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SUPPLEMENTARY MATERIAL
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ANTICANCER EFFECTS OF BRUCINE AND GEMCITABINE COMBINATION IN MCF-7
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HUMAN BREAST CANCER CELLS
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Mamatha, Serasanambati1,2, Shanmuga Reddy.Chilakapati1,2, Pavan Kumar. Manikonda 1,
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Jagadeeshwara Reddy.Kanala1, Damodar Reddy, Chilakapati 1*
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Sugen Life Sciences Pvt Ltd, Andhra Pradesh, India-517 505.
Sri Venkateswara University, Department of Biochemistry, Andhra Pradesh, India-517 502.
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*Corresponding author. E-mail: cdr@sugenlife.com
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Abstract
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The present study was designed to investigate combination effects of brucine and gemcitabine,
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each with anticancer properties, in MCF-7 human breast cancer cells in culture. With regards to cell
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viability, effects of both the drugs and their combinations were inversely proportional to dose and time.
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For various proportional drug combinations studied, combination effects were analyzed using CompuSyn
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software. The analyses revealed synergistic and/or additive effects regarding cell viability, anchorage
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independent growth and cell migration. Combination analyses exhibited diversified impacts of the
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format/mode of combination treatment, viz., pretreatment with either drug followed by exposure to the
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other, or treated with both drugs at the same time. Compared to untreated, combination treatment of
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asynchronised MCF-7 cells resulted in 17.2x decrease in G2 phase, increasing G1 (2.1x) and S (1.5x)
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phase cells in cell cycle analysis. Brucine, either individually or in combination, but not gemcitabine,
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inhibited NF-kB subunit (p65) expression in MCF-7 cells.
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Key words: Brucine; Gemcitabine; Cell cycle; NF-kB; Combination; Synergy; Breast cancer
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3. Experimental
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3.1. Cancer cell line and reagents
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Human breast cancer cell line (MCF-7) was purchased from American Type Culture Collection
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(ATCC, Manassas, VA) and was cultured as per vendor’s specifications. 50 mM brucine and 10 mM
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gemcitabine (Sigma-Aldrich, St. Louis, MO,USA) stocks were prepared in dimethylsulfoxide (DMSO;
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Merck, Darmstadt, Germany) and phosphate buffer saline (PBS) pH 7.2 respectively and stored in small
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aliquots at -20ºC until use. Anti NF-kB (p65) and goat anti-rabbit Ig G conjugated secondary antibody
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were purchased from Santa Cruz Biotechnology, Inc., USA. All other chemicals of analytical grade were
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purchased from Sigma-Aldrich, USA.
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3.2. Cell viability assay
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The cytotoxic effect of brucine and/or gemcitabine in MCF-7 cells was determined by 3-(4,5-
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dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as followed by Molnar et al., (2004).
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Briefly, 5000 cells/well were seeded in 96-well plate and treated with desired concentrations of brucine
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or gemcitabine. Combination treatments were performed in three formats. First: Cells were pre-treated
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with brucine for 12 h followed by exposure to gemcitabine for a total of 48 h. Second: Cells were pre-
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treated with gemcitabine for 12 h and followed by exposure to brucine for 48 h. Third: Cells were treated
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with both brucine and gemcitabine at the same time for 48 h. Cell viability was determined by adding 10
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µl of MTT (0.5 mg/ml in PBS) to each well and ultimately measuring A570 after 4 h incubation.
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3.3. Combination effect on cell cycle distribution
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The cell cycle analysis with regards to distribution of cell cycle phases (G1, S and G2) was
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evaluated by flow cytometry. MCF-7 cells (2×106) in 60 mm plates were treated with either brucine or
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gemcitabine, and in combination of both drugs added at the same time for 48 h. The cells were processed,
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stained with propidium iodine (1 µg/ml, BD Pharmingen) and analyzed by flow cytometry using a FACS
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(fluorescence-activated cell sorter) Callibur instrument (BD Bioscience, San Jose, CA) and analyzed
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using Cell Quest pro software (BD Biosciences, San Jose, USA) to determine the percentage distribution
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of cell cycle phases for untreated and drug treated groups.
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3.4. NF-kB expression analysis by Western blot
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MCF-7 cells (1×106) were seeded in 60 mm culture plates and allowed to adhere overnight at
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37ºC. The cells were treated either with brucine or gemcitabine or combinations of both drugs for 48 h.
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The cell lysates were analysed for NF-kB subunit expression by Western blot, using anti-NF-kB (p65)
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antibody following SDS-PAGE separation on an 8% gel. The protein bands were visualised by
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incubation with electro-chemiluminescent (ECL- Bio-Rad) reagent for 1 min and were quantified by
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image-J software (National Institutes of Health, Bethesda, MD, USA) (Lai et al., 2012).
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3.5. Soft agar colony formation assay
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Soft agar colony formation assay was performed to determine the anchorage dependent growth
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of the cells as reported by Keane et al., (1999).
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3.6. In vitro scratch assay
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MCF-7 cells (5×104) were seeded in 60 mm culture plates and the monolayer of 80% confluent
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cells were subjected to serum starvation for 3 h and treated for 48 h either with brucine or gemcitabine
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and their combinations. After 48 h incubation, the drug containing medium was removed, scratch was
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created by sterile p200 tip and washed (2X) with PBS to remove floating cells and 2% FBS media was
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added. Photographic images were taken at 0, 12, 24 and 48 h using inverted phase microscope (Zeiss,
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Axiovert 25, Germany). Cell migration was expressed as the percentage of the gap relative to the total
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area of the cell-free region using Image-J software (National Institutes of Health, Bethesda, MD, USA)
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(Cao et al, 2013)
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3.7. Analysis of drug combination using CompuSyn
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Conceptually, the interaction of two or more agents is described to be ‘synergistic’, when it
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produces a combined effect greater than the sum of their individual effects. However, such a mere
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concept is not good enough to precisely and scientifically evaluate any data of drug combination/s,
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especially, where the combination chemotherapy may have clinical implications. The cell viability effect
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of brucine and gemcitabine combination in MCF-7 cells was scientifically analyzed using CompuSyn
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software (Chou, 2006). This software was specifically developed to analyze drug combination effect
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based on the median effect equation of drug dose and the response and the algorithms developed using
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several aspects viz., law of mass action, biochemical kinetic phenomena such as association, dissociation,
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cooperation of molecules that participate in dynamic reactions. It basically needs the data of dose
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response of the individual drugs, and the different combinations of them. When the data were analyzed
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using this software, it generates a plot of the combination index (CI) against the fraction affected (Fa)
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with reference to the each drug combination tested in the experiment. For each of the specific drug
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combination tested, if the CI is <1, that combination effect is ‘synergistic’, and if CI=1, it is ‘additive’
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and if CI >1, it is ‘antagonistic’.
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3.8. Statistical analysis
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Data were expressed as mean ± SD obtained from three independent experiments. The data were
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processed by analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The level
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of significance was set at p ≤ 0.05.
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Figures:
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Figure S1: Dose and time course of (a) brucine and (b) gemcitabine effect on viability of MCF-7 cells.
Cell viability of untreated control was set as 100% to calculate viabilities of the rest of the treated groups.
Data were expressed as mean ± SD (n=3). *p ≤ 0.001.
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Figure S2: Combination effect of brucine (B) and gemcitabine (G) on viability of MCF-7 cells. Cell
viability of untreated control was set as 100% to calculate viabilities of the rest of the treated groups at 48
h. Data were expressed as mean ± SD (n=3).*p ≤ 0.001.
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Figure S3: Combination effect of brucine (B) and gemcitabine (G) on cell cycle in MCF-7 cells. From
the percentage distribution of cell cycle (Table S2), phase-wise comparative analysis showed an increase
of G1, S phase cells with 17.2x decrease of G2 phase cells compared to the corresponding phase of
untreated control. Data were expressed as mean ± SD (n=3). *p ≤ 0.001.
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Figure S4: Combination effect of brucine (B) and/or gemcitabine (G) on NF-kB subunit (p65)
expression in MCF-7 cells. (a) Western blot and (b) Quantification of NF-kB (p65). Data were expressed
as mean ± SD (n=4). *p≤ 0.001.
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Figure S5: Combination effect of brucine (B) and gemcitabine (G) on colony formation (anchorage
independent growth) of MCF-7 cells in soft agar. (a) Images of colonies that were stained with 0.5%
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crystal violet reagent in culture plates and (b) the percentage of colonies counted in plates was expressed
as mean ± SD (n=3). *p≤ 0.005; **p≤ 0.001.
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Figure S6: Combination effect of brucine (B) and gemcitabine (G) on cell migration was monitored at 0,
12, 24 and 48 h and the cell migration was analyzed as described in ‘Experimental’. Data were expressed
as mean ± SD (n=3).*p≤ 0.001.
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Tables Notes:
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Table S1: CI Values for combination effect on viability of MCF-7 cells
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Note: Combination effects of brucine (B) and gemcitabine (G) in MCF-7 cells with regards to cell
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viability were analysed using CompuSyn software. Combination Index (CI) values were listed for each
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combination of B+G tested in the experiment. If CI value is <1, the combination effect is ‘synergistic’;
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equals to 1, is ‘additive’; or >1, is ‘antagonistic’.
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Table S1. CI Values for combination effect on
viability in MCF-7 cells
B+G
Both drugs at
(µM)
the same time
50+25
136.152
50+50
55.331
50+100
11.090
100+25
6.377
100+50
4.879
100+100
1.798
300+25
0.585
Pretreatment with Pretreatment
Gemcitabine
with Brucine
120.232
12.148
119.690
9.370
82.968
10.074
9.424
3.406
6.700
3.254
1.939
3.085
1.127
2.180
300+50
300+100
0.384
0.166
0.315
0.149
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Table S2: Combination effect on % distribution of cell cycle in MCF-7 cells.
1.560
1.414
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Note: Cell cycle phase distributions of MCF-7 were expressed as mean ±SD (n=3). *p<0.0005.
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Table S2. Combination effect on % distribution of cell cycle in
MCF-7 cells
Group
G1
S
G2
Untreated Control
38.7±0.7
10.3±0.7
51.0±1.2
B 300 µM
60.1±1.8*
23.5±4.1*
16.4±3.2*
G 100 µM
64.0±1.5*
20.9±1.1*
15.1±0.7*
B+G 300+100 µM
81.3±0.8*
15.8±1.4*
3.0±1.4*
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Table S3: CI Values for brucine and gemcitabine combination effects in MCF-7 cells
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Note: Combination effects of brucine (B) and gemcitabine (G) in MCF-7 cells with regards to colony
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formation and cell migration were analysed using CompuSyn software. CI values were listed for each
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combination of B+G tested in the experiment. If CI value is <1, the combination effect is ‘synergistic’;
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equals to 1, is ‘additive’; or >1, is ‘antagonistic’.
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Table S3. CI Values for brucine and gemcitabine
combination effects in MCF-7 cells
Inhibition of
Inhibition of Cell
B+G (µM)
Colony Formation
Migration
50+25
1.147
0.916
50+50
1.038
1.498
100+25
0.494
1.209
100+50
0.620
1.803
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