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<!DOCTYPE html> <html lang="en" dir="ltr" xmlns="http://www.w3.org/1999/xhtml" xmlns:mml="http://www.w3.org/1998/Math/MathML"> <head prefix="og: http://ogp.me/ns# article: http://ogp.me/ns/article# book: http://ogp.me/ns/book#" >  <link rel="dns-prefetch" href="//cdn.jsdelivr.net" /> <link rel="dns-prefetch" href="//cdn.foxycart.com" /> <link rel="dns-prefetch" href="//scholar.google.com" /> <meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> <meta name="Generator" content="Drupal 7 (http://drupal.org)" /> <link rel="canonical" href="https://ar.iiarjournals.org/content/44/11/4751" /> <link rel="alternate" type="application/pdf" title="Full Text (PDF)" href="/content/44/11/4751.full.pdf" /> <link rel="alternate" type="text/plain" title="Full Text (Plain)" href="/content/44/11/4751.full.txt" /> <link rel="alternate" type="application/vnd.ms-powerpoint" title="Powerpoint" href="/content/44/11/4751.ppt" /> <meta name="issue_cover_image" content="https://ar.iiarjournals.org/sites/default/files/highwire/anticanres/44/11.cover-source.jpg" /> <meta name="type" content="article" /> <meta name="category" content="research-article" /> <meta name="HW.identifier" content="/anticanres/44/11/4751.atom" /> <meta name="HW.pisa" content="anticanres;44/11/4751" /> <meta name="DC.Format" content="text/html" /> <meta name="DC.Language" content="en" /> <meta name="DC.Title" content="Differential Effects of Punicic Acid on Cytotoxicity and Peroxiredoxin Expression in MCF-7 Breast Cancer and MCF-10A Normal Cells" /> <meta name="DC.Identifier" content="10.21873/anticanres.17301" /> <meta name="DC.Date" content="2024-11-01" /> <meta name="DC.Publisher" content="International Institute of Anticancer Research" /> <meta name="DC.Rights" content="Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0)." /> <meta name="DC.AccessRights" content="open-access" /> <meta name="DC.Description" content="Background/Aim: The pomegranate fruit has been associated with a variety of human health benefits based on its antioxidant and anti-inflammatory properties. Punicic acid (PA) is an omega-5 long chain polyunsaturated fatty acid that constitutes approximately 65-80% of the oil from pomegranate seeds and has been found to possesses anti-cancer activity in various cancer types. To better understand its cell specificity, we investigated the effects of punicic acid on both the MCF-7 human breast cancer cell line as well as the non-cancerous MCF-10A breast epithelial line. Materials and Methods: We treated both cell types with different concentrations of punicic acid and measured viable cell density, cytotoxicity and apoptosis, as well as peroxiredoxin (Prdx) antioxidant expression. Results: We found that punicic acid was cytotoxic to both lines, but MCF-10A cells demonstrated higher levels of cytotoxicity and sensitivity to lower concentrations. We further demonstrated that cytotoxicity was associated with apoptosis in both lines. Using real time PCR, we demonstrated induction of all six Prdx mRNAs in MCF-7 cells, ranging from a 1.4-fold increase with 2 μg/ml, to over a 5-fold increase with 10 μg/ml. In stark contrast, MCF-10A cells exhibited considerably higher induction of all six Prdx mRNAs at 10μg/ml, exceeding a 30-fold induction of Prdx1, Prdx2 and Prdx5. Conclusion: Our data are the first to demonstrate differential cytotoxicity and Prdx regulation by punicic acid in these two cell lines. These results may provide important insight into cell-specific mechanisms of punicic acid in breast cancer." /> <meta name="DC.Contributor" content="BRADEN QUITMEYER" /> <meta name="DC.Contributor" content="CHIEMELIE EMELIFE" /> <meta name="DC.Contributor" content="HANNAH KLAUSNER" /> <meta name="DC.Contributor" content="OLUWAFEMI GBAYISOMORE" /> <meta name="DC.Contributor" content="SHELLEY PHELAN" /> <meta name="article:published_time" content="2024-11-01" /> <meta name="article:section" content="Experimental Studies" /> <meta name="citation_title" content="Differential Effects of Punicic Acid on Cytotoxicity and Peroxiredoxin Expression in MCF-7 Breast Cancer and MCF-10A Normal Cells" /> <meta name="citation_abstract" lang="en" content="<p>Background/Aim: The pomegranate fruit has been associated with a variety of human health benefits based on its antioxidant and anti-inflammatory properties. Punicic acid (PA) is an omega-5 long chain polyunsaturated fatty acid that constitutes approximately 65-80% of the oil from pomegranate seeds and has been found to possesses anti-cancer activity in various cancer types. To better understand its cell specificity, we investigated the effects of punicic acid on both the MCF-7 human breast cancer cell line as well as the non-cancerous MCF-10A breast epithelial line. Materials and Methods: We treated both cell types with different concentrations of punicic acid and measured viable cell density, cytotoxicity and apoptosis, as well as peroxiredoxin (Prdx) antioxidant expression. Results: We found that punicic acid was cytotoxic to both lines, but MCF-10A cells demonstrated higher levels of cytotoxicity and sensitivity to lower concentrations. We further demonstrated that cytotoxicity was associated with apoptosis in both lines. Using real time PCR, we demonstrated induction of all six Prdx mRNAs in MCF-7 cells, ranging from a 1.4-fold increase with 2 μg/ml, to over a 5-fold increase with 10 μg/ml. In stark contrast, MCF-10A cells exhibited considerably higher induction of all six Prdx mRNAs at 10μg/ml, exceeding a 30-fold induction of Prdx1, Prdx2 and Prdx5. Conclusion: Our data are the first to demonstrate differential cytotoxicity and Prdx regulation by punicic acid in these two cell lines. These results may provide important insight into cell-specific mechanisms of punicic acid in breast cancer.</p>" /> <meta name="citation_journal_title" content="Anticancer Research" /> <meta name="citation_publisher" content="International Institute of Anticancer Research" /> <meta name="citation_publication_date" content="2024/11/01" /> <meta name="citation_mjid" content="anticanres;44/11/4751" /> <meta name="citation_id" content="44/11/4751" /> <meta name="citation_public_url" content="https://ar.iiarjournals.org/content/44/11/4751" /> <meta name="citation_abstract_html_url" content="https://ar.iiarjournals.org/content/44/11/4751.abstract" /> <meta name="citation_full_html_url" content="https://ar.iiarjournals.org/content/44/11/4751.full" /> <meta name="citation_pdf_url" content="https://ar.iiarjournals.org/content/anticanres/44/11/4751.full.pdf" /> <meta name="citation_issn" content="0250-7005" /> <meta name="citation_issn" content="1791-7530" /> <meta name="citation_doi" content="10.21873/anticanres.17301" /> <meta name="citation_pmid" content="39477324" /> <meta name="citation_volume" content="44" /> <meta name="citation_issue" content="11" /> <meta name="citation_article_type" content="Research Article" /> <meta name="citation_section" content="Experimental Studies" /> <meta name="citation_firstpage" content="4751" /> <meta name="citation_lastpage" content="4759" /> <meta name="citation_access" content="all" /> <meta name="citation_author" content="BRADEN QUITMEYER" /> <meta name="citation_author_institution" content="Department of Biology, Fairfield University, Fairfield, CT, U.S.A.;" /> <meta name="citation_author" content="CHIEMELIE EMELIFE" /> <meta name="citation_author_institution" content="Department of Biology, Fairfield University, Fairfield, CT, U.S.A.;" /> <meta name="citation_author" content="HANNAH KLAUSNER" /> <meta name="citation_author_institution" content="Univerisity of New England College of Osteopathic Medicine, Biddeford, ME, U.S.A.;" /> <meta name="citation_author" content="OLUWAFEMI GBAYISOMORE" /> <meta name="citation_author_institution" content="Meharry Medical College, Nashville, TN, U.S.A." /> <meta name="citation_author" content="SHELLEY PHELAN" /> <meta name="citation_author_institution" content="Department of Biology, Fairfield University, Fairfield, CT, U.S.A.;" /> <meta name="citation_author_email" content="sphelan@fairfield.edu" /> <meta name="citation_reference" content="citation_journal_title=Compr Rev Food Sci Food Saf;citation_author=M. 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Abu;citation_author=S. Punicic;citation_title=Punicic and Jacaric acids induce Mitochondrial dysfunction in prostate cancer cell lines;citation_pages=99-106;citation_volume=13;citation_year=2016;citation_issue=6;citation_doi=10.7537/marslsj13061613" /> <meta name="citation_fulltext_world_readable" content="" /> <meta name="twitter:title" content="Differential Effects of Punicic Acid on Cytotoxicity and Peroxiredoxin Expression in MCF-7 Breast Cancer and MCF-10A Normal Cells" /> <meta name="twitter:card" content="summary_large_image" /> <meta name="twitter:image" content="https://ar.iiarjournals.org/sites/default/files/highwire/anticanres/44/11.cover-source.jpg" /> <meta name="twitter:description" content="Background/Aim: The pomegranate fruit has been associated with a variety of human health benefits based on its antioxidant and anti-inflammatory properties. Punicic acid (PA) is an omega-5 long chain polyunsaturated fatty acid that constitutes approximately 65-80% of the oil from pomegranate seeds and has been found to possesses anti-cancer activity in various cancer types. To better understand its cell specificity, we investigated the effects of punicic acid on both the MCF-7 human breast cancer cell line as well as the non-cancerous MCF-10A breast epithelial line. Materials and Methods: We treated both cell types with different concentrations of punicic acid and measured viable cell density, cytotoxicity and apoptosis, as well as peroxiredoxin (Prdx) antioxidant expression. Results: We found that punicic acid was cytotoxic to both lines, but MCF-10A cells demonstrated higher levels of cytotoxicity and sensitivity to lower concentrations. We further demonstrated that cytotoxicity was associated with apoptosis in both lines. Using real time PCR, we demonstrated induction of all six Prdx mRNAs in MCF-7 cells, ranging from a 1.4-fold increase with 2 μg/ml, to over a 5-fold increase with 10 μg/ml. In stark contrast, MCF-10A cells exhibited considerably higher induction of all six Prdx mRNAs at 10μg/ml, exceeding a 30-fold induction of Prdx1, Prdx2 and Prdx5. Conclusion: Our data are the first to demonstrate differential cytotoxicity and Prdx regulation by punicic acid in these two cell lines. These results may provide important insight into cell-specific mechanisms of punicic acid in breast cancer." /> <meta name="og-title" property="og:title" content="Differential Effects of Punicic Acid on Cytotoxicity and Peroxiredoxin Expression in MCF-7 Breast Cancer and MCF-10A Normal Cells" /> <meta name="og-url" property="og:url" content="https://ar.iiarjournals.org/content/44/11/4751" /> <meta name="og-site-name" property="og:site_name" content="Anticancer Research" /> <meta name="og-description" property="og:description" content="Background/Aim: The pomegranate fruit has been associated with a variety of human health benefits based on its antioxidant and anti-inflammatory properties. Punicic acid (PA) is an omega-5 long chain polyunsaturated fatty acid that constitutes approximately 65-80% of the oil from pomegranate seeds and has been found to possesses anti-cancer activity in various cancer types. To better understand its cell specificity, we investigated the effects of punicic acid on both the MCF-7 human breast cancer cell line as well as the non-cancerous MCF-10A breast epithelial line. Materials and Methods: We treated both cell types with different concentrations of punicic acid and measured viable cell density, cytotoxicity and apoptosis, as well as peroxiredoxin (Prdx) antioxidant expression. Results: We found that punicic acid was cytotoxic to both lines, but MCF-10A cells demonstrated higher levels of cytotoxicity and sensitivity to lower concentrations. We further demonstrated that cytotoxicity was associated with apoptosis in both lines. Using real time PCR, we demonstrated induction of all six Prdx mRNAs in MCF-7 cells, ranging from a 1.4-fold increase with 2 μg/ml, to over a 5-fold increase with 10 μg/ml. In stark contrast, MCF-10A cells exhibited considerably higher induction of all six Prdx mRNAs at 10μg/ml, exceeding a 30-fold induction of Prdx1, Prdx2 and Prdx5. Conclusion: Our data are the first to demonstrate differential cytotoxicity and Prdx regulation by punicic acid in these two cell lines. These results may provide important insight into cell-specific mechanisms of punicic acid in breast cancer." /> <meta name="og-type" property="og:type" content="article" /> <meta name="og-image" property="og:image" content="https://ar.iiarjournals.org/sites/default/files/highwire/anticanres/44/11.cover-source.jpg" /> <link rel="shortlink" href="/node/81986" /> <link rel="shortcut icon" href="https://ar.iiarjournals.org/sites/default/files/images/favicon.ico" type="image/vnd.microsoft.icon" /> <meta name="viewport" content="width=device-width, initial-scale=1" /> <title>Differential Effects of Punicic Acid on Cytotoxicity and Peroxiredoxin Expression in MCF-7 Breast Cancer and MCF-10A Normal Cells | Anticancer Research</title> <link type="text/css" rel="stylesheet" href="/sites/default/files/advagg_css/css__QEy_BpbtMcsqv3xURK855GSs2fFjOibU10yA758Mn4c__NucWi_bEk5E3BNVDtTt2nhiPtE1A5I24YwRDQ2fojJ0__FTZ25lEcrtnzSW3JzKpn0j1z_ccWiSdhuJwjpWHa4ec.css" media="all" /> <link type="text/css" 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data-pisa="anticanres;44/11/4751" data-pisa-master="anticanres;44/11/4751" data-apath="/anticanres/44/11/4751.atom" data-hw-author-tooltip-instance="highwire_author_tooltip"><div class="highwire-cite highwire-cite-highwire-article highwire-citation-jcore-article-title-complete clearfix has-author-tooltip highwire-citation-highwire-article-top-a"> <div class="highwire-cite-overline"><span class="highwire-cite-metadata-article-type highwire-cite-metadata">Research Article</span><span class="separator-pipe"></span><span class="highwire-cite-metadata-art-cat highwire-cite-metadata"><span class="wrapper">Experimental Studies</span></span></div> <div class="highwire-cite-access"><span class="highwire-citation-access"><i class="highwire-access-icon highwire-access-icon-open-access open-access hw-icon-open-access" title="Open Access" aria-hidden="true"></i><span class="element-invisible highwire-access-icon highwire-access-icon-open-access" style="">Open Access</span></span></div> <h1 class="highwire-cite-title" id="page-title">Differential Effects of Punicic Acid on Cytotoxicity and Peroxiredoxin Expression in MCF-7 Breast Cancer and MCF-10A Normal Cells</h1> <div class="highwire-cite-authors"><span class="highwire-citation-authors"><span class="highwire-citation-author first" data-delta="0">BRADEN QUITMEYER</span>, <span class="highwire-citation-author" data-delta="1">CHIEMELIE EMELIFE</span>, <span class="highwire-citation-author" data-delta="2">HANNAH KLAUSNER</span>, <span class="highwire-citation-author" data-delta="3">OLUWAFEMI GBAYISOMORE</span> and <span class="highwire-citation-author" data-delta="4">SHELLEY PHELAN</span></span></div> <div class="highwire-cite-metadata"><span class="highwire-cite-metadata-journal highwire-cite-metadata">Anticancer Research </span><span class="highwire-cite-metadata-date highwire-cite-metadata">November 2024, </span><span class="highwire-cite-metadata-volume highwire-cite-metadata">44 </span><span class="highwire-cite-metadata-issue highwire-cite-metadata">(11) </span><span class="highwire-cite-metadata-pages highwire-cite-metadata">4751-4759; </span><span class="highwire-cite-metadata-doi highwire-cite-metadata">DOI: https://doi.org/10.21873/anticanres.17301 </span></div> <div class="highwire-cite-extras"><span class="highwire-foxycart-add-to-cart-ahah highwire-foxycart-add-to-cart-ahah" data-text="Add to Cart (%short-price)" data-apath="/anticanres/44/11/4751.atom" data-type="link" data-font-icon="" data-parent-id="81985"></span></div> </div> <div id="hw-article-author-popups-top-node-81986--61293416611" style="display: none;"><div class="author-tooltip-0"><div class="author-tooltip-name">BRADEN QUITMEYER </div><div class="author-tooltip-affiliation"><span class="author-tooltip-text"><div class='author-affiliation'><span class='nlm-sup'>1</span>Department of Biology, Fairfield University, Fairfield, CT, U.S.A.;</div></span></div><ul class="author-tooltip-find-more"><li class="author-tooltip-gs-link first"><a href="/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=BRADEN%2BQUITMEYER%2B" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on Google Scholar</a></li><li class="author-tooltip-pubmed-link"><a href="/lookup/external-ref?access_num=QUITMEYER%20B&link_type=AUTHORSEARCH" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on PubMed</a></li><li class="author-site-search-link last"><a href="/search/author1%3ABRADEN%2BQUITMEYER%2B" rel="nofollow" class="" data-icon-position="" data-hide-link-title="0">Search for this author on this site</a></li></ul></div><div class="author-tooltip-1"><div class="author-tooltip-name">CHIEMELIE EMELIFE </div><div class="author-tooltip-affiliation"><span class="author-tooltip-text"><div class='author-affiliation'><span class='nlm-sup'>1</span>Department of Biology, Fairfield University, Fairfield, CT, U.S.A.;</div></span></div><ul class="author-tooltip-find-more"><li class="author-tooltip-gs-link first"><a href="/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=CHIEMELIE%2BEMELIFE%2B" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on Google Scholar</a></li><li class="author-tooltip-pubmed-link"><a href="/lookup/external-ref?access_num=EMELIFE%20C&link_type=AUTHORSEARCH" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on PubMed</a></li><li class="author-site-search-link last"><a href="/search/author1%3ACHIEMELIE%2BEMELIFE%2B" rel="nofollow" class="" data-icon-position="" data-hide-link-title="0">Search for this author on this site</a></li></ul></div><div class="author-tooltip-2"><div class="author-tooltip-name">HANNAH KLAUSNER </div><div class="author-tooltip-affiliation"><span class="author-tooltip-text"><div class='author-affiliation'><span class='nlm-sup'>2</span>Univerisity of New England College of Osteopathic Medicine, Biddeford, ME, U.S.A.;</div></span></div><ul class="author-tooltip-find-more"><li class="author-tooltip-gs-link first"><a href="/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=HANNAH%2BKLAUSNER%2B" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on Google Scholar</a></li><li class="author-tooltip-pubmed-link"><a href="/lookup/external-ref?access_num=KLAUSNER%20H&link_type=AUTHORSEARCH" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on PubMed</a></li><li class="author-site-search-link last"><a href="/search/author1%3AHANNAH%2BKLAUSNER%2B" rel="nofollow" class="" data-icon-position="" data-hide-link-title="0">Search for this author on this site</a></li></ul></div><div class="author-tooltip-3"><div class="author-tooltip-name">OLUWAFEMI GBAYISOMORE </div><div class="author-tooltip-affiliation"><span class="author-tooltip-text"><div class='author-affiliation'><span class='nlm-sup'>3</span>Meharry Medical College, Nashville, TN, U.S.A.</div></span></div><ul class="author-tooltip-find-more"><li class="author-tooltip-gs-link first"><a href="/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=OLUWAFEMI%2BGBAYISOMORE%2B" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on Google Scholar</a></li><li class="author-tooltip-pubmed-link"><a href="/lookup/external-ref?access_num=GBAYISOMORE%20O&link_type=AUTHORSEARCH" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on PubMed</a></li><li class="author-site-search-link last"><a href="/search/author1%3AOLUWAFEMI%2BGBAYISOMORE%2B" rel="nofollow" class="" data-icon-position="" data-hide-link-title="0">Search for this author on this site</a></li></ul></div><div class="author-tooltip-4"><div class="author-tooltip-name">SHELLEY PHELAN </div><div class="author-tooltip-affiliation"><span class="author-tooltip-text"><div class='author-affiliation'><span class='nlm-sup'>1</span>Department of Biology, Fairfield University, Fairfield, CT, U.S.A.;</div></span></div><ul class="author-tooltip-find-more"><li class="author-tooltip-gs-link first"><a href="/lookup/google-scholar?link_type=googlescholar&gs_type=author&author%5B0%5D=SHELLEY%2BPHELAN%2B" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on Google Scholar</a></li><li class="author-tooltip-pubmed-link"><a href="/lookup/external-ref?access_num=PHELAN%20S&link_type=AUTHORSEARCH" target="_blank" class="" data-icon-position="" data-hide-link-title="0">Find this author on PubMed</a></li><li class="author-site-search-link"><a href="/search/author1%3ASHELLEY%2BPHELAN%2B" rel="nofollow" class="" data-icon-position="" data-hide-link-title="0">Search for this author on this site</a></li><li class="author-corresp-email-link last"><span>For correspondence: <a href="mailto:sphelan@fairfield.edu" class="" data-icon-position="" data-hide-link-title="0">sphelan@fairfield.edu</a></span></li></ul></div></div></div> </div> </div> </div> </div> </div> </div>  <div class="panel-row-wrapper clearfix"> <div class="main-content-wrapper grid-17 suffix-1 alpha"> <div class="panel-panel panel-region-content"> <div class="inside"><div class="panel-pane pane-highwire-panel-tabs pane-panels-ajax-tab-tabs" > <div class="pane-content"> <div class="item-list"><ul class="tabs inline panels-ajax-tab"><li class="first"><a href="/content/44/11/4751" class="panels-ajax-tab-tab" data-panel-name="jnl_iiar_tab_art" data-target-id="highwire_article_tabs" data-entity-context="node:81986" data-trigger="" data-url-enabled="1">Article</a><a href="/panels_ajax_tab/jnl_iiar_tab_art/node:81986/1" rel="nofollow" style="display:none" class="js-crawler-link"></a></li><li><a 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data-highwire-cite-ref-tooltip-instance="highwire_reflinks_tooltip" class="content-block-markup" xmlns:xhtml="http://www.w3.org/1999/xhtml"><div class="article fulltext-view "><span class="highwire-journal-article-marker-start"></span><div class="section abstract" id="abstract-1"><h2>Abstract</h2><p id="p-4">Background/Aim: The pomegranate fruit has been associated with a variety of human health benefits based on its antioxidant and anti-inflammatory properties. Punicic acid (PA) is an omega-5 long chain polyunsaturated fatty acid that constitutes approximately 65-80% of the oil from pomegranate seeds and has been found to possesses anti-cancer activity in various cancer types. To better understand its cell specificity, we investigated the effects of punicic acid on both the MCF-7 human breast cancer cell line as well as the non-cancerous MCF-10A breast epithelial line. Materials and Methods: We treated both cell types with different concentrations of punicic acid and measured viable cell density, cytotoxicity and apoptosis, as well as peroxiredoxin (Prdx) antioxidant expression. Results: We found that punicic acid was cytotoxic to both lines, but MCF-10A cells demonstrated higher levels of cytotoxicity and sensitivity to lower concentrations. We further demonstrated that cytotoxicity was associated with apoptosis in both lines. Using real time PCR, we demonstrated induction of all six Prdx mRNAs in MCF-7 cells, ranging from a 1.4-fold increase with 2 μg/ml, to over a 5-fold increase with 10 μg/ml. In stark contrast, MCF-10A cells exhibited considerably higher induction of all six Prdx mRNAs at 10μg/ml, exceeding a 30-fold induction of Prdx1, Prdx2 and Prdx5. Conclusion: Our data are the first to demonstrate differential cytotoxicity and Prdx regulation by punicic acid in these two cell lines. These results may provide important insight into cell-specific mechanisms of punicic acid in breast cancer.</p></div><span class="kwd-group-title">Key Words:</span><ul class="kwd-group"><li class="kwd"><a href="/keyword/breast-cancer" class="hw-term hw-article-keyword hw-article-keyword-breast-cancer" rel="nofollow">Breast cancer</a></li><li class="kwd"><a href="/keyword/peroxiredoxin" class="hw-term hw-article-keyword hw-article-keyword-peroxiredoxin" rel="nofollow">peroxiredoxin</a></li><li class="kwd"><a href="/keyword/mcf-7" class="hw-term hw-article-keyword hw-article-keyword-mcf-7" rel="nofollow">MCF-7</a></li><li class="kwd"><a href="/keyword/mcf-10a" class="hw-term hw-article-keyword hw-article-keyword-mcf-10a" rel="nofollow">MCF-10A</a></li><li class="kwd"><a href="/keyword/punicic-acid" class="hw-term hw-article-keyword hw-article-keyword-punicic-acid" rel="nofollow">punicic acid</a></li><li class="kwd"><a href="/keyword/pomegranate" class="hw-term hw-article-keyword hw-article-keyword-pomegranate" rel="nofollow">pomegranate</a></li></ul><p id="p-5">The pomegranate is an ancient fruit known for its many health benefits, some of which include antioxidant, antitumoral, antimicrobial, and anti-inflammatory properties. The pomegranate contains valuable compounds in each component of the fruit, including the seeds, peel, arils, and juice (<a id="xref-ref-1-1" class="xref-bibr" href="#ref-1">1</a>). Chemicals including hydrolysable tannins, condensed tannins, flavonols, anthocyanins, and phenolic and organic acids are linked to its health benefits (<a id="xref-ref-1-2" class="xref-bibr" href="#ref-1">1</a>, <a id="xref-ref-2-1" class="xref-bibr" href="#ref-2">2</a>). In particular, the anti-inflammatory and antioxidant properties of the pomegranate fruit have been demonstrated in both <em>in vivo</em> and <em>in vitro</em> studies and have been attributed to the bioactive polyphenols of the fruit (<a id="xref-ref-2-2" class="xref-bibr" href="#ref-2">2</a>). Pomegranate juice and its components have been used for the prevention of diseases, such as obesity and diabetes, by inhibiting α-glucosidase activity <em>in vitro</em> and reducing starch digestibility under simulated gastrointestinal conditions (<a id="xref-ref-3-1" class="xref-bibr" href="#ref-3">3</a>). In addition to its effect on metabolic disease, consumption of pomegranate has been associated with slowed progression of neurodegenerative diseases such as Alzheimer’s disease (<a id="xref-ref-4-1" class="xref-bibr" href="#ref-4">4</a>), and <em>in vitro</em> studies show that pomegranate and its derivates inhibit neuroinflammation, amyloidogenesis and lipopolysaccharide-induced memory impairment in different models (<a id="xref-ref-5-1" class="xref-bibr" href="#ref-5">5</a>-<a id="xref-ref-7-1" class="xref-bibr" href="#ref-7">7</a>).</p><p id="p-6">There is substantial evidence that the pomegranate also possesses anticancer activity. Pomegranate and its active constituents have been linked to preventive activity for multiple cancers including breast, prostate, colorectal, leukemia, bladder cancer, glioblastoma, and hepatocellular carcinoma (<a id="xref-ref-8-1" class="xref-bibr" href="#ref-8">8</a>, <a id="xref-ref-9-1" class="xref-bibr" href="#ref-9">9</a>). One study demonstrated that polyphenols from pomegranate are hydrolyzed in the gut to urolithin A, which dose-dependently inhibits proliferation in two human prostate cancer cell lines (<a id="xref-ref-10-1" class="xref-bibr" href="#ref-10">10</a>). Evidence for a beneficial effect of pomegranate compounds in breast cancer is growing (<a id="xref-ref-11-1" class="xref-bibr" href="#ref-11">11</a>, <a id="xref-ref-12-1" class="xref-bibr" href="#ref-12">12</a>), with several studies showing anti-proliferative or cytotoxic function in animal studies or cell lines (<a id="xref-ref-13-1" class="xref-bibr" href="#ref-13">13</a>, <a id="xref-ref-14-1" class="xref-bibr" href="#ref-14">14</a>). Of note, pomegranate extract also was shown to disrupt cellular replication and induce apoptosis of MCF-7 breast cancer cells (<a id="xref-ref-15-1" class="xref-bibr" href="#ref-15">15</a>).</p><p id="p-7">The most abundant conjugated fatty acid found in pomegranate seed is punicic acid, an omega-5 long chain polyunsaturated fatty acid that constitutes approximately 65-80% of the oil from pomegranate seed (<a id="xref-ref-16-1" class="xref-bibr" href="#ref-16">16</a>). While many studies have examined whole pomegranate seed extract, some research has been able to attribute particular properties to punicic acid (<a id="xref-ref-17-1" class="xref-bibr" href="#ref-17">17</a>). With respect to anticancer activity in breast cancer, punicic acid has been found to prevent proliferation and induce cytotoxicity in MCF-7 mammary cancer cells and increase apoptosis of the MDA-MB-435 human breast cancer cells (<a id="xref-ref-18-1" class="xref-bibr" href="#ref-18">18</a>, <a id="xref-ref-19-1" class="xref-bibr" href="#ref-19">19</a>). A recent study from our own lab also showed that punicic acid can induce cytotoxicity in the MCF-7 cell line (<a id="xref-ref-20-1" class="xref-bibr" href="#ref-20">20</a>), supporting a specific anti-cancer effect of punicic acid in these cells.</p><p id="p-8">Antioxidant genes play an important role in cancer cell survival, and many have been shown to be elevated in cancer cells. Since intracellular reactive oxygen species (ROS) are generated at high levels in cancer cells, overexpression of antioxidants is thought to provide protection against what would otherwise be ROS-induced toxicity. One highly conserved family of antioxidant genes elevated in cancer cells are the Peroxiredoxins (Prdx’s), which encode thiol specific antioxidant proteins that enable cancer cells to survive and proliferate under oxidative stress (<a id="xref-ref-21-1" class="xref-bibr" href="#ref-21">21</a>). Previous work from our lab has demonstrated elevated Prdx expression in breast tumor tissue, as compared to adjacent non-tumor breast tissue, in most patients analyzed (<a id="xref-ref-22-1" class="xref-bibr" href="#ref-22">22</a>). We also reported that the MCF-7 human breast cancer cells express significantly higher levels of five of the six Prdx genes as compared to MCF-10A cells (<a id="xref-ref-23-1" class="xref-bibr" href="#ref-23">23</a>). Furthermore, our lab reported that suppression of several Prdx genes in MCF-7 cells increases susceptibility to doxorubicin-induced cell death, supporting a protective role for these proteins in these cancer cells (<a id="xref-ref-24-1" class="xref-bibr" href="#ref-24">24</a>).</p><p id="p-9">While the mechanism of action of punicic acid is not well understood, a recent study from our lab demonstrated that after 24 hours of treatment, punicic-acid induced cytotoxicity in MCF-7 cells was associated with an increase in Prdx1 and Prdx6 expression (<a id="xref-ref-20-2" class="xref-bibr" href="#ref-20">20</a>), suggesting a possible role for these proteins in the cellular response to punicic acid. However, the effect of punicic acid on cytotoxicity and peroxiredoxin expression in MCF-10A cells has not yet been reported. Considering that prior research has shown evidence that supports the potential usage of punicic acid/pomegranates as a cancer remedy, it is important to consider the effect of the compound on healthy MCF-10A cells. In order to better understand the cell-specific effects of punicic acid and its therapeutic potential, we sought to measure and compare cell density, cytotoxicity and peroxiredoxin gene expression between the MCF-7 breast cancer cells and the MCF-10A breast epithelial cells. This work has pertinent implications in the field of natural products as a therapeutic option for cancer treatment.</p><div class="section materials-methods" id="sec-1"><h2 class="">Materials and Methods</h2><p id="p-10"><em>Cell lines and treatments</em>. Two cell lines were used for these studies. The human MCF-7 breast cancer cell line and MCF-10A non-cancerous breast epithelial cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 cells were cultured in EMEM media containing 5% fetal bovine serum (ATCC catalog # 30-2020) and 50 ng/ml insulin. MCF-10A cells were cultured in MEBM with the MEGM bullet kit supplements (ATCC). Both lines were grown and maintained at 37°C in a humidified 5% CO<sub>2</sub> incubator. Punicic Acid was purchased from Cayman Chemicals, Ann Arbor, MI, USA as a 10mg/ml stock dissolved in methanol and dissolved in 70% ethanol to make 0.4, 2 and 10 mg/ml stock solutions. For all cell experiments, viable cells were quantified by hemacytometer using a trypan blue exclusion assay. Viable cells were seeded into appropriate well plates for each assay and treated with equal volumes of stock solutions for final treatment concentrations of 2, 10 or 50 μg/ml punicic acid. An equal volume of 70% ethanol was used as a control treatment for all experiments. All stock punicic acid solutions were stored at −20°C and protected from light.</p><p id="p-11"><em>Analysis of cell morphology</em>. Cells were plated at a density of 1.0×10<sup>4</sup> cells per well in a 96-well plate in replicates of 6-10. Cells were treated with punicic acid or ethanol control, and they were monitored daily. At 72 hours post treatment, cells were imaged at 100X using phase contrast microscopy.</p><p id="p-12"><em>Measurement of viable cell density</em>. Cells were plated at a density of 1.0×10<sup>4</sup> cells per well in a 96-well plate in replicates of 6-8. They were treated with punicic acid or ethanol control. At 72 hours post treatment, media was removed, and cells were assayed for MTS conversion using the MTS Cell Proliferation assay (Cayman Chemicals) with final absorbance measured at 490 nm. Values were normalized to the control average, and averages for each treatment were calculated.</p><p id="p-13"><em>Measurement of cytotoxicity</em>. Cells were plated at a density of 1.0×10<sup>4</sup> cells per well in a 96-well plate in replicates of six. Cells were treated with punicic acid or ethanol control. At 72 hours post treatment, 50 μl of media was removed and measured for LDH levels using the colorimetric Cyquant Lactate Dehydrogenase (LDH) Assay (Thermo Fisher Scientific, Waltham, MA, USA), which quantifies cellular cytotoxicity by measuring the release of LDH into cell culture media when the plasma membrane is damaged. Per protocol instructions, specific absorbance was measured at 490 nm and background measured at 680. Specific LDH levels were calculated by subtracting A680 from A490. Values were normalized to control average and averages for each treatment were calculated.</p><p id="p-14"><em>Measurement of apoptosis</em>. Cells were plated at a density of 1.0×10<sup>4</sup> cells per well in a 96-well plate in replicates of three. Cells were treated with punicic acid or ethanol control. At 72 hours post treatment, cells were assayed for apoptosis using the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen, Carlsbad, CA, USA). Briefly, cells were incubated with the CellEvent Caspase 3/7 green reagent, a short peptide that fluoresces green when caspase 3/7 is active, and counterstained with the Sytox AAdvanced Dead Cell reagent (Thermo Fisher Scientific), a nucleic acid dead cell stain, which fluoresces red. Assayed cells were imaged with phase contrast microscopy and fluorescence microscopy for green and red fluorescence.</p><p id="p-15"><em>Measurement of peroxiredoxin expression</em>. To measure peroxiredoxin expression, cells were plated at a density of 1×10<sup>4</sup> cells/well in a 96-well plate in replicates of three. Cells were treated with punicic acid or ethanol for 48 h and subsequently lysed and RNA was isolated using the Cells-to-CT kit (Applied Biosystems, Foster City, CA, USA). RNA was reverse transcribed with Reverse Transcriptase enzyme and buffer (Applied Biosystems) and Taqman Real Time PCR Assays were conducted using Applied Biosystems primer/probes specific to each of the six mammalian peroxiredoxin genes. These included human-specific TaqMan assays for Prdx1 (Hs00602020), Prdx2 (Hs03044902), Prdx3 (Hs00428953), Prdx4 (Hs00197384), Prdx5 (Hs00738905) and Prdx6 (Hs00705355). An 18S rRNA primer/probe (Applied Biosystems cat#4333760F) was used as an endogenous control for all RT reactions. All reactions were set up in triplicate and were used to calculate relative quantification (RQ).</p><p id="p-16"><em>Statistical analysis</em>. For each experiment, the mean of each treatment group was statistically compared to the control group using a two-tailed student’s <em>t</em>-test, assuming equal variances. <em>p</em><0.05 was used for statistical significance.</p></div><div class="section results" id="sec-2"><h2 class="">Results</h2><p id="p-17">We first sought to examine the effects of punicic acid on the growth of MCF-7 and MCF-10A cells. To do this, we treated both cell lines with three different concentrations of punicic acid (2, 10, and 50 μg/ml), or ethanol control, for 72 h. Cell morphology was examined using phase contrast microscopy. As shown in <a id="xref-fig-1-1" class="xref-fig" href="#F1">Figure 1</a>, the 2 μg/ml dose of punicic acid had no noticeable effect on the morphology of either cell type. At 10 μg/ml, we continued to see no significant change in the MCF-7 cell morphology, but the MCF-10A cells had lost their elongated morphology and become small and spherical, suggesting a significant effect on the health of the cells. After 72 h of 50 μg/ml punicic acid treatment, the MCF-7 cells had lost their regular morphology and were completely rounded up, while the MCF-10 cells were no longer visible, suggesting total cell death.</p><div id="F1" class="fig pos-float odd"><div class="highwire-figure"><div class="fig-inline-img-wrapper"><div class="fig-inline-img"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F1.large.jpg?width=800&height=600&carousel=1" title="Effect of punicic acid on cell morphology in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 h. Cell morphology was assessed using phase contrast microscopy. Representative images are shown at 200× (Scale bar is 100 mm)." class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1047684150" data-figure-caption="<div class="highwire-markup">Effect of punicic acid on cell morphology in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 h. Cell morphology was assessed using phase contrast microscopy. Representative images are shown at 200× (Scale bar is 100 mm).</div>" data-icon-position="" data-hide-link-title="0"><span class="hw-responsive-img"><img class="highwire-fragment fragment-image lazyload" alt="Figure 1." src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" data-src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F1.medium.gif" width="440" height="179"/><noscript><img class="highwire-fragment fragment-image" alt="Figure 1." src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F1.medium.gif" width="440" height="179"/></noscript></span></a></div></div><ul class="highwire-figure-links inline"><li class="download-fig first"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F1.large.jpg?download=true" class="highwire-figure-link highwire-figure-link-download" title="Download Figure 1." data-icon-position="" data-hide-link-title="0">Download figure</a></li><li class="new-tab"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F1.large.jpg" class="highwire-figure-link highwire-figure-link-newtab" target="_blank" data-icon-position="" data-hide-link-title="0">Open in new tab</a></li><li class="download-ppt last"><a href="/highwire/powerpoint/82085" class="highwire-figure-link highwire-figure-link-ppt" data-icon-position="" data-hide-link-title="0">Download powerpoint</a></li></ul></div><div class="fig-caption" xmlns:xhtml="http://www.w3.org/1999/xhtml"><span class="fig-label">Figure 1.</span> <p id="p-18" class="first-child">Effect of punicic acid on cell morphology in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 h. Cell morphology was assessed using phase contrast microscopy. Representative images are shown at 200× (Scale bar is 100 mm).</p><div class="sb-div caption-clear"></div></div></div><div id="F2" class="fig pos-float odd"><div class="highwire-figure"><div class="fig-inline-img-wrapper"><div class="fig-inline-img"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F2.large.jpg?width=800&height=600&carousel=1" title="Effect of punicic acid on viable cell density in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 hours. Viable cell density was assessed using a dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium (MTS) assay and absorbances were measured at 490 nm, blank-corrected and normalized to ethanol control levels for each cell line. Averages of replicates are shown. Error bars indicate standard deviation. *p" class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1047684150" data-figure-caption="<div class="highwire-markup"><div xmlns="http://www.w3.org/1999/xhtml">Effect of punicic acid on viable cell density in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 hours. Viable cell density was assessed using a dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium (MTS) assay and absorbances were measured at 490 nm, blank-corrected and normalized to ethanol control levels for each cell line. Averages of replicates are shown. Error bars indicate standard deviation. *p<0.05, <sup>#</sup>p<0.001 using Student’s t-tests to compare each treatment group to the control for each cell line.</div></div>" data-icon-position="" data-hide-link-title="0"><span class="hw-responsive-img"><img class="highwire-fragment fragment-image lazyload" alt="Figure 2." src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" data-src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F2.medium.gif" width="440" height="288"/><noscript><img class="highwire-fragment fragment-image" alt="Figure 2." src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F2.medium.gif" width="440" height="288"/></noscript></span></a></div></div><ul class="highwire-figure-links inline"><li class="download-fig first"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F2.large.jpg?download=true" class="highwire-figure-link highwire-figure-link-download" title="Download Figure 2." data-icon-position="" data-hide-link-title="0">Download figure</a></li><li class="new-tab"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F2.large.jpg" class="highwire-figure-link highwire-figure-link-newtab" target="_blank" data-icon-position="" data-hide-link-title="0">Open in new tab</a></li><li class="download-ppt last"><a href="/highwire/powerpoint/82200" class="highwire-figure-link highwire-figure-link-ppt" data-icon-position="" data-hide-link-title="0">Download powerpoint</a></li></ul></div><div class="fig-caption"><span class="fig-label">Figure 2.</span> <p id="p-19" class="first-child">Effect of punicic acid on viable cell density in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 hours. Viable cell density was assessed using a dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium (MTS) assay and absorbances were measured at 490 nm, blank-corrected and normalized to ethanol control levels for each cell line. Averages of replicates are shown. Error bars indicate standard deviation. *p<0.05, <sup>#</sup>p<0.001 using Student’s t-tests to compare each treatment group to the control for each cell line.</p><div class="sb-div caption-clear"></div></div></div><p id="p-20">To gain a better understanding of the cellular effects of punicic acid on the two lines, we measured viable cell density after 72 h using an MTS assay. As shown in <a id="xref-fig-2-1" class="xref-fig" href="#F2">Figure 2</a>, we saw a slight effect on MCF-7 cells at the lowest concentration, and this dropped to approximately 30% and 20% viable cell density (compared to control) at 10 and 50 μg/ml punicic acid, respectively. In contrast, we found a very clear dose-dependent decrease in viable cell density in the MCF-10A cell line, with a reduction to about 50% at the 2 μg/ml dose, and a reduction to below 20% viable cell density at 10 and 50 μg/ml. These data show that MCF-10A cells are more sensitive to punicic acid treatment than MCF-7 cells.</p><p id="p-21">Since a reduction in viable cell density could be due to inhibition of proliferation, induction of cell death, or both, we wanted to measure cytotoxicity of punicic acid in both lines. After 72 h of treatment, cytotoxicity was measured using an LDH release assay. As shown in <a id="xref-fig-3-1" class="xref-fig" href="#F3">Figure 3</a>, we found no measurable cytotoxicity at the two lower concentrations, but we observed an approximately 40% increase in relative released LDH with the 50 μg/ml treatment. In contrast, we found significantly higher cytotoxicity in the MCF-10A cells at the 10 and 50 μg/ml doses, with over a 100% increase in both treatments. Together, these data show that both lines exhibit cytotoxicity in response to punicic acid, but with MCF-10A cells showing more susceptibility.</p><div id="F3" class="fig pos-float odd"><div class="highwire-figure"><div class="fig-inline-img-wrapper"><div class="fig-inline-img"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F3.large.jpg?width=800&height=600&carousel=1" title="Effect of punicic acid on cytotoxicity in MCF-7 and MCF-10A cells. MCF-7 and MCF-10A cells were plated at equal densities and were treated with control or the indicated concentrations of punicic acid and incubated for 72 h. Media was collected after 72 h and assayed for released lactate dehydrogenase (LDH) to measure cytotoxicity. Absorbances were blank-subtracted and normalized to the control levels for each cell line. Averages from replicates are shown. Error bars indicate standard deviation. *p" class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1047684150" data-figure-caption="<div class="highwire-markup">Effect of punicic acid on cytotoxicity in MCF-7 and MCF-10A cells. MCF-7 and MCF-10A cells were plated at equal densities and were treated with control or the indicated concentrations of punicic acid and incubated for 72 h. Media was collected after 72 h and assayed for released lactate dehydrogenase (LDH) to measure cytotoxicity. Absorbances were blank-subtracted and normalized to the control levels for each cell line. Averages from replicates are shown. Error bars indicate standard deviation. *p<0.05, **p<0.005 and #p<0.001 using student’s t-tests to compare each treatment group to the control for each cell line.</div>" data-icon-position="" data-hide-link-title="0"><span class="hw-responsive-img"><img class="highwire-fragment fragment-image lazyload" alt="Figure 3." src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" data-src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F3.medium.gif" width="440" height="311"/><noscript><img class="highwire-fragment fragment-image" alt="Figure 3." src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F3.medium.gif" width="440" height="311"/></noscript></span></a></div></div><ul class="highwire-figure-links inline"><li class="download-fig first"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F3.large.jpg?download=true" class="highwire-figure-link highwire-figure-link-download" title="Download Figure 3." data-icon-position="" data-hide-link-title="0">Download figure</a></li><li class="new-tab"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F3.large.jpg" class="highwire-figure-link highwire-figure-link-newtab" target="_blank" data-icon-position="" data-hide-link-title="0">Open in new tab</a></li><li class="download-ppt last"><a href="/highwire/powerpoint/82246" class="highwire-figure-link highwire-figure-link-ppt" data-icon-position="" data-hide-link-title="0">Download powerpoint</a></li></ul></div><div class="fig-caption"><span class="fig-label">Figure 3.</span> <p id="p-22" class="first-child">Effect of punicic acid on cytotoxicity in MCF-7 and MCF-10A cells. MCF-7 and MCF-10A cells were plated at equal densities and were treated with control or the indicated concentrations of punicic acid and incubated for 72 h. Media was collected after 72 h and assayed for released lactate dehydrogenase (LDH) to measure cytotoxicity. Absorbances were blank-subtracted and normalized to the control levels for each cell line. Averages from replicates are shown. Error bars indicate standard deviation. *p<0.05, **p<0.005 and #p<0.001 using student’s t-tests to compare each treatment group to the control for each cell line.</p><div class="sb-div caption-clear"></div></div></div><p id="p-23">We sought to further explore cell death by measuring apoptosis in our experiments. We used a caspase 3/7 assay to measure apoptosis after 72 h of treatment at the 10 and 50 μg/ml doses, since those were the doses that induced cell death. As shown in <a id="xref-fig-4-1" class="xref-fig" href="#F4">Figure 4</a>, the MCF-7 cells exhibited detectable but low levels of apoptosis in the 10 μg/ml treatment, and significantly higher levels with 50 μg/ml punicic acid, with all cells that were positive for the dead cell staining also staining positive for apoptosis. Likewise, we observed apoptosis of MCF-10A cells at both concentrations, but levels were markedly higher than MCF-7 cells. All dead cells were also apoptotic in this line. These data reveal that punicic acid induces apoptosis in both lines, with a stronger effect on MCF-10A cells consistent with our cytotoxicity results.</p><div id="F4" class="fig pos-float odd"><div class="highwire-figure"><div class="fig-inline-img-wrapper"><div class="fig-inline-img"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F4.large.jpg?width=800&height=600&carousel=1" title="Effect of punicic acid on apoptosis in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 h. Cells were assayed for apoptosis using the Caspase 3/7 CellEvent assay which includes a green, fluorescent dye for apoptotic cells, and a red fluorescent dye for dead cells. Assayed cells were imaged with phase contrast microscopy and fluorescence microscopy for green (apoptotic cells) and red (dead cells) fluorescence. Representative images for MCF-7 (A) and MCF-10A (B) are shown at 200×. Scale bar is 100 mm." class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1047684150" data-figure-caption="<div class="highwire-markup">Effect of punicic acid on apoptosis in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 h. Cells were assayed for apoptosis using the Caspase 3/7 CellEvent assay which includes a green, fluorescent dye for apoptotic cells, and a red fluorescent dye for dead cells. Assayed cells were imaged with phase contrast microscopy and fluorescence microscopy for green (apoptotic cells) and red (dead cells) fluorescence. Representative images for MCF-7 (A) and MCF-10A (B) are shown at 200×. Scale bar is 100 mm.</div>" data-icon-position="" data-hide-link-title="0"><span class="hw-responsive-img"><img class="highwire-fragment fragment-image lazyload" alt="Figure 4." src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" data-src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F4.medium.gif" width="304" height="440"/><noscript><img class="highwire-fragment fragment-image" alt="Figure 4." src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F4.medium.gif" width="304" height="440"/></noscript></span></a></div></div><ul class="highwire-figure-links inline"><li class="download-fig first"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F4.large.jpg?download=true" class="highwire-figure-link highwire-figure-link-download" title="Download Figure 4." data-icon-position="" data-hide-link-title="0">Download figure</a></li><li class="new-tab"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F4.large.jpg" class="highwire-figure-link highwire-figure-link-newtab" target="_blank" data-icon-position="" data-hide-link-title="0">Open in new tab</a></li><li class="download-ppt last"><a href="/highwire/powerpoint/82104" class="highwire-figure-link highwire-figure-link-ppt" data-icon-position="" data-hide-link-title="0">Download powerpoint</a></li></ul></div><div class="fig-caption"><span class="fig-label">Figure 4.</span> <p id="p-24" class="first-child">Effect of punicic acid on apoptosis in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 72 h. Cells were assayed for apoptosis using the Caspase 3/7 CellEvent assay which includes a green, fluorescent dye for apoptotic cells, and a red fluorescent dye for dead cells. Assayed cells were imaged with phase contrast microscopy and fluorescence microscopy for green (apoptotic cells) and red (dead cells) fluorescence. Representative images for MCF-7 (A) and MCF-10A (B) are shown at 200×. Scale bar is 100 mm.</p><div class="sb-div caption-clear"></div></div></div><p id="p-25">Based on our observed differences in cell responses to punicic acid, we were interested in whether antioxidant gene regulation also may be differentially regulated by punicic acid in these two lines. We measured mRNA expression of all six peroxiredoxin genes after 48 h of punicic acid treatment. Our results are shown in <a id="xref-fig-5-1" class="xref-fig" href="#F5">Figure 5</a>. In the MCF7 cell line, we found an increase in all six peroxiredoxins with the low concentration of punicic acid relative to control (<a id="xref-fig-5-2" class="xref-fig" href="#F5">Figure 5A</a>). These inductions ranged from 1.4X for Prdx1, Prdx3, Prdx5 and Prdx6, to 1.7X for Prdx2, and 1.6X for Prdx4. In the 10 μg/ml treatment, we found a more substantial elevation in several Prdx genes. These ranged from 3X-5X for Prdx1, Prdx2, Prdx4 and Prdx6, while Prdx3 mRNA increased by 1.9X. Interestingly, the 10 μg/ml concentration actually led to a decrease in Prdx5 mRNA to approximately half of control levels.</p><div id="F5" class="fig pos-float odd"><div class="highwire-figure"><div class="fig-inline-img-wrapper"><div class="fig-inline-img"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F5.large.jpg?width=800&height=600&carousel=1" title="Regulation of peroxiredoxins by punicic acid in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 48 h. qPCR was used to measure mRNA expression for Prdx1-Prdx6. Average Relative Quantification (RQ) values from triplicate reactions for MCF-7 cells (A) and MCF-10A cells (B) are shown, with numerical values indicated. Error bars indicate standard deviation. *p" class="highwire-fragment fragment-images colorbox-load" rel="gallery-fragment-images-1047684150" data-figure-caption="<div class="highwire-markup"><div xmlns="http://www.w3.org/1999/xhtml">Regulation of peroxiredoxins by punicic acid in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 48 h. qPCR was used to measure mRNA expression for Prdx1-Prdx6. Average Relative Quantification (RQ) values from triplicate reactions for MCF-7 cells (A) and MCF-10A cells (B) are shown, with numerical values indicated. Error bars indicate standard deviation. *p<0.05, **p<.005 and <sup>#</sup>p<0.001 using student’s t-tests to compare each treatment group to the control group for each gene.</div></div>" data-icon-position="" data-hide-link-title="0"><span class="hw-responsive-img"><img class="highwire-fragment fragment-image lazyload" alt="Figure 5." src="data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7" data-src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F5.medium.gif" width="362" height="440"/><noscript><img class="highwire-fragment fragment-image" alt="Figure 5." src="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F5.medium.gif" width="362" height="440"/></noscript></span></a></div></div><ul class="highwire-figure-links inline"><li class="download-fig first"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F5.large.jpg?download=true" class="highwire-figure-link highwire-figure-link-download" title="Download Figure 5." data-icon-position="" data-hide-link-title="0">Download figure</a></li><li class="new-tab"><a href="https://ar.iiarjournals.org/content/anticanres/44/11/4751/F5.large.jpg" class="highwire-figure-link highwire-figure-link-newtab" target="_blank" data-icon-position="" data-hide-link-title="0">Open in new tab</a></li><li class="download-ppt last"><a href="/highwire/powerpoint/82266" class="highwire-figure-link highwire-figure-link-ppt" data-icon-position="" data-hide-link-title="0">Download powerpoint</a></li></ul></div><div class="fig-caption"><span class="fig-label">Figure 5.</span> <p id="p-26" class="first-child">Regulation of peroxiredoxins by punicic acid in MCF-7 and MCF-10A cells. Cells were treated with ethanol or the indicated concentrations of punicic acid and incubated for 48 h. qPCR was used to measure mRNA expression for Prdx1-Prdx6. Average Relative Quantification (RQ) values from triplicate reactions for MCF-7 cells (A) and MCF-10A cells (B) are shown, with numerical values indicated. Error bars indicate standard deviation. *p<0.05, **p<.005 and <sup>#</sup>p<0.001 using student’s t-tests to compare each treatment group to the control group for each gene.</p><div class="sb-div caption-clear"></div></div></div><p id="p-27">Interestingly, we found a very different peroxiredoxin response to punicic acid in the MCF-10A cells (<a id="xref-fig-5-3" class="xref-fig" href="#F5">Figure 5B</a>). We noted two distinct differences in patterns in this cell line. First, the low concentration resulted in little to no effect on Prdx levels in the MCF-10 line, and they were more gene specific. At this concentration, Prdx1 and Prdx2 expression did not change, while Prdx3, Prdx4 and Prdx6 were slightly lower than control levels (at 74%, 61% and 78%, respectively). The only Prdx that was induced by this treatment was Prdx5, which was 1.34X higher than the control. In contrast, 10 μg/ml punicic acid caused a profound induction of all six peroxiredoxins, with a magnitude of induction far exceeding that found in MCF-7 cells. The fold induction for the six peroxiredoxins were 37.4X for Prdx1, 32.2X for Prdx2, 9.3X for Prdx3, 8.2X for Prdx4, 70.2X for Prdx5, and 12.9X for Prdx6.</p></div><div class="section discussion" id="sec-3"><h2 class="">Discussion</h2><p id="p-28">In this study, we compared the effect of punicic acid treatment on the MCF-7 and MCF-10A cell lines. We found that both cell lines were susceptible to punicic acid, but that the MCF-7 cancer cells were more resistant to punicic acid-induced cytotoxicity than the non-cancerous MCF-10A cells. MCF-10A cells exhibited lower viability, higher cytotoxicity and higher levels of apoptosis than MCF-7 cells. Examination of peroxiredoxin expression in both lines revealed a modest and dose-dependent induction of all six Prdx mRNAs in MCF-7 cells, and a nearly ten-fold higher induction of all six Prdx mRNAs observed in MCF-10A cells at the higher concentration. These data demonstrate unique cell-specific effects on the two lines and suggest that antioxidant gene regulation may be an important downstream effect of punicic acid in a cell-specific manner.</p><p id="p-29">Our findings that MCF-7 cells are susceptible to punicic acid at a high concentration are consistent with other reports and support possible growth-inhibitory and apoptotic activity of punicic acid in the cancer line. Our previous study also found that a high concentration of punicic acid reduces viable cell density and increases cytotoxicity in these cells (<a id="xref-ref-20-3" class="xref-bibr" href="#ref-20">20</a>), and Shirode <em>et al.</em> reported an anti-proliferative effect of punicic acid on MCF-7 cells (<a id="xref-ref-15-2" class="xref-bibr" href="#ref-15">15</a>). Although most of the published research has used pomegranate extract to examine its biological properties on mammalian cells, a 2011 study examining the effect of pomegranate fruit peel extract also found a decrease in cell proliferation and increase in apoptosis in MCF-7 cells (<a id="xref-ref-16-2" class="xref-bibr" href="#ref-16">16</a>). In contrast to our observations in MCF-7 cells, our data demonstrating that the MCF-10A non-cancerous breast epithelial cell line exhibited much higher susceptibility to punicic acid is the first comparative study of the two lines. Since many proliferation and apoptotic signaling pathways are altered in MCF-7 cells, the different cellular responses may be a product of these altered pathways. This new data is important as the effect of punicic acid on normal tissue should be considered in the context of its therapeutic potential for cancer treatment. The differential effects of punicic acid on these two lines reveals a significant difference in the cellular responses and may suggest that punicic acid has more viability as a preventative agent than a treatment.</p><p id="p-30">While the mechanism of action of punicic acid these cells is not known, some insight may come from studies in other cell lines. One study reported that punicic acid-induced cytotoxicity in prostate cancer cells was associated with DNA fragmentation after just 24 hours of treatment (<a id="xref-ref-25-1" class="xref-bibr" href="#ref-25">25</a>). Grossman <em>et al.</em> also showed that punicic acid-induced proliferation, inhibition, and apoptosis in MDA-MB-231 and MDA-ERα7 cells was dependent on lipid peroxidation and the PKC pathway (<a id="xref-ref-18-2" class="xref-bibr" href="#ref-18">18</a>). This is consistent with another study in carcinoma cells demonstrating that punicic acid induces lipid peroxidation and ferroptotic cell death in these cells (<a id="xref-ref-26-1" class="xref-bibr" href="#ref-26">26</a>). A 2011 study demonstrated that pomegranate extract induced cell cycle arrest and anti-tumor phenotypic changes in PANC-1 and AsPC-1 human pancreatic cancer cells (<a id="xref-ref-27-1" class="xref-bibr" href="#ref-27">27</a>). This study also tested specific phytochemicals within the pomegranate extract and concluded that ellagic acid, luteolin and ursolic acid played minimal roles in this effect, while punicic acid was not tested. A more recent <em>in vivo</em> study with pomegranate seed extract reported that pomegranate seed nano-emulsion in mice results in tumor inhibition and apoptosis, with induction of DNA damage and pro-apoptotic gene expression and a reduction in Bcl2 expression (<a id="xref-ref-28-1" class="xref-bibr" href="#ref-28">28</a>). Gafar <em>et al.</em> showed that treatment with punicic acid at 30 and 100 μM concentrations disrupted and damaged mitochondrial membrane and function within the prostate cancer lines LNCaP and its androgen independent counterpart PC-3 (<a id="xref-ref-29-1" class="xref-bibr" href="#ref-29">29</a>). Together, these data continue to support a potential anti-cancer role for punicic acid.</p><p id="p-31">There is very little known about the cellular pathways regulated by punicic acid in normal and cancerous breast tissue, or the mechanism by which punicic acid elicits its cytotoxic activity. An analysis of downstream gene expression is one way of revealing key pathways and targets of its action. Our observations of a moderate increase in Prdx expression in MCF-7 cells and a much more robust increase in MCF-10A cells further illustrates the cell-specific responses to punicic acid. Since punicic acid has been shown to alter signal transduction pathways, it is not surprising that it could induce differential regulation of antioxidant pathways in these two lines. An examination of key transcription factors that regulate peroxiredoxin activity in both cell lines would provide important insight into the mechanism of regulation. As to the role of Prdx upregulation in the differences in punicic acid-induced cytotoxicity between the two lines, this remains unknown. It is possible that Prdx upregulation in MCF-7 cells, which already possess elevated Prdx levels, is sufficient to protect these cells from cytotoxicity, whereas the excessive induction in MCF-10A cells may reveal their inability to withstand the elevated ROS or altered cell signaling. An investigation of the role of peroxiredoxin upregulation in the punicic acid induced cellular response in each line will be critical for our understanding of its potential therapeutic applications.</p></div><div class="section conclusion" id="sec-4"><h2 class="">Conclusion</h2><p id="p-32">In conclusion, our observations that the MCF-7 breast cancer cell line is susceptible to punicic acid at high concentrations but is significantly more resistant than the non-cancerous MCF-10A cells, support its role as an anti-cancer agent, but reveal potential toxicity to normal breast tissue. Since punicic acid is gaining interest for therapeutic potential, it will be important to further dissect the mechanism of action of punicic acid in normal and cancer cells, and better understand its efficacy in cancer-prevention and cancer treatment.</p></div><div class="section ack" id="ack-1"><h2 class="">Acknowledgements</h2><p id="p-37">This work was supported by the Department of Biology and the Science Institute at Fairfield University.</p></div><div class="section fn-group" id="fn-group-1"><h2>Footnotes</h2><ul><li class="fn" id="fn-1"><p id="p-1"><strong>Authors’ Contributions</strong></p><p id="p-2">F.G., H.K., and S.P. contributed to the study design. All authors contributed to the experimental studies. B.Q., C.E., and S.P. contributed to the data analysis. All Authors contributed to the manuscript writing and editing, and all Authors read and approved the final manuscript.</p></li><li class="fn" id="fn-2"><p id="p-33"><strong>Conflicts of Interest</strong></p><p id="p-34">There are no financial or non-financial conflicts of interest.</p></li><li class="fn" id="fn-3"><p id="p-35"><strong>Funding</strong></p><p id="p-36">This work was funded by the Department of Biology and the Science Institute at Fairfield University.</p></li></ul></div><ul class="history-list"><li xmlns:hwp="http://schema.highwire.org/Journal" class="received" hwp:start="2024-08-02"><span class="received-label">Received </span>August 2, 2024.</li><li xmlns:hwp="http://schema.highwire.org/Journal" class="rev-recd" hwp:start="2024-09-11"><span class="rev-recd-label">Revision received </span>September 11, 2024.</li><li xmlns:hwp="http://schema.highwire.org/Journal" class="accepted" hwp:start="2024-09-16"><span class="accepted-label">Accepted </span>September 16, 2024.</li></ul><ul class="copyright-statement"><li class="fn" id="copyright-statement-1">Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.</li></ul><div class="license" id="license-1"><p id="p-3">This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (<a href="https://creativecommons.org/licenses/by-nc-nd/4.0" rel="license">https://creativecommons.org/licenses/by-nc-nd/4.0</a>).</p></div><div class="section ref-list" id="ref-list-1"><h2 class="">References</h2><ol class="cit-list"><li><a class="rev-xref-ref" href="#xref-ref-1-1" title="View reference 1 in text" id="ref-1">↵</a><div class="cit ref-cit ref-journal" id="cit-44.11.4751.1" data-doi="10.1111/j.1541-4337.2010.00131.x"><div class="cit-metadata"><ol class="cit-auth-list"><li><span class="cit-auth"><span class="cit-name-surname">Viuda-Martos</span> <span class="cit-name-given-names">M</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Fernández-López</span> <span class="cit-name-given-names">J</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Pérez-Álvarez</span> <span class="cit-name-given-names">JA</span></span></li></ol><cite>: <span class="cit-article-title">Pomegranate and its many functional components as related to human health: a review</span>. <abbr class="cit-jnl-abbrev">Compr Rev Food Sci Food Saf</abbr> <span class="cit-vol">9</span>(<span class="cit-issue">6</span>): <span class="cit-fpage">635</span>-<span class="cit-lpage">654</span>, <span class="cit-pub-date">2010</span>.<span class="cit-pub-id-sep cit-pub-id-doi-sep"> </span><span class="cit-pub-id-scheme">DOI: </span><span class="cit-pub-id cit-pub-id-doi">10.1111/j.1541-4337.2010.00131.x</span></cite></div><div class="cit-extra"><a href="{openurl}?query=rft.jtitle%253DCompr%2BRev%2BFood%2BSci%2BFood%2BSaf%26rft.volume%253D9%26rft.spage%253D635%26rft_id%253Dinfo%253Adoi%252F10.1111%252Fj.1541-4337.2010.00131.x%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx" class="cit-ref-sprinkles cit-ref-sprinkles-openurl cit-ref-sprinkles-open-url"><span>OpenUrl</span></a><a href="/lookup/external-ref?access_num=10.1111/j.1541-4337.2010.00131.x&link_type=DOI" class="cit-ref-sprinkles cit-ref-sprinkles-doi cit-ref-sprinkles-crossref"><span>CrossRef</span></a></div></div></li><li><a class="rev-xref-ref" href="#xref-ref-2-1" title="View reference 2 in text" id="ref-2">↵</a><div class="cit ref-cit ref-book" id="cit-44.11.4751.2"><div class="cit-metadata"><ol class="duplicate"><li><span class="cit-ed"><span class="cit-name-surname">Preedy</span> <span class="cit-name-given-names">V</span></span></li></ol><ol class="cit-auth-list"><li><span class="cit-auth"><span class="cit-name-surname">Nuncio-Jáuregui</span> <span class="cit-name-given-names">N</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Calín-Sánchez</span> <span class="cit-name-given-names">Á</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Vázquez-Araújo</span> <span class="cit-name-given-names">L</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Pérez-López</span> <span class="cit-name-given-names">AJ</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Frutos-Fernández</span> <span class="cit-name-given-names">MJ</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Carbonell-Barrachina</span> <span class="cit-name-given-names">AA</span></span></li></ol><cite>: <span class="cit-chapter-title">Processing pomegranates for juice and impact on bioactive components. 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seed nano-emulsion in mice bearing solid Ehrlich carcinoma cells</span>. <abbr class="cit-jnl-abbrev">Sci Rep</abbr> <span class="cit-vol">13</span>(<span class="cit-issue">1</span>): <span class="cit-fpage">5525</span>, <span class="cit-pub-date">2023</span>.<span class="cit-pub-id-sep cit-pub-id-doi-sep"> </span><span class="cit-pub-id-scheme">DOI: </span><span class="cit-pub-id cit-pub-id-doi">10.1038/s41598-023-32488-6</span></cite></div><div class="cit-extra"><a href="{openurl}?query=rft.jtitle%253DSci%2BRep%26rft.volume%253D13%26rft.spage%253D5525%26rft_id%253Dinfo%253Adoi%252F10.1038%252Fs41598-023-32488-6%26rft_id%253Dinfo%253Apmid%252F37016062%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx" class="cit-ref-sprinkles cit-ref-sprinkles-openurl cit-ref-sprinkles-open-url"><span>OpenUrl</span></a><a 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class="cit-name-given-names">S</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Khalifa</span> <span class="cit-name-given-names">M</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Shair</span> <span class="cit-name-given-names">W</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Abu</span> <span class="cit-name-given-names">W</span></span>, </li><li><span class="cit-auth"><span class="cit-name-surname">Punicic</span> <span class="cit-name-given-names">S</span></span></li></ol><cite>: <span class="cit-article-title">Punicic and Jacaric acids induce Mitochondrial dysfunction in prostate cancer cell lines</span>. <abbr class="cit-jnl-abbrev">Life Sci J</abbr> <span class="cit-vol">13</span>(<span class="cit-issue">6</span>): <span class="cit-fpage">99</span>-<span class="cit-lpage">106</span>, <span class="cit-pub-date">2016</span>.<span class="cit-pub-id-sep cit-pub-id-doi-sep"> </span><span 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