The Effect of Myrica Rubra Essential Oil and Its Components Α-Humulene and Trans-Nerolidol on Adhesion and Apoptosis of Colorectal Cancer Cells

RESEARCH ARTICLE

The effect of Myrica rubra essential oil and its components α-humulene and trans-nerolidol on adhesion and apoptosis of colorectal cancer cells

Veronika Hanušová1, Lenka Skálová2, Martin Ambrož2, Věra Králová1, Lenka Langhansová3, Petra Matoušková2

1Department of Medical Biology and Genetics, Charles University in Prague, Faculty of Medicine, Šimkova 870, Hradec Králové, CZ-500 38, Czech Republic 2Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy, Heyrovského 1203, Hradec Králové, CZ-500 05, Czech Republic 3Laboratory of Plant Biotechnologies, Institute of Experimental Botany, Czech Academy of Sciences, Rozvojová 263, Prague 6, CZ-165 02, Czech Republic

Correspondence: Veronika Hanušová E-mail: [email protected] Received: October 08, 2015 Published online: November 19, 2015

Essential oil from leaves of Myrica rubra (MEO), a subtropical Asian fruit tree with traditional use in folk medicines, had significant antiproliferative effect in several intestinal cancer cell lines. In present study, we tested the influence of MEO and its most effective compounds α-humulene and trans-nerolidol on the cell adhesion, expression of adhesion molecules (ICAM-1; E-cadherin; β-catenin) and apoptotic molecules (NF-κB, caspases) in colorectal cancer cell line HT29. All parameters were followed up and compared in presence or absence of pro-inflammatory agent TNFα. The results showed that MEO was able to decrease adhesion of colon cancer HT29 cells to collagen. Furthermore MEO, α-humulene and trans-nerolidol significantly suppressed adhesion of TNFα-induced cells probably due to down-regulation of ICAM-1. Moreover, MEO and α-humulene could diminish tumor invasion and metastasis via up-regulation of E-cadherin. In presence of TNFα, MEO and trans-nerolidol decreased activation (phosphorylation) of NF-κB, increased activity of caspases and by this way induced apoptosis of cancer cells. More pronounced effects of MEO than those of α-humulene and trans-nerolidol indicate synergism and/or contribution of other components. Keywords: alpha-humulene; adhesion molecules; apoptosis; Myrica rubra (Myricaceae); leaf extract; trans-nerolidol To cite this article: Veronika Hanušová, et al. The effect of Myrica rubra essential oil and its components α-humulene and trans-nerolidol on adhesion and apoptosis of colorectal cancer cells. Can Cell Microenviron 2015; 2: e1058. doi: 10.14800/ccm.1058.

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Introduction number of essential oils and sesquiterpenes, their main components, have been found to inhibit proliferation, induce In folk medicines, plant essential oils have been used for apoptosis, suppress angiogenesis, retard metastasis and centuries in various indications including cancer [1]. A enhance chemotherapy both in vitro and in vivo [2]. For

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Cancer Cell & Microenvironment 2015; 2: e1058. doi: 10.14800/ccm.1058; © 2015 by Veronika Hanušová, et al. http://www.smartscitech.com/index.php/ccm example, sesquiterpene zerumbone increased the apoptosis sesquiterpenes (α-humulene and trans-nerolidol) were and inhibited the invasion of cancer cells [3-5]. Other supplied by Sigma-Aldrich. Stock solutions were prepared in sesquiterpene β-caryophyllene oxide suppressed tumor cell dimethyl sulfoxide (DMSO) and stored in the dark at 4 °C. proliferation and invasion via down-regulation of nuclear All other chemicals used were of HPLC or analytical grade factor κB (NF-κB) [6]. In our previous studies, essential oil and provided by Sigma-Aldrich. from Myrica rubra leaves (MEO) and some of its sesquiterpenes significantly decreased the proliferation of Plant material, extraction and analysis several colorectal cancer cell lines. Moreover, MEO and its components were able to enhance the efficacy of anticancer MEO was prepared from the leaves of Myrica rubra (Sieb. drug doxorubicin through the increase of et Zucc), using hydro-distillation. The composition of MEO doxorubicin-mediated oxidative stress and accumulation of was analyzed using GC×GC-TOFMS as described previously doxorubicin in cancer cells [7-9]. [9]. The batch of MEO used in present experiments was the same as in our previous study [9]. MEO was pre-dissolved in Essential oils as well as sesquiterpenes as highly lipophilic DMSO at concentration of 100 mg/mL and stored at 4 °C, compounds can affect membrane structure and thus facilitate before being added to cell cultures. drug accumulation within cells. Moreover, they could also change cell adhesion and migration. For example, zerumbone Cell culture was able to decrease expression of intercellular adhesion molecule-1 (ICAM-1), which correlated with the inhibition Human epithelial colorectal adenocarcinoma line HT29 of tumor necrosis factor alpha (TNFα) induced invasion by was purchased from ATCC (supplier for Czech Rep.: LGC zerumbone [10]. Sesquiterpene derivative istanbulin Standards, Poland). Cells were multiplied in three passages, significantly affected expression of E-cadherin, which play frozen in aliquots and stored in liquid nitrogen. The absence crucial role in cell-cell adhesion and recognition [11]. of mycoplasma in all cell lines used in the laboratory was Sesquiterpene lactone xanthatin remarkably changed periodically checked. For every set of experiments (lasted expression of β-catenin, another important protein in cell-cell 3-9 weeks) new storage cells were resuscitated. The HT29 adhesion [12]. Cell adhesion is essential in all aspects of cell cells were cultured in DMEM supplemented with 10% FBS growth, cell migration and cell differentiation in vertebrate and 0.5% penicillin/streptomycin. Cells were grown in T-75 cells. Metastasis is facilitated by cell-cell interactions cm2 culture flasks in a humidified atmosphere containing 5% between tumor cells and the endothelium in distant tissues CO2 at 37 ºC. and adhesion of cancer cells to endothelial cells is an essential step. Tumor cells in circulation interact also with Western blot analysis platelets and leukocytes that further contribute to tumor cell adhesion, extravasation, and the establishment of metastatic Cells were treated for 8 and 24 h with 10 ng/ml TNFα and lesions [13,14]. Therefore new agents which could stop these 30 µg/ml MEO and/or 30 µg/ml trans-nerolidol and/or 10 processes have been intensively searched for. ng/ml TNFα and/or 30 µg/ml α-humulene. Treated and control cells were washed with PBS and harvested at various In present study, we tested the influence of MEO and its time intervals in ice-cold lysis buffer (50 mM Tris/HCl, 150 most effective compounds α-humulene and trans-nerolidol on mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 the cell adhesion, expression of adhesion molecules mM EGTA, β-glycerolphosphate, 50 mM NaF, 10 mM (ICAM-1; E-cadherin; β-catenin) and apoptotic molecules sodium pyrophosphate, 200 µM sodium orthovanadate, 2 (NF-κB, caspases) in colorectal cancer cell line HT29. All mM DTT). The lysates were resuspended and the amount of parameters were followed up and compared in presence or protein in supernatant was determined by BCA assay. The absence of pro-inflammatory agent TNFα. whole cell lysates were boiled for 5 min/95 °C in SDS sample buffer (Tris-HCl pH 6.8, 40% glycerol, 6% SDS, 0.2 Experimental section M DTT, 0.1 g bromphenol blue) and thereafter 30 µg of sample were loaded onto SDS/polyacrylamide gel. After Chemicals and Reagents electrophoresis, proteins were transferred to a PVDF membrane (100 V, 90 min) and incubated at 25 °C for 1.5 h Dulbecco’s modified Eagle medium (DMEM) was with a solution containing 5% nonfat dry milk, 10 mM supplied by Sigma-Aldrich (Prague, Czech Republic). Fetal Tris-HCl (pH 8.0), 150 mM sodium chloride, and 0.1% bovine serum and gentamicin sulfate were purchased from Tween 20 (TBST). Membranes were incubated with primary Invitrogen (Carlsbad, CA, USA) and bovine serum albumin antibody polyclonal rabbit anti CD54/ICAM1 (1:5 000, Cell (BSA) from Fluka (Prague, Czech Republic). Pure signaling technology), monoclonal mouse anti-EpCAM (1:2

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000, Cell signaling technology), monoclonal mouse anti- E-cadherin (1: 1 500, BD Biosciences), monoclonal mouse anti-NF-κB (1: 3 000, Cell signaling technology), polyclonal anti-phospho NF-κB p65 (Ser 536) (1: 3 000, Cell signaling technology) polyclonal rabbit anti - β-catenin (1:5 000, Cell signaling technology) at 4 °C overnight followed by six 5 min washes in TBST. Next, the membranes were incubated with secondary peroxidase-conjugated antibodies (1:20 000, 2 h, 25 °C), washed with TBST and the signal was developed with a chemiluminescence ECL Prime Western Blotting Detection Reagent (Amersham, GE Healthcare Life Science). Quantity of chemiluminescence was detected using Imaging System (Gel Logic 2200 Pro).

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis

Total RNA was isolated from treated and untreated cells using TriReagent according to manufacturer’s instructions (Biotech, Prague, Czech Republic). Cells were treated for 8 and 24 h with 10 ng/ml TNFα and/or 30 µg/ml MEO and/or 30 µg/ml trans-nerolidol and/or 10 ng/ml TNFα and/or 30 µg/ml α-humulene. RNA concentration and purity were determined spectrophotometrically, and the RNA integrity was checked by gel electrophoresis. First-strand cDNA was synthesized from 1 µg total RNA using ProtoScript II reverse transcriptase (New England Biolabs, Ipswich, MA, USA) and random hexamers. qPCR analyses were performed in iQ5 thermocycler (Bio-Rad, Prague, Czech Republic) using qPCR Core kit for SYBR Green I (Eurogentec, Seraing, Belgium) following manufacturer’s protocol. Primers were designed manually and are available upon request.

Calculations were based on the “Delta-Delta Ct method”. The data were expressed as fold change of the treated groups relative to the control. Hypoxanthine-phosphoribosyl transferase 1 (HPRT1) and beta-2-microglobulin (B2M) were used as a reference gene.

Apoptosis luminescent assay

The activities of initiator caspases 2, 8 and 9 and effector caspases 3/7 were assayed using Promega Caspase-Glo Assays (Madison, USA). The HT29 cells were cultivated in 96-well plates. After 12, 24 and 48 h incubation in the medium with tested substances, cells with medium were Figure 1. Effect of TNFα on the adhesion of HT29 colon cancer cells to collagen type IV. HT29 cells treated with 10 lysed using buffer containing 50 mM HEPES, 5 mM CHAPS ng/ml TNFα and 30 µg/ml MEO (A) 10 ng/ml TNFα and 30 and 5 mM DTT. The control cells were incubated in medium µg/ml trans-nerolidol (B) and 10 ng/ml TNFα and 30 µg/ml containing only solvent DMSO (0.1%). The lysates were α-humulene (C). Impedance (corresponding to cell adhesion) collected into microtubes. All caspases reagents were was measured every 10 min for 12 hours. Adhesions were expressed as a percentage of untreated controls. Data are prepared as described in manufacturer's instruction. The expressed as the mean ± SD of averages from at least three lysates (25 μL in each well) were transferred into experiments. white-walled 384-well-plate for luminometer. Then, 25 μL of

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Figure 2. Effect of MEO on the TNFα-induced HT29 cell adhesion molecules expression in HT29 cells. HT29 cells were treated with TNFα (10 ng/ml) and MEO (10 or 30µg/ml) for 8 or 24 h. E-cadherin, ICAM-1 and β-actin expressions were determined by RT-PCR (A) or western blot analysis (B). Expressions were expressed as a fold increase. Data are expressed as the mean ± SD of averages from at least two experiments. * p<0.05 versus control α p<0.05 versus TNFα-treated cells. caspases reagents were added to each well. Plates were Statistical analysis gently mixed for 30 s and then incubated for 30 min at room temperature. The luminescence was measured using Data are expressed as the mean ± SD of averages from at luminometer Tecan Infinite M200. least two experiments. Statistical analysis was carried out using two way analysis of variance (ANOVA) followed by Cell adhesion assay Bonferroni´s test significant at a level of p˂0.05. Data analysis was performed using GraphPad Prism 6.0. The measurement of cellular adhesion was carried out in Results x-CELLigence Real-Time Cell Analyzer. The bottom of E-plates was coated with 20 µg/ml concentration of collagen Effect of MEO, α-humulene and trans-nerolidol on the type IV (Sigma-Aldrich). Serum free medium supplemented adhesion of cancer cells in presence or absence of TNFα with 1% BSA and tested substances (final concentrations: TNFα - 10 ng/ml; MEO - 30 µg/ml; α-humulene - 30 µg/ml; HT29 cells were seeded into E-plates coated with collagen trans-nerolidol - 30 µg/ml) was placed into each chamber and type IV and incubated in serum-free medium with MEO, background was measured. Optimal amount of the cells α-humulene and trans-nerolidol in presence or absence of (40,000/100 µl), resuspended in serum free medium with TNFα. Cell indexes were monitored for 12 hours. As shown tested substances, were added into each chamber and cell in Fig. 1, pro-inflammatory cytokine TNFα increased cell index values were monitored. Data were calculated as a adhesion of HT29 cells. MEO was able to decrease cell percent of untreated control. adhesion to collagen in comparison to control, furthermore MEO significantly suppressed cell adhesion of TNFα-induced HT29 cells. On the other hand, α-humulene and trans-nerolidol decreased adhesion only in TNFα-treated

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Figure 3. Effect of α-humulen and trans-nerolidol on the TNFα-induced HT29 cell adhesion molecules expression in HT29 cells. HT29 cells were treated with TNFα (10 ng/ml) and α-humulen, resp. trans-nerolidol (30 µg/ml) for 8 or 24 h. E-cadherin, ICAM-1 and β-actin expressions were determined by RT-PCR (A) or western blot analysis (B). Expressions were expressed as a fold increase. Data are expressed as the mean ± SD of averages from at least two experiments. * p<0.05 versus control. cells. show any significant changes. The mRNA as well as protein expression of β-catenin didn´t show any changes in neither Effect of MEO on expression of adhesion proteins in tested time intervals by any treatment (Fig.2.). presence and absence of TNFα Effect of α-humulene and trans-nerolidol on adhesion The relative amounts of mRNA and protein of three proteins in presence and absence of TNFα important adhesion molecules (E-cadherin, β-catenin, ICAM-1) were measured and compared in cells incubated The relative of mRNA and protein amounts of E-cadherin, with MEO alone (10 and 30 µg/ml), TNFα alone and MEO + β-catenin and ICAM-1 were measured and compared in cells TNFα combinations for 8 and 24 hours. The results are incubated with sesquiterpenes α-humulene and presented in Fig. 2. The mRNA level of E-cadherin was not trans-nerolidol alone (30 µg/ml), TNFα alone and affected by any treatment. Protein level of E-cadherin was sesquiterpenes + TNFα combinations for 8 and 24 hours. The significantly increased after MEO alone treatment (at both results are summarized in Fig. 3. Although mRNA levels of concentrations), and also after MEO + TNFα combinations E-cadherin didn´t changed after any treatment, E-cadherin treatment, while TNFα itself didn´t affect expression of protein was significantly increased by α-humulene and E-cadherin in neither test time intervals. On the other hand, trans-nerolidol treatment in both tested intervals. (Fig. 3). TNFα increased expression of ICAM-1 after 8-hour treatment on mRNA level, which was accompanied by Expression levels of ICAM-1 increased after 8 h of TNFα significant increase of ICAM-1 expression on protein level treatment on mRNA level and after 24 h treatment on protein after 24-hour treatment. MEO was able to decrease protein level. Both, α-humulene and trans-nerolidol alone were able levels of TNFα-induced ICAM-1 after 24-hour treatment, to increase protein levels of ICAM-1 after 8 h, but this effect especially in 10 µg/ml concentration. MEO alone didn´t was not further observed after 24 h treatment.

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Figure 4. Effect of MEO (A,C) and α-humulene and trans-nerolidol (B,D) on expression of NF-κB (protein and mRNA level), resp. phospho-NF-κB (protein level) in TNFα-induced HT29 cells. HT29 cells were treated with TNFα (10 ng/ml) and α-humulen, resp. trans-nerolidol (30 µg/ml) for 8 or 24 h. NF-κB, phospho-NF-κB and β-actin expressions were determined by RT-PCR (A, B) or western blot analysis (C, D). Expressions were expressed as a fold increase. Data are expressed as the mean ± SD of averages from at least two experiments. * p<0.05 versus control.

Interestingly, α-humulene and trans-nerolidol had different phosphorylation in cells with or without TNFα treatment. effect on TNFα induced ICAM-1 protein level after 24-hour treatment of cells. Using α-humulene + TNFα combinations In our experiments, TNFα increased transcription of resulted in increased protein levels, but trans-nerolidol + NF-B after 8 h and 24 h, whereas protein level of NF-B TNFα combinations caused decrease of ICAM-1 protein was not significantly changed. On the other hand, NF-B level in comparison to TNFα alone. Expression of β-catenin was activated by phosphorylation after TNFα treatment in was not changed by any treatment (Fig.3.). both tested time intervals. MEO was able to decrease TNFα-induced activation of NF-B after 24h treatment (Fig Effect of MEO, α-humulen and trans-nerolidol on NF-B 4). Similarly, trans-nerolidol treatment reduced expression in presence and absence of TNFα TNFα-mediated increase of phospho-NF-B (Fig. 4). On the other hand, α-humulene increased NF-B phosphorylation in We have also investigated the effects of MEO, TNFα-induced cells after 8 and 24 h treatment (Fig. 4). α-humulene and trans-nerolidol on NF-B expression and its

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Figure 5. Effect of MEO, α-humulene and trans-nerolidol on activity of iniciator capsases (caspase 2, 8 and 9) and effector caspases (caspase 3/7) in TNFα-induced HT29 cells. HT29 cells were treated with TNFα (10 ng/ml) and α-humulen, resp. trans-nerolidol (30 µg/ml) for 12 and 24 h. Activity of caspases were expressed as a percentage of untreated controls. Data are expressed as the mean ± SD of averages from at least three experiments. * p<0.05 versus TNFα.

Effect of MEO, α-humulen and trans-nerolidol on the are particularly important processes in colorectal cancer TNFα-induced apoptosis (CRC) progression and metastasis. CAMs are implicated not only in malignant cell detachment from the primary The activities of caspase 2, 3/7, 8 and 9 were measured in carcinoma, but also in tumor cell attachment to distant tissue. cells incubated with MEO, α-humulene and trans-nerolidol In the former case, adhesion is down-regulated, mainly by alone, TNFα alone. MEO + TNFα combination and modification of the cadherin-catenin system; in the latter, cell sesquiterpenes + TNFα combinations for 12 and 24 hours. surface molecules mediate the tumor-host attachment. The results are summarized in Fig. 5. The results suggested Interestingly, CAMs may also regulate the organ selectivity the potential of tested substances to enhance the apoptosis of metastasis, through their altered expression on various induced by TNFα through the activation of initiator and epithelia and the presence of different adhesion ligands on effector caspases. Especially, activation of initiator caspase 9 various CRC cell lines [16]. and effector caspases 3/7 were significantly higher after 24 h treatment with MEO and trans-nerolidol in TNFα-induced Adhesion of circulation tumor cells to vascular cells, in comparison with cells treated with TNFα alone. endothelium becomes a crucial starting point of metastasis that precedes invasion and extravasation of circulation tumor Discussion cells, and formation of micrometastasis foci [14]. Recently, CAMs have become primary targets for cancer therapy. Cellular adhesion molecules (CAMs) are important Sesquiterpenes and other natural substances are able to participants in cell-cell interactions and interactions between modulate activity and expression of CAMs in several types cells and components of the extracellular matrix. These of cancer. For example, fumagillin was found to suppress molecules have been implicated in a wide variety of cellular CRC growth and metastasis by suppressing angiogenesis functions including signal transduction, cellular through activated leukocyte cell adhesion molecule, and communication and recognition, embryogenesis, intercellular adhesion molecule-1 (ICAM-1) genes [17]. inflammatory and immune responses, and apoptosis. Flavonoid curcumin suppressed metastasis, through the Evidence to date suggests that the CAMs may be associated regulation of metastatic proteins, MMP-2, MMP-9 and with invasion and metastasis in a variety of human intercellular adhesion molecules [18]. Polyphenol resveratrol malignancies [15]. (50 µM) blocked significantly ICAM-1 expression and cell adhesion between PMA-induced HT1080 and ECV 304 The maintenance, promotion or disruption of cell adhesion umbilical cells [19]. Sesquiterpene β-caryophyllene oxide (30

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µM) was able to decrease TNFα-induced expression of well as α-humulene, effective compound of MEO, showed ICAM-1 and MMP-9 in several time intervals and similar effect. The expression of β-catenin, another important suppressed invasion of the H1299 lung cancer cells [6]. protein in cell-cell adhesion, was not affected by any treatment in our experiments. In our previous experiments, MEO and its two sesquiterpenes, α-humulene and trans-nerolidol showed With aim to bring more light in MEO and sesquiterpenes significant antiproliferative effect in several intestinal cancer action in cancer cells, their effect on NF-B expression and cell lines including HT29 cells. As the anti-proliferative activation was studied. NF-B acts as a regulator of genes effect of MEO was less pronounced in end-point tests than in that control cell proliferation and cell survival. It is activated x-Celligence system, which is based on the impedance of by various carcinogens and inflammatory stimuli, including adhering cells, we hypothesized that MEO and its cigarette smoke, TNFα, IL-1, receptor activator of NF-B components can affect cell adhesion. For that reason, present ligand (RANKL), PMA and lipopolysaccharide (LPS) [10]. study was designed to test the effect of MEO, α-humulene Aberrant activation of NF-κB is frequently observed in many and trans-nerolidol on adhesion and CAMs expression in cancers. Moreover, suppression of NF-κB limits the HT29 cells. As pro-inflammatory cytokine TNFα proliferation of cancer cells. Many natural products with up-regulates CAMs expression and play important role in anti-cancer and anti-inflammatory activity have also been cell adhesion, all our experiments were followed up and shown to inhibit NF-κB. Group of Yoon [25] showed that compared in presence and absence of TNFα. essential oil from Neolitsea sericea leaves can inhibit NF-κB activation induced by LPS. β-Caryophyllene oxide, one of Our results showed that MEO was able to decrease HT29 Neolitsea sericea essential oil component, down-regulated cell adhesion to collagen IV. Collagen type IV is a most of the gene products, which may account for the sheet-forming collagen and a major constituent of the vessel [20] potentiation of the cytotoxic effects of TNFα, paclitaxel and wall . Moreover, MEO significantly limited cell adhesion doxorubicin. In further studies, β-caryophyllene oxide was increased by TNFα. This is important result indicating found to block the activation of NF-κB without directly promising property of MEO. Its components, α-humulene interfering with the DNA binding ability of NF-B and and trans-nerolidol decreased adhesion only in caryophyllene oxide inhibited TNFα-induced activation of TNFα-induced cells. With aim to determine which adhesion IκB kinase (IKK) [6]. molecules are affected by MEO, the expression of three important adhesion molecules was analyzed. As expected, TNFα is known to be regulated by NF-B and the TNFα significantly induced ICAM-1 expression. ICAM-1 levels are enhanced by inflammatory cytokines and down- activation of NF-B has been shown to inhibit regulated by anti-inflammatory agents in general [21]. MEO TNFα-induced apoptosis. For example zerumbone was able to significantly decrease TNFα-induced ICAM-1 down-regulated NF-B dependent gene products involved in expression. Thus, suppression of the TNFα-induced adhesion cell proliferation (cyclin D1, c-myc) and anti-apoptosis of tumor cells could be due to down-regulation of ICAM-1. (surviving, IAP1, IAP2, XIAP, Bcl-2, BclXL). This Similarly the decrease of ICAM-1expression after down-regulation led to potentiation of apoptosis induced by trans-nerolidol treatment (30 µg/ml) was observed. However, cytokines and chemotherapeutic agents [10]. As well as this component can´t be solely responsible for the cell caryophyllene oxide significantly down-regulated expression adhesion lowering effect of MEO as seen in cell adhesion of NF-B and simultaneously caused enhancement of assay. apoptosis induced by TNFα, paclitaxel and doxorubicin [6]. In our experiments, we observed decreased phosphorylation of Further molecule involved in cell adhesion processes is NF-B (Ser536) in TNFα-induced HT29 cells after MEO (30 E-cadherin, which mediates cell contact and acts as an µg/ml) and trans-nerolidol (30 µg/ml) treatment. These important suppressor of epithelial tumor cell invasiveness findings correlate with significant increased activation of and metastasis [22]. Reduced expression of complex caspases. Especially after 24 h, the induction of initiator E-cadherin-catenin in malignant disease is associated with caspases (2, 9) and effector caspases (3/7) by TNFα was tumor invasion, metastasis and unfavorable prognosis [23]. found significantly increased after MEO and trans-nerolidol Sesquiterpene β-elemene was reported to positively regulate treatment. the expression of E-cadherin in MCF-7 cells [24]. Consistently, β-elemene induced a switch in cell morphology Conclusions and reduced cell migration and invasion. Our study revealed that MEO was able to significantly increased protein level of MEO was able to decrease adhesion of colon cancer HT29 E-cadherin after 24 h, even in TNFα-induced HT29 cells, as cells to collagen. Furthermore MEO, α-humulene and

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Cancer Cell & Microenvironment 2015; 2: e1058. doi: 10.14800/ccm.1058; © 2015 by Veronika Hanušová, et al. http://www.smartscitech.com/index.php/ccm trans-nerolidol significantly suppressed adhesion of 6. Kim C, Cho SK, Kim KD, Nam D, Chung WS, Jang HJ, et al. TNFα-induced cells probably due to down-regulation of beta-Caryophyllene oxide potentiates TNFalpha-induced apoptosis and inhibits invasion through down-modulation of ICAM-1. Moreover, MEO and α-humulene may diminish NF-kappaB-regulated gene products. Apoptosis 2014,19:708-718. tumor invasion and metastasis via up-regulation of E-cadherin. In presence of TNFα, MEO and trans-nerolidol 7. Ambroz M, Bousova I, Skarka A, Hanusova V, Kralova V, Matouskova P, et al. The Influence of Sesquiterpenes from Myrica decreased activation (phosphorylation) of NF-B and rubra on the Antiproliferative and Pro-Oxidative Effects of increased activity of caspases, thus inducing apoptosis of Doxorubicin and Its Accumulation in Cancer Cells. Molecules cancer cells. More pronounced effects of MEO than those of 2015,20:15343-15358. α-humulene and trans-nerolidol, indicate synergism and/or 8. Ambroz M, Hanusova V, Skarka A, Bousova I, Kralova V, contribution of other components. Langhasova L, et al. Essential Oil from Myrica rubra Leaves Potentiated Antiproliferative and Prooxidative Effect of Doxorubicin and its Accumulation in Intestinal Cancer Cells. Conflicting interests Planta Med 2015. The authors declare that they have no conflict of interest. 9. Langhansova L, Hanusova V, Rezek J, Stohanslova B, Ambroz M, Kralova V, et al. Essential oil from Myrica rubra leaves inhibits cancer cell proliferation and induces apoptosis in several human Acknowledgements intestinal lines. Industrial Crops and Products 2014,59:20-26.

This work was supported by the program PRVOUK 10. Takada Y, Murakami A, Aggarwal BB. Zerumbone abolishes NF-kappaB and IkappaBalpha kinase activation leading to P37/01 and by Czech Science Foundation, grant No. suppression of antiapoptotic and metastatic gene expression, P303/12/G163. upregulation of apoptosis, and downregulation of invasion. Oncogene 2005,24:6957-6969. Abbreviations 11. Fu JJ, Wang S, Lu H, Ma JC, Ke XQ, Liu T, et al. In vitro inhibitory effects of terpenoids from Chloranthus multistachys on CAM: cell adhesion molecule; ICAM-1: intercellular epithelial-mesenchymal transition via down-regulation of Runx2 adhesion molecule; MEO: Myrica rubra essential oil; NF-κB: activation in human breast cancer. Phytomedicine 2015,22:165-172. nuclear factor kappa-light-chain-enhancer of activated B cells; TNFα: tumor necrosis factor alpha. 12. Li WD, Wu Y, Zhang L, Yan LG, Yin FZ, Ruan JS, et al. Characterization of xanthatin: anticancer properties and mechanisms of inhibited murine melanoma in vitro and in vivo. Author contributions Phytomedicine 2013,20:865-873.

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