The Effect of Myrica Rubra Essential Oil and Its Components Α-Humulene and Trans-Nerolidol on Adhesion and Apoptosis of Colorectal Cancer Cells
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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 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. Copyright: © 2015 The Authors. Licensed under a Creative Commons Attribution 4.0 International License which allows users including authors of articles to copy and redistribute the material in any medium or format, in addition to remix, transform, and build upon the material for any purpose, even commercially, as long as the author and original source are properly cited or credited. 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 Page 1 of 10 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 Page 2 of 10 Cancer Cell & Microenvironment 2015; 2: e1058. doi: 10.14800/ccm.1058; © 2015 by Veronika Hanušová, et al. http://www.smartscitech.com/index.php/ccm 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