Omega-3 DHA- and EPA–dopamine conjugates induce PPARγ-dependent breast cancer cell death through autophagy and apoptosis

    Omega-3 DHA- and EPA-Dopamine Conjugates induce PPARγ-dependent breast cancer cell death through autophagy and apoptosis Daniela Rovito, Cinzia Giordano, Pierluigi Plastina, Ines Barone, Francesca De Amicis, Loredana Mauro, Pietro Rizza, Marilena Lanzino, Stefania Catalano, Daniela Bonofiglio, Sebastiano And`o PII: DOI: Reference:

S0304-4165(15)00213-5 doi: 10.1016/j.bbagen.2015.08.004 BBAGEN 28255

To appear in:

BBA - General Subjects

Received date: Revised date: Accepted date:

3 March 2015 30 July 2015 9 August 2015

Please cite this article as: Daniela Rovito, Cinzia Giordano, Pierluigi Plastina, Ines Barone, Francesca De Amicis, Loredana Mauro, Pietro Rizza, Marilena Lanzino, Stefania Catalano, Daniela Bonofiglio, Sebastiano And` o, Omega-3 DHA- and EPA-Dopamine Conjugates induce PPARγ-dependent breast cancer cell death through autophagy and apoptosis, BBA - General Subjects (2015), doi: 10.1016/j.bbagen.2015.08.004

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Omega-3 DHA- and EPA-Dopamine Conjugates induce PPAR-dependent breast cancer cell death through autophagy and apoptosis

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*Daniela Rovito1, *Cinzia Giordano2, Pierluigi Plastina1, Ines Barone1,2, Francesca De Amicis1,2, Loredana Mauro1, Pietro Rizza1, Marilena Lanzino1,2, Stefania Catalano1,2, #Daniela Bonofiglio1,2, #Sebastiano Andò1,2 1

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende (CS), Italy Centro Sanitario, University of Calabria, Arcavacata di Rende (CS), Italy

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*Contributed equally to this work.

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#Joint Senior Authors

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Correspondence and requests should be addressed to: Sebastiano Andò: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende (CS), Italy. Phone: +39 0984 496201. Fax: 39 0984 492929. e-mail: [email protected].

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Daniela Bonofiglio: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende (CS), Italy. Phone: +39 0984 496208. Fax: 39 0984 496203. e-mail: [email protected].

Running title: DHADA and EPADA induce breast cancer cell death via PPAR

This work was supported by MURST and Ex 60%, Associazione Italiana Ricerca sul Cancro (AIRC) grant IG 11595, Lilli Funaro Foundation. Key words: Apoptosis, Autophagy, Beclin-1, Breast cancer, Omega-3 polyunsaturated fatty acid conjugates, Peroxisome Proliferator-Activated Receptor gamma.

ACCEPTED MANUSCRIPT Abstract Background: The omega-3 Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) may

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form conjugates with amines that have potential health benefits against common diseases including

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cancers. Here we synthesized DHA-dopamine (DHADA) and EPA-dopamine (EPADA) conjugates

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and studied their biological effects on different breast cancer cell lines.

Methods and Results: MTT assays indicated that increasing concentrations of DHADA and

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EPADA significantly affected viability in MCF-7, SKBR3 and MDA-MB-231 breast cancer cells, whereas no effect was observed in MCF-10A non-tumorigenic epithelial breast cells. DHADA and

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EPADA enhanced Beclin-1 expression, as evidenced by immunoblotting, real-time-PCR and functional analyses. Chromatin Immunoprecipitation (ChIP) and Re-ChIP assays revealed that both

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compounds induced recruitment of Peroxisome-Proliferator-Activated-Receptor gamma (PPAR)

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and RNA Polymerase-II at the Retinoic-X-Receptor binding region on Beclin-1 promoter.

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Moreover, both compounds enhanced autophagosome formation, evaluated by LC-3 and monodansylcadaverine labelling, that was prevented by the PPAR antagonist GW9662, addressing the direct involvement of PPAR. Noteworthy, long-term treatment with DHADA and EPADA

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caused the blockade of autophagic flux followed by apoptotic cell death as evidenced by PARP cleavage and DNA fragmentation in all breast cancer cells. Conclusions: We have provided new insights into the molecular mechanism through which PPAR, as a central molecule in the cross talk between autophagy and apoptosis, mediates DHADA- and EPADA-induced cell death in breast cancer cells. General significance: Our findings suggest that omega-3 DHADA- and EPADA activation of PPAR may assume biological relevance in setting novel adjuvant therapeutic interventions in breast carcinoma.

ACCEPTED MANUSCRIPT Key words: Apoptosis, Autophagy, Beclin-1, Breast cancer, Omega-3 polyunsaturated fatty acid

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conjugates, Peroxisome Proliferator-Activated Receptor gamma.

ACCEPTED MANUSCRIPT 1. Introduction Dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) are known to be beneficial in the

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treatment of several types of disease, including cancers of mammary, lung and colonic origin by

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increasing tumor cell susceptibility to apoptosis and thereby enhancing chemotherapy efficacy [1-

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6]. The two main n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), naturally present in cold-water fish as well as in fish oil supplements, are the most promising in inhibiting carcinogenesis and reducing risk for breast cancer [7-12]. Antineoplastic activities of n-3

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PUFAs include alteration of membrane fluidity and cell surface receptor function, modulation of

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COX activity and increased cellular oxidative stress [13]. Besides, the biological effects of DHA and EPA can also be exerted by their derivative molecules widely present in nature and endogenously metabolized in a tissue-specific manner [14,15]. In the last years N-acyl amines of

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DHA and EPA and other n-3 PUFAs-derived conjugates with amino acids and neurotransmitters have attracted much attention because of their potential roles in pathophysiological conditions

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suggesting that they could represent a new additional class of endogenous signaling molecules [16]. Chemically, these compounds are amphiphilic molecules, characterized by a head group that may

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be charged or neutral and a hydrocarbon tail, that allows them to penetrate into cell membranes, potentially affecting their functional properties [17]. Moreover, it has been demonstrated that some of these molecules show affinity for different receptors, including cannabinoid receptors (CB1 and CB2), several G protein-coupled receptors (GPRs), transient receptor potential channel type V1 (TRVP1) and Peroxisome Proliferator-Activated Receptors (PPARs) [17 and references therein]. Particularly, it has been reported that the n-3 acyl-ethanolamines, DHEA and EPEA, show antiinflammatory or general immune modulating properties [18,19] and possess anti-tumoral activities in prostate cancer cells [20]. We have recently found that DHEA and EPEA induce autophagy through PPAR activation in human breast cancer cells [21], highlighting the importance of these compounds from a pharmacological perspective. Apart from DHEA and EPEA, other conjugates of n-3 PUFAs with serotonin, L-alanine, L-serine, histidine, GABA, glutamic acid or dopamine have

ACCEPTED MANUSCRIPT been found in mammals [16 and references therein]. However, relatively little is known about their biological significance or pharmacological potential. A relatively small number of long-chain N-

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fatty acyl-dopamines (e.g. N-palmitoyl-, N-stearoyl-, N-oleoyl- and N-arachidonoyl-dopamine)

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have been isolated and characterized from the mammalian brain, with the highest concentrations in

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the striatum, hippocampus and cerebellum, attracting a special interest for their activity in nervous system [23]. The biological activity of these compounds could be regulated by the interaction with different enzymes involved in their metabolism. Indeed, the half-life of all these endogenous fatty

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acids amides is very short due to rapid deactivation by fatty acid amide hydrolase (FAAH) [24,25].

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For instance, N-acyl dopamines were first described as potent inhibitors of 5-lipoxygenase [26], while N-arachidonoyl dopamine was synthesized as an inhibitor of FAAH [27]. Synthesis and biological evaluation of some of N-acyl-dopamines including DHA-Dopamine (DA) and EPA-DA

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have been firstly described by Bezuglov et al. [28]. The authors reported that both compounds inhibited locomotion and induced catalepsy, hypothermia and analgesia in rats, demonstrating their

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ability to produce cannabinoid-like effects in vivo via CB1 [28]. Moreover, it has been reported that some N-acyl-dopamines are able to exert positive effects on hypoxic-ischaemic injury or brain

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inflammatory processes and to inhibit cancer cell proliferation [29,30], suggesting N-acyldopamines as potential antiinflammatory and antitumor leads. In the present study, we have synthesized omega-3 DHADA and EPADA conjugates and tested their biological effects on normal and different human breast cancer cells. We have demonstrated that both compounds inhibit growth and induce autophagy and subsequent apoptosis by activating PPAR in all breast cancer cells.

ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Reagents

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BRL49653 (BRL) was from Alexis (San Diego, CA), GW9662 (GW), DA, DHA, EPA and

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Hydroxychloroquine sulfate (HCQ) from Sigma–Aldrich (Milan, Italy); UVI3003 (UVI) from

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Tocris Bioscience (Bristol, United Kingdom). 2.2. Synthesis of DHADA and EPADA

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Synthesis of DHADA and EPADA was attained by modifying a previously reported lipase-

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catalyzed N-acylation method [31]. Briefly, dopamine hydrochloride, triethylamine and the appropriate fatty acid (molar ratio 1:1.5:1) were incubated at 50 °C in 2-methyl-2-butanol as solvent for 48 hours, using immobilised Candida antarctica Lipase B as the catalyst. After evaporating the

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solvent under reduced pressure, the products were purified by column chromatography on silica gel. Authenticity of the products was verified by ESI-MS and NMR.

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2.3. Plasmids

The human Beclin-1 gene promoter (p-644, p-277 and p-58) and the PPRE sequence plasmids were

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a gift from Prof. M. Zhao (Institute of Biochem & Cell Biology, SIBS, Shanghai, China) and Dr. R. Evans (The Salk Institute, San Diego, CA, USA), respectively. 2.4. Cell culture MCF-7 human breast cancer cells were cultured in DMEM/F-12 medium plus glutamax supplemented with 5% newborn calf serum (Invitrogen, Carlsbad, CA, USA), and 1mg/ml penicillin–streptomycin at 37 C with 5% CO2 air. SKBR3 human breast cancer cells were grown in phenol red free RPMI 1640, containing 10% fetal bovine serum (FBS), the triple-negative MDAMB-231 human breast cancer cells were cultured in DMEM/F-12 plus glutamax containing 10% FBS and 1mg/ml penicillin–streptomycin. MCF-10A non tumorigenic breast epithelial cells were

ACCEPTED MANUSCRIPT grown in DMEM-F12 plus glutamax containing 5% horse serum (Invitrogen), 1mg/ml penicillin– streptomycin, 0.5mg/ml hydrocortisone, and 10mg/ml insulin. For experimental purposes, cells

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were grown in phenol red-free media containing 5% charcoal-treated FBS (CT-FBS) for 24h and

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then treated as described.

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2.5. Cell viability assay

Cell viability was determined with the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium (MTT)

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assay. Cells (30,000 cells/well) were grown in 24-well plates and exposed to treatments as indicated. MTT (2mg/ml, Sigma) were added to each well, and the plates were incubated for 2h at

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37°C followed by medium removal and solubilization in 500µl DMSO. The absorbance was measured at 570nm. The IC50 values were calculated using GraphPad Prism 4 (GraphPad Software,

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2.6. DNA Flow Cytometry

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Inc., San Diego, CA, USA) as described [32].

To determine cell cycle distribution analysis, cells were harvested by trypsinization, fixed and stained with Propidium iodide (100 g/ml) after treatment with RNase A (20 g/ml). The DNA

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content was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and the data acquired using CellQuest software. Cell cycle profiles were determined using ModFit LT. 2.7. Anchorage-independent soft agar growth assays Soft-agar anchorage-independent growth assay was assessed as described [32]. 2.8. Immunoblot analysis Cells were treated as indicated before lysis for total protein extraction [38]. Equal amounts of cell extract proteins were resolved on 8-11% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-PPAR, -Beclin-1, -LC3, -PARP 1/2, -Bid, -Cyt c, -caspase-9, -

ACCEPTED MANUSCRIPT GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), - Beclin-1 (BD Biosciences, San Jose, CA, USA) and -p62/SQSTM1 (Cell Signalling Technology, Danvers, MA, USA) antibodies.

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The antigen–antibody complex was detected as previously described [38].

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2.9. RT-PCR/real-time PCR

Analysis of PPAR and Beclin-1 gene expression was performed using real-time RT-PCR. Two micrograms of total RNA were reverse transcribed with the RETROscript Kit (Applied Biosystems,

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Monza, Italy); cDNA was diluted 1:3 in nuclease-free water and 5µl were analyzed in triplicates by real-time-PCR in a iCycler iQ Detection System (Bio-Rad, Milan, Italy) as previously described

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[21]. Negative control contained water instead of first strand cDNA was used. Each sample was normalized on its GAPDH mRNA content. For PPAR, Beclin-1 and GAPDH the primers were: 5'-

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GGCTTCATGACAAGGGAGTTTC-3' (PPARforward) and 5'-

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AACTCAAACTTGGGCTCCATAAAG-3' (PPARreverse), 5’-

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ACAGTGGACAGTTTGGCACA-3’ (Beclin-1-forward), 5’-CGGCAGCTCGTTAGATTTGT-3’ (Beclin-1-reverse) 5'-CCCACTCCTCCACCTTTGAC-3' (GAPDH-forward), 5'-

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TGTTGCTGTAGCCAAATTCGTT-3' (GAPDH-reverse). 2.10. Transient transfection assay Cells were transiently transfected using X-TREME (Roche, Indianapolis, IN, USA) reagent with 3XPPRE-TK reporter gene (PPRE-Luciferase). Another set of experiments was performed trasfecting BECN1 promoter-luciferase constructs. After transfection, cells were treated as described. Luciferase activity was assayed as described [21]. 2.11. Immunofluorescence Cells were fixed with 4% paraformaldehyde, permeabilized with PBS 0.2% Triton X-100 followed by blocking with 5% bovine serum albumin, and incubated with anti-Beclin-1, anti-LC3 (Santa

ACCEPTED MANUSCRIPT Cruz), primary antibodies and with fluorescein isothiocyanate-conjugated secondary antibodies. IgG primary antibody was used as negative control. 4’,6-Diamidino-2-phenylindole (DAPI; Sigma)

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staining was used for nuclei detection. Fluorescence was photographed with OLYMPUS BX51

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microscope, 100X objective.

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2.12. DNA fragmentation

Cells were collected and washed with PBS and pelleted at 1,800 rpm for 5 min. The samples were

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resuspended in 0.5 ml of extraction buffer (50mmol/L Tris–HCl, pH 8; 10mmol/L EDTA, 0.5% SDS) for 20 min in rotation at 4°C. DNA was extracted three times with phenol–chloroform and

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one time with chloroform. The aqueous phase was used to precipitate nucleic acids with 0.1 volumes of 3M sodium acetate and 2.5 volumes cold ethanol overnight at 20°C. The DNA pellet

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was resuspended in 15µl of H2O treated with RNase A for 30 min at 37°C. The extracted DNA was

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subjected to electrophoresis on 2% agarose gels, stained with ethidium bromide and then

2.13. TUNEL assay

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photographed.

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Apoptosis was determined by enzymatic labelling of DNA strand breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). TUNEL labelling was conducted using APO-BrdUTM TUNEL Assay Kit (Invitrogen) and performed according to the manufacturer’s instructions as described [38]. 2.14. Monodansylcadaverine staining Monodansylcadaverine (MDC; Sigma–Aldrich) was used to visualize autophagic vacuoles [33]. Cells were stained using 0.05mM MDC in PBS at 37°C for 10 min. After incubation, cells were washed with PBS and analyzed by fluorescence microscopy. 2.15. Chromatin Immunoprecipitation Assay

ACCEPTED MANUSCRIPT Cells were treated with DHADA and EPADA for 1h and then DNA/protein complexes were extracted as described [39]. The precleared chromatin was immunoprecipitated with specific anti-

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PPAR, anti-RXR, and anti-Polymerase II antibodies. The anti-PPAR and anti-RXR

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immunoprecipitated samples were re-immunoprecipitated (Re-ChIP) with an anti-RXR and anti-

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PPAR antibodies, respectively. A 5l of each sample and input were used for real-time-PCR. The primers flanking the RXR sequence present in the Beclin-1 promoter region were the following:

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5’- GGG ATT TAA CCA TTT TGG CCA GGC-3’ and 5’- GCA ACA ACA AAA GGC CGG GC3’. Final results were calculated using the Ct method using input Ct values instead of the

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GAPDH. The basal sample was used as calibrator.

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2.16. BECN1 knock-down

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Cells were transiently transfected with short hairpin (sh) RNA control or sh-BECN1 vectors (Santa Cruz Biotechnology) using X-TREME reagent. Twenty-four hours after transfection, cells were

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harvested and used for different experiments.

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2.17. Statistical analysis

Each datum point represents the mean±SD of three different experiments. Data were analyzed by Student’s t-test using the GraphPad Prism4 software program. p<0.05was considered as statistically significant.

ACCEPTED MANUSCRIPT 3. Results 3.1. Omega-3 DHA- and EPA-Dopamine conjugates inhibit breast cancer cell growth

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Omega-3 EPA- and DHA-Dopamine (DA) conjugates were synthesized in our laboratory by a

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lipase-catalyzed N-acylation method [31] (structures of DHADA and EPADA are indicated in Fig. 1A and B). First, we examined the effects of DHADA and EPADA on proliferation of ER/PRpositive MCF-7, ER/PR double negative and HER2 overexpressing SKBR3 and triple-negative

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MDA-MB-231 human breast cancer cells. Using MTT assays, we observed that both compounds significantly reduced cell viability in a dose- and time-dependent manner in all cell lines tested (Fig.

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1C, D and E). It is worth noting that DHADA and EPADA exerted their inhibitory effects at lower doses in MDA-MB-231 cells as evidenced by the half-maximal inhibitory concentration (IC50)

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values reported in Table 1. According to IC50 values, for all experiments we used both molecules at

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25M in MCF-7 and SKBR3 cells, while at 10M in MDA-MB-231 cells. Interestingly, the highest

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concentrations of both conjugates did not elicit any inhibitory effects in non-tumorigenic breast epithelial cells, MCF-10A (Supplementary Fig. 1A). Consistently with MTT assay results, DHADA and EPADA treatment significantly reduced colony formation, evaluated by anchorage-independent

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soft agar growth assays, in breast cancer cells (Supplementary Fig. 1B). The specificity of biological activities exerted by DHADA and EPADA is highlighted by the observation that dopamine (DA) itself did not exert any effect on cell proliferation and did not change the efficacy of EPA and DHA treatment on MCF-7 cell growth (Supplementary Fig. 1C). Moreover, to investigate the effects of DHADA and EPADA on cell cycle progression, Flow Cytometric analysis was performed in all breast cancer cell lines. Twenty-four hour treatment caused a cell cycle arrest concomitant with a reduced fraction of cells in S-phase compared with untreated cells (Table 2). Taken together, our results indicate the ability of DHADA and EPADA to induce growth inhibition and cell cycle arrest in breast cancer cells, independently of ER/PR/HER2 status.

ACCEPTED MANUSCRIPT 3.2. The antiproliferative effects of DHADA and EPADA are mediated by PPAR activation in breast cancer cells

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Since either DHA or EPA as well as their conjugates [21,34,35] have been demonstrated to

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modulate PPAR expression and activity, we wondered whether DHADA and EPADA could be

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able to activate PPAR in breast cancer cells. We found an increased expression of PPAR at both protein and mRNA levels in cells treated with DHADA and EPADA (Fig. 2A and B). To assess the

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ability of both compounds to transactivate endogenous PPAR, we performed in MCF7 cells functional assay using a PPAR response element (PPRE) reporter plasmid. As shown in Figure 2C,

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a significantly enhanced transcriptional activation of the reporter plasmid was induced by DHADA and EPADA, similarly to that observed upon treatment with the PPAR ligand rosiglitazone (BRL).

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Induction of PPRE reporter activity by BRL or both compounds was abolished by the PPAR

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antagonist GW9662 (GW) (Fig. 2C), addressing the direct involvement of PPAR. Having

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established that DHADA and EPADA are PPAR activators, we investigated their effects in combination with GW on MCF-7 cell proliferation. We found that GW abolished the reduction on

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cell growth induced by DHADA and EPADA treatments, supporting the role of PPAR in mediating these effects (Fig. 2D). 3.3. DHADA and EPADA upregulate Beclin-1 expression and its gene promoter in breast cancer cells Next, we wondered whether the DHADA/EPADA-antiproliferative effects could be associated to cell death processes. Our previous studies demonstrated the ability of PPARγ activation to induce both extrinsic and intrinsic apoptotic pathways in breast cancer cells [36-38]. Thus, to investigate if DHADA and EPADA may induce cell death by apoptosis through PPAR activation in our experimental system, we evaluated the typical hallmarks of cells undergoing apoptosis after 24h exposure to both compounds. At this time point, we did not observe either PARP cleavage or DNA

ACCEPTED MANUSCRIPT laddering in all cell lines tested (Supplementary Fig. 2). These latter results prompted us to explore the potential involvement of autophagy as another type of cell death in which PPARγ could be

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involved. It is well known that autophagy is a catabolic process orchestrated by numerous distinct

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autophagy-related genes (Atg) and their encoded proteins, among which Beclin-1 (the mammalian

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orthologue of yeast Atg6) plays a key role in the initiation of the process. We found that either DHADA or EPADA up-regulated Beclin-1 expression, as evaluated by immunofluorescence, immunoblotting and real-time PCR in MCF-7 (Fig. 3A), SKBR3 (Fig. 3B) and MDA-MB-231 (Fig.

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3C) cells. Next, we evaluated whether DHADA and EPADA can modulate Beclin-1 transcriptional

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activity. To this aim, we transiently transfected MCF-7 cells with a plasmid, p644 (-644/+95), containing Beclin-1 regulatory sequences and found that DHADA and EPADA significantly

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increased luciferase activity (Fig. 4B). The Beclin-1 promoter contains multiple transcription factor

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binding motifs, among which NFkB, Sp1 and RXR sites, representing potential PPAR binding sequences [39-41]. To determine which elements in the Beclin-1 promoter can mediate the above

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described effects, Beclin-1 promoter–deleted constructs p277 (-277/+95) and p58 (-58/+95) were tested in transient transfection experiments (Schematically reported in Fig. 4A). By using p277

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construct, missing RXR, Sp1 and NFkB sites the responsiveness to DHADA and EPADA was reduced but still present, whereas when we used the construct p58, in which the RXR site closer to the initiation transcription site is deleted, upregulatory effects were no longer noticeable (Fig. 4B), suggesting that RXR site is required for Beclin-1 promoter activation induced by both conjugates. The involvement of either PPAR or RXR in the stimulatory effect of DHADA and EPADA was evidenced by the ability of the PPAR antagonist GW as well as the RXR antagonist UVI to abolish the Beclin-1 promoter activation (Fig. 4C). To provide insights into the molecular mechanism by which DHADA and EPADA modulate Beclin-1 promoter activity through RXR motif, we performed chromatin immunoprecipitation (ChIP) and Re-ChIP assays. Using anti-RXR and/or anti-PPAR antibodies, protein-chromatin complexes were immunoprecipitated from MCF-7 cells treated with DHADA and EPADA. Real-time PCR was used to determine the recruitment of RXR

ACCEPTED MANUSCRIPT and/or PPAR to the Beclin-1 region containing the RXR site. An enhanced occupancy Beclin-1 promoter by both nuclear receptors in the presence of DHADA and EPADA was observed in both

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ChIP and Re-ChIP assays (Fig. 4D). These results well correlated with an increased association of

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RNA-polymerase II to the Beclin-1 regulatory region in cells treated with both compounds (Fig.

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4D).

3.4. DHADA and EPADA trigger autophagy in a PPAR-dependent manner in breast cancer cells

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Next, to evaluate if DHADA and EPADA were able to trigger the process of autophagy, we evaluated the accumulation of microtubule-associated protein 1 light-chain 3 (LC3), a specific

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membrane marker for the detection of early autophagosome formation, in cells treated with both compounds. As shown in Figure 5B, upon 24h treatment of DHADA or EPADA a significant

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increase in LC3 immunofluorescence was observed along with normal nuclei, as evidenced by

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DAPI staining in all cell lines. Finally, the formation of autophagosomes was confirmed by using

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monodansylcadaverine (MDC) staining. Cells treated with both conjugates showed an enhanced accumulation of MDC-labeled vacuoles compared to untreated MCF-7, SKBR3 and MDA-MB-231

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cells (Fig. 5C). Interestingly, the DHADA- and EPADA-induced autophagy was dependent of the activation of PPAR, since GW was able to prevent the upregulation of Beclin-1 as well as the accumulation of LC3 and MDC-labeled vacuoles in breast cancer cells (Fig. 5A, B and C). To provide convincing support to the concern that autophagy plays a crucial role in the antiproliferative action of these compounds, we examined the effects of 24 h treatment with DHADA and EPADA on cell viability under conditions of autophagy inhibition. Using MDC staining, we first demonstrated that either the pharmacological autophagy inhibitor, hydroxychloroquine (HCQ), or genetic silencing of Beclin-1 expression actually inhibit autophagy in all breast cancer cells (Supplementary Fig. 3). We therefore observed that when autophagy is blocked DHADA and EPADA did not exert any inhibitory effects on cell viability evaluated by MTT assays, highlighting

ACCEPTED MANUSCRIPT autophagy as the mechanism responsible for the observed effects exerted by both conjugates in all breast cancer cells (Fig. 5D).

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3.5. DHADA and EPADA maximize death of breast cancer cells by inducing apoptosis

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In the complex interplay existing between autophagy and apoptosis, autophagic process may precede apoptosis [42]. To define the dialogue between autophagy and cell death pathways we monitored autophagic flux in breast cancer cells treated with DHADA and EPADA at 24 and 48h.

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Besides LC3, levels of several specific substrates preferentially degraded by autophagy can be used to monitor autophagic flux. Among these, the best studied is p62 (also known as

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SQSTM1/sequestome 1), which expression levels inversely correlate with autophagic activity. According to our previous findings after 24h treatment with DHADA and EPADA, immunoblotting

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analyses revealed increased LC3 levels concomitantly with decreased levels of p62. Interestingly,

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after 48h treatment p62 appeared significantly accumulated, indicating that autophagy was inhibited

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in all breast cancer cells (Fig. 6A). It has been reported that among autophagy protein fragments resulting from caspases cleavage, proteolytic form of Beclin-1 localizes to mitochondria and causes the release of cytochrome c which in turn facilitates apoptosis, representing a critical step in the

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apoptotic cascade [43]. We observed that both compounds after 48h treatment induced a nice increase of the cleaved active fragment of Caspase 9 together with an enhanced Beclin-1 cleavage (Fig. 6B), providing evidences into the meet of key elements of apoptosis and autophagy in our breast cancer cell lines. Indeed, the significant reduction in Beclin-1 expression is associated with enhanced levels of proapoptotic Bid, released cytochrome C from mitochondria into the cytosol and induced PARP cleavage (Fig. 6C). Moreover, DNA laddering after exposure to DHADA and EPADA in all breast cancer cells revealed changes in the internucleosomal fragmentation profile of genomic DNA, which is a diagnostic hallmark of cells undergoing apoptosis (Fig. 6D). It is worth to note that this latter effect was also PPAR-mediated since it was completely abolished in the presence of GW (Supplementary Fig. 4). In addition, by TUNEL assay the percentage of the cell

ACCEPTED MANUSCRIPT population undergoing apoptosis was evaluated. As shown in Fig. 6E, treatments with DHADA and EPADA significantly increased the percentage of apoptotic cells in all breast cancer cell lines.

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Finally, we blocked autophagy either pharmacologically, by using HCQ or genetically, by knocking

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down Beclin-1 expression and evaluated apoptosis by DNA fragmentation assay. Our results

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showed that, in cells exposed to HCQ as well as in cells in which Beclin-1 was silenced, the apoptosis induced by 48h treatment with DHADA and EPADA was completely prevented,

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suggesting that autophagy precedes apoptosis in our experimental models (Fig.7A and B).

ACCEPTED MANUSCRIPT 4. Discussion N-acylamines of n–3 PUFAs are endogenous mediators whose biological significance has to be

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fully established. Although diet is one of the main environmental factor modulating their formation,

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the association between dietary intake of n–3 PUFAs and the formation of their respective fatty acid

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amides, their role and significance in mediating the alleged health effects of fish oil remain speculative. Evidence is accumulating that N-acyldopamines possess several properties that deserve further research in relation not only to inflammatory and neural disorders, but also to cancer

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[29,30].

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In the present study we demonstrated the inhibitory effects of DHADA and EPADA on cell viability in ER/PR-positive MCF-7, ER/PR-negative SKBR3 and triple-negative MDA-MB-231 human breast cancer cells. Particularly, MDA-MB-231 cells exhibited the lowest IC50 values for

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both ligands compared with the other cell lines, suggesting a higher sensitivity by the more aggressive cellular phenotype. The translational relevance of these effects is pointed out by the

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observation that both compounds do not affect proliferation of the non-tumorigenic breast epithelial MCF-10A cell line. Moreover, the anticancer efficacy and potency of DHADA and EPADA

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compared with parent fatty acids and the reported potential ability to inhibit FAAH, that increases their (local) concentrations [16], makes them of interest to both the pharmacological and nutritional research fields.

Previous findings demonstrated that n-3 PUFAs and their derivatives act as natural ligands of PPAR which mediates their effects on cell function [44-47]. Here, we demonstrated for the first time that DHADA and EPADA are PPAR-inducers able to activate the endogenous PPAR and to up-regulate its expression in breast cancer cells. Many current lines of evidence highlight the existence of a crosstalk between PPAR activity and death signalling pathways leading to antiproliferative effects, cell-cycle arrest, apoptosis and autophagy in human breast cancer cells [21,36,37,48,49]. In the present study, we demonstrated that, after 24h treatment, DHADA and EPADA, induce cell cycle arrest and trigger, through PPAR activation, autophagy as the

ACCEPTED MANUSCRIPT mechanism responsible for the reduction of cell viability in different breast cancer cells, as confirmed by the lack of these effects under conditions of autophagy inhibition. The DHADA- and

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EPADA-induced autophagy occurs through the capability of both compounds to upregulate Beclin-

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1 expression, which is the first identified mammalian gene inducing autophagy [50,51]. Of interest,

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in all the investigated breast cancer cells PPAR activation by DHADA and EPADA administration increases Beclin-1 levels by regulating its gene promoter transcriptional activity. Activation of PPAR is a multistep process that involves ligand binding, heterodimerization with RXR,

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interaction with cognate DNA sequences, and recruitment of coregulatory proteins [52]. The human

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Beclin-1 promoter contains multiple transcription regulatory elements, including Sp1, NFkB, RAR and RXR sites. We documented that the region spanning from -277 to -58, which includes the RXR

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site, is required for the responsiveness to DHADA and EPADA. Transactivation of Beclin-1

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promoter by DHADA and EPADA directly involves either PPAR or RXR since activation of Beclin-1 promoter was prevented by treatment with the specific antagonist of both receptors. This

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well fit with the evidence that the RXR/PPAR occupancy of the DHADA/EPADA-responsive promoter region, induced by treatment with both compounds, is concomitant with an enhancement

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in RNA Pol II recruitment consistent with the observed increase in Beclin-1 transcriptional activity. Induction of Beclin-1 gene expression in breast cancer cells upon 24h treatment with DHADA and EPADA triggers autophagic pathways as evidenced by the recruitment of LC3 to autophagosomes whose enhanced formation was confirmed by MDC fluorescence. The use of a selective inhibitor of PPAR provides a distinct role of this receptor in DHADA- and EPADA-induced autophagy in breast cancer cells. At the same time point, we did not detect any characteristic hallmarks of cells undergoing apoptosis, confirming that in our experimental conditions autophagy is the first early event governing cell death exerted by DHADA and EPADA. Under sustained stress conditions, caspase-mediated cleavage of Beclin-1 not only abrogates the autophagic function of Beclin-1, but also induces its delocalization to the mitochondria sensitizing cells to apoptotic signals [43]. In line with previous findings, after long-term treatment (48h) with DHADA and EPADA we found the

ACCEPTED MANUSCRIPT enhanced cleaved fragment of Caspase 9 and the increased Beclin-1 cleavage concomitant with the blockade of autophagic flux. These events ultimately cause cell death by apoptosis as evidenced by

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the increased levels of the proapoptotic Bid, the release of cytochrome C, the cleavage of PARP

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leading to changes in the internucleosomal fragmentation profile of genomic DNA. Again, either

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pharmacological or genetic inhibition of autophagy prevented cell death at late time point of treatment with both conjugates. Overall, our results demonstrated that upon DHADA and EPADA exposure autophagy precedes and facilitates the activation of apoptotic cell death in breast cancer

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cells. In conclusion, in the present study we have provided new insights into the molecular

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mechanism through which PPAR, as a central molecule in the cross talk between autophagy and apoptosis, mediates DHADA- and EPADA-induced cell death in breast cancer cells. Our findings

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suggest that omega-3 DHADA- and EPADA activation of PPAR may assume biological relevance

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in setting novel adjuvant therapeutic interventions in breast cancer and particularly in triple negative

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breast carcinoma exhibiting major aggressiveness and invasiveness.

ACCEPTED MANUSCRIPT Conflict of Interest

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All authors declare no conflict of interest.

ACCEPTED MANUSCRIPT Acknowledgments The Authors thank Prof. M. Zhao and Dr. R. Evans for generously providing Beclin-1 promoter and

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PPRE plasmids, respectively.

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(AIRC) grant IG 11595, Lilli Funaro Foundation.

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This work was supported by MURST and Ex 60%, Associazione Italiana Ricerca sul Cancro

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derivatives. Cell Mol Life Sci 2002;59:790-8. [47] Allred CD, Talbert DR, Southard RC, Wang X, Kilgore MW. PPARgamma1 as a molecular target of eicosapentaenoic acid in human colon cancer (HT-29) cells. J Nutr 2008;138:250-6. [48] Schmidt MV, Brune B, von Knethen A. The nuclear hormone receptor PPARgamma as a therapeutic target in major diseases. Sci World J 2010;10:2181-97. [49] Bonofiglio D, Gabriele S, Aquila S, Catalano S, Gentile M, Middea E, et al. Estrogen receptor alpha binds to peroxisome proliferator-activated receptor (PPAR) response element and negatively interferes with PPARgamma signalling in breast cancer cells. Clin Cancer Res 2005;11:6139-47.

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[51] Liang XH, Yu J, Brown K, Levine B. Beclin 1 contains a leucine-rich nuclear export signal

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. Antiproliferative effects of DHADA and EPADA in breast cancer cell growth. Structures of

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docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-Dopamine (DA) conjugates (A and

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B, respectively). Cell viability was determined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-

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diphenyltetrazolium (MTT) assays in MCF-7 (C), SKBR3 (D) and MDA-MB-231 (E) cells untreated (-) or treated with increasing concentrations (100nM, 1, 10, 25, 50, 100M) of DHADA

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(upper panels) or EPADA (lower panels) for 24, 48 and 72h as indicated. The results are expressed as fold change respect to untreated cells (-). The values represent the mean±SD of three different

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experiments, each performed with triplicate samples. *P<0.05 treated vs untreated cells (-). Fig. 2. Activation of PPAR by DHADA and EPADA in breast cancer cells. (A) Immunoblots of

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PPAR expression from total extracts of cells untreated (-) or treated with DHADA or EPADA 25M (MCF-7 and SKBR3 cells) or 10M (MDA-MB-231 cells) for 12h. GAPDH was used as a

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loading control. Numbers on the bottom of the blot represent the average fold change relative to control normalized for GAPDH. (B) PPAR mRNA content in MCF-7, SKBR3 and MDA-MB-231

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cells untreated (-) or treated with DHADA or EPADA, as in A, for 6h was evaluated by real-time PCR. Each sample was normalized on its GAPDH mRNA content. (C) Luciferase activity measured in MCF-7 cells transiently transfected with a PPRE-luciferase reporter gene (PPRE, PPARResponse Element) and untreated (-) or treated for 24h with BRL 10M, DHADA 25M, EPADA 25M and/or GW9662 (GW) 10M. The results are expressed as fold change respect to the untreated cells (-). (D) MTT assays in MCF-7 cells untreated (-) or treated with DHADA 25M, EPADA 25M, and/or GW9662 (GW) 10M for 24h. The results are expressed as fold change respect to untreated cells (-). The values represent the mean±SD of three different experiments, each performed with triplicate samples. *P<0.05 treated vs untreated cells (-). n.s. = not significant. Fig. 3. Upregulation of Beclin-1 expression by DHADA and EPADA in MCF-7 (A), SKBR3 (B)

ACCEPTED MANUSCRIPT and MDA-MB-231 (C) cells. Upper panel, Immunofluorescence of Beclin-1 (BECN1) in cells untreated (-) or treated with DHADA or EPADA for 24h. Small squares, negative controls. 4’,6-

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Diamidino-2-phenylindole (DAPI) was used for the determination of the nuclei. Scale bar, 25m.

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Middle panel, Immunoblots of BECN1 expression in cells untreated (-) or treated with DHADA or

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EPADA for 24h. GAPDH was used as loading control. Numbers on the bottom of the blots represent the average fold change relative to untreated cells (-) normalized for GAPDH. Lower panel, BECN1 mRNA content in cells untreated (-) or treated with DHADA or EPADA for 12h was

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evaluated by real-time PCR. Each sample was normalized on its GAPDH mRNA content. The

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results are expressed as fold change respect to the untreated cells (-). The values represent the mean±SD of three different experiments, each performed with triplicate samples. *P<0.05 treated

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vs untreated cells (-).

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Fig. 4. DHADA and EPADA upregulate Beclin-1 transcriptional activity in MCF-7 cells. (A)

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Schematic representation of the Beclin-1 promoter constructs (promBECN1) used in this study. (B) Cells were transiently transfected with luciferase plasmids containing the BECN1 promoter (prom BECN1-luc-644, p-644) and its deletions (p-277 and p-58) and then untreated (-) or treated with

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DHADA or EPADA 25M for 12h. The results are expressed as fold change respect to the untreated cells (-). (C) Cells were transiently transfected with luciferase plasmids containing the BECN1 promoter (p-644) and then untreated (-) or treated with DHADA 25M, EPADA 25M and/or GW 10M, and/or UVI 10M for 12h. The results are expressed as fold change respect to the untreated cells (-). (D) Chromatin Immunoprecipitation (ChIP) with the anti-PPAR, anti-RXR, and anti-Pol II antibodies in cells untreated (-) or treated with DHADA or EPADA 25M for 1h. ChIP with the anti-PPAR or anti-RXR antibodies was re-immunoprecipitated (re-ChIP) with the anti-RXR or anti-PPAR antibodies, respectively. The Beclin-1 promoter sequence including the RXR site was detected by real-time-PCR with specific primers (see Materials and Methods section). The results are mean±SD of three different experiments, each performed with triplicate samples.

ACCEPTED MANUSCRIPT *P<0.05 treated vs untreated cells

(-). n.s. = not significant.

Fig. 5. PPAR-mediated autophagy by DHADA and EPADA in breast cancer cells. (A)

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Immunoblots of BECN1 protein expression from total extracts of MCF-7, SKBR3 and MDA-MB-

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231cells untreated (-) or treated with DHADA, EPADA and/or GW for 24h. GAPDH was used as

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loading control. (B) Immunofluorescence of microtubule-associated protein 1 light-chain 3 (LC3) in MCF-7, SKBR3 and MDA-MB-231 cells untreated (-) or treated with DHADA, EPADA and/or

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GW for 24h. Small squares, negative controls. DAPI was used for the determination of the nuclei. Scale bar, 25m. (C) Monodansylcadaverine staining (MDC) in MCF-7, SKBR3 and MDA-MB-

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231 cells untreated (-) or treated as in B. One of three similar experiments is presented. (D) MTT assays in MCF-7, SKBR3 and MDA-MB-231 cells exposed to Hydroxychloroquine sulfate (HCQ)

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10µM (upper panels) or transfected with control shRNA (sh-control) or BECN1 shRNA (sh-

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BECN1) (middle panels) vectors and then untreated (-) or treated for 48h with DHADA and

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EPADA. The results are mean±SD of three different experiments, each performed with triplicate samples. *P<0.05 treated vs untreated cells (-). n.s. = not significant. Immunoblots of BECN1 protein expression (lower panels) from total extracts of MCF-7, SKBR3 and MDA-MB-231 cells

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transiently transfected with sh-control or sh-BECN1 vectors. GAPDH was used as loading control. One of three similar experiments is presented. Fig. 6. DHADA and EPADA induce apoptotic cell death in breast cancer cells. (A) Immunoblots of LC3 and p62/SQSTM1 protein levels from total extracts of MCF-7, SKBR3 and MDA-MB-231 cells untreated (-) or treated for 24 and 48h with DHADA and EPADA. (B) Immunoblots of caspase 9 and BECN1 protein expression from total extracts of MCF-7, SKBR3 and MDA-MB-231 cells untreated (-) or treated with DHADA or EPADA for 48h. (C) Immunoblots of BECN1, Bid, Cytochrome C (Cyt C) and PARP protein expression from total extracts of MCF-7, SKBR3 and MDA-MB-231 cells untreated (-) or treated with DHADA or EPADA for 48h. GAPDH was used as a loading control. Numbers on the bottom of the blots represent the average fold change relative to

ACCEPTED MANUSCRIPT untreated cells (-) normalized for GAPDH. (D) DNA laddering performed in MCF-7, SKBR3 and MDA-MB-231 cells untreated (-) or treated with DHADA or EPADA for 48h. One of three similar

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experiments is presented. (E) Terminal deoxynucleotidyl transferase-mediated dUTP Nick End

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Labeling (TUNEL) staining for detection of apoptosis in MCF-7, SKBR3 and MDA-MB-231 cells

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treated with DHADA and EPADA for 48h. Columns represent quantitation of apoptotic cells from two independent experiments performed in triplicate; bars, ±SD. *p < 0.05 treated (-) vs untreated

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cells.

Fig.7. Effects of autophagy inhibition on DHADA- and EPADA-induced apoptosis in breast cancer

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cells. DNA laddering in MCF-7, SKBR3 and MDA-MB-231 cells exposed to Hydroxychloroquine sulfate (HCQ) 10µM (A) or transfected with control shRNA (sh-control) or BECN1 shRNA (sh-

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BECN1) vectors (B) and then untreated (-) or treated for 48h with DHADA and EPADA.

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Immunoblots of BECN1 protein expression from total extracts of MCF-7, SKBR3 and MDA-MB231 cells transiently transfected with sh-control or sh-BECN1 vectors. GAPDH was used as loading

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ACCEPTED MANUSCRIPT Table 1. IC50 values of DHADA and EPADA for breast cancer cells.

compounds

IC (µM)

95% Confidence Interval

DHADA

25

19,3-30

EPADA

19

14-23

DHADA

20

13,5-25

EPADA

22

15,5-27

DHADA

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4,4-7,1

EPADA

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6,1-10,3

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MCF-7

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SKBR3

ACCEPTED MANUSCRIPT Table 2. DHADA and EPADA effects on cell cycle in breast cancer cells.

-

65±2

30,8±2

4,2±1

DHADA

68,9±1,8*

23±1,5*

8,1±0,8*

EPADA

68,4±2,1*

22,7±2*

8,9±1,2*

69,2±1,5

25,7±1

5,1±0,5

74,8±1,5*

13,5±0,8*

11,6±1*

EPADA

77,9±2*

15,7±0,7*

6,4±1,2*

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76,7±1,2

20±1,5

3,2±0,5

DHADA

83±1*

14,4±1*

3,5±0,7

EPADA

86,3±1,3*

8,9±0,5*

4,8±0,8

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MDA-MB-231

DHADA

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SKBR3

*P< 0.05 DHADA- or EPADA-treated vs untreated cells (-).

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G2/M (%)

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Highlights Omega-3 DHA- and EPA-Dopamine (DA) conjugates inhibit breast cancer cell growth.

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DHADA and EPADA as PPAR inducers up-regulate Beclin-1 expression at transcriptional level.

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DHADA and EPADA trigger autophagy and apoptosis through PPAR in breast cancer cells