branched chain fatty acids exert growth inhibiting and apoptosis

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Bioactive Constituents, Metabolites, and Functions

Iso- but not anteiso- branched chain fatty acids exert growth inhibiting and apoptosis-inducing effects in MCF-7 cells Payam Vahmani, Vivien Salazar, David C. Rolland, Katherine E. Gzyl, and Michael E. R Dugan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03549 • Publication Date (Web): 17 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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Journal of Agricultural and Food Chemistry

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Iso- but not anteiso- branched chain fatty acids exert growth inhibiting and apoptosis-

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inducing effects in MCF-7 cells

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Payam Vahmani, †,‡ Vivien Salazar, ‡ David C. Rolland, ‡ Katherine E. Gzyl, ‡ and Michael E.

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R. Dugan‡,*

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† Department of Animal Science, University of California, 2251 Meyer Hall, Davis, CA, 95616,

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‡ Lacombe Research and Development Centre, Agriculture and Agri-Food Canada, Lacombe,

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Alberta T4L 1W1, Canada

USA

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*Corresponding author:

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Michael E. R. Dugan E-mail: [email protected] Tel: 403-782-8125 Fax : 403-782-6120

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ABSTRACT

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The present study compared the growth-inhibitory effects of four common branched chain fatty

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acids (BCFA) found in beef and dairy fats including iso 15:0, anteiso 15:0, iso 17:0, and anteiso

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17:0. MCF-7 human breast cancer cells were exposed for 72 h to media containing increasing

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doses (50 to 400 µM) of the four BCFA. Cell viability was not affected by any of the BCFA

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treatments at doses less than 200 μM. Culturing cells with 200 μM of iso-15:0 or iso-17:0

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reduced cell viability by 27 ± 2.8% and 43 ± 8.3% at 24h, 35 ± 4.6% and 49 ± 9.1% at 48h and,

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44 ± 6.8% and 57 ± 8.8% at 72h post-treatment. In contrast, culturing cells with 200 μM of

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anteiso- 15:0 or anteiso-17:0 did not affect cell viability for any durations tested. The

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incorporation of iso 15:0 and iso 17:0 into cells (19.1 ± 1.3 and 21.2 ± 1.4 μmol /mg protein

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respectively) were greater (P < 0.01) than that of anteiso 15:0 and anteiso 17:0 (11.8 ± 0.7 and

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13.8 ± 0.8 μmol /mg protein respectively). Iso-15:0 and iso-17:0 down-regulated (P < 0.01) the

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expression of anti-apoptotic Bcl-2 (0.71 ± 0.6 and 0.64± 0.09 fold respectively) and up-regulated

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(P < 0.01) the expression of pro-apoptotic Bax (1.72± 0.14 and 2.15± .24 fold respectively)

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compared to the control, whereas their corresponding anteiso isomers did not affect the

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expression of any apoptosis-related genes. Our findings suggest that the branching structure

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influences anticarcinogenic effects of BCFA with iso being more potent than anteiso.

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KEYWORDS: ruminant fat, fatty acids, iso, anteiso, cancer cells

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INTRODUCTION

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Branched chain fatty acids (BCFA) are saturated fatty acids with one or more methyl branches

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on the carbon chain. The methyl branches are predominantly located at the methyl end resulting

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in either a propan-2-yl group (iso) or butan-2-yl group (anteiso) of fatty acid. In humans, BCFA

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are mainly found in vernix caseosa and gastrointestinal tracts of normal healthy newborns,1-2 and

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they have long been known to be a natural bioactive component of colostrum and breast-milk.3-4

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BCFA are also found in meat and milk from ruminants (e.g. cattle, goat and sheep), which are

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derived from the cell membranes of bacteria leaving the rumen.5-7 Fermented soybean products

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such as natto, and ruminant milk and meat are the major source of BCFA in the human diet, with

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dairy and beef being the primary sources of BCFA intake in North America.8-9 In the US, the

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consumption of BCFA from dairy products and beef is estimated to be 487 mg/day, 8 which is

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5.5 times greater than the current consumption of long-chain omega-3 fatty acids including EPA

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and DHA (88 mg/day).10 The BCFA in dairy products and beef range from 14 to 18 carbons,

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with branched 15:0 (iso 15:0, anteiso 15:0) and 17:0 (iso 17:0 and anteiso 17:0) making up about

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78% of total BCFA.5, 9, 11

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Interest in BCFA as bioactive fatty acids originated two decades ago when iso 15:0 purified from

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a soy fermentation products exerted potent anticarcinogenic effects in various cancer cell lines

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(breast cancer MCF7, colon carcinoma HCT116, prostate cancer DU145, leukemia K562, lung

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small cell carcinoma NCI-H1688, pancreatic adenocarcinoma BxPC3, gastric carcinoma NCI-

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SNU-1, and liver carcinoma SNU-423), and in an orthotopic tumor mouse model (i.e. nude mice

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implanted with human prostate tumor xenografts).12 Further studies showed that BCFA have

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anti-tumor effects on lymphomas13 and carcinomas, 14 and improve pancreatic ß-cell function,15



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decrease necrotizing enterocolitis incidence in newborns,16 and reduce inflammatory response in

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intestinal epithelial cells.17

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Studies on the anti-carcinogenic properties of BCFA have been mainly limited to iso 15:0 12-13, 18-

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BCFA with different carbon chain and branching structure. Wongtangtintharn et al.22 reported

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that both iso and anteiso BCFA exerted anti-breast tumor activity, with the growth inhibitory

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effects dependent on the chain length of BCFA. To date, no study has compared the

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anticarcinogenic activities of common BCFA found in ruminant fats (i.e. beef and dairy

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products) including iso 15:0, anteiso 15:0, iso 17:0, and anteiso 17:0. Therefore, the objective of

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the current study was to compare the effects of these four BCFA on the fatty acid composition,

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growth and viability of MCF-7 human breast adenocarcinoma cells. To further validate

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underlying mechanisms associated with the inhibition in cell growth detected for different

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BCFA, we performed apoptosis assays and analyzed the expression key genes involved in the

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regulation (BAX), initiation (p53, AIF and BAD) inhibition (Bcl-2), and execution (Caspase 3)

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of apoptosis. We hypothesized that individual BCFA would have distinct effects on the viability,

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induction of apoptosis and apoptosis related gene expression in MCF-7cells.

and anteiso 15:0. 14, 20-21 There are very limited data comparing growth inhibitory effects of

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MATERIALS AND METHODS

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Chemicals. Pure BCFA (purity ≥ 98%) including iso 15:0, anteiso 15:0, iso 17:0, and anteiso

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17:0 were purchased from Larodan Fine Chemicals (Malmö, Sweden). Cis10-17:1 and GC

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reference standards (GLC-603) were purchased from Nu-Chek Prep. Inc. (Elysian, MN, USA).

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Eagle’ minimum essential medium (EMEM) was purchased from the American Type Culture

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Collection (ATCC). Fetal bovine serum was purchased from Sigma-Aldrich (St. Louis,


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MO,USA). Penicillin-streptomycin was purchased from Life Technologies (Burlington, ON,

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Canada) and insulin-like growth factor-1 was sourced from Corning (Tewkesbury, MA, USA).

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Fatty Acid Treatments and Cell Culture. BCFA were dissolved in ethanol to yield 100 mM

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stock solutions. For all experiments, these stocks were diluted with cell culture medium to

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provide the desired BCFA concentrations (50 to 400 μM). For control cells, the cell culture

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medium was supplemented with 0.25% ethanol only. The human breast adenocarcinoma cell line

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MCF-7 (ATCC_ HTB22TM) was purchased from the American Type Culture Collection

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(ATCC), and maintained in a growth medium containing EMEM supplemented with 10% fetal

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bovine serum, 1% penicillin-streptomycin and 0.01mg/ml insulin-like growth factor-1. Cells

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were seeded in T-75 cm2 flasks (VWR, Radnor, PA, USA) at a density of 5 × 105 cells/flask, and

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cultured under standard conditions at 37 °C and 5% CO2 in a humidified incubator.

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Fatty Acid Analysis. Cell lipids were extracted using hexane:isopropanol (3:1) 23 and then

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methylated using 0.5 M sodium methoxide in methanol (15 minutes at 50°C) with the inclusion

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of cis10-17:1 as an internal standard. Fatty acid methyl esters (FAME) were dissolved in hexane

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(1 mg/mL) and analysed using a a ZB-WAXplus capillary column (30 m, 0.25 mm ID, 0.25 µm

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film thickness) in a Varian 3800 gas chromatograph equipped with a flame ionization detector

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and a Varian 8400-series autosampler (Varian Inc., Walnut Creek, CA, USA). Hydrogen was

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used as the carrier gas (initial pressure 15 psi) and injector and detector temperatures set at

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250°C. For each analysis, 1 μl of sample was injected, and a 20:1 split was used. The initial

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column temperature was 160 oC, held for 1 min, then ramped at 5 oC per min to 250 oC and held

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for 11 min. Individual peaks were identified using reference standards (GLC-603) and peak order

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and retention times reported in the literature 24-25. The FAME were quantified using

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chromatographic peak area and internal standard based calculations. The amount of fatty acids



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per well (μg/well) were converted to μmole/well and subsequently expressed as μmole/mg

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protein.

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Protein Assay. Cells were washed with phosphate-buffered saline and then lysed using NP40

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cell lysis buffer (Invitrogen, CA, USA). Protein contents of cell lysates were determined using a

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Bicinchoninic Acid (BCA) protein assay kit (Sigma-Aldrich) according to the manufacturer’s

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protocol, and used to normalize fatty acid data and expressed as µM fatty acid/mg protein.

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Cell Viability Assay. To measure cell viability, MCF-7 cells were seeded into 96-well plates

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(NunclonTM D surface, Nunc, Denmark) at 2500 cells/well in the culture medium (described

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above) containing 50, 100, 200 or 400 μM of the BCFA treatments. Cell viability was

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determined using RealTime-Glo™ MT Cell Viability Assay (Promega, MI, USA) as per

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manufacturer's instruction. Luminescence signal (i.e. the indicator of cell viability) was measured

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over 96 hours using a Spectramax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA).

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The viability results for BCFA treatments were expressed relative to ethanol control.

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Apoptosis and Necrosis Assays. Apoptosis and necrosis assays were performed to determine

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the potential mechanism of cell death following treatment of cells with BCFA. Cells were plated

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at a density of 5000 cells/well in 96-well plates in 50 μL culture medium and allowed to attach

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for 4 h before 200 μL of medium containing assay reagents plus BCFA treatments (50, 100, 200

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and 400 μM) were added. Apoptosis and necrosis were measured using RealTime-Glo Annexin

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V Apoptosis and Necrosis Assay kits (Promega) according to manufacturer’s instructions.

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Luminescence (indicative of apoptosis) and fluorescence (indicative of necrosis) signals were

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measured at 0, 12, 24, 48, 72 and 96 h following treatment using a Spectramax M5 plate reader

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(Molecular Devices).


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RNA Extraction, Reverse Transcription and Quantitative PCR. Total cellular RNA was

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extracted from MCF-7 cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the

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manufacturer’s instructions. The RNA concentrations were determined using a Nanodrop ND-

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1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE), and the RNA purity was

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evaluated using the 260:280 and 260:230 absorbance ratios. All the samples had both ratios

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between 1.8 and 2.1. Integrity of RNA was confirmed using an Agilent Bioanalyzer 2100

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(Agilent Technologies, Palo Alto, CA) with an Agilent RNA 600 nano kit. The RNA integrity

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number of the samples ranged between 7.7 and 9.1. cDNA was synthesized from 1 μg of RNA

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using Maxima cDNA Synthesis kit (Thermo Scientific, Carlsbad, CA, USA) in the presence of

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both random hexamers and oligo(dT) primer in a total reaction volume of 20 μl. Real-time PCR

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was performed in duplicate in 96-well plates, and each reaction contained 6 μl cDNA diluted at

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1:50, 0.4 μl of each forward and reverse primes (10 mM), 8 μl Green-2-Go qPCR Mastermix

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(BioBasic, Markham, ON, Canada), and 5.2 μl of nuclease-free water. Real-time PCR was done

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using a Stratagene Mx3005P QPCR system (Agilent Technologies) using the following protocol:

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enzyme activation 95oC for 10 min, initial denaturation at 95oC for 15 s, and annealing/extension

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at 60oC for 60 s, repeated for 40 cycles. This was followed by a melt curve analysis as per the

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manufacturer’s settings to ensure specific amplification.The amplification efficiency for each

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primer pair was calculated from the slope of the standard curve generated with serial dilutions of

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a pooled cDNA sample using the formula (E = 10(−1/slope)). The amplification efficiencies were

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between 90 and 105% for all primer pairs used in this study. Primer sequences for the internal

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control gene (β-actin) and target genes involved in the apoptosis induction (apoptosis inducing

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factor [AIF], caspase 3 [Cas3], Bax, Bad, Bcl2 and p53) were taken from Vahmani et al.26 and

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Wongtangtintharn et al.18 respectively. Relative mRNA expression of target genes was



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calculated using the ΔCt method with β-actin as the internal control gene. Each plate was set up

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to include reactions for both target gene and the internal control gene for each cDNA sample.

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Target gene cycle threshold (Ct) values were normalized to that of β-actin using 2 –ΔCt where ΔCt

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= Ct target gene Ct β-actin27. Statistical analysis was performed on 2 –ΔCt data and the results

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were expressed as fold change relative to control.

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Statistical Analysis. Data were analyzed using the mixed models procedure of SAS (v 9.3; SAS

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Institute, Cary, IN) with treatment and dose as main effects and time as a repeated measure when

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applicable (i.e. for viability, apoptosis and necrosis data). The experimental unit was the

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individual well from five independent cell culture plates (n=5/treatment). Prior to analysis, data

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were checked for normality using the Anderson–Darling test and all data were normally

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distributed. Differences between means were considered to be significant at P 0.05).

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DISCUSSION

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BCFA have been shown to exert anti-cancer properties in several cancer cell lines including

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breast, prostate, leukemia, hepatocellular carcinoma, and bladder cancer.12-13, 18-19, 28 In the

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present study, we examined the effects of four common BCFA found in ruminant fats (iso 15:0,

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anteiso 15:0, iso 17:0, anteiso 17:0) on the fatty acid composition, growth of MCF-7 cells and

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induction of apoptosis and expression of key apoptosis regulatory genes. A number of studies

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have reported on apoptosis of cancer lines treated with BCFA, specifically iso 15:0 (isolated

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from soy fermentation products), but there is no report available in the literature comparing the

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effects of ruminant-derived BCFA with different carbon chain and branching structure (i.e. iso

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vs. anteiso) on cancer cells. Our results demonstrated that iso 15:0 and iso 17:0 could effectively

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inhibit MCF-7 cell growth and induce apoptosis in a dose and time dependant manner. In

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contrast, anteiso 15:0 and 17:0 did not induce apoptosis in MCF-7 cells, and only slightly

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reduced cell growth after 72 h treatment when applied at the highest dose (400 µM). Our results

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for the first time indicate that the branching structure influences growth inhibiting effects of

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BCFA on MCF-7 cells with iso being more potent than anteiso.

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The absence of BCFA in the control cells confirms that all BCFA in treated cells originated

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through supplementation with BCFA. The incorporation of BCFA into cellular lipids was mainly

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at the expense of saturated and monounsaturated fatty acids. The greater incorporation (1.5-1.6

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fold) of iso than anteiso BCFA into cells in the present study was consistent with Wang et al. 20


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who showed that the uptake of iso 18:0 by MC7 cells was 1.7 fold greater than that of anteiso

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15:0. The reason for the greater incorporation of iso than anteiso BCFA into MC7 cells is not

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clear, but might be in part related to potential differences in their cellular uptake and rate of

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metabolism.

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The increased growth inhibitory effects of iso 15:0 and iso 17:0 were consistent with their

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greater incorporation into MCF-7 cells compared to their corresponding anteiso isomers. Thus it

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is plausible that greater iso BCFA incorporation into cellular lipids modulate membrane fluidity

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(e.g. through reduced membrane PUFA contents) leading to cell dysfunction and consequently

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reduced cell viability.18, 22

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Both iso 15:0 and iso 17:0 reduced cell viability at 200 µM starting at 24 h (Figure 1) which is

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consistent with previous studies where iso 15:0 was used to treat breast, leukemia, and bladder

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cancer cell lines.12-13, 18-19 However, neither anteiso 15:0 nor anteiso 17:0 led to significant

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growth inhibition in MCF-7 cells. In contrast to our findings, Wongtangtintharn et al.22 found

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that the cytotoxicity of anteiso 15:0 was comparable to that of iso 15:0 in breast cancer cells.

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The growth inhibiting effects of iso 15:0 have been mainly attributed to induction apoptosis in

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cancer cells.12-13, 18-19 Similarly, we observed a time-dependent increase in apoptosis related

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luminescence starting at 12 h in cells treated with both iso 15:0 and iso 17:0 (Figure 2). The

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increased luminescence is related to luciferase tagged annexin V binding to phosphatidylserine

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residues in the cell membrane, which is considered an early marker of apoptosis.29 The increased

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luminescence (i.e. apoptosis) in cells treated with iso 15:0 or iso 17:0 was followed by a delayed

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increase in fluorescence starting at 60h (Figure 2). The increased fluorescence is due to entry of a

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profluorescent DNA dye into the cells upon loss of cell membrane integrity, which is indicative

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of necrosis. The delay between apoptosis and necrosis is a marker of post-apoptotic necrosis



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(also called secondary necrosis), which can lead to cancer cell death.30 In the present study, we

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observed the strongest apoptotic and necrotic effects in cells treated with iso 15:0 or iso 17:0,

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which was inversely related to cell viability results. Therefore, the reduced viability in cells

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treated with iso 15:0 or iso 17:0 was likely caused by post-apoptotic necrosis.

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To explore the molecular mechanism of apoptosis induced by BCFA, we studied the mRNA

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expression of six key proteins involved in apoptosis including AIF, Cas3, Bax, Bad, Bcl2 and

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p53. Among these, only Bax and Bcl2 expression were affected by BCFA (P < 0.01; Figure 3).

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Both Bcl2 and Bax proteins are involved in mitochondrial-mediated apoptosis (MMA) which is

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triggered by mitochondrial outer membrane permeabilization (MOMP) resulting in release of

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cytochrome c (cyt c) into the cytosol where it activates caspases leading to apoptosis and

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subsequent cell death.31 During MMA, the pro-apoptotic Bax mediates the release of cyt c from

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mitochondria by forming large pores in the outer mitochondrial membrane.32 In opposition, the

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anti-apoptotic Bcl-2 inhibits the action of Bax and thereby prevents release of cyt c from

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mitochondria into cytoplasm which is the key step in MMA.33 The balance between Bcl-2 and

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Bax determines the start of MMA, with an increased ratio of Bax/Bcl-2 triggering the apoptosis

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process.34 In the present study, both iso 15:0 and iso 17:0 upregulated Bax expression and

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downregulated Bcl2 expression, which in turn could trigger the mitochondrial apoptotic pathway

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(release of cyt c and caspase activation) and subsequent death of MCF-7 cells. Our results are

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consistent with Lin et al.19 who showed that iso 15:0 induced apoptosis in bladder cancer cells

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through the MMA rather than the death receptor induced pathway (i.e. the other major apoptosis

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pathway).

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Our results showed that culturing with iso 15:0 or iso 17:0 induced apoptosis in MCF-7 cells

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leading to notable reduction in cell viability. However, neither anteiso 15:0 nor anteiso 17:0 led


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to significant growth inhibition in MCF-7 cells. In contrast to our findings, Wongtangtintharn et

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al.22 found that the cytotoxicity of anteiso 15:0 was comparable to that of iso 15:0 in breast

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cancer cells.

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Our findings suggest that the anti-carcinogenic effects of BCFA depend on the branching

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structure with the iso exerting stronger growth-inhibiting effects than anteiso. Additional studies

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are required to further investigate the isomer specific effect of BCFA in other cancer cells and in

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in vivo models, including investigating the interaction of iso and anteiso BCFA with different

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regulatory proteins and transcription factors involved in apoptosis and cell death. These studies

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will eventually help guide production practices to limit or increase specific BCFA in ruminant

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meat and milk, as well as fermented soybean foods for optimal human health outcomes.

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ACKNOWLEDGEMENT

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Study funding was provided through Agriculture and Agri-food Canada Peer-Review program.

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The authors thank Ms. Katelyn Le for her assistance in cell culture assays.

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REFERENCES

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(20) Wang, Z.; Wang, D. H.; Park, H. G.; Yan, Y.; Goykhman, Y.; Lawrence, P.; Kothapalli, K. S. D.; Brenna, J. T. Identification of genes mediating branched chain fatty acid elongation. FEBS Lett. 2019, doi: 10.1002/1873-3468.13451. (21) Yang, P.; Collin, P.; Madden, T.; Chan, D.; Sweeney-Gotsch, B.; McConkey, D.; Newman, R. A. Inhibition of proliferation of PC3 cells by the branched-chain fatty acid, 12methyltetradecanoic acid, is associated with inhibition of 5-lipoxygenase. Prostate. 2003, 55 (4), 281-291. (22) Wongtangtintharn, S.; Oku, H.; Iwasaki, H.; Toda, T. Effect of branched-chain fatty acids on fatty acid biosynthesis of human breast cancer cells. J. Nutr. Sci. Vitaminol (Tokyo). 2004, 50 (2), 137-43. (23) Hörl, G.; Wagner, A.; Cole, L. K.; Malli, R.; Reicher, H.; Kotzbeck, P.; Köfeler, H.; Höfler, G.; Frank, S.; Bogner-Strauss, J. G.; Sattler, W.; Vance, D. E.; Steyrer, E. Sequential synthesis and methylation of phosphatidylethanolamine promote lipid droplet biosynthesis and stability in tissue culture and in vivo. J. Biol. chem. 2011, 286 (19), 17338-17350. (24) Alves, S. P.; Bessa, R. J. The trans-10,cis-15 18:2: a missing intermediate of trans-10 shifted rumen biohydrogenation pathway? Lipids 2014, 49 (6), 527-41. (25) Kramer, J. K.; Hernandez, M.; Cruz-Hernandez, C.; Kraft, J.; Dugan, M. E. Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation. Lipids 2008, 43 (3), 259-73. (26) Vahmani, P.; Meadus, W. J.; da Silva, M. L. P.; Mitchell, A. D.; Mapiye, C.; Duff, P.; Rolland, D. C.; Dugan, M. E. R. A trans10-18:1 enriched fraction from beef fed a barley grainbased diet induces lipogenic gene expression and reduces viability of HepG2 cells. Biochem. Biophys. Rep 2016, 7, 84-90. (27) Schmittgen, T. D.; Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protocols 2008, 3 (6), 1101-1108. (28) Mobley, J. A.; Leav, I.; Zielie, P.; Wotkowitz, C.; Evans, J.; Lam, Y. W.; L'Esperance, B. S.; Jiang, Z.; Ho, S. M. Branched fatty acids in dairy and beef products markedly enhance alphamethylacyl-CoA racemase expression in prostate cancer cells in vitro. Cancer. Epidemiol. Biomarkers Prev. 2003, 12 (8), 775-83. (29) Fadok, V. A.; Voelker, D. R.; Campbell, P. A.; Cohen, J. J.; Bratton, D. L.; Henson, P. M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 1992, 148 (7), 2207-16. (30) Andreau, K.; Perfettini, J. L.; Castedo, M.; Metivier, D.; Scott, V.; Pierron, G.; Kroemer, G. Contagious apoptosis facilitated by the HIV-1 envelope: fusion-induced cell-to-cell transmission of a lethal signal. J. Cell Sci. 2004, 117 (Pt 23), 5643-53. (31) Xiong, S.; Mu, T.; Wang, G.; Jiang, X. Mitochondria-mediated apoptosis in mammals. Protein & cell. 2014, 5 (10), 737-749. (32) Korsmeyer, S. J.; Wei, M. C.; Saito, M.; Weiler, S.; Oh, K. J.; Schlesinger, P. H. Proapoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 2000, 7 (12), 1166-73. (33) Thomenius, M. J.; Distelhorst, C. W. Bcl-2 on the endoplasmic reticulum: protecting the mitochondria from a distance. J. Cell. Sci. 2003, 116 (22), 4493-4499. (34) Ghoneum, M.; Matsuura, M.; Braga, M.; Gollapudi, S. S. cerevisiae induces apoptosis in human metastatic breast cancer cells by altering intracellular Ca2+ and the ratio of Bax and Bcl2. Int. J. Oncol. 2008, 33 (3), 533-9. 15 ACS Paragon Plus Environment


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Table 1. Major fatty acid (FA) composition (μmol FA/mg protein) of MCF7 cells treated with 200 µM of different branched chain FAs for 24h. FA CTR1 iso 15:0 iso 17:0 anteiso 15:0 anteiso 17:0 SEM2 14:0 1.02a 0.71a 0.65a 1.04a 0.86a 0.20 iso 15:0 19.10 0.68 anteiso 15:0 11.84 0.73 a b b ac c 16:0 9.09 5.91 5.82 7.13 6.98b 1.03 a b b ac c cis9-16:1 5.89 2.49 2.76 5.04 4.71 0.66 iso 17:0 21.24 0.93 anteiso 17:0 13.75 0.71 18:0 4.32a 2.68b 2.18b 3.44c 3.02c 0.18 a b b c c cis9-18:1 17.59 11.72 11.96 13.98 14.02 0.64 a b b c c cis11-18:1 6.08 3.01 2.91 5.07 4.08 0.30 a b c a a 18:2n-6 0.94 0.68 0.78 0.99 0.99 0.03 20:4n-6 1.72a 1.49b 1.35b 1.69a 1.79a 0.10 22:6n-3

0.91a 0.81b 0.80b 0.99a 0.98a 0.03 1 Control cells (CTR) were cultured with 0.25% ethanol (medium control). 2Standard error of the mean. nd not detected. a-c Means within a row not sharing common letters are significantly different (P < 0.05). 393


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Figure 1. Effects of different branched chain fatty acids on viability of MCF-7 cells at 24 h (A), 48 h (B) and 72 h (C) determined by RealTime-Glo™ MT Cell Viability Assay (Promega, MI,. USA). MCF-7 cells were supplemented without fatty acids 0.25% ethanol (medium control) or with increasing doses (50 to 400 µM) of iso 15:0, anteiso 15:0, iso 17:0 and anteiso 17:0. Values are means ± SD, n = 5. Significantly different from medium control: * P < 0.05, ** P < 0.01, *** P < 0.001.



Figure 2. Apoptosis and necrosis following treatment of MCF-7 cells with 0.25% ethanol (medium control) or with 200 µM of different branched chain fatty acids (iso 15:0, anteiso 15:0, iso 17:0 and anteiso 17:0). Apoptosis and necrosis were measured using RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay kits (Promega Inc. WI, USA). Increased Luminescence (RLU) demonstrates Annexin V binding to phosphatidylserine residues in the cell membrane as an early marker of apoptosis. Increased fluorescence (RFU) demonstrates entering of the profluorescent DNA dye into the cells upon loss of membrane integrity, as a marker of secondary necrosis. The time delay between RLU and RFU peaks is indicative of an apoptotic phenotype leading to secondary necrosis. Values are means ± SD, n = 5. Significantly different from medium control: * P < 0.05, ** P < 0.01, *** P < 0.001.


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Figure 3. Effect of culturing MCF-7 cells for 24 hr with 200 µM of different branched chain fatty acids (iso 15:0, anteiso 15:0, iso 17:0 and anteiso 17:0) on relative mRNA expression of key regulatory proteins involved in the apoptosis induction including apoptosis inducing factor [AIF], caspase 3 (Cas3), Bax, Bad, Bcl2 and p53. Values (mean ± SE; n = 5/treatment) are expressed as fold changes relative to the ethanol control. Within each gene, means without common letters are significantly different (P

branched chain fatty acids exert growth inhibiting and apoptosis

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