher extent than that of the chemotherapeutic drug alone (Fig. 4).
4 Discussion The well-established classification of breast tumor subtypes according to the expression of ER-␣, progesterone receptor and HER2 and the discovery of agents targeted to these receptors allows the tailoring of breast cancer treatments in www.electrophoresis-journal.com
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Figure 4. Enhancement of the cell viability inhibitory effect of antitumor drugs commonly used in breast cancer therapy by SFRE. The effect of the antitumor drugs tamoxifen (A), paclitaxel (B), and trastuzumab (C) on cell viability of T-47D (ER+), MDA-MB231 (TNBC), and SK-BR-3 (HER2+) breast cancer cells, respectively (according to their target tumor subtype), was determined by MTT assay after 48 h (A and B) or 72 h (C) treatment with both the drug alone and in combination with SFRE. The results are expressed as the mean ± SEM of at least two independent experiments, each performed in quadruplicate. Student’s t-test was applied to determine the statistically significant differences between the effect of the drug alone and the effect of the drug combined with SFRE (*p < 0.05, **p < 0.01, ***p < 0.001).
order to avoid needless toxicities and to improve patient outcome. Nevertheless, breast cancer is still the leading cause of cancer deaths among women worldwide [1], and therefore the development of new therapeutic strategies for breast cancer patients is urgently needed. Rosemary (Rosmarinus officinalis L.) extract has been reported to possess chemopreventive activities in 7,12-dimethylbenz(a)anthracene-induced breast tumorigenesis [2], and to inhibit breast cancer cell viability in vitro [3,4]. Although the more sensitivity to rosemary extract of ER− breast cancer cells in comparison to ER+ cells has been suggested [3], little is known about its effect in each C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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breast tumor subtype. Since breast cancer patients are usually receiving chemotherapy, besides the determination of the specific sensitivity of the different tumor subtypes to the effect of rosemary, it is crucial to determine the effect of the combination of rosemary with the antitumor drugs currently used in breast cancer therapy in order to properly bring up the application of the rosemary extract as a complementary approach in breast cancer therapy. In this work we reported that low doses of SFRE, which probably correspond to the achievable concentrations in vivo, inhibits cell viability more strongly in HER2+ breast tumor cells. However, the effect of higher doses of SFRE did not show any pattern regarding the breast tumor classification, and apoptosis induction assessed by monitoring PARP1 cleavage was observed in SK-BR-3 (HER2+) and T-47D (ER+) cells (Fig. 1C), while cell death induction of MDA-MB-231 (TNBC), MCF-7 (ER+), and UACC-812 (HER2+) cells by SFRE, observed in the MTT assay (Fig. 1B), appears to be mediated by a different type of cell death, since PARP1 cleavage was not observed in this cell lines. Moreover, we found that the induction of apoptosis by SFRE correlated with the modulation of FOS gene, which was significantly upregulated (fold change ⬎ 2.0, and p ⬍ 0.05) in T-47D and SK-BR-3 at 40 g/mL treatment (Fig. 2B), and the degree of upregulation clearly correlated with induction of PARP1 cleavage in both cell lines (Fig. 2C), whereas this gene was not similarly modulated in the three other cell lines. Although FOS is traditionally known as oncogene, several references in the literature reported the relationship between the overexpression of FOS and the induction of cell death [20–22]. Indeed, the cell death induced by quercetin, a dietary compound with antitumor properties, has been also associated with the increase of FOS gene levels [20]. Due to the involvement of ER and HER2 signaling pathways in breast tumor proliferation and drug response, we determined the effect of SFRE on ER-␣ and HER2 expression. Although the sensitivity to SFRE appears to be not only determined by the ER+/ER− breast cancer classification, the markedly higher modulation of the ER signaling pathway in ER+ cells in comparison to ER− cells suggests the distinct molecular mechanism of SFRE in both tumor subtypes (Fig. 2A). Moreover, the decrease in the ER-␣ expression at both the mRNA (Fig. 3A) and protein (Fig. 3B) levels by SFRE points to a potential inhibitory effect of the extract on ER+ tumor proliferation, and indicates its utility in the ER+ breast cancer subtype. Additionally, the decrease of HER2 expression by SFRE at both gene and protein levels (Fig. 3C, D), indicates the effectiveness of SFRE in HER2+ breast cancer therapy. The different degree of HER2 oncoprotein inhibition among the two HER2+ cell lines, which is higher in SK-BR-3 cells than in UACC-812 cells, correlates with their different sensitivity to the SFRE regarding the inhibition of cell viability and apoptosis induction, which might support the role of HER2 modulation in the antitumor mechanism of SFRE in breast cancer. This results are consistent with the observation by Einbond et al. that MCF-7 transfected for HER2 overexpression are more sensitive to carnosic acid than www.electrophoresis-journal.com
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nontransfected MCF-7 cells [5]. Despite low doses more strongly affect to HER2+ than to HER2- breast cancer cells, the different sensitivity to SFRE among the different breast cancer cell lines showed that additional cellular and/or molecular characteristics are involved in the degree of sensitivity to the antitumor effect of SFRE, and remain to be elucidated. Furthermore, in order to propose the application of SFRE as a complementary agent in breast cancer therapy, it is essential to explore its effect in combination with the most frequently used chemotherapy in this type of cancer, to discard antagonistic effects, and to confirm the improvement of the antitumor effect with the combination with respect to the drug alone. In this regard, we assayed the combination of SFRE with the antitumor drugs tamoxifen (targeted to the ER+ breast cancer subtype), trastuzumab (targeted to the HER2+ breast cancer subtype), and paclitaxel (general antitumor drug frequently used in the TNBC subtype). Although the observed effect between SFRE and these drugs was not synergistic, as that previously found by our group in the combination of SFRE with 5-fluorouracil in colon cancer cells [14], every combination assayed showed a significant increase in the antitumor activity in comparison with the effect of the drug alone (Fig. 4). The confirmation of these results in the clinical setting would permit to maintain the efficacy of the treatment with the use of lower doses of these drugs, thus reducing the side effects associated to them. Moreover, HER2 overexpression can also be found in a subset of gastric, ovarian, and salivary gland tumors [23], and therefore trastuzumab may constitute a therapeutic option in these cases. On the other hand, paclitaxel is also used in ovarian and non-small-cell lung cancer therapy [24]. Consequently, the complementary use of SFRE might be beneficial not only in breast cancer, but also in the treatment of other cancer types in which these drugs are applied. In summary, SFRE showed antitumor activities against breast cancer ER+, HER2+, and TNBC cells regarding cell viability and cell death induction. Since ER-␣ and HER2 signaling pathways are key players in breast cancer development, SFRE might exert its antitumor effect, at least in part, by inhibiting ER-␣ in ER+ cells, and by decreasing HER2 in HER2+ cells. In TNBC cells, other mechanisms must mediate the antitumor action of SFRE. Although further studies are needed to better understand the additional cellular and molecular characteristics responsible for the different sensitivity of the breast cancer cell lines to SFRE, our results point to SFRE as a promising complementary agent in breast cancer therapy. This work has been supported by the Spanish Ministry of Science and Innovation (Plan Nacional I+D+i AGL201021565, RyC 2008-03734; IPT-2011-1248-060000); Comunidad de Madrid (ALIBIRD, S2009/AGR-1469); and European Union Structural Funds. We thank Bel´en Garc´ıa Carrasco for her technical help. We are also grateful to Genentech, Inc. (South San Francisco, CA) for providing the trastuzumab R ). (Herceptin C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The authors have declared no conflict of interest.
5 References [1] Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., Forman, D., CA Cancer J. Clin. 2011, 61, 69–90. [2] Singletary, K., MacDonald, C., Wallig, M., Cancer Lett. 1996, 104, 43–48. [3] Yesil-Celiktas, O., Sevimli, C., Bedir, E., Vardar-Sukan, F., Plant Foods Hum. Nutr. 2010, 65, 158–163. [4] Cheung, S., Tai, J., Oncol. Rep. 2007, 17, 1525–1531. [5] Einbond, L. S., Wu, H. A., Kashiwazaki, R., He, K., Roller, M., Su, T., Wang, X., Goldsberry, S., Fitoterapia 2012, 83, 1160–1168. [6] Yesil-Celiktas, O., Sevimli, C., Bedir, E., Vardar-Sukan, F., Plant Foods Hum. Nutr. 2010, 65, 158–163. [7] Aguilar, F., Autrup, H., Barlow, S., Castle, L., Crebelli, R., Dekant, W., Engel, K. H., Gontard, N., Gott, D., Grilli, S., EFSA J. 2008, 721, 1–29. [8] Allred, D. C., Mod. Pathol. 2010, 23 (Suppl 2), S52–S59. [9] Foulkes, W. D., Smith, I. E., Reis-Filho, J. S., New Engl. J. Med. 2010, 363, 1938–1948. [10] Zhu, B. T., Loder, D. P., Cai, M. X., Ho, C. T., Huang, M. T., Conney, A. H., Carcinogenesis 1998, 19, 1821–1827. [11] Zhao, P. W., Lee, D. Y., Ma, Z. Z., Huang, L. S., Sun, L. P., Li, Y. D., Chen, J. X., Niu, J. Z., Biol. Pharm. Bull. 2012, 35, 1947–1955. [12] Johnson, J. J., Syed, D. N., Suh, Y., Heren, C. R., Saleem, M., Siddiqui, I. A., Mukhtar, H., Cancer Prev. Res. 2010, 3, 1112–1123. ´ C., Garc´ıa-Canas, ˜ ˜ [13] Herrero, M., Simo, V., Iba´ nez, E., Cifuentes, A., Mass Spectrom. Rev. 2012, 31, 49–69. ´ [14] Gonzalez-Vallinas, M., Molina, S., Vicente, G., de la Cueva, A., Vargas, T., Santoyo, S., Garc´ıa-Risco, M. R., Fornari, T., Reglero, G., Ram´ırez de Molina, A., Pharmacol. Res. 2013, 72, 61–68. ´ [15] Vicente, G., Molina, S., Gonzalez-Vallinas, M., Garc´ıaRisco, M. R., Fornari, T., Reglero, G., Ram´ırez de Molina, A., J. Supercrit. Fluids 2013, 79, 101–108. [16] Boyd, M. R., Paull, K. D., Drug Dev. Res. 1995, 34, 91–109. [17] Ram´ırez de Molina, A., Gallego-Ortega, D., Sarmentero, ˜ J., Banez-Coronel, M., Mart´ın-Cantalejo, Y., Lacal, J. C., Cancer Res. 2005, 65, 5647–5653. [18] Livak, K. J., Schmittgen, T. D., Methods 2001, 25, 402–408. [19] Soldani, C., Scovassi, A. I., Apoptosis 2002, 7, 321–328. [20] Iwao, K., Tsukamoto, I., Biochim. Biophys. Acta 1999, 1427, 112–120. [21] Smeyne, R. J., Vendrell, M., Hayward, M., Baker, S. J., Miao, G. G., Schilling, K., Robertson, L. M., Curran, T., Morgan, J. I., Nature 1993, 363, 166–169. [22] Colotta, F., Polentarutti, N., Sironi, M., Mantovani, A., J. Biol. Chem. 1992, 267, 18278–18283. [23] Baselga, J., Swain, S. M., Nat. Rev. Cancer 2009, 9, 463–475. [24] Rodriguez-Antona, C., Pharmacogenomics 2010, 11, 621–623.
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Electrophoresis 2014, 35, 1719–1727 1 ´ Margarita Gonzalez-Vallinas 1 Susana Molina Gonzalo Vicente2 ´ Ruth Sanchez-Mart´ ınez1 Teodoro Vargas1 ´ Monica R. Garc´ıa-Risco2 Tiziana Fornari2 Guillermo Reglero1,2 Ana Ram´ırez de Molina1 1 IMDEA-Food
Institute, CEI UAM + CSIC, Madrid, Spain 2 Instituto de Investigacion ´ en ´ Ciencias de la Alimentacion (CIAL), CEI UAM+CSIC, Madrid, Spain
Received November 14, 2013 Revised February 6, 2014 Accepted February 7, 2014
Research Article
Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells Breast cancer is the leading cause of cancer-related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER-␣ and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER-␣ and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen-dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy. Keywords: Breast cancer / Estrogen receptor alpha / Human epidermal growth factor receptor-2 / Paclitaxel / Rosemary DOI 10.1002/elps.201400011
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Breast cancer is the most frequently diagnosed tumor among females in both economically developed and developing countries [1]. Although breast cancer death rates have been diminishing in some developed countries, mostly due to early detection and improved treatment, this type of cancer is still the leading cause of cancer-related mortality in females worldwide, thus being responsible for 14% of the total cancer deaths Correspondence: Dr. Ana Ram´ırez de Molina IMDEA-Food Institute. CEI UAM+CSIC. Ctra. de Canto Blanco 8. 28049 Madrid. Spain E-mail: [email protected] Fax: +34-91-1880756
Abbreviations: ER, estrogen receptor; ER+, estrogendependent; ER−, estrogen-independent; ER-␣, estrogen receptor alpha; HER2+, overexpression of human epidermal growth factor receptor-2; MTT, 3-(4,5-dimethyl-thyazol-2-yl)2,5-diphenyl-tetrazolium; qRT-PCR, quantitative reverse transcription PCR; SFRE, supercritical fluid rosemary extract; TNBC, triple negative breast cancer C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in 2008 [1]. Consequently, the development of novel antitumor treatments, ideally nontoxic and cost-effective, continues to be of the utmost importance. The utility of rosemary (Rosmarinus officinalis L.) extracts and their components in breast cancer prevention and treatment has been suggested by different studies. Regarding chemoprevention, a commercial extract (Herbalox type O, Kalsec Inc.), containing 40% of crude rosemary extract, showed an inhibitory effect on mammary inducedtumorigenesis in vivo, by either oral or intraperitoneal administration [2]. The effect was explained by the decrease of 7,12-dimethylbenz(a)anthracene-DNA adduct formation and the induction of glutathione S-transferase and quinone reductase activities, and mostly attributed to the carnosol content of the extract [2]. Moreover, rosemary extracts obtained by different procedures (including ethanolic, methanolic, and supercritical fluid extracts) were reported to inhibit the viability of several tumor cell types, including breast cancer [3–5], and carnosic acid was found to be the most active component regarding this activity [5, 6]. The comparison between supercritical fluid and methanolic rosemary extracts indicated that the former contain more active compounds, and are www.electrophoresis-journal.com
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therefore more effective in inhibiting cell viability [6]. Moreover, rosemary extracts obtained by supercritical fluid technology have been declared as safe for human use by the European Food Safety Authority (EFSA) [7]. Nevertheless, breast cancer is a heterogeneous disease that involves different tumor subtypes that should be differentially treated. Breast tumors are classified as ER+ subtype (estrogen-dependent tumor, which expresses estrogen receptor alpha (ER-␣) and responds to estrogens, in contrast to estrogen-independent (ER−) tumors), human epidermal growth factor receptor-2 (HER2)+ subtype (HER2 overexpressing tumor, which could be either ER+ or ER−) and triple negative breast cancer (TNBC) subtype (which lacks the expression of the three biomarkers: ER-␣, progesterone receptor, and HER2). This classification allows the prediction of tumor sensitivity to the different chemotherapeutic drugs and it is therefore used for drug selection in the clinical practice [8]. Accordingly, patients with ER+ tumors are expected to respond to hormonal therapy (e.g. tamoxifen), whereas patients with HER2+ tumors will be candidates to receive trastuzumab treatment, a mAb that targets the extracellular domain of HER2 thus inhibiting tumor cell proliferation induced by HER2 signaling pathway. In contrast, there is no targeted chemotherapy for TNBC and this tumor subtype is commonly treated with the natural antitumor agent paclitaxel in combination with anthracyclines [9]. In view of that, it is necessary to assay the activity and mechanism of rosemary extract in the different breast tumor subtypes in order to establish the potential use of rosemary in breast cancer therapy. Some investigations suggest the role of estrogen action and metabolism in the molecular mechanism of rosemary. On the one hand, dietary administration of methanolic rosemary extract increased the liver metabolism (microsomal oxidation and glucuronidation) of estradiol and estrone and inhibited their uterotropic action, thus pointing to a beneficial effect in tumors that frequently respond to estrogens such as endometrial, ovarian, and breast cancers [10]. On the other hand, the antioxidant activity of the rosemary component carnosol was reported to be at least partially mediated through the ER-␣ signaling pathway [11], and this diterpene was proved to antagonize ER-␣ with no agonist effects [12]. These results suggest a potential differential effect of rosemary against ER+ and ER− breast cancer cells. Accordingly, previous reports suggested the higher cell sensitivity to rosemary extract of ER− in comparison to ER+ cell lines [3]; however, this suggestion was based on the use of MCF-7 cells as the unique model of ER+ breast cancer [3]. In this work, we aimed to determine the sensitivity of several breast cancer cell lines from different subtypes to the antitumor activities of a supercritical fluid rosemary extract (SFRE). In addition, due to the clinical relevance of ER-␣ and HER2 signaling pathways in breast cancer cells, we used a genomic approach to explore their potential modulation by this bioactive agent. Thus, the results of this work contribute to the global Foodomics strategy for the study of the health properties attributed to rosemary, which includes the investigation of the interaction between this bioactive food component and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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gene expression [13]. Finally, we assayed the antitumor effect of SFRE in combination with some of the antitumor drugs most frequently used in each breast cancer subtype, in order to determine the patients who will most benefit from the use of SFRE within their cancer therapy.
2 Materials and methods 2.1 Rosemary extract Rosemary extract was obtained from 0.5 kg of dried and grinded rosemary leaves by supercritical fluid extraction technology in a supercritical fluid pilot plant (Thar Technology, model SF2000) as previously described [14]. The CO2 flow rate was 60 g/min and a temperature equal to 40°C was maintained in extraction cell and separators. The pressure of the extractor was 300 bar and for the first 60 min the extracted material was fractionated by setting 100 bar of pressure in the first separator (S1), and 50 bar (the pressure of the recirculation system) in the second separator (S2). Afterwards, extraction continued without fractionation for 300 min. SFRE was collected from S1, dissolved in absolute ethanol at 50 mg/mL, and stored at −20°C. The major components of SFRE were 19.56% w/w carnosic acid, 1.90% w/w carnosol, and 15.52% w/w volatile oils. Carnosic acid and carnosol were quantified by HPLC analysis, and volatile oil components were determined by GC-MS analysis, as previously described [15]. 2.2 Cell culture Breast cancer cell lines MCF-7 (ER+), T-47D (ER+), MDA-MB-231 (TNBC), UACC-812 (HER2+), and SK-BR3 (HER2+), as well as CCD 841 cells (normal epithelial cells) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured under standard conditions of temperature (37°C), humidity (95%), and carbon dioxide (5%) in DMEM (MDA-MB-231 and MCF-7), RPMI (T-47D and SK-BR-3), or EMEM (CCD 841) supplemented with 10% FBS, 2 mM glutamine, and 1% of antibioticantimycotic solution (containing 10 000 units/mL penicillin base, 10 000 g/mL streptomycin base, and 25 000 ng/mL amphotericin B; Gibco, Grand Island, NY, USA). UACC-812 cells were maintained in DMEM supplemented with 20% FBS and 20 ng/mL epidermal growth factor, as recommended by the cell supplier. The main molecular characteristics of the breast cancer cell lines are collected in Table 1. 2.3 MTT assay Cell viability was assessed by the 3-(4,5-dimethyl-thyazol-2yl)-2,5-diphenyl-tetrazolium (MTT) assay as previously described [14]. Breast cancer cells (20 000–70 000 per well, optimized according to their proliferation rate) were seeded in 24-well plates. After overnight attachment, the cells underwent the corresponding treatment with SFRE and/or the www.electrophoresis-journal.com
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Electrophoresis 2014, 35, 1719–1727 Table 1. Molecular characterization of breast cancer cell lines
Breast cancer cell line
ER (+/−)a)
HER2 (+/−)b)
PgR (+/−)c)
Tumor subtype
MCF-7 T-47D UACC-812 SK-BR-3 MDA-MB-231
+ + − − −
− − + + −
+ + − − −
ER+ ER+ HER2+ HER2+ TNBCd)
a) ER: Estrogen responsiveness. b) HER2: Her-2/ErbB2/neu overexpression. c) PgR: Progesterone receptor expression. d) TNBC: Triple Negative Breast Cancer.
antitumor drugs paclitaxel (Sigma-Aldrich, St. Louis, MO, USA), 4-hydroxy-tamoxifen (Sigma-Aldrich), or trastuzumab R , kindly provided by GenenTech, San Francisco, (Herceptin CA, USA). Following 48 h or 72 h treatment, cells were incubated with MTT solution (5 mg/mL in PBS) at 1:10 dilution for 3 h, and the MTT metabolic product (formazan) was dissolved in 200 L DMSO. The quantity of formazan obtained, which correlates with cell viability, was determined by measuring the absorbance at 560 nm. The experiments involving the use of 4-hydroxy-tamoxifen were performed in culture medium without phenol red and with 5% charcoal/dextrantreated FBS (instead of 10% FBS), in order to minimize the level of estrogens and thus avoiding possible interferences with the results. The parameters IC50 (50% cell viability inhibition), GI50 (50% growth inhibition), TGI (total growth inhibition), and LC50 (50% cell death) were calculated according to the NIH definitions using a logistic regression [16]. In order to calculate the parameters related to cell proliferation and cell death (GI50, TGI, and LC50), the cell viability before the treatment (time zero) was determined.
2.4 Anchorage-independent growth in soft agar Breast cancer MCF-7 cells (90 000 cells), previously treated with 50 g/mL (treatment A and B), or vehicle (control) during 24 h, were suspended in culture medium containing 0.4% agar and seeded on top of a solidified layer of culture medium containing 0.5% agar in p60 plates. Cell suspension was treated with 50 g/mL SFRE (treatment B) or vehicle (treatment A and control). These plates were incubated under standard conditions to allow the formation of colonies, and irrigated twice a week with 200 L of culture medium supplemented with 100 g/mL SFRE (treatment A), 50 g/mL SFRE (treatment B), or the equivalent volume of vehicle (control) for 4–5 weeks. Colonies were dyed with Crystal Violet as previously described [17]. Representative regions of 4 mm2 of each plate (16 regions per plate) were selected and the colonies they contained were counted under a microscope. The total number of colonies in each plate was calculated according to the total surface of p60 plates. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.5 Western blot analysis Cell lysates from breast cancer cells were obtained by dissolving in Laemmli buffer (60 mM Tris-HCl pH 6.8, 10% glycerol, and 2% SDS) the cell pellet previously washed with PBS, and boiling at 95°C for 5 min. Protein concentrations were determined by the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacture’s procedure. Equal amounts of protein (35–50 g) from cell lysates were fractioned by SDS-PAGE in 8% polyacrylamide gels for HER2 analysis or 10% polyacrylamide gels for ER-␣ and poly(ADP-ribose) polymerase 1 (PARP1) analyses, and transferred onto a 0.2 m nitrocellulose membrane (Bio-Rad). The membranes were incubated with 5% BSA in TTBS (TBS supplemented with 0.05% Tween-20) in order to block unspecific sites. Antibodies against PARP1 (1:125 dilution, BD Pharmingen), ER-␣ (1:200 dilution, Santa Cruz Biotechnology), HER2 (antibody 29D8, 1:1000 dilution, Cell Signaling Technology, Beverly, MA, USA), and -actin (1:2000 dilution, SigmaAldrich) were used as primary antibodies. Horseradish peroxidase conjugated antibodies anti-mouse (antibody AP130P, 1:40 000 dilution, Millipore, Billerica, MA, USA) or anti-rabbit (antibody AP106P, 1:20 000 dilution, Millipore) were used as secondary antibodies. Detection was performed using the Clarity Western ECL Substrate (Bio-Rad). Molecular weights of protein bands and densitometry were determined by the TotalLab software (TotalLab, Newcastle, UK). -actin determination was used as an endogenous control of total protein quantity.
2.6 Immunofluorescence Cells were fixed with 4% buffered paraformaldehyde for 10 min at room temperature and then permeabilized for 30 min with −20°C methanol, and stained with a 1:100 dilution of anti-HER2 (29D8, Cell Signaling) followed by incubation with Alexa 488-conjugated anti-rabbit antibody (1:1000) and/or with 4,6 diaminophenylindole (DAPI; Prolong Gold antifade, Invitrogen) to visualize nuclei. Images were captured using a Leica DM IL microscope with a 40X Plan Fluotar objective.
2.7 Gene expression analysis To analyze the expression of 84 key genes of the estrogen receptor (ER) signaling pathway, total RNA from breast cancer cells, treated with 40 g/mL SFRE or vehicle for 48 h, was extracted using the RNeasy Mini Kit with RNAse-Free DNAse kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). The quantity and purity of the obtained RNA samples were determined by UV-spectroscopy (NanoDropTM 2000 Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). Samples with ratio A260:A230 above 1.7, ratio A260:A280 between 1.8 and 2.0, and concentration higher than 40 ng/L were accepted for subsequent www.electrophoresis-journal.com
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analysis. Gene expression was determined by using the Human Estrogen Receptor Signaling RT2 Profiler PCR Array (SABiosciences). For each PCR array, 500 ng were retrotranscribed with RT2 First Strand Kit, and RT2 qPCR SYBR Green/ROX Master mix was used to perform quantitative PCR (qPCR) in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer’s protocol. Results were analyzed using the RT2 ProfilerTM PCR Array Data Analysis (SABiosciences), applying the mean of B2M, HPRT1, RPL13A, GAPDH, and ACTB gene expression as endogenous control for each plate. Absolute fold change ⬎ 2 and p value ⬍ 0.05 cutoffs were used to consider a gene to be significantly modulated. Individual gene expression analysis was also performed by quantitative reverse transcription-PCR (qRT-PCR). To that end, 400 ng RNA was reverse-transcribed to cDNA using the High Capacity RNA-to-cDNA Master Mix system (Applied Biosystems, Carlsba, CA, USA), as directed by the manufacturer. qPCR was performed in the 7900HT Real-Time PCR System (Applied Biosystems), according to the manufacturer’s protocol using the specific Taqman gene expression assays (Applied Biosystems; Hs01001580_m1 for ERBB2, Hs00174860_m1 for ESR1, Hs00170630_m1 for FOS, and Hs99999901_s1 for 18S as endogenous control). qRT-PCR data extraction was performed using the RQ Manager software (Applied Biosystems), and the 2−࢞࢞Ct method was applied to calculate the relative expression of each gene as previously described [18].
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SFRE, cell viability of normal CCD 841 cells was 39.3 ± 9.4% (mean ± SEM), whereas viability of tumor cells decreased on average until 7.8 ± 1.9% (mean ± S.E.M.) after the same treatment. This result indicates that there is a therapeutic range for the use of SFRE in the clinical practice. Regarding the differences among tumor cells, MCF-7 and UACC-812 seemed to be the most resistant cell lines at higher doses of SFRE, although the treatment at low SFRE concentrations showed the higher sensitivity of HER2+ breast cancer cells (Fig. 1A). The comparison of the parameters regarding the inhibition of 50% cell proliferation (GI50) of the different cell lines confirmed the higher sensitivity of HER2+ cell subtype to low doses of SFRE, due to GI50 values of SFRE in SK-BR-3 and UACC-812 cells are lower than those in the other breast cancer cell lines assayed (Fig. 1B). Since the concentration of phenolic compounds that can be achieved in vivo are usually low, it is probable that the sensitivity of the different cancer cell subtypes to low doses of SFRE in vitro most reliably reflects the different sensitivity of the tumor subtypes in vivo. Apoptosis induction assessed by PARP1 cleavage analysis (which is considered a prominent marker of apoptosis [19]) showed that SFRE dose-dependently induced apoptosis in T47D and SK-BR-3 cell lines, while cleaved PARP1 is almost absent in the rest of treated breast cancer cell lines (Fig. 1C). Since LC50 parameters suggest that 80 g/mL of SFRE is able to induce cell death in the breast cancer cell lines assayed, the absence of PARP1 cleavage at that concentration indicates that SFRE may also induce a different type of cell death in breast cancer cells.
2.8 Statistical analysis Results are shown as the mean ± SEM of three independent experiments, each performed in triplicate, unless otherwise indicated. Comparisons between two groups were analyzed by Student’s t-test, whereas differences among several groups were determined by one-way ANOVA with Bonferroni’s post hoc test. Statistically significant values are indicated by asterisks as follows: *p ࣘ 0.05; **p ࣘ 0.01; ***p ࣘ 0.001. IBM SPSS Statistics version 20 (SPSS, Chicago, IL) was used for statistical analysis.
3 Results 3.1 Differential effect of SFRE on cell viability inhibition and apoptosis induction of human tumor cells from distinct breast cancer subtypes Human breast cancer cells from the subtypes ER+ (MCF-7 and T-47D), HER2+ (SK-BR-3 and UACC-812), and TNBC (MDA-MB-231) were treated with increasing concentrations of SFRE, and cell viability was determined by MTT assay. SFRE decreased cell viability of the five breast cancer cell lines in a dose-dependent manner (Fig. 1A). Normal epithelial cells (CCD 841) were used to compare the effect of SFRE on tumor and normal cells. After treatment with 120 g/mL of C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Inhibitory effect of SFRE on cell transformation of human breast cancer cells Since anchorage-independent proliferation is a hallmark of malignant transformation, the effect of SFRE on cell transformation was assessed by determining the capacity of the less sensitive breast cancer MCF-7 cells seeded in suspension in a semisolid medium to form colonies. Two different patterns of SFRE treatment (A and B) were compared to control (vehicletreated) cells. Both treatment A and treatment B plates involved the 24 h pretreatment with SFRE and the irrigation with SFRE twice a week, and treatment B additionally contained SFRE in the semisolid medium. As it can be observed in Fig. 1D, both SFRE treatment patterns significantly inhibited the transformation of human breast cancer cells, and the effect was markedly higher when the semisolid medium also contained SFRE, suggesting a specific dose-dependent effect.
3.3 Different modulation by SFRE of the ER signaling pathway in ER+ and ER− breast cancer cells In order to determine the different molecular mechanism of SFRE in ER+ and ER− breast cancer cells, the modulation www.electrophoresis-journal.com
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Figure 1. Differential antitumor effects of SFRE in human tumor cell lines from several breast cancer subtypes regarding cell viability, apoptosis induction, and cell transformation. (A) Cell viability inhibition (assessed by the MTT assay) of different breast cancer cell lines after 48 h treatment with increasing concentrations of SFRE. The results are expressed as the mean ± SEM of at least two independent experiments, each performed in quadruplicate. The first part of the graph was enlarged (square on the right) in order to better visualize the different sensitivity of the breast cancer cell lines to low doses of SFRE. (B) Parameters representing the SFRE concentrations (in g/mL) responsible for the 50% cell viability inhibition (IC50), 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% cell death (LC50) after 48 h treatment of the different breast cancer cell lines. The results shown are the mean ± SEM of at least four independent experiments, each performed in quadruplicate. (C) Western blot analysis of PARP1 cleavage (and -actin as endogenous control of total protein quantity) after 48 h treatment with vehicle (Cntrl) or increasing concentrations of SFRE (expressed as g/mL above each lane) of breast cancer cell lines. (D) Capacity of colony formation in soft agar of MCF-7 cells treated with SFRE compared to control (vehicle-treated) cells. Treatment A consisted of 50 g/mL SFRE pretreatment for 24 h and irrigation of plates with 200 L of culture medium containing 100 g/mL SFRE twice a week, whereas treatment B consisted of 50 g/mL SFRE pretreatment for 24 h, 50 g/mL SFRE treatment of soft agar and irrigation of plates with 200 L of culture medium containing 50 g/mL SFRE twice a week. Results are expressed as mean ± SEM of two independent experiments, each performed in triplicate. Statistically significant differences were determined by the Student’s t-test (***p < 0.001).
of the ER signaling pathway was studied by low density arrays in both ER+ (MCF-7) and ER− (MDA-MB-231) breast cancer cell lines. SFRE significantly modulated 14 genes in ER+ cells, whereas only four genes were modulated in ER− cells (Fig. 2A), suggesting a relevant role of ER signaling pathway in the antitumor action of SFRE in ER+ cells. Interestingly, SFRE downregulated the expression of ESR1, the gene encoding the receptor ER-␣, which is responsible for the proliferation of ER+ tumors. Moreover, SFRE also modulated the expression of FOS. Due to the controversial role of this gene, which has been reported to precede cell death in some cases [20, 21], we assayed its modulation in the five breast cancer cell lines (Fig. 2B). FOS gene showed to be upregulated by SFRE in T-47D and SK-BR-3, and the upregu-
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lation correlated with the induction of apoptosis assessed by PARP1 cleavage (Fig. 2C).
3.4 SFRE induces the downregulation of ER-␣ in ER+ breast cancer cells Due to the relevant role of the ER-␣ signaling pathway in breast cancer, and concretely in the ER+ tumor subtype, we validated the effect of SFRE in mRNA and protein levels of ER-␣ in MCF-7 and T-47D (ER+) breast cancer cell lines by qRT-PCR and Western blot analysis, respectively. The results showed that gene (Fig. 3A) and protein (Fig. 3B) levels of
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Figure 2. Modulation of the expression of the estrogen receptor (ER) signaling pathway genes by SFRE in ER− and ER+ human breast cancer cells. (A) Panels (left) and graphs (right) representing the modulation of the expression of genes involved in the ER signaling pathway in MDA-MB-231 (ER−) and MCF-7 (ER+) breast cancer cell lines after 48 h treatment with 40 g/mL SFRE in relation to control (vehicle-treated) cells, assessed by qRT-PCR. Results are expressed as the mean ± SD of three independent experiments. Student’s t-test was applied to assess statistically significant differences. (B and C) Modulation of FOS gene after 48 h SFRE treatment in five different breast cancer cell lines (B) and comparison with the densitometry of Western blot analysis of PARP1 cleavage after the same treatment duration (C). FOS modulation is expressed as the mean ± SEM of three independent experiments, each performed with biological triplicates, and statistically significant differences were determined by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 3. Downregulation of ER-␣ and HER2 receptors by SFRE in breast cancer cells. (A and B) Modulation of ESR1 gene (encoding ER-␣) (A) and ER-␣ protein (B) expressions in ER+ breast cancer cell lines (MCF-7 and T-47D) after 48 h treatment with increasing concentrations of SFRE in relation to control (vehicle-treated) cells. (C and D) Modulation of ERBB2 gene (encoding HER2) (C) and HER2 protein (D) expression in HER2+ breast cancer cell lines (SK-BR-3 and UACC-812) after 48 h treatment with vehicle (control) or increasing concentrations of SFRE. (A, C) Results of qRT-PCR are expressed as the mean ± SD of biological triplicates, and statistically significant differences in relation to the control (vehicle-treated cells) were determined by Student’s t-test (**p < 0.01, ***p < 0.001). (B, D) Western blot analysis of ER-␣ and HER2 was performed after treatment with increasing concentrations of SFRE (expressed in g/mL above each lane), and -actin determination was used as endogenous control of total protein quantity. Cntrl: control.
ER-␣ are inhibited by SFRE in both tumor cell lines in a dose-dependent manner.
3.5 SFRE induces the downregulation of HER2 in HER2+ breast cancer cells Since HER2-dependent signaling pathway is involved in the increased proliferative capacity of HER2+ breast cancer cells, HER2 gene and protein expression was analyzed in SK-BR3 and UACC-812 (HER2+) breast cancer cells by qRT-PCR and Western blot analysis, respectively, after 48 h treatment with increasing concentrations of SFRE. Figure 3 shows that SFRE dose-dependently decreased the HER2 gene (Fig. 3C) and protein (Fig. 3D) levels in both HER2+ breast cancer cell lines. Moreover, immunofluorescence was performed to determine the effect of SFRE on the localization of HER2 protein, due to only the receptor located at the cellular membrane is able to activate the proliferative HER2 signaling pathway. The results showed that the decrease in total HER2 levels induced by SFRE correlates with the decrease of the receptor at the plasma membrane (Supporting Information Fig. 1). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.6 SFRE enhances the antitumor effect of commonly used breast cancer chemotherapy The inhibitory effect of antitumor drugs currently used in breast cancer therapy on tumor cell viability was assessed by MTT assay and compared to the effect of the drug combined with SFRE. Each chemotherapeutic molecule was assayed in the tumor subtype in which it is applied in the clinical setting. Accordingly, tamoxifen was assayed in the ER+ subtype (T-47D cells), while trastuzumab activity was examined in the HER2+ subtype (SK-BR-3 cells) and the effect of paclitaxel was analyzed in the TNBC subtype (MDA-MB-231 cells). The results showed that every combination assayed inhibited tumor cell viability to a significantly higher extent than that of the chemotherapeutic drug alone (Fig. 4).
4 Discussion The well-established classification of breast tumor subtypes according to the expression of ER-␣, progesterone receptor and HER2 and the discovery of agents targeted to these receptors allows the tailoring of breast cancer treatments in www.electrophoresis-journal.com
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Figure 4. Enhancement of the cell viability inhibitory effect of antitumor drugs commonly used in breast cancer therapy by SFRE. The effect of the antitumor drugs tamoxifen (A), paclitaxel (B), and trastuzumab (C) on cell viability of T-47D (ER+), MDA-MB231 (TNBC), and SK-BR-3 (HER2+) breast cancer cells, respectively (according to their target tumor subtype), was determined by MTT assay after 48 h (A and B) or 72 h (C) treatment with both the drug alone and in combination with SFRE. The results are expressed as the mean ± SEM of at least two independent experiments, each performed in quadruplicate. Student’s t-test was applied to determine the statistically significant differences between the effect of the drug alone and the effect of the drug combined with SFRE (*p < 0.05, **p < 0.01, ***p < 0.001).
order to avoid needless toxicities and to improve patient outcome. Nevertheless, breast cancer is still the leading cause of cancer deaths among women worldwide [1], and therefore the development of new therapeutic strategies for breast cancer patients is urgently needed. Rosemary (Rosmarinus officinalis L.) extract has been reported to possess chemopreventive activities in 7,12-dimethylbenz(a)anthracene-induced breast tumorigenesis [2], and to inhibit breast cancer cell viability in vitro [3,4]. Although the more sensitivity to rosemary extract of ER− breast cancer cells in comparison to ER+ cells has been suggested [3], little is known about its effect in each C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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breast tumor subtype. Since breast cancer patients are usually receiving chemotherapy, besides the determination of the specific sensitivity of the different tumor subtypes to the effect of rosemary, it is crucial to determine the effect of the combination of rosemary with the antitumor drugs currently used in breast cancer therapy in order to properly bring up the application of the rosemary extract as a complementary approach in breast cancer therapy. In this work we reported that low doses of SFRE, which probably correspond to the achievable concentrations in vivo, inhibits cell viability more strongly in HER2+ breast tumor cells. However, the effect of higher doses of SFRE did not show any pattern regarding the breast tumor classification, and apoptosis induction assessed by monitoring PARP1 cleavage was observed in SK-BR-3 (HER2+) and T-47D (ER+) cells (Fig. 1C), while cell death induction of MDA-MB-231 (TNBC), MCF-7 (ER+), and UACC-812 (HER2+) cells by SFRE, observed in the MTT assay (Fig. 1B), appears to be mediated by a different type of cell death, since PARP1 cleavage was not observed in this cell lines. Moreover, we found that the induction of apoptosis by SFRE correlated with the modulation of FOS gene, which was significantly upregulated (fold change ⬎ 2.0, and p ⬍ 0.05) in T-47D and SK-BR-3 at 40 g/mL treatment (Fig. 2B), and the degree of upregulation clearly correlated with induction of PARP1 cleavage in both cell lines (Fig. 2C), whereas this gene was not similarly modulated in the three other cell lines. Although FOS is traditionally known as oncogene, several references in the literature reported the relationship between the overexpression of FOS and the induction of cell death [20–22]. Indeed, the cell death induced by quercetin, a dietary compound with antitumor properties, has been also associated with the increase of FOS gene levels [20]. Due to the involvement of ER and HER2 signaling pathways in breast tumor proliferation and drug response, we determined the effect of SFRE on ER-␣ and HER2 expression. Although the sensitivity to SFRE appears to be not only determined by the ER+/ER− breast cancer classification, the markedly higher modulation of the ER signaling pathway in ER+ cells in comparison to ER− cells suggests the distinct molecular mechanism of SFRE in both tumor subtypes (Fig. 2A). Moreover, the decrease in the ER-␣ expression at both the mRNA (Fig. 3A) and protein (Fig. 3B) levels by SFRE points to a potential inhibitory effect of the extract on ER+ tumor proliferation, and indicates its utility in the ER+ breast cancer subtype. Additionally, the decrease of HER2 expression by SFRE at both gene and protein levels (Fig. 3C, D), indicates the effectiveness of SFRE in HER2+ breast cancer therapy. The different degree of HER2 oncoprotein inhibition among the two HER2+ cell lines, which is higher in SK-BR-3 cells than in UACC-812 cells, correlates with their different sensitivity to the SFRE regarding the inhibition of cell viability and apoptosis induction, which might support the role of HER2 modulation in the antitumor mechanism of SFRE in breast cancer. This results are consistent with the observation by Einbond et al. that MCF-7 transfected for HER2 overexpression are more sensitive to carnosic acid than www.electrophoresis-journal.com
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nontransfected MCF-7 cells [5]. Despite low doses more strongly affect to HER2+ than to HER2- breast cancer cells, the different sensitivity to SFRE among the different breast cancer cell lines showed that additional cellular and/or molecular characteristics are involved in the degree of sensitivity to the antitumor effect of SFRE, and remain to be elucidated. Furthermore, in order to propose the application of SFRE as a complementary agent in breast cancer therapy, it is essential to explore its effect in combination with the most frequently used chemotherapy in this type of cancer, to discard antagonistic effects, and to confirm the improvement of the antitumor effect with the combination with respect to the drug alone. In this regard, we assayed the combination of SFRE with the antitumor drugs tamoxifen (targeted to the ER+ breast cancer subtype), trastuzumab (targeted to the HER2+ breast cancer subtype), and paclitaxel (general antitumor drug frequently used in the TNBC subtype). Although the observed effect between SFRE and these drugs was not synergistic, as that previously found by our group in the combination of SFRE with 5-fluorouracil in colon cancer cells [14], every combination assayed showed a significant increase in the antitumor activity in comparison with the effect of the drug alone (Fig. 4). The confirmation of these results in the clinical setting would permit to maintain the efficacy of the treatment with the use of lower doses of these drugs, thus reducing the side effects associated to them. Moreover, HER2 overexpression can also be found in a subset of gastric, ovarian, and salivary gland tumors [23], and therefore trastuzumab may constitute a therapeutic option in these cases. On the other hand, paclitaxel is also used in ovarian and non-small-cell lung cancer therapy [24]. Consequently, the complementary use of SFRE might be beneficial not only in breast cancer, but also in the treatment of other cancer types in which these drugs are applied. In summary, SFRE showed antitumor activities against breast cancer ER+, HER2+, and TNBC cells regarding cell viability and cell death induction. Since ER-␣ and HER2 signaling pathways are key players in breast cancer development, SFRE might exert its antitumor effect, at least in part, by inhibiting ER-␣ in ER+ cells, and by decreasing HER2 in HER2+ cells. In TNBC cells, other mechanisms must mediate the antitumor action of SFRE. Although further studies are needed to better understand the additional cellular and molecular characteristics responsible for the different sensitivity of the breast cancer cell lines to SFRE, our results point to SFRE as a promising complementary agent in breast cancer therapy. This work has been supported by the Spanish Ministry of Science and Innovation (Plan Nacional I+D+i AGL201021565, RyC 2008-03734; IPT-2011-1248-060000); Comunidad de Madrid (ALIBIRD, S2009/AGR-1469); and European Union Structural Funds. We thank Bel´en Garc´ıa Carrasco for her technical help. We are also grateful to Genentech, Inc. (South San Francisco, CA) for providing the trastuzumab R ). (Herceptin C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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The authors have declared no conflict of interest.
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