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Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species C. Weidner a, M. Rousseau a, A. Plauth a, S.J. Wowro a, C. Fischer a, H. Abdel-Aziz b, S. Sauer a,∗
Q1
a b
Otto Warburg Laboratory, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany Scientific Department, Steigerwald Arzneimittelwerk GmbH, 64295 Darmstadt, Germany
a r t i c l e
i n f o
Article history: Received 25 September 2014 Revised 15 December 2014 Accepted 15 December 2014 Available online xxx Keywords: Colon cancer Apoptosis Functional food Melissa officinalis Reactive oxygen species Prevention
a b s t r a c t Purpose: Efficient strategies for the prevention of colon cancer are extensively being explored, including dietary intervention and the development of novel phytopharmaceuticals. Safe extracts of edible plants contain structurally diverse molecules that can effectively interfere with multi-factorial diseases such as colon cancer. In this study, we describe the antiproliferative and proapoptotic effects of ethanolic lemon balm (Melissa officinalis) leaves extract in human colon carcinoma cells. We further investigated the role of extraand intracellular reactive oxygen species (ROS). Methods: Antitumor effects of lemon balm extract (LBE) were investigated in HT-29 and T84 human colon carcinoma cells. Inhibition of proliferation was analyzed by DNA quantification. The causal cell cycle arrest was determined by flow cytometry of propidium iodide-stained cells and by immunoblotting of cell cycle regulator proteins. To investigate apoptosis, cleavage of caspases 3 and 7 was detected by immunoblotting and fluorescence microscopy. Phosphatidylserine externalization was measured by Annexin V assays. Mechanistic insights were gained by measurement of ROS using the indicator dyes CM-H2 DCFDA and Cell ROX Green. Results: After 3 and 4 days of treatment, LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC50 ) of 346 and 120 μg/ml, respectively. Antiproliferative effects were associated with a G2/M cell cycle arrest and reduced protein expression of cyclin dependent kinases (CDK) 2, 4, 6, cyclin D3, and induced expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21). LBE (600 μg/ml) induced cleavage of caspases 3 and 7 and phosphatidylserine externalization. LBEinduced apoptosis was further associated with formation of ROS, whereas quenching of ROS by antioxidants completely rescued the colon carcinoma cells from LBE-induced apoptosis. Conclusions: Lemon balm (Melissa officinalis) extract inhibits the proliferation of colon carcinoma cells and induces apoptosis through formation of ROS. Taken together, LBE or subfractions thereof could be used for the prevention of colon cancer. © 2015 Published by Elsevier GmbH.
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Abreviations
Introduction
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LBE ROS MD TBHP NAC GSH D3T α TOC AA AUC CRC
Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer-related death, accounting for over one million new cases per year (Brenner et al. 2014). The global incidence of CRC is increasing, and due to rapid acquirement of western lifestyle habits such as high caloric nutrition and sedentariness, CRC increasingly also affects people in newly industrialized countries (Center et al. 2009). Effective treatment of colorectal cancer is hampered by low compliance with screening recommendations, late stage diagnosis of new CRC cases, severe chemo- and radiotherapy toxicity, and by therapy resistance and cancer recurrences (Braun and Seymour 2011; Shekhar 2011). These limitations require novel approaches for effective prevention and treatment of CRC. Besides dietary and lifestyle interventions, complementation with safe preventive drugs, phytotherapeutics or tailored food supplements are
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∗
lemon balm extract reactive oxygen species menadione tert-butyl hydroperoxide N-acetyl cysteine glutathione 3H-1, 2-dithiole-3-thione α -Tocopherol ascorbic acid area under the curve colorectal cancer
Corresponding author. Tel.: +49 30 8413 1661; fax: +49 30 8413 1960. E-mail address: [email protected] (S. Sauer).
http://dx.doi.org/10.1016/j.phymed.2014.12.008 0944-7113/© 2015 Published by Elsevier GmbH.
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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needed to combat the epidemic of colorectal cancer and metabolic disorders (Ko and Auyeung 2013; Weidner et al. 2013). Generally, complex mixtures of phytochemicals of diverse chemical nature, such as phenolic acids, polyphenols, flavonoids and terpenoids, combine the advantage of targeting multiple molecular pathways that are often involved in the pathobiology of complex disorders with a considerable reduction in toxic side effects (Wagner and Ulrich-Merzenich 2009; Weidner et al. 2014). Recent studies have shown that herbal extracts and phytochemicals possess promising antitumor effects in CRC, including extracts from ginger, turmeric, grapes, green tea, soy and garlic (Ko and Auyeung 2013). In this study, we demonstrate that a hydroethanolic Melissa officinalis extract induces apoptosis and proliferation inhibition in HT-29 and T84 colon cancer cells. We further demonstrated for the first time that the antitumor effects of Melissa officinalis extracts can be directly attributed to formation of ROS, and that prevention of ROS formation by antioxidants protects the cancer cells from apoptosis.
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Methods
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Materials
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Chemical compounds were purchased from the following sources: oxaliplatin (OX) from Cayman Chemical (Biomol, Hamburg, Germany), staurosporine (STN) and irinotecan (IRI) from LKT Laboratories (Biomol). The following compounds were purchased from Sigma Aldrich (Taufkirchen, Germany): paclitaxel (PTX), 5-Fluorouracil (5-FU), menadione (MD), tert-butyl hydroperoxide (TBHP), N-acetyl cysteine (NAC), glutathione (GSH), 3H-1, 2-dithiole-3-thione (D3T), α -Tocopherol (α -TOC), ascorbic acid (AA). The hydroethanolic (31% (v/v); DER 1:2.5–3.5) lemon balm (Melissa officinalis L.) leaves extract (LBE) used in the current study is a well characterized extract that was provided by Steigerwald Arzneimittelwerk GmbH (Darmstadt, Germany). The extract was produced according to a standardized procedure and quality was controlled by means of HPLC fingerprinting and determination of its rosmarinic acid content as described earlier (Kroll and Cordes 2006). The specific batch used in this study (number 82142) contained 7.48 mg/ml rosmarinic acid (see Supplementary Fig. 1 for HPLC chromatogram). Prior to in vitro experiments, the hydroethanolic extract was centrifuged at 10,000g for 5 min to remove precipitates, and further lyophilized (yield 68.1 mg/ml) and resolubilized in 31% (v/v) ethanol to produce 272 mg/ml stock concentration. For in vitro studies the hydroethanolic extract was diluted in cell culture medium. Ethanol was always used as vehicle control. For all experiments the total ethanol concentration did not exceed 0.07% (v/v) (0.3% for dilution series during proliferation studies).
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Cell culture
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Human HT-29 (ACC-299, DSMZ, Braunschweig, Germany) and T84 colon cancer cells (CCL-248, ATCC, LGC Promochem, Wesel, Germany) were used as cellular model for CRC. Whereas HT-29 cells were established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma, T84 cells were derived from a lung metastasis of a colon carcinoma in a 72-year-old male. Both cell types were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, #11330-057, Gibco, Life Technologies, Darmstadt, Germany) including 2.5 mM l-glutamine and 15 mM HEPES, with 5% fetal bovine serum (FBS, Biochrom, Berlin, Germany) and 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom) at 37 °C in a humidified 5% CO2 atmosphere. Cells were treated with indicated compounds dissolved in DMSO, with hydroethanolic LBE or with the corresponding vehicle control.
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Proliferation assay
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For determination of cellular proliferation HT-29 and T84 cells were seeded in black 384-well plates (#3712, Corning, Fisher Scientific, Schwerte, Germany) with a density of 750 cells/well (HT29) and 900 cells/well (T84), respectively, in a final volume of 50 μl/well. Cells were then treated with the indicated compound concentrations by adding 10 μl of a 6 times stock concentration. After 72 h (HT-29) and 96 h (T84) of treatment, respectively, cells were quantified using the CyQUANT NF Cell Proliferation Assay Kit (Life Technologies) according to the manufacturer’s instructions. For co-treatment with antioxidants, treatment time was reduced to 48 h. Fluorescence intensity was measured with the POLARstar Omega (BMG LABTECH, Offenburg, Germany). For dilution series, data were fitted using GraphPad Prism 5.0 according to equation: Y = top + (bottom − top)/(1 + 10^((log IC50 − X) × Hill slope)) with variable Hill slope. Fluorescence intensity values were transformed to relative number of cells. Efficiency is the maximal observed induction of cell death after treatment relative to nontreated cells (set to 0%).
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Cell cycle analysis
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Analyses of cell cycle regulation were performed in HT-29 cells treated with the indicated compounds for 24 h or 48 h. Trypsinized cells were fixed in 70% ethanol and incubated on ice for 15 min. Fixed cells were then resuspended in propidium iodide (PI)/RNase staining solution (#4087, Cell Signaling Technology, New England Biolabs, Frankfurt, Germany), incubated for 15 min at room temperature and frozen at −20 °C until analysis. Finally, cells were measured in the FACS Aria II flow cytometer (BD Biosciences, Heidelberg, Germany). Data analysis was performed using the Dean–Jett–Fox model in FlowJo 7.6 (Tree Star). Histograms were visualized in GraphPad Prism 5.0.
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Phosphatidylserine externalization (Annexin V) assay
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Externalization of phosphatidylserine of HT-29 and T84 cells treated with the indicated compounds for 48 h was determined by staining with Annexin-V-FLUOS and propidium iodide using the Annexin-V-FLUOS Staining Kit (Roche Diagnostics, Mannheim, Germany) and subsequent flow cytometry (Accuri C6, BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions. Analysis was performed using FlowJo 7.6 (Tree Star). Apoptosis comprised early (Annexin positive, PI negative) and late stage apoptotic events (Annexin positive, PI positive).
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Measurement of extracellular reactive oxygen species (ROS)
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Formation of extracellular reactive oxygen species (ROS) was quantified using the CellROX Green (Life Technologies) dye. This probe is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. Since it is compatible with full medium conditions and requires no cellular activation, CellROX Green was chosen for measuring extracellular ROS. For that purpose, the dye was diluted to 10 μM in full cell culture medium in presence of 1 μg/ml lambda DNA (Life Technologies), and the compound to be tested was added as indicated. Measurement was performed in black 96-well plates (#655090, Greiner Bio-One, Frickenhausen, Germany) in a final volume of 150 μl/well. To increase assay sensitivity, the dye was protected against atmospheric oxygen by adding a sealing layer of 100 μl mineral oil (Luxcel Biosciences, Cork, Ireland) to each well. Fluorescence intensity (490/530 nm) was recorded at 37 °C for 21 h of incubation in the POLARstar Omega (BMG Labtech). For data analyses in GraphPad Prism 5.0, fluorescence values at time zero and signals from background wells were subtracted.
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Fig. 1. Inhibition of proliferation of HT-29 (A) and T84 (B) colon carcinoma cells after treatment with LBE (top) and reference anticancer drugs (bottom) for 3 days (A) and 4 days (B), respectively. Cells were treated with indicated compounds and the relative number of cells was determined by use of a DNA-binding fluorescent dye. LBE, lemon balm extract; IRI, irinotecan; PTX, paclitaxel; 5-FU, 5-Fluorouracil; OX, oxaliplatin.
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Measurement of intracellular reactive oxygen species (ROS)
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Formation of intracellular reactive oxygen species (ROS) was measured using the ROS-sensitive dye 5-(and-6)-chloromethyl-2 ,7 dichlorodihydrofluorescein diacetate (CM-H2 DCFDA, Life Technologies) and CellROX Green (Life Technologies) according to the manufacturer’s instructions. Briefly, for the CM-H2 DCFDA assay, one day before treatment HT-29 cells were seeded in 96-well plates (TPP, Biochrom) with a density of 15,000 cells/well. Before treatment, adherent cells were washed once with pre-warmed PBS and loaded with 50 μM dye diluted in PBS. Cells were then incubated for 30 min at 37 °C to allow incorporation and activation of CM-H2 DCFDA, followed by removing of free dye and washing with pre-warmed PBS. PhenolRed-free DMEM/F-12 medium (11039-021, Life Technologies) was added and cells were again incubated at 37 °C for 60 min. Compounds were added as indicated, and fluorescence (485/530 nm) was measured with the POLARstar Omega (BMG Labtech) at 37 °C for 22 h of treatment. Data were analyzed using GraphPad Prism 5.0. For the CellROX Green assay, one day before treatment HT-29 cells were seeded in 12-well plates (TPP) with a density of 250,000 cells/well. Cells were then treated for 24 h with the indicated compounds. Subsequently, trypsinized cells were labelled with 5 μM of the cell-permeable CellROX Green dye, incubated at 37 °C for 20 min, washed and resuspended in PBS and finally analyzed by flow cytometry (Accuri C6). Data were analyzed using FlowJo 7.6 (Tree Star) and GraphPad Prism 5.0.
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Fluorescence microscopy
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HT-29 cells were seeded on 13 mm cover slips (Sarstedt, Nürnbrecht, Germany) placed in 24 well plates (Nunc, Wiesbaden, Germany). One day later, adherent cells were treated for 48 h with the indicated compounds. Cells were washed with PBS, fixed in 4% formaldehyde/PBS for 15 min, and permeabilized in 0.3% Triton X100/PBS (PBS-T) for additional 10 min at room temperature. Blocking was performed in 5% goat serum (Sigma Aldrich) in PBST (0.3%) for
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60 min at room temperature. Cells were then incubated with primary β -actin antibody (C4, sc-47778, Santa Cruz, Heidelberg, Germany) and primary cleaved caspase 3 antibody (#9664, Cell Signaling Technology) diluted (1:200) in 1% BSA/PBST (0.3%) at 4 °C overnight. Subsequently, washed cells were stained with anti-mouse IgG (H+L) F(ab )2 fragment Alexa Fluor 555 Conjugate (#4409, Cell Signaling Technology) and anti-rabbit IgG (H+L), F(ab )2 fragment Alexa Fluor 488 Conjugate (#4409, Cell Signaling Technology) diluted (1:1,000) in 1% BSA/PBST (0.3%) for 1 h at room temperature. Finally, cover slips were counterstained with ProLong Gold Antifade Mountant solution (containing DAPI) and incubated on room temperature for 24 h. Fluorescence microscope imaging was performed on the LSM700 (Zeiss, Jena, Germany).
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Immunoblotting
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HT-29 cells were lysed in 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 1% SDS with protease inhibitors (Roche Diagnostics) and phosphatase inhibitors (Sigma Aldrich), and sonicated (Bandelin electronic, Berlin, Germany). After centrifugation for 10 min at 10,000g, the supernatants were stored at −80 °C until use. Samples were denaturated and separated using a NuPAGE Novex 4–12% Bis–Tris gel (Life Technologies) and blotted onto Hybond ECL nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Membranes were blocked for 1 h at 4 °C according to the manufacturer’s protocol and washed in PBS-T (0.1%). Primary antibodies against cyclin A2 (#4656), cyclin D1 (#2926), cyclin D3 (#2936), CDK 2 (#2546), CDK 4 (#2906), CDK 6 (#3136), p18 (#2596), p21 (#2947), p27 (#2552), caspase 3 (#9962), cleaved caspase 3 (#9964), caspase 7 (#9492), cleaved caspase 7 (#8438, all from Cell Signaling Technology) and GAPDH (#sc-48167, Santa Cruz) were diluted in PBS-T (0.1%) with milk powder and BSA, respectively, according to the manufacturer’s protocols. Membranes were shaken at 4 °C overnight, washed with PBS-T (0.1%) and subsequently incubated with anti-rabbit IgG-HRP (#sc2004), anti-mouse IgG-HRP (#sc-2005) and anti-goat IgG-HRP (#sc2020, all Santa Cruz), respectively, according to the manufacturer’s
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Fig. 2. Cell cycle analysis of HT-29 colon carcinoma cells after treatment with LBE for 24 h (A) or 48 h ((B) and (C)). Cell cycle was analyzed by flow cytometry of propidium iodide (PI) stained cells ((A) and (B)) and immunoblotting of whole cell lysates (C). Histograms show one representative experiment. Bars represent mean ± SEM (n = 3). n.s., not significant, ∗∗p ࣘ 0.01, ∗∗∗p ࣘ 0.001 vs. control. LBE, lemon balm extract.
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protocols. Detection was carried out with Western Lightning ECL solution (Perkin Elmer, Rodgau, Germany). Membranes were stripped with Restore Plus Western Blot Stripping Buffer (Thermo Scientific) for 7 min. Densitometry was performed with FusionCapt Advanced (Peqlab, Erlangen, Germany).
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Statistical analyses
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Data are expressed as mean ± standard error of mean (SEM) if not otherwise denoted. Statistical tests were performed in GraphPad Prism 5.0. For comparison of two groups statistical significance was examined by unpaired two-tailed Student’s t-test. For multiple comparisons data were analyzed by one-way ANOVA with subsequent Dunnett’s post test. A p value ࣘ 0.05 was defined as statistically significant.
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Results
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LBE concentration-dependently inhibited proliferation of human colon carcinoma cells
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To investigate the antiproliferative effects of LBE on colorectal cancer, HT-29 and T84 cells were treated with varying LBE concentrations for 72 and 96 h, respectively. Proliferation was determined by measurement of cellular DNA content via fluorescent dye binding. LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells in a concentration-dependent manner with IC50 values (mean ± SD) of 346 ± 19 μg/ml (Fig. 1A) and 120 ± 7 μg/ml (Fig. 1B), respectively. IC50 values of LBE were higher than these of the optimized anticancer drugs irinotecan, paclitaxel, 5-Fluorouracil and oxaliplatin (Fig. 1A and B, Table 1). On the other hand, in contrast to most of the reference anticancer drugs tested, LBE treatment led to complete death of colon carcinoma cells.
Fig. 3. Caspase cleavage in HT-29 colon carcinoma cells after treatment with LBE for 48 h. (A) Cellular expression of proteins was determined by immunoblotting of whole cell lysates. (B) Fluorescence microscopy. LBE, lemon balm extract.
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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Fig. 4. Annexin V assays of HT-29 (A) and T84 (B) colon carcinoma cells after treatment with LBE for 48 h. Apoptosis was determined by flow cytometry of Annexin V-FLUOS and propidium iodide stained cells. Scatter plots show one representative experiment. Bars represents mean ± SEM (n = 4). ∗∗p ࣘ 0.01, ∗∗∗p ࣘ 0.001 vs. control. LBE, lemon balm extract; Ctrl, control; IRI, irinotecan; PTX, paclitaxel.
Table 1 Effect of LBE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells. Compound
HT-29
T84
IC50 (μg/ml) LBE Cisplatin Oxaliplatin 5-FU Paclitaxel
346 3 0.6 0.7 0.001
± ± ± ± ±
19 1 0.1 0.2 0.0001
Efficacy (%) 99 91 70 68 76
± ± ± ± ±
4 6 2 5 1
IC50 (μg/ml) 120 4 0 1 0.01
± ± ± ± ±
7 0 0 1 0.001
Efficacy (%) 99 87 87 76 84
± ± ± ± ±
2 3 4 18 4
Efficiency is the maximal observed induction of cell death after treatment relative to nontreated cells (set to 0%).
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LBE induced G2/M cell cycle arrest in HT-29 colon carcinoma cells
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Proliferating cells generally require cell cycle progression. In order to determine if the antiproliferative effects of LBE were associated with changes in cell cycle regulation we treated HT-29 colon carcinoma cells with 600 μg/ml LBE for 24 and 48 h and analyzed propidium iodide stained cells in a flow cytometer. LBE significantly induced accumulation of HT-29 cells in the G2/M phase with concomitant reduction in G0/G1 and S phase after 24 h (Fig. 2A) and 48 h (Fig. 2B).
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Accordingly, LBE treatment reduced the protein expression of cyclin dependent kinases (CDK) 2, 4, 6 and cyclin D3. LBE further induced the expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21) (Fig. 2C). These results corroborate the observed inhibitory effects of LBE on cell cycle progression.
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LBE treatment led to cleavage of caspases 3 and 7 in HT-29 colon carcinoma cells
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Cleavage of inactive procaspases to active caspase enzymes is a hallmark of apoptosis. The caspases 3 and 7 serve as central effectors linking upstream apoptotic activation and downstream execution processes (Elmore 2007). Treatment of HT-29 cells with 600 μg/ml LBE for 48 h induced cleavage of caspases 3 and 7 approximately 2fold (Fig. 3A), suggesting activation of apoptosis. Cleavage of caspase 3 was also validated by fluorescence microscopy (Fig. 3B), confirming the pro-apoptotic effects of LBE in colon cancer cells.
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LBE induced phosphatidylserine externalization in HT-29 and T84 colon carcinoma cells
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In healthy cells phosphatidylserine (PS) is generally restricted to the inner leaflet of the cell membrane. Exposure of phosphatidylserine
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Fig. 5. Extracellular and intracellular detection of reactive oxygen species (ROS). (A) Extracellular formation of ROS in full cell culture medium was kinetically detected by use of CellROX Green fluorescence. Data are expressed as mean ± SEM (n = 8). (B) After treatment of HT-29 colon carcinoma cells for 24 h, intracellular ROS were fluorimetrically detected by use of the CellROX Green dye and flow cytometry. Histograms (left) are representative for each treatment condition. Data are expressed as mean ± SEM (n = 4). (C) Intracellular ROS were kinetically detected by use of CM-H2 DCFDA in living HT-29 colon carcinoma cells. Data are expressed as mean ± SEM (n = 4–8). (D) HT-29 cells were incubated with LBE in presence of known antioxidants, and intracellular ROS were kinetically detected by use of CM-H2DCFDA in living cells. Data are expressed as mean ± SEM (n = 7). ∗∗∗p ࣘ 0.001 vs. control; ###p ࣘ 0.001 vs. LBE-only treated cells. LBE, lemon balm extract (600 μg/ml); MD, menadione (100 μM); TBHP, tert-butyl hydroperoxide (200 μM); NAC, N-acetyl cysteine (1 mM); GSH, glutathione (5 mM); D3T, 3H-1, 2-dithiole-3-thione (50 μM); α TOC, α -Tocopherol (50 μM); AA, ascorbic acid (1 mM); AUC, area under the curve.
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on the outer leaflet is an effect that is commonly observed during apoptosis (Fadok et al. 1998). We determined PS externalization by flow cytometry of Annexin V-FLUOS/propidium iodide-labelled cells that were treated with 600 μg/ml LBE for 48 h. LBE significantly increased the number of apoptotic cells from 5 to 16% in HT-29 (Fig. 4A) and from 43 to 55% in the T84 colon cancer model (Fig. 4B). Similar observations were made with common doses of the known anticancer drugs paclitaxel (5 nM) and irinotecan (10 μM) (Fig. 4A and B).
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LBE induced formation of ROS in HT-29 colon carcinoma cells
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Cancer cells are generally characterized by an imbalance of reactive oxygen species (ROS). Increasing cellular oxidative stress is a common strategy of current anticancer therapies (Yang et al. 2013). To investigate the role of ROS formation during LBE-treatment, we first measured the fluorescence of the ROS-sensitive probe CellROX Green in cell culture medium with 600 μg/ml LBE in absence of cells. Strikingly, LBE induced the formation of ROS (2-fold) already during the first hours of incubation (Fig. 5A), but less than the known strong oxidant tert-butyl hydroperoxide (TBHP, 5-fold). In order to detect ROS in living cells, HT-29 cells were treated with 600 μg/ml LBE for 24 h and labelled with CellROX Green prior to flow cytometry. Noteworthy, LBE significantly increased intracellular ROS levels similar to TBHP, but less than the free radical generator menadione (MD, Fig. 5B). To confirm these observations, we kinetically measured the fluorescence of CM-H2 DCFDA-labelled cells during treatment. Again, LBE significantly increased intracellular ROS levels (3-fold) during the first hours of treatment similar to TBHP (4-fold), but less than MD (16fold) (Fig. 5C). These results show that, first, hydroethanolic Melissa officinalis extracts exerted prooxidative rather than antioxidative effects, and second, ROS formation occurred inside and outside of the HT-29 colon carcinoma cells. To test for the specificity of LBE-induced ROS formation we further co-treated the HT-29 cells with LBE and one of the antioxidants N-acetyl cysteine (NAC), glutathione (GSH), 3H-1, 2-dithiole-3-thione (D3T), α -Tocopherol (α TOC) or ascorbic acid (AA), and subsequently measured the formation of ROS in living cells. Noteworthy, all antioxidants significantly reduced the ROS formation, with NAC (60% reduction) and GSH (96% reduction) having the strongest effects (Fig. 5D). These data demonstrate that the observed increase in intracellular ROS levels was reversible and no technical artifact.
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Formation of ROS is causal for antiproliferative and proapoptotic effects of LBE in HT-29 colon carcinoma cells
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In order to investigate if ROS formation is the cause or rather the result of the LBE-induced apoptosis we co-treated HT-29 cells with LBE and antioxidants and measured again cellular DNA content and PS externalization. Strikingly, NAC, GSH and AA significantly diminished the LBE-induced antiproliferative effects (Fig. 6). Moreover, NAC, GSH, AA and α TOC completely or partly protected the HT-29 cells from LBEinduced apoptosis (Fig. 7). These results indicate that ROS formation is the underlying main mechanism of LBE-induced apoptosis, and that HT-29 cancer cells can be rescued from LBE-induced apoptosis by antioxidative conditions.
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Discussion
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Melissa officinalis (lemon balm) plants are native to the Mediterranean region and are also common in western Asia, the United States and Europe. Melissa extracts contain a large amount of detectable phytochemicals, including phenolic acids (rosmarinic acid, coumaric acid, caffeic acid, protocatechuic acid, ferulic acid, chlorogenic acid), flavonoids (quercetin, luteolin, apigenin, and their glucosides), sesquiterpenes (β -caryophyllene and germacrene),
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Fig. 6. Inhibition of proliferation of HT-29 colon carcinoma cells after co-treatment with antioxidants and 450 μg/ml LBE for 48 h. Cells were treated with indicated compounds and the relative number of cells was determined by use of a DNA-binding fluorescent dye. Bars represent mean ± SEM (n = 4). ∗∗∗p ࣘ 0.001 vs. untreated cells (control); ###p ࣘ 0.001 vs. LBE-only treated cells. LBE, lemon balm extract; NAC, Nacetyl cysteine (1 mM); GSH, glutathione (2.5 mM); D3T, 3H-1, 2-dithiole-3-thione (25 μM); α TOC, α -Tocopherol (50 μM); AA, ascorbic acid (0.5 mM).
monoterpenes (β -pinene) and triterpenes (Chung et al. 2010; Fecka and Turek 2007; Ulbricht et al. 2005a). Melissa officinalis is a popular herb that has been used for centuries in various cultures worldwide for several applications, including antiviral treatment, sleep disorders, anxiety and gastrointestinal disorders (Ulbricht et al. 2005b). Several recent reports further showed cytotoxic and antiproliferative effects in several tumor cell lines, but the underlying mechanisms of action are still poorly understood. Antiproliferative effects of Melissa officinalis essential oil in several cancer cell lines were originally described (de Sousa et al. 2004), and these observations were associated to antioxidative effects measured in a cell-free DPPH (1,1-diphenyl2-picryl-hydrazyl) assay. Other studies (Canadanovic-Brunet et al. 2008) demonstrated antiproliferative activities of different Melissa officinalis extracts in HeLa and MCF-7 cells, and further observed radical scavenging properties using cell-free electron spin resonance spectroscopy. Additional reports (Encalada et al. 2011) showed antiproliferative effects of Melissa officinalis extracts and one of its components rosmarinic acid in HCT-116 colon cancer cells, and linked these observations to antioxidative effects using again cell-free ABTS (2,2azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH assays. Moreover, activation of apoptotic events including caspase activation, DNA fragmentation and PS externalization was described in breast cancer cells in vitro and in vivo (Saraydin et al. 2012). Similar results were obtained in leukemia cells (Ebrahimnezhad Darzi and Amirghofran 2013) and glioblastoma cells (Queiroz et al. 2014). In general, many effects of LBE can be a result of multiple extra- and intracellular interactions of known and potentially unknown compounds of this extract with numerous molecular targets playing important role in regulation of apoptosis. However, the role of ROS formation by Melissa officinalis extracts in cancer cells and its direct role in causing antiproliferative and pro-apoptotic effects have not yet been reported. In this study, we show that a hydroethanolic extract of Melissa officinalis exerted prooxidative rather than antioxidative effects due to ROS formation in treated colon cancer cells. Furthermore, we demonstrated for the first time that increased ROS levels were required for the antiproliferative and apoptotic effects of Melissa officinalis extracts, which were reverted by simultaneous application of strong antioxidants such as glutathione and N-acetyl cysteine. The role of many phenolic natural products in modulating the balance between oxidation and antioxidation is controversially discussed, especially for (poly)phenols and flavonoids (Bouayed and Bohn 2010; Halliwell 2008; Khan et al. 2012). Generally, these redoxactive molecules have the potential to play dual roles in the cellular redox state – on the one hand, as an antioxidant that scavenge free radicals and maintain the levels of antioxidant enzymes, and on the
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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Fig. 7. Annexin V assays of HT-29 colon carcinoma cells after co-treatment with antioxidants and 600 μg/ml LBE for 48 h. Apoptosis was determined by flow cytometry of Annexin V-FLUOS and propidium iodide stained cells. Scatter plots (A) show one representative experiment. Bars (B) represent mean ± SEM (n = 4). ∗∗∗p ࣘ 0.001 vs. untreated cells (control); ###p ࣘ 0.001 vs. LBE-only treated cells. LBE, lemon balm extract; NAC, N-acetyl cysteine (1 mM); GSH, glutathione (5 mM); D3T, 3H-1, 2-dithiole-3-thione (50 μM); α TOC, α -Tocopherol (50 μM); AA, ascorbic acid (1 mM).
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other hand, as pro-oxidant due to its strong reducing power on metal ions, which may drive the Fenton reaction leading to formation of ROS (e.g. hydrogen peroxide, hydroxyl radicals). The behavior to act as anti- or prooxidant depends mainly on the microoxygen environment, the levels of metal ions (especially iron and copper), the presence of other pro-/antioxidants, and the concentration of the natural product itself (Bouayed and Bohn 2010). Based on these considerations different observations regarding the redox effects of phenolic natural products can mostly be explained by taking different experimental conditions into account. ROS have important roles in regulating cell signaling and homeostasis, but an imbalance of ROS can contribute to the develop-
ment of various disorders including cancer. Due to their accelerated metabolism, cancer cells exhibit increased ROS levels compared with normal cells. The high ROS levels in cancer cells renders them more vulnerable to oxidative stress-induced cell death, so that further increasing cellular oxidative stress is a common strategy of current anticancer therapies (Nogueira and Hay 2013). In the present study, we demonstrated that Melissa officinalis extract has the potential to kill colon cancer cells, at least in part through a ROS-inducing mechanism. Many compounds that are contained in Melissa extracts have been reported to change the ROS balance and to exert antiproliferative and cytotoxic effects, particularly rosmarinic acid (Petersen and Simmonds 2003), caffeic acid
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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(Touaibia et al. 2011), ferulic acid (Mancuso and Santangelo 2014), quercetin, luteolin and apigenin (Ross and Kasum 2002) among others. However, given the complexity of plant extracts, it seems not reasonable to explain the effects of the whole plant extracts by the action of single compounds. Instead, in particular the powerful concept of multidrug/multitarget interactions that is the basis for many phytomedical approaches has the potential to address the polyetiological background of complex diseases such as cancer (Wagner and Ulrich-Merzenich 2009). The application of Melissa extracts may thus represent an interesting approach for anticancer phytotherapies, particularly for preventively reducing the risk for colon cancer and cancer recurrences. Since in the gut local concentrations of orally applied plant extracts are in general high, even microgram per milliliter concentrations as used in this exploratory in vitro study are promising starting points for following investigations. The Melissa extracts are generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA) (Ulbricht et al, 2005b), and are widely used as herb and tea preparations. In a next step, preclinical in vivo analyses as well as clinical studies are needed to further investigate the beneficial health effects of Melissa extracts observed in the context of cancer prevention. Phytomedical and nutraceutical applications of Melissa officinalis may provide promising approaches for alleviating cancer diseases.
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Acknowledgments
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Our work was supported by the German Ministry for Education and Research (BMBF, grant no. 0315082 (01EA1303)).
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Supplementary materials
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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2014.12.008.
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Ebrahimnezhad Darzi, S., Amirghofran, Z., 2013. Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines. Immunopharmacol. Immunotoxicol. 35, 313–320. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495–516. Encalada, M.A., Hoyos, K.M., Rehecho, S., Berasategi, I., de Ciriano, M.G., Ansorena, D., Astiasaran, I., Navarro-Blasco, I., Cavero, R.Y., Calvo, M.I., 2011. Anti-proliferative effect of Melissa officinalis on human colon cancer cell line. Plant Foods Hum. Nutr. 66, 328–334. Fadok, V.A., Bratton, D.L., Frasch, S.C., Warner, M.L., Henson, P.M., 1998. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ. 5, 551–562. Fecka, I., Turek, S., 2007. Determination of water-soluble polyphenolic compounds in commercial herbal teas from Lamiaceae: peppermint, melissa, and sage. J. Agric. Food Chem. 55, 10908–10917. Halliwell, B., 2008. Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?. Arch. Biochem. Biophys. 476, 107–112. Khan, H.Y., Zubair, H., Ullah, M.F., Ahmad, A., Hadi, S.M., 2012. A prooxidant mechanism for the anticancer and chemopreventive properties of plant polyphenols. Curr. Drug Targets 13, 1738–1749. Ko, J.K., Auyeung, K.K., 2013. Target-oriented mechanisms of novel herbal therapeutics in the chemotherapy of gastrointestinal cancer and inflammation. Curr. Pharm. Des. 19, 48–66. Kroll, U., Cordes, C., 2006. Pharmaceutical prerequisites for a multi-target therapy. Phytomedicine 13 (Suppl. 5), 12–19. Mancuso, C., Santangelo, R., 2014. Ferulic acid: pharmacological and toxicological aspects. Food Chem. Toxicol. 65, 185–195. Nogueira, V., Hay, N., 2013. Molecular pathways: reactive oxygen species homeostasis in cancer cells and implications for cancer therapy. Clin. Cancer Res. 19, 4309–4314. Petersen, M., Simmonds, M.S., 2003. Rosmarinic acid. Phytochemistry 62, 121–125. Queiroz, R.M., Takiya, C.M., Guimaraes, L.P., Rocha Gda, G., Alviano, D.S., Blank, A.F., Alviano, C.S., Gattass, C.R., 2014. Apoptosis-inducing effects of Melissa officinalis L. essential oil in glioblastoma multiforme cells. Cancer Invest. 32, 226–235. Ross, J.A., Kasum, C.M., 2002. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annu. Rev. Nutr. 22, 19–34. Saraydin, S.U., Tuncer, E., Tepe, B., Karadayi, S., Ozer, H., Sen, M., Karadayi, K., Inan, D., Elagoz, S., Polat, Z., Duman, M., Turan, M., 2012. Antitumoral effects of Melissa officinalis on breast cancer in vitro and in vivo. Asian Pac. J. Cancer Prev. 13, 2765– 2770. Shekhar, M.P., 2011. Drug resistance: challenges to effective therapy. Curr. Cancer Drug Targets 11, 613–623. Touaibia, M., Jean-Francois, J., Doiron, J., 2011. Caffeic acid, a versatile pharmacophore: an overview. Mini Rev. Med. Chem. 11, 695–713. Ulbricht, C., Brendler, T., Gruenwald, J., Kligler, B., Keifer, D., Abrams, T.R., Woods, J., Boon, H., Kirkwood, C.D., Hackman, D.A., Basch, E., Lafferty, H.J., 2005a. Lemon balm (Melissa officinalis L.): an evidence-based systematic review by the Natural Standard Research Collaboration. J. Herb. Pharmacother. 5, 71–114. Ulbricht, C., Brendler, T., Gruenwald, J., Kligler, B., Keifer, D., Abrams, T.R., Woods, J., Boon, H., Kirkwood, C.D., Hackman, D.A., Basch, E., Lafferty, H.J., Natural Standard Research Collaboration, 2005b. Lemon balm (Melissa officinalis L.): an evidencebased systematic review by the Natural Standard Research Collaboration. J. Herb. Pharmacother. 5, 71–114. Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine 16, 97–110. Weidner, C., Wowro, S.J., Freiwald, A., Kawamoto, K., Witzke, A., Kliem, M., Siems, K., Muller-Kuhrt, L., Schroeder, F.C., Sauer, S., 2013. Amorfrutin B is an efficient natural peroxisome proliferator-activated receptor gamma (PPARgamma) agonist with potent glucose-lowering properties. Diabetologia 56, 1802–1812. Weidner, C., Wowro, S.J., Freiwald, A., Kodelja, V., Abdel-Aziz, H., Kelber, O., Sauer, S., 2014. Lemon balm extract causes potent antihyperglycemic and antihyperlipidemic effects in insulin-resistant obese mice. Mol. Nutr. Food Res. 58, 903– 907. Yang, Y., Karakhanova, S., Werner, J., Bazhin, A.V., 2013. Reactive oxygen species in cancer biology and anticancer therapy. Curr. Med. Chem. 20, 3677–3692.
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species C. Weidner a, M. Rousseau a, A. Plauth a, S.J. Wowro a, C. Fischer a, H. Abdel-Aziz b, S. Sauer a,∗
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a b
Otto Warburg Laboratory, Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany Scientific Department, Steigerwald Arzneimittelwerk GmbH, 64295 Darmstadt, Germany
a r t i c l e
i n f o
Article history: Received 25 September 2014 Revised 15 December 2014 Accepted 15 December 2014 Available online xxx Keywords: Colon cancer Apoptosis Functional food Melissa officinalis Reactive oxygen species Prevention
a b s t r a c t Purpose: Efficient strategies for the prevention of colon cancer are extensively being explored, including dietary intervention and the development of novel phytopharmaceuticals. Safe extracts of edible plants contain structurally diverse molecules that can effectively interfere with multi-factorial diseases such as colon cancer. In this study, we describe the antiproliferative and proapoptotic effects of ethanolic lemon balm (Melissa officinalis) leaves extract in human colon carcinoma cells. We further investigated the role of extraand intracellular reactive oxygen species (ROS). Methods: Antitumor effects of lemon balm extract (LBE) were investigated in HT-29 and T84 human colon carcinoma cells. Inhibition of proliferation was analyzed by DNA quantification. The causal cell cycle arrest was determined by flow cytometry of propidium iodide-stained cells and by immunoblotting of cell cycle regulator proteins. To investigate apoptosis, cleavage of caspases 3 and 7 was detected by immunoblotting and fluorescence microscopy. Phosphatidylserine externalization was measured by Annexin V assays. Mechanistic insights were gained by measurement of ROS using the indicator dyes CM-H2 DCFDA and Cell ROX Green. Results: After 3 and 4 days of treatment, LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC50 ) of 346 and 120 μg/ml, respectively. Antiproliferative effects were associated with a G2/M cell cycle arrest and reduced protein expression of cyclin dependent kinases (CDK) 2, 4, 6, cyclin D3, and induced expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21). LBE (600 μg/ml) induced cleavage of caspases 3 and 7 and phosphatidylserine externalization. LBEinduced apoptosis was further associated with formation of ROS, whereas quenching of ROS by antioxidants completely rescued the colon carcinoma cells from LBE-induced apoptosis. Conclusions: Lemon balm (Melissa officinalis) extract inhibits the proliferation of colon carcinoma cells and induces apoptosis through formation of ROS. Taken together, LBE or subfractions thereof could be used for the prevention of colon cancer. © 2015 Published by Elsevier GmbH.
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Abreviations
Introduction
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LBE ROS MD TBHP NAC GSH D3T α TOC AA AUC CRC
Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer-related death, accounting for over one million new cases per year (Brenner et al. 2014). The global incidence of CRC is increasing, and due to rapid acquirement of western lifestyle habits such as high caloric nutrition and sedentariness, CRC increasingly also affects people in newly industrialized countries (Center et al. 2009). Effective treatment of colorectal cancer is hampered by low compliance with screening recommendations, late stage diagnosis of new CRC cases, severe chemo- and radiotherapy toxicity, and by therapy resistance and cancer recurrences (Braun and Seymour 2011; Shekhar 2011). These limitations require novel approaches for effective prevention and treatment of CRC. Besides dietary and lifestyle interventions, complementation with safe preventive drugs, phytotherapeutics or tailored food supplements are
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lemon balm extract reactive oxygen species menadione tert-butyl hydroperoxide N-acetyl cysteine glutathione 3H-1, 2-dithiole-3-thione α -Tocopherol ascorbic acid area under the curve colorectal cancer
Corresponding author. Tel.: +49 30 8413 1661; fax: +49 30 8413 1960. E-mail address: [email protected] (S. Sauer).
http://dx.doi.org/10.1016/j.phymed.2014.12.008 0944-7113/© 2015 Published by Elsevier GmbH.
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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needed to combat the epidemic of colorectal cancer and metabolic disorders (Ko and Auyeung 2013; Weidner et al. 2013). Generally, complex mixtures of phytochemicals of diverse chemical nature, such as phenolic acids, polyphenols, flavonoids and terpenoids, combine the advantage of targeting multiple molecular pathways that are often involved in the pathobiology of complex disorders with a considerable reduction in toxic side effects (Wagner and Ulrich-Merzenich 2009; Weidner et al. 2014). Recent studies have shown that herbal extracts and phytochemicals possess promising antitumor effects in CRC, including extracts from ginger, turmeric, grapes, green tea, soy and garlic (Ko and Auyeung 2013). In this study, we demonstrate that a hydroethanolic Melissa officinalis extract induces apoptosis and proliferation inhibition in HT-29 and T84 colon cancer cells. We further demonstrated for the first time that the antitumor effects of Melissa officinalis extracts can be directly attributed to formation of ROS, and that prevention of ROS formation by antioxidants protects the cancer cells from apoptosis.
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Methods
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Materials
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Chemical compounds were purchased from the following sources: oxaliplatin (OX) from Cayman Chemical (Biomol, Hamburg, Germany), staurosporine (STN) and irinotecan (IRI) from LKT Laboratories (Biomol). The following compounds were purchased from Sigma Aldrich (Taufkirchen, Germany): paclitaxel (PTX), 5-Fluorouracil (5-FU), menadione (MD), tert-butyl hydroperoxide (TBHP), N-acetyl cysteine (NAC), glutathione (GSH), 3H-1, 2-dithiole-3-thione (D3T), α -Tocopherol (α -TOC), ascorbic acid (AA). The hydroethanolic (31% (v/v); DER 1:2.5–3.5) lemon balm (Melissa officinalis L.) leaves extract (LBE) used in the current study is a well characterized extract that was provided by Steigerwald Arzneimittelwerk GmbH (Darmstadt, Germany). The extract was produced according to a standardized procedure and quality was controlled by means of HPLC fingerprinting and determination of its rosmarinic acid content as described earlier (Kroll and Cordes 2006). The specific batch used in this study (number 82142) contained 7.48 mg/ml rosmarinic acid (see Supplementary Fig. 1 for HPLC chromatogram). Prior to in vitro experiments, the hydroethanolic extract was centrifuged at 10,000g for 5 min to remove precipitates, and further lyophilized (yield 68.1 mg/ml) and resolubilized in 31% (v/v) ethanol to produce 272 mg/ml stock concentration. For in vitro studies the hydroethanolic extract was diluted in cell culture medium. Ethanol was always used as vehicle control. For all experiments the total ethanol concentration did not exceed 0.07% (v/v) (0.3% for dilution series during proliferation studies).
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Cell culture
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Human HT-29 (ACC-299, DSMZ, Braunschweig, Germany) and T84 colon cancer cells (CCL-248, ATCC, LGC Promochem, Wesel, Germany) were used as cellular model for CRC. Whereas HT-29 cells were established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma, T84 cells were derived from a lung metastasis of a colon carcinoma in a 72-year-old male. Both cell types were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, #11330-057, Gibco, Life Technologies, Darmstadt, Germany) including 2.5 mM l-glutamine and 15 mM HEPES, with 5% fetal bovine serum (FBS, Biochrom, Berlin, Germany) and 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom) at 37 °C in a humidified 5% CO2 atmosphere. Cells were treated with indicated compounds dissolved in DMSO, with hydroethanolic LBE or with the corresponding vehicle control.
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Proliferation assay
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For determination of cellular proliferation HT-29 and T84 cells were seeded in black 384-well plates (#3712, Corning, Fisher Scientific, Schwerte, Germany) with a density of 750 cells/well (HT29) and 900 cells/well (T84), respectively, in a final volume of 50 μl/well. Cells were then treated with the indicated compound concentrations by adding 10 μl of a 6 times stock concentration. After 72 h (HT-29) and 96 h (T84) of treatment, respectively, cells were quantified using the CyQUANT NF Cell Proliferation Assay Kit (Life Technologies) according to the manufacturer’s instructions. For co-treatment with antioxidants, treatment time was reduced to 48 h. Fluorescence intensity was measured with the POLARstar Omega (BMG LABTECH, Offenburg, Germany). For dilution series, data were fitted using GraphPad Prism 5.0 according to equation: Y = top + (bottom − top)/(1 + 10^((log IC50 − X) × Hill slope)) with variable Hill slope. Fluorescence intensity values were transformed to relative number of cells. Efficiency is the maximal observed induction of cell death after treatment relative to nontreated cells (set to 0%).
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Cell cycle analysis
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Analyses of cell cycle regulation were performed in HT-29 cells treated with the indicated compounds for 24 h or 48 h. Trypsinized cells were fixed in 70% ethanol and incubated on ice for 15 min. Fixed cells were then resuspended in propidium iodide (PI)/RNase staining solution (#4087, Cell Signaling Technology, New England Biolabs, Frankfurt, Germany), incubated for 15 min at room temperature and frozen at −20 °C until analysis. Finally, cells were measured in the FACS Aria II flow cytometer (BD Biosciences, Heidelberg, Germany). Data analysis was performed using the Dean–Jett–Fox model in FlowJo 7.6 (Tree Star). Histograms were visualized in GraphPad Prism 5.0.
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Phosphatidylserine externalization (Annexin V) assay
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Externalization of phosphatidylserine of HT-29 and T84 cells treated with the indicated compounds for 48 h was determined by staining with Annexin-V-FLUOS and propidium iodide using the Annexin-V-FLUOS Staining Kit (Roche Diagnostics, Mannheim, Germany) and subsequent flow cytometry (Accuri C6, BD Biosciences, Heidelberg, Germany) according to the manufacturer’s instructions. Analysis was performed using FlowJo 7.6 (Tree Star). Apoptosis comprised early (Annexin positive, PI negative) and late stage apoptotic events (Annexin positive, PI positive).
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Measurement of extracellular reactive oxygen species (ROS)
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Formation of extracellular reactive oxygen species (ROS) was quantified using the CellROX Green (Life Technologies) dye. This probe is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. Since it is compatible with full medium conditions and requires no cellular activation, CellROX Green was chosen for measuring extracellular ROS. For that purpose, the dye was diluted to 10 μM in full cell culture medium in presence of 1 μg/ml lambda DNA (Life Technologies), and the compound to be tested was added as indicated. Measurement was performed in black 96-well plates (#655090, Greiner Bio-One, Frickenhausen, Germany) in a final volume of 150 μl/well. To increase assay sensitivity, the dye was protected against atmospheric oxygen by adding a sealing layer of 100 μl mineral oil (Luxcel Biosciences, Cork, Ireland) to each well. Fluorescence intensity (490/530 nm) was recorded at 37 °C for 21 h of incubation in the POLARstar Omega (BMG Labtech). For data analyses in GraphPad Prism 5.0, fluorescence values at time zero and signals from background wells were subtracted.
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Fig. 1. Inhibition of proliferation of HT-29 (A) and T84 (B) colon carcinoma cells after treatment with LBE (top) and reference anticancer drugs (bottom) for 3 days (A) and 4 days (B), respectively. Cells were treated with indicated compounds and the relative number of cells was determined by use of a DNA-binding fluorescent dye. LBE, lemon balm extract; IRI, irinotecan; PTX, paclitaxel; 5-FU, 5-Fluorouracil; OX, oxaliplatin.
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Measurement of intracellular reactive oxygen species (ROS)
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Formation of intracellular reactive oxygen species (ROS) was measured using the ROS-sensitive dye 5-(and-6)-chloromethyl-2 ,7 dichlorodihydrofluorescein diacetate (CM-H2 DCFDA, Life Technologies) and CellROX Green (Life Technologies) according to the manufacturer’s instructions. Briefly, for the CM-H2 DCFDA assay, one day before treatment HT-29 cells were seeded in 96-well plates (TPP, Biochrom) with a density of 15,000 cells/well. Before treatment, adherent cells were washed once with pre-warmed PBS and loaded with 50 μM dye diluted in PBS. Cells were then incubated for 30 min at 37 °C to allow incorporation and activation of CM-H2 DCFDA, followed by removing of free dye and washing with pre-warmed PBS. PhenolRed-free DMEM/F-12 medium (11039-021, Life Technologies) was added and cells were again incubated at 37 °C for 60 min. Compounds were added as indicated, and fluorescence (485/530 nm) was measured with the POLARstar Omega (BMG Labtech) at 37 °C for 22 h of treatment. Data were analyzed using GraphPad Prism 5.0. For the CellROX Green assay, one day before treatment HT-29 cells were seeded in 12-well plates (TPP) with a density of 250,000 cells/well. Cells were then treated for 24 h with the indicated compounds. Subsequently, trypsinized cells were labelled with 5 μM of the cell-permeable CellROX Green dye, incubated at 37 °C for 20 min, washed and resuspended in PBS and finally analyzed by flow cytometry (Accuri C6). Data were analyzed using FlowJo 7.6 (Tree Star) and GraphPad Prism 5.0.
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Fluorescence microscopy
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HT-29 cells were seeded on 13 mm cover slips (Sarstedt, Nürnbrecht, Germany) placed in 24 well plates (Nunc, Wiesbaden, Germany). One day later, adherent cells were treated for 48 h with the indicated compounds. Cells were washed with PBS, fixed in 4% formaldehyde/PBS for 15 min, and permeabilized in 0.3% Triton X100/PBS (PBS-T) for additional 10 min at room temperature. Blocking was performed in 5% goat serum (Sigma Aldrich) in PBST (0.3%) for
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60 min at room temperature. Cells were then incubated with primary β -actin antibody (C4, sc-47778, Santa Cruz, Heidelberg, Germany) and primary cleaved caspase 3 antibody (#9664, Cell Signaling Technology) diluted (1:200) in 1% BSA/PBST (0.3%) at 4 °C overnight. Subsequently, washed cells were stained with anti-mouse IgG (H+L) F(ab )2 fragment Alexa Fluor 555 Conjugate (#4409, Cell Signaling Technology) and anti-rabbit IgG (H+L), F(ab )2 fragment Alexa Fluor 488 Conjugate (#4409, Cell Signaling Technology) diluted (1:1,000) in 1% BSA/PBST (0.3%) for 1 h at room temperature. Finally, cover slips were counterstained with ProLong Gold Antifade Mountant solution (containing DAPI) and incubated on room temperature for 24 h. Fluorescence microscope imaging was performed on the LSM700 (Zeiss, Jena, Germany).
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Immunoblotting
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HT-29 cells were lysed in 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 1% SDS with protease inhibitors (Roche Diagnostics) and phosphatase inhibitors (Sigma Aldrich), and sonicated (Bandelin electronic, Berlin, Germany). After centrifugation for 10 min at 10,000g, the supernatants were stored at −80 °C until use. Samples were denaturated and separated using a NuPAGE Novex 4–12% Bis–Tris gel (Life Technologies) and blotted onto Hybond ECL nitrocellulose membranes (GE Healthcare, Freiburg, Germany). Membranes were blocked for 1 h at 4 °C according to the manufacturer’s protocol and washed in PBS-T (0.1%). Primary antibodies against cyclin A2 (#4656), cyclin D1 (#2926), cyclin D3 (#2936), CDK 2 (#2546), CDK 4 (#2906), CDK 6 (#3136), p18 (#2596), p21 (#2947), p27 (#2552), caspase 3 (#9962), cleaved caspase 3 (#9964), caspase 7 (#9492), cleaved caspase 7 (#8438, all from Cell Signaling Technology) and GAPDH (#sc-48167, Santa Cruz) were diluted in PBS-T (0.1%) with milk powder and BSA, respectively, according to the manufacturer’s protocols. Membranes were shaken at 4 °C overnight, washed with PBS-T (0.1%) and subsequently incubated with anti-rabbit IgG-HRP (#sc2004), anti-mouse IgG-HRP (#sc-2005) and anti-goat IgG-HRP (#sc2020, all Santa Cruz), respectively, according to the manufacturer’s
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Fig. 2. Cell cycle analysis of HT-29 colon carcinoma cells after treatment with LBE for 24 h (A) or 48 h ((B) and (C)). Cell cycle was analyzed by flow cytometry of propidium iodide (PI) stained cells ((A) and (B)) and immunoblotting of whole cell lysates (C). Histograms show one representative experiment. Bars represent mean ± SEM (n = 3). n.s., not significant, ∗∗p ࣘ 0.01, ∗∗∗p ࣘ 0.001 vs. control. LBE, lemon balm extract.
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protocols. Detection was carried out with Western Lightning ECL solution (Perkin Elmer, Rodgau, Germany). Membranes were stripped with Restore Plus Western Blot Stripping Buffer (Thermo Scientific) for 7 min. Densitometry was performed with FusionCapt Advanced (Peqlab, Erlangen, Germany).
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Statistical analyses
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Data are expressed as mean ± standard error of mean (SEM) if not otherwise denoted. Statistical tests were performed in GraphPad Prism 5.0. For comparison of two groups statistical significance was examined by unpaired two-tailed Student’s t-test. For multiple comparisons data were analyzed by one-way ANOVA with subsequent Dunnett’s post test. A p value ࣘ 0.05 was defined as statistically significant.
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Results
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LBE concentration-dependently inhibited proliferation of human colon carcinoma cells
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To investigate the antiproliferative effects of LBE on colorectal cancer, HT-29 and T84 cells were treated with varying LBE concentrations for 72 and 96 h, respectively. Proliferation was determined by measurement of cellular DNA content via fluorescent dye binding. LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells in a concentration-dependent manner with IC50 values (mean ± SD) of 346 ± 19 μg/ml (Fig. 1A) and 120 ± 7 μg/ml (Fig. 1B), respectively. IC50 values of LBE were higher than these of the optimized anticancer drugs irinotecan, paclitaxel, 5-Fluorouracil and oxaliplatin (Fig. 1A and B, Table 1). On the other hand, in contrast to most of the reference anticancer drugs tested, LBE treatment led to complete death of colon carcinoma cells.
Fig. 3. Caspase cleavage in HT-29 colon carcinoma cells after treatment with LBE for 48 h. (A) Cellular expression of proteins was determined by immunoblotting of whole cell lysates. (B) Fluorescence microscopy. LBE, lemon balm extract.
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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Fig. 4. Annexin V assays of HT-29 (A) and T84 (B) colon carcinoma cells after treatment with LBE for 48 h. Apoptosis was determined by flow cytometry of Annexin V-FLUOS and propidium iodide stained cells. Scatter plots show one representative experiment. Bars represents mean ± SEM (n = 4). ∗∗p ࣘ 0.01, ∗∗∗p ࣘ 0.001 vs. control. LBE, lemon balm extract; Ctrl, control; IRI, irinotecan; PTX, paclitaxel.
Table 1 Effect of LBE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells. Compound
HT-29
T84
IC50 (μg/ml) LBE Cisplatin Oxaliplatin 5-FU Paclitaxel
346 3 0.6 0.7 0.001
± ± ± ± ±
19 1 0.1 0.2 0.0001
Efficacy (%) 99 91 70 68 76
± ± ± ± ±
4 6 2 5 1
IC50 (μg/ml) 120 4 0 1 0.01
± ± ± ± ±
7 0 0 1 0.001
Efficacy (%) 99 87 87 76 84
± ± ± ± ±
2 3 4 18 4
Efficiency is the maximal observed induction of cell death after treatment relative to nontreated cells (set to 0%).
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LBE induced G2/M cell cycle arrest in HT-29 colon carcinoma cells
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Proliferating cells generally require cell cycle progression. In order to determine if the antiproliferative effects of LBE were associated with changes in cell cycle regulation we treated HT-29 colon carcinoma cells with 600 μg/ml LBE for 24 and 48 h and analyzed propidium iodide stained cells in a flow cytometer. LBE significantly induced accumulation of HT-29 cells in the G2/M phase with concomitant reduction in G0/G1 and S phase after 24 h (Fig. 2A) and 48 h (Fig. 2B).
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Accordingly, LBE treatment reduced the protein expression of cyclin dependent kinases (CDK) 2, 4, 6 and cyclin D3. LBE further induced the expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21) (Fig. 2C). These results corroborate the observed inhibitory effects of LBE on cell cycle progression.
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LBE treatment led to cleavage of caspases 3 and 7 in HT-29 colon carcinoma cells
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Cleavage of inactive procaspases to active caspase enzymes is a hallmark of apoptosis. The caspases 3 and 7 serve as central effectors linking upstream apoptotic activation and downstream execution processes (Elmore 2007). Treatment of HT-29 cells with 600 μg/ml LBE for 48 h induced cleavage of caspases 3 and 7 approximately 2fold (Fig. 3A), suggesting activation of apoptosis. Cleavage of caspase 3 was also validated by fluorescence microscopy (Fig. 3B), confirming the pro-apoptotic effects of LBE in colon cancer cells.
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LBE induced phosphatidylserine externalization in HT-29 and T84 colon carcinoma cells
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In healthy cells phosphatidylserine (PS) is generally restricted to the inner leaflet of the cell membrane. Exposure of phosphatidylserine
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Fig. 5. Extracellular and intracellular detection of reactive oxygen species (ROS). (A) Extracellular formation of ROS in full cell culture medium was kinetically detected by use of CellROX Green fluorescence. Data are expressed as mean ± SEM (n = 8). (B) After treatment of HT-29 colon carcinoma cells for 24 h, intracellular ROS were fluorimetrically detected by use of the CellROX Green dye and flow cytometry. Histograms (left) are representative for each treatment condition. Data are expressed as mean ± SEM (n = 4). (C) Intracellular ROS were kinetically detected by use of CM-H2 DCFDA in living HT-29 colon carcinoma cells. Data are expressed as mean ± SEM (n = 4–8). (D) HT-29 cells were incubated with LBE in presence of known antioxidants, and intracellular ROS were kinetically detected by use of CM-H2DCFDA in living cells. Data are expressed as mean ± SEM (n = 7). ∗∗∗p ࣘ 0.001 vs. control; ###p ࣘ 0.001 vs. LBE-only treated cells. LBE, lemon balm extract (600 μg/ml); MD, menadione (100 μM); TBHP, tert-butyl hydroperoxide (200 μM); NAC, N-acetyl cysteine (1 mM); GSH, glutathione (5 mM); D3T, 3H-1, 2-dithiole-3-thione (50 μM); α TOC, α -Tocopherol (50 μM); AA, ascorbic acid (1 mM); AUC, area under the curve.
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on the outer leaflet is an effect that is commonly observed during apoptosis (Fadok et al. 1998). We determined PS externalization by flow cytometry of Annexin V-FLUOS/propidium iodide-labelled cells that were treated with 600 μg/ml LBE for 48 h. LBE significantly increased the number of apoptotic cells from 5 to 16% in HT-29 (Fig. 4A) and from 43 to 55% in the T84 colon cancer model (Fig. 4B). Similar observations were made with common doses of the known anticancer drugs paclitaxel (5 nM) and irinotecan (10 μM) (Fig. 4A and B).
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LBE induced formation of ROS in HT-29 colon carcinoma cells
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Cancer cells are generally characterized by an imbalance of reactive oxygen species (ROS). Increasing cellular oxidative stress is a common strategy of current anticancer therapies (Yang et al. 2013). To investigate the role of ROS formation during LBE-treatment, we first measured the fluorescence of the ROS-sensitive probe CellROX Green in cell culture medium with 600 μg/ml LBE in absence of cells. Strikingly, LBE induced the formation of ROS (2-fold) already during the first hours of incubation (Fig. 5A), but less than the known strong oxidant tert-butyl hydroperoxide (TBHP, 5-fold). In order to detect ROS in living cells, HT-29 cells were treated with 600 μg/ml LBE for 24 h and labelled with CellROX Green prior to flow cytometry. Noteworthy, LBE significantly increased intracellular ROS levels similar to TBHP, but less than the free radical generator menadione (MD, Fig. 5B). To confirm these observations, we kinetically measured the fluorescence of CM-H2 DCFDA-labelled cells during treatment. Again, LBE significantly increased intracellular ROS levels (3-fold) during the first hours of treatment similar to TBHP (4-fold), but less than MD (16fold) (Fig. 5C). These results show that, first, hydroethanolic Melissa officinalis extracts exerted prooxidative rather than antioxidative effects, and second, ROS formation occurred inside and outside of the HT-29 colon carcinoma cells. To test for the specificity of LBE-induced ROS formation we further co-treated the HT-29 cells with LBE and one of the antioxidants N-acetyl cysteine (NAC), glutathione (GSH), 3H-1, 2-dithiole-3-thione (D3T), α -Tocopherol (α TOC) or ascorbic acid (AA), and subsequently measured the formation of ROS in living cells. Noteworthy, all antioxidants significantly reduced the ROS formation, with NAC (60% reduction) and GSH (96% reduction) having the strongest effects (Fig. 5D). These data demonstrate that the observed increase in intracellular ROS levels was reversible and no technical artifact.
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Formation of ROS is causal for antiproliferative and proapoptotic effects of LBE in HT-29 colon carcinoma cells
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In order to investigate if ROS formation is the cause or rather the result of the LBE-induced apoptosis we co-treated HT-29 cells with LBE and antioxidants and measured again cellular DNA content and PS externalization. Strikingly, NAC, GSH and AA significantly diminished the LBE-induced antiproliferative effects (Fig. 6). Moreover, NAC, GSH, AA and α TOC completely or partly protected the HT-29 cells from LBEinduced apoptosis (Fig. 7). These results indicate that ROS formation is the underlying main mechanism of LBE-induced apoptosis, and that HT-29 cancer cells can be rescued from LBE-induced apoptosis by antioxidative conditions.
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Discussion
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Melissa officinalis (lemon balm) plants are native to the Mediterranean region and are also common in western Asia, the United States and Europe. Melissa extracts contain a large amount of detectable phytochemicals, including phenolic acids (rosmarinic acid, coumaric acid, caffeic acid, protocatechuic acid, ferulic acid, chlorogenic acid), flavonoids (quercetin, luteolin, apigenin, and their glucosides), sesquiterpenes (β -caryophyllene and germacrene),
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Fig. 6. Inhibition of proliferation of HT-29 colon carcinoma cells after co-treatment with antioxidants and 450 μg/ml LBE for 48 h. Cells were treated with indicated compounds and the relative number of cells was determined by use of a DNA-binding fluorescent dye. Bars represent mean ± SEM (n = 4). ∗∗∗p ࣘ 0.001 vs. untreated cells (control); ###p ࣘ 0.001 vs. LBE-only treated cells. LBE, lemon balm extract; NAC, Nacetyl cysteine (1 mM); GSH, glutathione (2.5 mM); D3T, 3H-1, 2-dithiole-3-thione (25 μM); α TOC, α -Tocopherol (50 μM); AA, ascorbic acid (0.5 mM).
monoterpenes (β -pinene) and triterpenes (Chung et al. 2010; Fecka and Turek 2007; Ulbricht et al. 2005a). Melissa officinalis is a popular herb that has been used for centuries in various cultures worldwide for several applications, including antiviral treatment, sleep disorders, anxiety and gastrointestinal disorders (Ulbricht et al. 2005b). Several recent reports further showed cytotoxic and antiproliferative effects in several tumor cell lines, but the underlying mechanisms of action are still poorly understood. Antiproliferative effects of Melissa officinalis essential oil in several cancer cell lines were originally described (de Sousa et al. 2004), and these observations were associated to antioxidative effects measured in a cell-free DPPH (1,1-diphenyl2-picryl-hydrazyl) assay. Other studies (Canadanovic-Brunet et al. 2008) demonstrated antiproliferative activities of different Melissa officinalis extracts in HeLa and MCF-7 cells, and further observed radical scavenging properties using cell-free electron spin resonance spectroscopy. Additional reports (Encalada et al. 2011) showed antiproliferative effects of Melissa officinalis extracts and one of its components rosmarinic acid in HCT-116 colon cancer cells, and linked these observations to antioxidative effects using again cell-free ABTS (2,2azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH assays. Moreover, activation of apoptotic events including caspase activation, DNA fragmentation and PS externalization was described in breast cancer cells in vitro and in vivo (Saraydin et al. 2012). Similar results were obtained in leukemia cells (Ebrahimnezhad Darzi and Amirghofran 2013) and glioblastoma cells (Queiroz et al. 2014). In general, many effects of LBE can be a result of multiple extra- and intracellular interactions of known and potentially unknown compounds of this extract with numerous molecular targets playing important role in regulation of apoptosis. However, the role of ROS formation by Melissa officinalis extracts in cancer cells and its direct role in causing antiproliferative and pro-apoptotic effects have not yet been reported. In this study, we show that a hydroethanolic extract of Melissa officinalis exerted prooxidative rather than antioxidative effects due to ROS formation in treated colon cancer cells. Furthermore, we demonstrated for the first time that increased ROS levels were required for the antiproliferative and apoptotic effects of Melissa officinalis extracts, which were reverted by simultaneous application of strong antioxidants such as glutathione and N-acetyl cysteine. The role of many phenolic natural products in modulating the balance between oxidation and antioxidation is controversially discussed, especially for (poly)phenols and flavonoids (Bouayed and Bohn 2010; Halliwell 2008; Khan et al. 2012). Generally, these redoxactive molecules have the potential to play dual roles in the cellular redox state – on the one hand, as an antioxidant that scavenge free radicals and maintain the levels of antioxidant enzymes, and on the
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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Fig. 7. Annexin V assays of HT-29 colon carcinoma cells after co-treatment with antioxidants and 600 μg/ml LBE for 48 h. Apoptosis was determined by flow cytometry of Annexin V-FLUOS and propidium iodide stained cells. Scatter plots (A) show one representative experiment. Bars (B) represent mean ± SEM (n = 4). ∗∗∗p ࣘ 0.001 vs. untreated cells (control); ###p ࣘ 0.001 vs. LBE-only treated cells. LBE, lemon balm extract; NAC, N-acetyl cysteine (1 mM); GSH, glutathione (5 mM); D3T, 3H-1, 2-dithiole-3-thione (50 μM); α TOC, α -Tocopherol (50 μM); AA, ascorbic acid (1 mM).
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other hand, as pro-oxidant due to its strong reducing power on metal ions, which may drive the Fenton reaction leading to formation of ROS (e.g. hydrogen peroxide, hydroxyl radicals). The behavior to act as anti- or prooxidant depends mainly on the microoxygen environment, the levels of metal ions (especially iron and copper), the presence of other pro-/antioxidants, and the concentration of the natural product itself (Bouayed and Bohn 2010). Based on these considerations different observations regarding the redox effects of phenolic natural products can mostly be explained by taking different experimental conditions into account. ROS have important roles in regulating cell signaling and homeostasis, but an imbalance of ROS can contribute to the develop-
ment of various disorders including cancer. Due to their accelerated metabolism, cancer cells exhibit increased ROS levels compared with normal cells. The high ROS levels in cancer cells renders them more vulnerable to oxidative stress-induced cell death, so that further increasing cellular oxidative stress is a common strategy of current anticancer therapies (Nogueira and Hay 2013). In the present study, we demonstrated that Melissa officinalis extract has the potential to kill colon cancer cells, at least in part through a ROS-inducing mechanism. Many compounds that are contained in Melissa extracts have been reported to change the ROS balance and to exert antiproliferative and cytotoxic effects, particularly rosmarinic acid (Petersen and Simmonds 2003), caffeic acid
Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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(Touaibia et al. 2011), ferulic acid (Mancuso and Santangelo 2014), quercetin, luteolin and apigenin (Ross and Kasum 2002) among others. However, given the complexity of plant extracts, it seems not reasonable to explain the effects of the whole plant extracts by the action of single compounds. Instead, in particular the powerful concept of multidrug/multitarget interactions that is the basis for many phytomedical approaches has the potential to address the polyetiological background of complex diseases such as cancer (Wagner and Ulrich-Merzenich 2009). The application of Melissa extracts may thus represent an interesting approach for anticancer phytotherapies, particularly for preventively reducing the risk for colon cancer and cancer recurrences. Since in the gut local concentrations of orally applied plant extracts are in general high, even microgram per milliliter concentrations as used in this exploratory in vitro study are promising starting points for following investigations. The Melissa extracts are generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA) (Ulbricht et al, 2005b), and are widely used as herb and tea preparations. In a next step, preclinical in vivo analyses as well as clinical studies are needed to further investigate the beneficial health effects of Melissa extracts observed in the context of cancer prevention. Phytomedical and nutraceutical applications of Melissa officinalis may provide promising approaches for alleviating cancer diseases.
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Acknowledgments
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Our work was supported by the German Ministry for Education and Research (BMBF, grant no. 0315082 (01EA1303)).
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Supplementary materials
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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2014.12.008.
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Please cite this article as: C. Weidner et al., Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2014.12.008
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