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Introduction
Breast cancer is the most frequently diagnosed cancer among women internationally and is the second highest cause of cancer-related death. The incidence of breast cancer varies hugely around the world, both in developed and developing countries (1). In Thailand, breast cancer is currently the most common cancer and involves 43%
of all cancers diagnosed in women (2).
Estrogen receptors, when binding with estrogen, have important physiological roles in cardiovascular protec- tion, the humoral immune response, neuroprotection, and bone remodeling. In breast cancer cells, estrogen and/or progesterone receptor status is highly linked to the patients’ prognosis after surgery (3). Although many selective estrogen receptor modulators have been studied (4, 5), at present, hormonal therapy with tamoxifen is considered a gold standard when preventing tumor recurrence in women with hormone-responsive breast cancer. Tamoxifen, a non-steroidal selective estrogen receptor modulator, is commonly used for the hormone therapy of receptors-positive breast cancers in pre-
In Vitro and In Vivo Effects of Xanthorrhizol on Human Breast Cancer MCF-7 Cells Treated With Tamoxifen
Nattanant Noomhorm1, Chun-Ju Chang2, Che-Sheng Wen3, Jir-You Wang4, Jiun-Liang Chen5,6, Ling-Ming Tseng7,8, Wei-Shone Chen9,10, Jen-Hwey Chiu1,7,11,*, and Yi-Ming Shyr7
1Institute of Traditional Medicine, 8Department of Surgery, School of Medicine, National Yang-Ming University, Taipei, 112, Taiwan, ROC
2Department of Food Science, National Taiwan Ocean University, Keelung, 202, Taiwan, ROC
3Department of Orthopedics, 11Division of General Surgery, Cheng-Hsin General Hospital, Taipei, 112, Taiwan, ROC
4Department of Orthopedics, 7Division of General Surgery, Department of Surgery, 9Division of Colorectal Surgery, Department of Surgery, 10Experimental Surgery of the Department of Surgery, Taipei Veterans General Hospital, Taipei, 112, Taiwan, ROC
5School of Traditional Chinese Medicine, Chang-Gung University, Taoyuan, 333, Taiwan, ROC
6Center for Traditional Chinese Medicine, Chang-Gung Memorial Hospital, Taoyuan, 333, Taiwan, ROC Received February 2, 2014; Accepted May 8, 2014
Abstract. This study investigated the herb–drug interaction of xanthorrhizol and tamoxifen in human breast cancer cells. Using MCF-7 cell line as an in vitro model, the herb–drug interaction between xanthorrhizol and tamoxifen was measured by MTT assay, luciferase reporter assay, and cell cycle analysis. The effects of xanthorrhizol on growth/autophagy related signaling were determined by immunostaining, western blotting, and real time RT-PCR. Additionally, the in vivo effect of xanthorrhizol and tamoxifen on athymic nude mice implanted with MCF-7 cells was evaluated. When MCF-7 cells were co-treated with tamoxifen and xanthorrhizol, there were no significant changes in terms of cell number, luciferase activity, percentage S-phase cells and LC3- II expression. However, using the MCF-7 implanted nude mice model, it was possible to detect significantly increased tumor volumes, a larger tumor size, and increased protein expression of P38 and P27(Kip1) in the xanthorrhizol + tamoxifen group compared to the tamoxifen-alone group. It can be concluded that while there is no significant herb–drug interaction between xanthorrhizol and tamoxifen in vitro, there is such an interaction in tumor-bearing mice, which provides important information that affects breast cancer treatment translational research.
Keywords: breast cancer, tamoxifen, interaction, xanthorrhizol
*Corresponding author. chiujh@mailsrv.ym.edu.tw Published online in J-STAGE
doi: 10.1254/jphs.14024FP
menopausal and post-menopausal women. In breast tissue, tamoxifen acts as an estrogen antagonist and competitively inhibits estrogen binding to ERs, which blocks the actions of estrogen on breast cancer cells (6).
Recent investigations have shown that tamoxifen promptly stimulates the activation of ERK1/2 in an ER-positive breast cancer cell line (MCF-7) (7), and this causes both cell-cycle arrest and apoptosis via an induction of signaling that leads to a modulation of the MAPK and mitochondria/caspase pathways in breast cancer cells (8). In addition, tamoxifen stimulates auto- phagy by increasing the intracellular level of ceramide, which then inhibits mTOR activation and/or stimulates expression of Atg genes (9). Although tamoxifen brings about a significant reduction in breast cancer occurrence at rates of 43% – 69% in women across all age groups, it frequently causes both mild and serious adverse effects.
The most common side effect is hot flashes, which occurs in up to 64% of women who take tamoxifen (10).
For decades, Complementary and Alternative Medicine (CAM) has offered cancer patients a chance to alleviate discomfort induced by conventional medical treatment (11), which have prevalence rates between 30% and 83%. These therapies are particularly popular among breast cancer patients where the aim is an improvement in the patient’s quality of life (12).
Curcuma xanthorrhiza Roxb., an indigenous herb of Southeast Asia countries, is commonly used to bring about uterus tightening (13), particularly in pregnant women after childbirth or when patients suffer from postpartum uterine swelling (14). Xanthorrhizol, a sesquiterpene compound isolated from Curcuma xanthorrhiza Roxb., has been reported to possess not only anticandidal, antibacterial, antimetastatic, and anti-inflammatory activity (15 – 17). Furthermore, xanthorrhizol, either alone or in combination with curcumin, has been shown to have potent antiproliferative activity in a MDA-MB-231 cell system via the induction of cell apoptosis (18, 19).
Previous studies have shown that plants such as Curcuma xanthorrhiza Roxb., which have traditionally been used for treatment of female disorders, act via their estrogenic activity. Patients often seek complementary and alternative therapy that includes Curcuma xanthorrhiza Roxb. in order to relieve their discomforts after tamoxifen treatment for their ER(+)/PR(+) breast cancers. However, the possibility of an in vitro and/or in vivo interaction between xanthorrhizol and tamoxifen remains un- explored. Accordingly, we investigated the herb–drug interaction between xanthorrhizol and tamoxifen, both in vitro and in vivo, using a human breast cancer MCF-7 cell model.
Materials and Methods Cell culture
The human breast cancer MCF-7 cell line was obtained from Food Industry Research and Development Institute (Taiwan, ROC). The cells were cultured in Dulbecco’s modified Eagle medium DMEM (Invitrogen Co., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.3 g/L l-glutamine, 1 g/L NaHCO3, and 3 g/L HEPES (Invitrogen, Grand Island, NY, USA) and maintained at 37°C with 5%
CO2 in a humidified atmosphere. Cells were kept in the logarithmic growth phase by routine passage every 2 – 3 days using 0.025% trypsin–EDTA treatment.
Assessment of cell cytotoxicity and cell proliferation The antiproliferative activity of xanthorrhizol on MCF-7 cells was determined by the colorimetric assay using 3-(4,5-diethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT assay). Cells (1 × 104/well) were seeded 24 h prior to treatment in a 96-well plate. For the single shot protocol, after 24 h of attachment, the cells were treated with various concentrations (0, 0.3, 1, 3 mM) of xanthorrhizol (Enzo Life Sciences, Inc., NY, USA) (Fig. 1) once for 48 and 72 h, respectively. For the repeated administration protocol, the cells were treated with xanthorrhizol daily for 48 and 72 h, respectively.
The xanthorrhizol was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co., St. Louis, MO, USA) and the drug treatment includes 0.001% DMSO. After incubation, MTT solution was added to each well.
The plate was then incubated for 4 h, after which the remaining MTT solution was removed and the purple formazan crystal formed at the bottom of the wells was dissolved using DMSO for 15 min. The absorbance at 570 nm was read on a spectrophotometric plate reader.
The study protocol for the in vitro interaction between xanthorrhizol, and tamoxifen involved pretreatment with xanthorrhizol (0, 1, 3 mM) for 30 min, before the MCF-7 cells were treated with tamoxifen (Sigma- Aldrich) (0, 2.5, 5 mg/mL) for 72 h. The cell number was evaluated using the MTT method.
In addition to the above, the trypan blue dye exclusion method was also used. In brief, MCF7 cells (1 × 105) were seeded on a 24-well plate. After treatment for 48
Fig. 1. Structure of Xanthorrhizol (Molecular weight: 218.3).
and 72 h, the cells were washed with phosphate-buffered saline (PBS) and trypsinized using 0.5 mL Trypsin- EDTA. The cell suspension was then suspended in fresh culture medium. Cell death analysis was performed by trypan blue dye exclusion by a hemocytometer. The percentage of cells taking up blue dye was used to determine relative cell viability.
Cell cycle analysis
MCF-7 cells (3 × 105) were seeded for 24 h and the culture medium was then replaced overnight with the same medium but containing 0.1% fetal bovine serum.
This was followed by pretreatment with xanthorrhizol (0, 1, 3 mM) for 30 min, after which the cells were treated with tamoxifen (0, 2.5, 5 mg/mL) for another 24 h. After treatment, the cells were harvested using trypsin and rinsed with PBS. They were then fixed with 75% ethanol solution and filtered to allow cell cycle analysis by flow cytometry. The WinMDI program was used to determine the percentage of cells stalled at each phase of the cell cycle, namely, the G0/G1 phase, S-phase, and G2/M phase.
Luciferase reporter assay
For the luciferase reporter assays, MCF-7 cells (1.6 × 105) were seeded onto 24-well plates for 24 h. Cells were then co-transfected (PolyJetTM-DNA) with pGL4- phESR1 and c-ERBB2 promoter(533)-pGL2-basic. All vectors were allowed to express in the MCF-7 cells for 5 h and then the culture media were replaced with dextran charcoal-treated FBS (CDFBS) medium for 24 h. This was followed by treatment with xanthorrhizol (0, 1, 3, 10 mM) for 24 h. The activities of the firefly and Renilla luciferases were measured using a dual luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The data are presented as the relative optic density ratio, namely, the specific luciferase/Renilla luciferase ratio.
Total RNA extraction and real-time quantitative reverse trancription-PCR
After MCF-7 cells (8 × 105 in a six-well plate) had been treated with xanthorrhizol (0, 1, 3 mM) for 24 h, they were harvested and cellular RNA was extracted by the TRIzol method (20) (Life Technologies, Rockville, MD, USA). In order to avoid contamination with DNA, RNase-free DNase (Qiagen Inc., Valencia, CA, USA) was used to treat the samples. The extracted RNA was quantified by spectrophotometry at 260 nm. Comple- mentary DNA (cDNA) was prepared from the total RNA by the SuperScript III first strand synthesis system. The synthesis and polymerase chain reaction were carried out using a SensiFAST SYBR Hi-ROX Kit (Bioline, MA, USA) with 3-step cycling. Since ESR1, ERBB2, and PS2
play critical roles in breast cancer cell growth, their gene expression levels were investigated and quantified by RT-PCR. The relative expression levels of the ESR1, ERBB2, and PS2 mRNAs were normalized against the amount of b-actin mRNA in the same RNA extract.
Western blot analysis
MCF-7 cells were cultured overnight in 6-well plates at a density of 3 × 105/well. Next the culture media were replaced with one containing 0.1% fetal bovine serum overnight and this was followed by pretreatment with xanthorrhizol (0, 1, 3 mM). After 30-min incubation, the cells were treated with tamoxifen (0, 2.5, 5 mg/mL) for 4 h or 24 h. The collected samples were lysed in lysis buffer containing 1.5 M KCl, 0.1 M Tris (pH7.4), 1%
Triton-X-100, and protease inhibitors for 30 min with vortexing every 10 min. Next, the lysates were centri- fuged at 12000 × g at 4°C for 10 min and the supernatant collected. Protein concentration was determined by Bradford protein assay (21). Equal amounts of protein (30 – 50 mg) were boiled in SDS sample-loading buffer for 5 min and then separated by electrophoresis on a 10%
SDS–polyacrylamide gel. The blots were transferred from the SDS–polyacrylamide gel to a PVDF membrane (Millipore, Bedford, MA, USA), which was followed by non-specific blocking using blocking buffer (Hycell Biotechnology, Inc., USA) for 1 min. Next, the mem- branes were incubated with one of the following anti- bodies for 1 h at a room temperature: anti-Phospho-AKT (1:1000X, Ser473; Cell Signaling Technology, Beverly, MA, USA), anti-AKT (Cell Signaling Technology), anti-phospho-ERK1/2 (1:1000X, Thr202/Tyr204; Cell Signaling Technology) anti-ERK1/2 (1:1000X, Cell Signaling Technology), anti-phospho-p38 (1:500X, Thr180/Tyr182; Cell Signaling Technology), anti-p38 (1:1000X, Cell Signaling Technology), anti-phospho- p27(kip1) (1:1000X, pThr187; Cell Signaling Technol- ogy), anti-LC3-II (1:1000X, Cell Signaling, Beverly), anti-b-actin (1:8000X; Gene Tex, CA, USA), and anti-a- tubulin (1:5000X; Abfrontier, Seoul, Korea). The blots were washed with washing buffer 3 times for 5 min and then incubated with anti-Rabbit IgG HRP–linked secondary antibodies (1:5000X, Cell Signaling Technology).
After 1 h of incubation, the blots were washed 3 times with washing buffer and then developed using an ECL detection kit (Amersham Pharmacia Biotech. Inc., NJ, USA), which was followed by quantification by Multi- Gauge software analysis (Fuji Photo Film Co., Ltd., Tokyo) according to the manufacturer’s protocol.
Normalization was carried out using either b-actin or a- tubulin as the internal control.
Immnohistochemistry for autophagy (LC3-II expression) For IHC staining, MCF-7 cells were cultured for 24 h and then pretreated with xanthorrhizol (0, 1, 3 mM) for 30 min followed by tamoxifen (0, 2.5, 5 mg/mL) treat- ment for another 24 h. The cells were then fixed with 4% formaldehyde for 15 min. After washing with PBS, the cells were blocked with 0.4% Triton X100 and 2%
BSA for 1 h, washed with PBS, and incubated with rabbit anti-LC3-II antibody (Cell Signaling Technology) at a dilution of 1:50 for 2 h at room temperature. Subse- quently, goat anti-rabbit IgG (Sigma-Aldrich) conjugated with FITC (1:80X) was applied for 1 h at room tempera- ture. After rinsing with PBS and sealing the slide with 50% glycerol, the stained samples were mounted and visualized under glass coverslips using a fluorescent microscope.
In vivo animal model
Six-week-old BALB/c athymic female nude mice were obtained from National Laboratory Animal Center (Taipei, Taiwan, ROC). The animals were maintained in specific pathogen-free environment with a 12 h/12 h light–dark cycle at 22°C – 24°C and 50% humidity and given ad libitum access to food and water as regulated by the Guide for the Care and Use of Laboratory Animals (National Academy Press, 2011). The study was approved by animal care committee of National Yang- Ming University with license number #1000907. All mice were inoculated subcutaneously with 2 × 106 MCF-7 cells in 50 mL per site in Martigel 0.2 mL (BD Biosciences, San Jose, CA, USA) on dorsal aspect of the mice. After 3 days, the mice were randomly allocated to 5 groups. These were the vehicle (300 mL sterile water, n = 4) group, the tamoxifen (4.6 mg/kg of tamoxifen, n = 4) group, the xanthorrhizol (0.1 mg/kg) + tamoxifen (4.6 mg/kg) group (n = 4), the xanthorrhizol (0.2 mg/kg) + tamoxifen (4.6 mg/kg) group (n = 4) and the xanthorrhizol (0.4 mg/kg) + tamoxifen (4.6 mg/kg) group (n = 4). The xanthorrhizol (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was dissolved in DMSO and diluted in PBS to its final concentration before use and then injected intraperitoneally (i.p.). The tamoxifen was administrated orally (AstraZeneca, London, UK) every 2 days for 21 days (22).
After mice were sacrificed with adequate anesthesia (40 mg/kg) (Virbac Lab., Carros, France), their body weights, tumor weights, and tumor volumes were measured. Tumor size was calculated using calipers to measure the length and width of the tumor. Tumor volume was then calculated according to the following formula: width2 × length × 0.5. The tumors were frozen in liquid nitrogen before storage at −70°C.
Statistics
All data are expressed as the mean ± S.E.M. of at least 4 independent experiments. Statistical analysis was per- formed using one-way analysis of variance (ANOVA) followed by Dunnett’s correction performed post-hoc.
Statistical comparisons between two independent vari- ables were evaluated by two-way ANOVA followed by the Bonferroni comparison test. A P-value < 0.05 was considered statistically significant.
Results
Effects of xanthorrhizol on MCF-7 cell line growth The MCF-7 (ER+, HER-2 low) mammary duct cell line was used as an in vitro model, using different concentrations (0, 0.3, 1, 3 mM) of xanthorrhizol for 48 h and 72 h, and cell numbers and cell viability were mea- sured. The results showed that there was no significant cytotoxicity effect or cell proliferation effect as a result of the xanthorrhizol treatment (Fig. 2A).
We also examined the effects of different concentra- tions of xanthorrhizol for 24 h on the expression levels of ERBB2 and ESR1 using luciferase reporter vectors containing either the human HER2 or human ESR1 promoter region. The results showed that there was no significant change in either ERBB2 (Fig. 2B) or ESR1 (Fig. 2C) expression compared to the vehicle group.
In vitro effects of xanthorrhizol on tamoxifen-treated MCF-7 cell line growth
To assess in vitro the interaction between xanthorrhizol and tamoxifen, we co-treated MCF-7 breast cancer cells with different concentrations of xanthorrhizol (0, 1, 3 mM) and tamoxifen (0, 2.5, 5 mg/mL) and assessed cell growth and the cell cycle profile of the MCF-7 cells.
Tamoxifen significantly decreased cell numbers and caused cell cytotoxicity activity in a dose dependent manner. There was no change in the growth profile of the xanthorrhizol + tamoxifen group compared to the tamoxifen-alone group; however, there was a trend towards an increase in cell numbers (Fig. 3A). The cell cycle analysis showed that tamoxifen induced cell cytotoxicity via G1-S arrest and there was no significant change in this when the xanthorrhizol + tamoxifen group was compared to the tamoxifen alone group (Fig. 3:
B and C).
In vitro effects of xanthorrhizol on growth-related sig- naling pathways in MCF-7 cells treated with tamoxifen
The effects of xanthorrizol and tamoxifen on growth related signaling at the protein level were analyzed by western blotting. The results showed that there was no significant change in the phosphorylation of AKT, ERK,
and P38 at 4 h after xanthorrhizol + tamoxifen treatment compared with the tamoxifen-alone group. Although tamoxifen decreased the phosphorylation of P27(Kip1) dose-dependently, there was no significant change of this protein at 24 h after xanthorrhizol + tamoxifen treatment compared with the tamoxifen-alone group The relative expression levels of each phospho-protein were normal- ized against the total amount of protein present (Fig. 4A).
Since tamoxifen is a strong inducer of autophagy, we investigated autophagy in MCF-7 cells when they were
treated with both xanthorrhizol and tamoxifen. The autophagosome was detected by immunofluorescent staining (Fig. 4B) and quantified by western blotting using anti-LC3-II antibody (Fig. 4C). The results showed that there was no significant difference, both qualitatively and quantitatively, in LC3-II expression between the xanthorrhizol + tamoxifen group and tamoxifen-alone groups.
Effects of xanthorrhizol (repeated administration) on cell growth and cell growth-related gene expression of the MCF-7 human breast cancer cell line
MCF-7 cells were repeatedly treated with a range of concentrations (0, 0.3, 1, 3 mM) of xanthorrhizol. At 48 h and 72 h after xanthorrhizol treatment, the results showed that the repeated administration xanthorrhizol dose-dependently stimulated cell proliferation of MCF-7 cells at 72 h. The relative cell numbers compared to the control group at the various xanthorrhizol concentrations of 0, 0.3, 1, and 3 mM were 1.22 ± 0.10, 1.51 ± 0.14, 1.62 ± 0.19, and 1.69 ± 0.18, respectively (Fig. 5A).
Real-time PCR analysis after xanthorrhizol treatment for 24 h showed that the PS2 gene expression level of the vehicle, xanthorrhizol (1 mM), and xanthorrhizol (3 mM) groups were 1, 1.22 ± 0.03, and 1.28 ± 0.098, respectively, using the vehicle group as the control (Fig. 5B). Thus it would seem that xanthorrhizol signifi- cantly up-regulates PS2 gene expression.
In vivo effects of xanthorrhizol on tamoxifen-treated MCF-7–implanted athymic nude mice
When athymic BALB/c nude mice were used as an in vivo model to assess the interaction between xanthorrhizol and tamoxifen (Fig. 6), it can be seen that the tumors in the xanthorrizol (0.2 mg/kg) + tamoxifen group were larger than those in tamoxifen-alone group (Fig. 7A) with tumor weight being 38.38 ± 5.60 mg and 21.17 ± 3.26 mg, respectively (Fig. 7B), while the tumor volumes in the xanthorrizol (0.2 mg/kg) + tamoxifen group and tamoxifen-alone group were 51.654 ± 7.96 mL and 21.58 ± 3.16 mL, respectively (Fig. 7C). These findings demonstrated that tumor weight and tumor volume were significantly increased in the xanthorrizol (0.2 mg/kg) + tamoxifen group compared with the tamoxifen- alone group.
In vivo effects of xanthorrhizol on various signaling pathways using the tamoxifen-treated MCF-7–implanted athymic nude mouse model
Protein expression of P38 and P27(kip1) were analyzed by western blotting and the results showed that there were significant increases in both p-P38 (Fig. 8A) and p-P27(kip1) (Fig. 8B) expression levels in the
Fig. 2. Effects of xanthorrhizol (single shot) on cell growth and ERBB2/ESR1 luciferase activity in MCF-7 cell line. Cell number (A), ERBB2 (B), and ESR1 (C) luciferase activity were measured by MTT at 0, 48, and 72 h (A) and luciferase reporter system at 24 h (B, C) after xanthorrhizol treatment, respectively. Data are presented as the mean ± S.E.M. (n = 4). The results showed that there was no significant cytotoxicity, cell proliferation, or increased luciferase activity by one-shot xanthorrhizol treatment.
xanthorrhizol (0.1 mg/kg) + tamoxifen group compared to the tamoxifen-alone group. The relative expression levels of each phospho-protein were normalized against the total P38 or P27 protein as appropriate. Thus xanthor- rhizol seems to attenuate the tamoxifen-induced cell cytotoxicity effects on tumor weight and tumor volume and to increase the phosphorylation of P38 and P27(kip1) in the xanthorrhizol + tamoxifen group compared with the tamoxifen-alone group. This suggests that xanthorrhizol-enhanced cell proliferation occurs via increased phosphorylation of the cell cycle controlling protein P27(kip1) and the anti-apoptosis protein P38.
Discussion
In this study, we adopted an appropriate animal model, MCF-7 implanted athymic nude mice, to elucidate the interaction between xanthorrhizol and tamoxifen (22).
To our knowledge, this is the first study to explore the interaction between these two compounds. Previously, it has been shown that xanthorrhizol significantly enhances expression of PS2 mRNA in MCF-7 cells via
the classical ER pathway (23). In this study, xanthorrhizol significantly increased Gal-4/ER luciferase activity in a dose-dependent manner (1 – 5 mM) and induced the endogenous ER-estrogen response element (ERE) interaction in MCF-7 cells. However, the fact that xanthorrhizol inhibited the proliferation of the human breast cancer MCF-7 cells with an IC50 value of 1.71 mg/mL suggests the biphasic effects of this compound, namely, enhancing cell proliferation at low concentration (< 5 mM) while inhibiting cell growth at high concentration (> 5 mM) (18). By using a low concentration (0 – 3 mM), our results showed that there was no growth stimulating activity by one-shot treatment of xanthorrhizol, and repeated administration significantly enhances cell pro- liferation and up-regulates PS2 gene expression. In our animal model, xanthorrhizol was administered every other day, which mimics repeated dosing in vitro, to MCF-7–implanted nude mice; and tumor size, tumor weight, and tumor volume were found to be significantly increased in the xanthorrizol + tamoxifen groups com- pared with the tamoxifen-alone group. The action of repeated dosing is higher than that of a single shot
Fig. 3. Effect of xanthorrhizol (single shot) on cell growth and cell cycle in MCF-7 cells treated with tamoxifen. Cell number (A) and cell cycle analysis (B, C) were measured by MTT assay at 72 h (A) and flow cytometry at 24 h (B, C) after treatment. Data are reported as the mean ± S.E.M. (n = 10). The results showed that tamoxifen significantly decreased the cell number through G1-S arrest, while there was no significant change in terms of cell number and cell cycle analysis in the xanthorrhizol + tamoxifen group compared with the tamoxifen-alone group. *P < 0.05 vs. vehicle group.
of xanthorrhizol, suggesting the possibility of an accu- mulating effect of this compound in enhancing the proliferative activity in MCF-7 cells. Based on the pharmacological consideration, we can speculate the reason why the middle dose (2 mg/kg) had a stronger
action against tumor proliferation compared to the higher (4 mg/kg) one is that the latter action actually reaches to the plateau phase. Taking these findings as a whole, the results are in agreement with a previous report demonstrating xanthorrhizol has estrogenic activity (16).
Fig. 4. Effects of xanthorrhizol on growth- and autophagy-related signaling pathways in MCF-7 cells treated with tamoxifen.
The protein expressions of growth- (A) and autophagy-related signaling pathways were detected by immunofluorescent staining (B) and quantified by western blotting using anti-LC3-II antibody (C). The results showed that there was no significant difference, both qualitatively and quantitatively, in LC3-II expression between xanthorrhizol + tamoxifen group and tamoxifen-alone groups.
Relative OD ratio = LC3-II/b-tubulin.
In normal cells, the cell cycle is regulated by the cyclin- dependent kinases (CDKs). The CDKs are themselves controlled by various phosphorylation reactions and two groups of proteins, the cyclins and cyclin-dependent kinase inhibitors (CDKIs). The cyclin proteins bind to the CDKs to activate them, while the CDKIs inhibit the CDKs, which include P27(kip1) (24). P27(kip1), a potential tumor suppressor, binds and inhibits cyclin–
CDK complexes, which leads to a block between the G1 and S phases of the cell cycle. Once P27(kip1) is phosphorylated at Ser (10), it is excluded from the nucleus and cannot inhibit cell growth. A recent study has suggested that P27(kip1) levels are predictive in premenopausal and postmenopausal women with early- stage hormone-responsive breast cancer in relation to adjuvant endocrine therapies such as tamoxifen (25).
Besides, tamoxifen is a strong inducer of autophagy, resulting in the increased expression of LC3-II and the decreased G2/M fraction in cell cycle analysis. Our results showed that there was no evidence of interaction between tamoxifen and xanthorrhizol treatment. In spite of the fact that there was no in vitro interaction between the two treatments, our in vivo study showed that there was an increase of p-P27(kip1) expression in the xanthorrhizol + tamoxifen group as compared to the tamoxifen-alone group (Fig. 8B), suggesting the possibility of functional inactivation of P27(Kip1) by
Fig. 5. Effects of xanthorrhizol (repeated administration) on cell growth and growth-related gene expression in MCF-7 human breast cancer cell line. The cell number (5A) and mRNA transcription level (5B) were determined by MTT and real-time PCR, respectively. The results showed that repeated administration of xanthorrhizol signifi- cantly enhanced cell proliferation (A) and up-regulated PS2 gene expression. Data are presented as the mean ± S.E.M. (n = 4). *P < 0.05 vs. vehicle group.
Fig. 6. In vivo study design for determining xanthorrhizol (Xan)-tamoxifen (Tam) interaction. By using athymic BALB/c nude mice as an in vivo model, 2 × 106 MCF-7 cells were implanted on the back of the mouse (4 sites/mouse), subcutaneously. Mice were randomly separated into 5 groups: vehicle group (n = 4), Tam (n = 4), xanthorrhizol (0.1 mg/kg) + Tam (n = 4), xanthorrhi- zol (0.2 mg/kg + Tam) (n = 4), and xanthorrhizol (0.4 mg/kg) + Tam (n = 4). Xanthorrhizol was administered by i.p. injection administration and tamoxifen given by oral administration. The body weight, tumor volume, and tumor weight were assessed after 21 days of treatment.
xanthorrizol.
Evidence is available showing that there is a dis- crepancy between the pro-apoptotic and anti-apoptotic roles of P38 MAPKs across various cell types (26). The mechanisms by which P38 contributes to an enhanced anti-apoptotic response include the phosphorylation and translocation of proteins from the Bcl-2 family, which leads to the release of cytochrome c from the mitochon- dria (27). Our results demonstrated that xanthorrhizol reverses tamoxifen-induced cytotoxicity in MCF-7 implanted nude mice and this might be partly explained through activation of the P38/MAPK signaling pathway.
In Thailand, there are two main species of Wan-Chak- Mod-Luk, namely, Curcuma xanthorrhiza Roxb. and Curcuma comosa Roxb. These plants are indigenous to Thailand and are widely used as foods, flavoring, coloring, and traditional herbs. They have also been
commonly used to treat women suffering from post- partum uterine swelling, postpartum uterine bleeding, peri-menopausal bleeding, uterine inflammation, and as a general tonic after childbirth. A previous study extracted the essential oils from the rhizomes of Curcuma xanthorrhiza Roxb., grown in Thailand by hydrodistilla- tion and the oils were analyzed by capillary GC and GC/MS. Xanthorrhizol making up 18.70% of these oils appeared to be a major constituent of Curcuma xanthorrhiza Roxb. (28). This was then converted concentration to animal dose as described previously (29).
For decades, there has been an increasing trend in the use of herbal medicines as complementary treatments to alleviate discomfort and to improve the quality of life in patients with breast cancer who are receiving conven- tional therapies such as chemotherapy and hormonal therapy. In this study, the interaction of xanthorrhizol
Fig. 7. Effects of xanthorrhizol in tamoxifen-treated MCF-7 tumor–implanted athymic BALB/c nude mice. MCF-7 human breast cancer cells were subcutaneously injected and the tumor-bearing mice were sacrificed on day 21; representative photo- graphs (A) of gross tumors in mice in the vehicle, Tam, xanthorrhizol 0.1 mg/kg + Tam, xanthorrhizol 0.2 mg/kg + Tam and Xanthorrhizol 0.4 mg/kg + Tam group. Tumor weight (B) and tumor volume (C) were significantly increased in xanthorrhizol + tamoxifen groups compared with the tamoxifen alone group. *P < 0.05 vs. vehicle group, #P < 0.05 vs. tamoxifen group.
and tamoxifen was evaluated. Our in vitro findings indicate that there is no significant herb–drug interaction when a single-dose approach is used. However, it was possible to detect herb–drug interactions between xanthorrhizol and tamoxifen in tumor-bearing mice using chronic administration and an animal model.
Consequently, we suggested that the appropriate admin- istration of Wan-Chak-Mod-Luk should involve only single doses and the herb should not be used for the long-term treatment of breast cancer patients being treated with tamoxifen. In summary, our findings pro- vides important information in relation to breast cancer treatment translational research, especially for those patients with ER(+)/PR(+) breast cancer.
Acknowledgments
This work was supported by grants from Taipei Veterans General Hospital (V101C-022), Cheng-Hsin & Yang-Ming project (101F195CY22), and Ministry of health and welfare (Center of Excellence for Cancer Research at Taipei Veterans General Hospital phase II, MOHW103-TD-B-111-02). The luciferase reporter vectors, RDB no. 2839 and RDB no.7528, used in this study were deposited by Dr. Masatoshi Tagawa at Chiba Cancer Center and Gene Engineering Division of RIKEN BioResource Center, respectively. These two
clones were provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was assisted by the Division of Experimental Surgery, Department of Surgery, Taipei Veterans General Hospital. We are in debt to Miss Chiang for her technical assistance.
Conflicts of Interest
We declare that there were no financial and personal relationships with other people or organizations that could inappropriately influ- ence this work.
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