SUPPLEMENTARY MATERIAL
In vitro evaluation of antiproliferative effect of ethyl gallate against human oral squamous
carcinoma cell line KB
Mohan Shalini, Thiagarajan Kalaivani and Chandrasekaran Rajasekaran*
Plant Biotechnology Division, School of Bio Sciences and Technology, VIT University, Vellore – 632014,
Tamil Nadu, India.
*Corresponding author:
Dr. C. Rajasekaran,
School of Bio Sciences and Technology,
VIT University
Tel: +91-416-2202542
Fax: +91-416-2243092
Mob: +91-9442994888
E-mail: drcrs70@gmail.com
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In vitro evaluation of antiproliferative effect of ethyl gallate against human oral squamous
carcinoma cell line KB
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Although some polyphenols are known to possess anticancer activity in different cancer
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cell lines through induction of apoptosis, the mode of antiproliferative effect of ethyl
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gallate against human oral squamous carcinoma cell line KB was not studied until now.
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Therefore, the antiproliferative effect of ethyl gallate was evaluated by 3-(4,5-
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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in comparison with
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the reference drug paclitaxel. Generation of reactive oxygen species (ROS),
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mitochondrial membrane potential (MMP) loss, DNA damage and apoptosis were
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determined using 2,7-diacetyldichlorofluorescein fluorescence, uptake of rhodamine-123
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by mitochondria, comet assay and acridine orange/ethidium bromide dual dye staining
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method. Both ethyl gallate and paclitaxel showed cytotoxicity in a dose-dependent
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manner. The 50% inhibitory concentration for ethyl gallate was 30 µg/mL and 20 µg/mL
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for paclitaxel. 50 µg/mL of ethyl gallate was found to be significantly effective (P < 0.05)
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in controlling the cancer cell proliferation leading to acute apoptosis.
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Keywords: paclitaxel; human oral squamous carcinoma cells; antiproliferation; ROS;
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apoptosis; DNA damage
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Experimental
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Materials
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Paclitaxel, Rhodamine
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123 (Rh-123), 2,7-diacetyldichlorofluorescein (DCFH-DA), Dulbecco’s Modified Eagle’s
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Medium (DMEM), 10% FBS, 1% glutamine, penicillin, streptomycin, EDTA and trypsin were
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purchased from Sigma-Aldrich Co. (St. Louis, USA). All other chemicals used were of analytical
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grade.
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Plant material and isolation of ethyl gallate
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Fresh leaves of Acacia nilotica (L.) Wild. ex. Delile subsp. indica (Benth.) Brenan was collected
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from Vellore district, Tamil Nadu, India and authenticated by Dr. G. V. S. Murthy, Scientist-in-
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charge, Botanical Survey of India, Southern Regional Centre, Tamil Nadu Agricultural
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University (TNAU), Coimbatore, India (Voucher specimen number: 1035). We have previously
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reported the extraction and isolation of ethyl gallate from leaves of A. nilotica. The shade-dried
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leaves were sequentially extracted through maceration using chloroform, ethanol, ammonium
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acetate buffer and water as previously described (Kalaivani and Mathew, 2010). Further, ethyl
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gallate was isolated from A. nilotica leaves by bioactivity-guided fractionation using column
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chromatography. The purity was confirmed by thin layer chromatography (TLC) and high
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performance liquid chromatography (HPLC). The yield of ethyl gallate was 0.26174 µg/g leaves
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of A. nilotica (Kalaivani et al, 2011).
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Cell culture
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Human squamous carcinoma cell line (KB) was purchased from the National Centre for Cell
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Science (NCCS), Pune, India. Cells were grown as monolayer at 37°C in 5% CO2 atmosphere in
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Dulbecco’s Modified Eagle’s Medium (DMEM) along with 10% FBS, 1% glutamine and 100
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U/mL penicillin-streptomycin. Cell numbers were counted and seeded at 5x104 cells per well in
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24-well tissue culture plates. 1 mg/mL stock of ethyl gallate or paclitaxel was prepared in 0.5%
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dimethyl sulphoxide (DMSO) (w/v) and further diluted in the culture medium till the DMSO
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concentration becomes 0.01% (v/v).
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Cytotoxicity assay
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Cytotoxicity of ethyl gallate and paclitaxel was determined by the MTT assay (Mosmann, 1983).
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5x104 cells per well were seeded in 24-well tissue culture plates prior to the exposure of test
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substances. Effects of different concentrations of ethyl gallate and paclitaxel (5-120 µg/mL) were
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evaluated for 24 h at 37°C in 5% CO2 incubator. After this period, the cells were incubated with
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MTT (0.5 mg/mL) for 4 h and centrifuged. The formazan crystals formed were dissolved in 200
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µL of DMSO in all the wells. Absorbance was measured at 570 nm and the percentage cell
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viability was calculated using the formula [(Test/Control) x 100] and percentage cytotoxicity by
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[1-(Test/Control)] x 100.
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ROS measurement
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ROS generation was measured in KB cells seeded at 5x104 cells per well using a non-fluorescent
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probe, DCFH-DA (Jesudason et al, 2008). After the treatment of KB cells with ethyl gallate (20,
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30, 40 and 50 µg/mL) and paclitaxel (20 µg/mL), the cells were harvested and suspended in PBS
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for incubation with DCFH-DA (13 mM) for 30 minutes at 37°C in 5% CO2 incubator.
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Fluorescence was measured with excitation and emission filters set at 485±10 and 530±12.5 nm
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respectively. Quantification was done using a spectrofluorimeter and fluorescence microscopic
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images were captured using a blue filter (450-490 nm) in Nikon TS500 fluorescence microscope.
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Mitochondrial membrane potential loss
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Rhodamine 123 (Rh-123) is a lipophilic cationic dye specific to mitochondria and used to
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measure the membrane potential loss in KB cells (Bhosle et al, 2005). After the cells (5x104 cells
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per well) were exposed to ethyl gallate (20, 30, 40 and 50 µg/mL) and paclitaxel (20 µg/mL) for
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24 h, the fluorescent dye Rh-123 (10 µg/mL) was added and incubated for 30 min. The polarized
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and depolarized mitochondria are viewed under a fluorescence microscope using a blue filter
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after washing with PBS. Orange-red fluorescence inside the cell indicates polarized mitochondria
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while the green fluorescence indicates the depolarized ones.
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DNA damage
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DNA damage was analyzed in KB cells treated with ethyl gallate (20, 30, 40 and 50 µg/mL) and
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paclitaxel (20 µg/mL), using alkaline single cell gel electrophoresis (comet assay) (Singh et al,
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1988). Glass slides coated with 1% of normal melting agarose was overlaid with 50 µL of cells
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mixed with 200 µL of low melting point agarose (1%). These slides were maintained in a cold
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lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100 and 10% DMSO) at
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pH 10, 4°C for 1 h. These slides were maintained in the alkaline electrophoresis solution of pH
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13 containing 300 mM NaOH and EDTA for 25 min. Electrophoresis were carried out in a fresh
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buffer for 20 min. The slides were neutralized using 0.4 M tris of pH 7.5 for 5 min and stained
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with ethidium bromide. DNA damage was visualized and the comet images were captured using
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an epifluorescence microscope and analyzed using comet assay software project (CASP) (Konca
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et al, 2003). 100 cells were analyzed for each sample and categorized visually into one of the
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five classes according to the intensity of fluorescence in the comet tail. Quantification of DNA
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damage was done by measuring the tail moment, tail length and olive tail moment (Olive et al,
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1990).
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Apoptosis
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Condensation and fragmentation of DNA are the characteristics of apoptosis in a cell. Cells that
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are undergoing apoptosis can be visualized by staining the DNA with acridine orange (AO) and
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ethidium bromide (EtBr). AO, a membrane-permeable fluorescent dye has specific binding to
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apoptotic cells. Cells showing the features of apoptosis in a particular field were considered as a
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part of the entire number of cells present in total. After 24 h of incubation of KB cells with test
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compounds, they were fixed with glacial acetic acid and methanol in the ratio of 1:3 at room
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temperature for 1 h. These cells were stained with AO/EtBr at 100 µg/mL concentrations in PBS
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at the ratio of 1:1 for 5 minutes (Lakshmi et al, 2008). After which, the cells were again washed
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with PBS to remove the excess binding. Images were captured using UV illumination in 40x
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objective under Nikon fluorescence microscope.
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Statistical analysis
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Statistical analysis was done using one-way analysis of variance (ANOVA) followed by
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Duncan’s multiple range test (DMRT) in statistical package for social sciences (SPSS). Mean
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values (n=6) were considered statistically significant if P < 0.05. EC50 values for each parameter
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was calculated using graph pad prism software.
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Acknowledgements
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The authors are thankful to the VIT management for providing the infrastructure, constant support and
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encouragement.
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References
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Table S1. EC50 values exhibited by ethyl gallate in human oral cancer KB cells
Assay
EC50 (μg/mL)
Reactive oxygen species generation
21.54±1.40
Mitochondrial membrane potential loss
33.67±1.45
Tail length
41.75±1.00
Tail moment
42.00±1.20
Olive tail moment
39.82±0.80
Apoptosis
29.59±1.20
Note: EC50, dose inducing a 50% response in ethyl gallate treated KB cells
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Figure S1. Structure of ethyl gallate
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Figure S2. Effect of ethyl gallate in KB cells (a) intracellular reactive oxygen species (ROS)
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generation (b) mitochondrial membrane potential (MMP) loss (c) DNA damage (d) apoptotic
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morphology. (G1: 0 µg/mL) untreated control, (G2-G5: 20, 30, 40 and 50 µg/mL) ethyl gallate
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treatment, (G6: 20 µg/mL) paclitaxel treatment respectively.
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