Abstract
Several studies have demonstrated that polyphenolics from pomegranate (Punica granatum L.) are potent inhibitors of cancer cell proliferation and induce apoptosis, cell cycle arrest, and also decrease inflammation in vitro and vivo. There is growing evidence that botanicals exert their cytotoxic and anti-inflammatory activities, at least in part, by decreasing specificity protein (Sp) transcription factors. These are overexpressed in breast tumors and regulate genes important for cancer cell survival and inflammation such as the p65 unit of NF-κB. Moreover, previous studies have shown that Pg extracts decrease inflammation in lung cancer cell lines by inhibiting phosphatidylinositol-3,4,5-trisphosphate (PI3K)-dependent phosphorylation of AKT in vitro and inhibiting the activation of NF-kB in vivo. The objective of this study was to investigate the roles of miR-27a–ZBTB10–Sp and miR-155–SHIP-1–PI3K on the anti-inflammatory and cytotoxic activity of pomegranate extract. Pg extract (2.5-50 μg/ml) inhibited growth of BT-474 and MDA-MB-231 cells but not the non-cancer MCF-10F and MCF-12F cells. Pg extract significantly decreased Sp1, Sp3, and Sp4 as well as miR-27a in BT474 and MDA-MB-231 cells and increased expression of the transcriptional repressor ZBTB10. A significant decrease in Sp proteins and Sp-regulated genes was also observed. Pg extract also induced SHIP-1 expression and this was accompanied by downregulation of miRNA-155 and inhibition of PI3K-dependent phosphorylation of AKT. Similar results were observed in tumors from nude mice bearing BT474 cells as xenografts and treated with Pg extract. The effects of antagomirs and knockdown of SHIP-1 by RNA interference confirmed that the anti-inflammatory and cytotoxic effects of Pg extract were partly due to the disruption of both miR-27a–ZBTB10 and miR-155–SHIP-1. In summary, the anticancer activities of Pg extract in breast cancer cells were due in part to targeting microRNAs155 and 27a. Both pathways play an important role in the proliferative/inflammatory phenotype exhibited by these cell lines.
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Prakobwong SG, Gupta SC, Kim JH, Sung B, Pinlaor P, Hiraku Y, Wongkham S, Sripa B, Pinlaor S, Aggarwal BB (2011) Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways. Carcinogenesis 32:1372–1380
Kim ND, Mehta R, Yu W, Neeman I, Livney T, Amichay A, Poirier D, Nicholls P, Kirby A, Jiang W, Mansel R, Ramachandran C, Rabi T, Kaplan B, Lansky E (2002) Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast Cancer Res Treat 71:203–217
Khan N, Afaq F, Kweon MH, Kim K, Mukhtar H (2007) Oral consumption of pomegranate fruit extract inhibits growth and progression of primary lung tumors in mice. Cancer Res 67:3475–3482
Khan N, Hadi N, Afaq F, Syed DN, Kweon MH, Mukhtar H (2007) Pomegranate fruit extract inhibits prosurvival pathways in human A549 lung carcinoma cells and tumor growth in athymic nude mice. Carcinogenesis 1:163–173
Khan GN, Gorin MA, Rosenthal D, Pan Q, Bao LW, Wu ZF, Newman RA, Pawlus AD, Yang P, Lansky EP, Merajver SD (2009) Pomegranate fruit extract impairs invasion and motility in human breast cancer. Integr Cancer Ther 8:242–253
Jutooru I, Chadalapaka G, Lei P, Safe S (2010) Inhibition of NFkB and pancreatic cancer cell and tumor growth by curcumin is dependent on specificity protein down-regulation. J Biol Chem 285:25332–25344
Adams LS, Zhang Y, Seeram NP, Heber D, Chen S (2010) Pomegranate ellagitannin-derived compounds exhibit antiproliferative and anti-aromatase activity in breast cancer cells in vitro. Cancer Prev Res (Phila) 3:108–113
Hakimuddin F, Tiwari K, Paliyath G, Meckling K (2008) Grape and wine polyphenols down-regulate the expression of signal transduction genes and inhibit the growth of estrogen receptor-negative MDA-MB231 tumors in nu/nu mouse xenografts. Nutr Res 28:702–713
Chadalapaka G, Jutooru I, Chintharlapalli S, Papineni S, Smith R 3rd, Li X, Safe S (2008) Curcumin decreases specificity protein expression in bladder cancer cells. Cancer Res 68:5345–5354
Adhami VM, Khan N, Mukhtar H (2009) Cancer chemoprevention by pomegranate: laboratory and clinical evidence. Nutr Cancer 61:811–815
Adhami VM, Siddiqui IA, Syed DN, Lall RK, Mukhtar H (2012) Oral infusion of pomegranate fruit extract inhibits prostate carcinogenesis in the TRAMP model. Carcinogenesis 33:644–651
Kunnumakkara AB, Anand P, Aggarwal BB (2008) Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Lett 269:199–225
Khan N, Afaq F, Mukhtar H (2008) Cancer chemoprevention through dietary antioxidants: progress and promise. Antioxid Redox Signal 10:475–510
Kang NJ, Shin SH, Lee HJ, Lee KW (2011) Polyphenols as small molecular inhibitors of signaling cascades in carcinogenesis. Pharmacol Ther 130:310–324
Noratto GD, Kim Y, Talcott ST, Mertens-Talcott SU (2011) Flavonol-rich fractions of yaupon holly leaves (Ilex vomitoria, Aquifoliaceae) induce microRNA-146a and have anti-inflammatory and chemopreventive effects in intestinal myofibroblast CCD-18Co cells. Fitoterapia 82:557–569
Banerjee S, Kambhampati S, Haque I, Banerjee SK (2011) Pomegranate sensitizes Tamoxifen action in ER-α positive breast cancer cells. J Cell Commun Signal 5:317–324
Grossmann ME, Mizuno NK, Schuster T, Cleary MP (2010) Punicic acid is an omega-5 fatty acid capableof inhibiting breast cancer proliferation. Int J Oncol 36:421–426
Jurenka J (2008) Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev 13:128–144
Afaq F, Saleem M, Krueger CG, Reed JD, Mukhtar H (2005) Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-kB pathways and inhibits skin tumorigenesis in CD-1 mice. Int J Cancer 113:423–433
Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG, Heber D (2005) In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. J Nutr Biochem 16:360–367
Kawaii S, Lansky EP (2004) Differentiation-promoting activity of pomegranate (Punica granatum) fruit extracts in HL-60 human promyelocytic leukemia cells. J Med Food 7:13–18
Sreeja S, Santhosh Kumar TR, Lakshmi BS, Sreeja S (2011) Pomegranate extract demonstrate a selective estrogen receptor modulator profile in human tumor cell lines and in vivo models of estrogen deprivation. J Nutr Biochem 23:725–732
Bishayee A, Bhatia D, Thoppil RJ, Darvesh AS, Nevo E, Lansky EP (2011) Pomegranate-mediated chemoprevention of experimental hepatocarcinogenesis involves Nrf2-regulated antioxidant mechanisms. Carcinogenesis 32:888–896
Kohno H, Suzuki R, Yasui Y, Hosokawa M, Miyashita K, Tanaka T (2004) Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats. Cancer Sci 95:481–486
Malik A, Afaq F, Sarfaraz S, Adhami VM, Syed DN, Mukhtar H (2005) Pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer. Proc Natl Acad Sci USA 102:14813–14818
Kumar AP, Bhaskaran S, Ganapathy M, Crosby K, Davis MD, Kochunov P, Schoolfield J, Yeh IT, Troyer DA, Ghosh R (2007) Akt/cAMP-responsive element binding protein/cyclin D1 network: a novel target for prostate cancer inhibition in transgenic adenocarcinoma of mouse prostate model mediated by Nexrutine, a Phellodendron amurense bark extract. Clin Cancer Res 13:2784–2794
Wang L, Wei D, Huang S, Peng Z, Le X, Wu TT, Yao J, Ajani J, Xie K (2003) Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clin Cancer Res 9:6371–6380
Yao JC, Wang L, Wei D, Gong W, Hassan M, Wu TT, Mansfield P, Ajani J, Xie K (2004) Association between expression of transcription factor Sp1 and increased vascular endothelial growth factor expression, advanced stage, and poor survival in patients with resected gastric cancer. Clin Cancer Res 10:4109–4117
Hosoi Y, Watanabe T, Nakagawa K, Matsumoto Y, Enomoto A, Morita A, Nagawa H, Suzuki N (2004) Up-regulation of DNA-dependent protein kinase activity and Sp1 in colorectal cancer. Int J Oncol 25:461–468
Mertens-Talcott SU, Chintharlapalli S, Li X, Safe S (2007) The oncogenic microRNA-27a targets genes that regulate specificity protein transcription factors and the G2-M checkpoint in MDA-MB-231 breast cancer cells. Cancer Res 67:11001–11011
Mertens-Talcott SU, Noratto G, Li X, Angel-Morales G, Bertoldi MC, Safe S (2012) Betulinic acid decreases ER-negative breast cancer cell growth in vitro and in vivo: role of sp transcription factors and MicroRNA-27a:ZBTB10. Mol Carcinog 10. doi: 10.1002/mc.21893
Chintharlapalli S, Papineni S, Ramaiah SK, Safe S (2007) Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res 67:2816–2823
Chintharlapalli S, Papineni S, Abdelrahim M, Abudayyeh A, Jutooru I, Chadalapaka G, Wu F, Mertens-Talcott S, Vanderlaag K, Cho SD et al (2009) Oncogenic microRNA-27a is a target for anticancer agent methyl 2-cyano-3,11-dioxo-18 beta-olean-1,12-dien-30-oate in colon cancer cell. Int J Cancer 125:1965–1974
Chintharlapalli S, Papineni S, Lei P, Pathi S, Safe S (2011) Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors. BMC Cancer 11:371
Li X, Mertens-Talcott SU, Zhang S, Kim K, Ball J, Safe S (2010) MicroRNA-27a indirectly regulates estrogen receptor alpha expression and hormone responsiveness in MCF-7 breast cancer cells. Endocrinology 151:2462–2473
Weisburg JH, Schuck AG, Silverman MS, Ovits-Levy CG, Solodokin LJ, Zuckerbraun HL, Babich H (2010) Pomegranate extract, a prooxidant with antiproliferative and proapoptotic activities preferentially towards carcinoma cells. Anti-Cancer Agents Med Chem 10:634–643
Prasad S, Ravindran J, Aggarwal BB (2010) NF-kappaB and cancer: how intimate is this relationship. Mol Cell Biochem 336:25–37
Baran CP, Tridandapani S, Helgason CD, Humphries RK, Krystal G, Marsh CB (2003) The inositol 5′-phosphatase SHIP-1 and the Src kinase Lyn negatively regulate macrophage colony-stimulating factor-induced Akt activity. J Biol Chem 278:38628–38636
Chao X, Zao J, Xiao-Yi G, Li-Jun M, Tao S (2010) Blocking of PI3K/AKT induces apoptosis by its effect on NF-kappaB activity in gastric carcinoma cell line SGC7901. Biomed Pharmacother 64:600–604
Boesch-Saadatmandia C, Loboda A, Wagnera AE, Stachurskab A, Jozkowiczb A, Dulakb J, Döringa F, Wolfframc S, Rimbacha G (2011) Effect of quercetin and its metabolites isorhamnetin and quercetin-3-glucuronide on inflammatory gene expression: role of miR-155. J Nutr Biochem 22:293–299
Tili E, Michaille JJ, Adair B, Alder H, Limagne E, Taccioli C, Ferracin M, Delmas D, Latruffe N, Croce CM (2010) Resveratrol decreases the levels of miR-155 by upregulating miR-663, a microRNA targeting JunB and JunD. Carcinogenesis 31:1561–1566
Wagner AE, Boesch-Saadatmandi C, Dose J, Schultheiss G, Rimbach G (2012) Anti-inflammatory potential of allyl-isothiocyanate-role of Nrf2, NFκB and microRNA-155. J Cell Mol Med 16:836–843
Kleemann R, Verschuren L, Morrison M, Zadelaar S, van Erk MJ, Wielinga PY, Kooistra T (2011) Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models. Atherosclerosis 218:44–52
Bhattacharyya S, Balakathiresan N, Dalgard C, Gutti U, Armistead D, Jozwik C, Srivastava M, Pollard HB, Biswas R (2011) Elevated miR-155 promotes inflammation in cystic fibrosis by driving hyperexpression of interleukin-8. J Biol Chem 286:11604–11615
Gil MI, Tomás-Baberán FA, Hess-Pierce B, Holcroft DM, Kader AA (2000) Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 48:4581–4589
Brauns SC, Dealtry G, Milne P, Naude R, Van de Venter M (2005) Caspase-3 activation and induction of PARP cleavage by cyclic dipeptide Cyclo(Phe-Pro) in HT-29 cells. Anticancer Res 25:4197–4202
Ruan K, Fang X, Ouyang G (2009) MicroRNAs: novel regulators in the hallmarks of human cancer. Cancer Lett 285:116–126
Wang V, Wu W (2007) MicroRNA: a new player in breast cancer development. J Cancer Mol 3:133–138
Fu SW, Chen L, Man YG (2011) miRNA biomarkers in breast cancer detection and management. J Cancer 2:116–122
George GP, Mittal RD (2010) MicroRNAs: potential biomarkers in cancer. Indian J Clin Biochem 25:4–14
O’Connella RM, Chaudhuria AA, Raoa DS, Baltimore D (2009) Inositol phosphatase SHIP1 is a primary target of miR-155. Proc Natl Acad Sci USA 106:7113–7118
Hong MY, Seeram NP, Heber D (2008) Pomegranate polyphenols down-regulate expression of androgen-synthesizing genes in human prostate cancer cells overexpressing the androgen receptor. J Nutr Biochem 19:848–855
Ozbaya T, Nahta R (2011) Delphinidin inhibits HER2 and Erk1/2 signaling and suppresses growth of HER2-overexpressing and triple negative breast cancer cell lines. Breast Cancer 5:143–154
Pandey PR, Okuda H, Watabe M, Pai SK, Liu W, Kobayashi A, Xing F, Fukuda K, Hirota S, Sugai T, Wakabayashi G, Koeda K, Kashiwaba M, Suzuki K, Chiba T, Endo M, Fujioka T, Tanji S, Mo YY, Cao D, Wilber AC, Watabe K (2011) Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase. Breast Cancer Res Treat 130:387–398
Zhu H, Wu H, Liu X, Evans BR, Medina DJ, Liu CG, Yang JM (2008) Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. Biochem Pharmacol 76:582–588
Liu T, Tang H, Lang Y, Liu M, Li X (2009) MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Lett 2:233–242
Jutooru I, Chadalapaka G, Abdelrahim M, Basha MR, Samudio I, Konopleva M, Andreeff M, Safe S (2010) Methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate decreases specificity protein transcription factors and inhibits pancreatic tumor growth: role of MicroRNA-27a. Mol Pharmacol 78:226–236
Wang Y, Keogh RJ, Hunter MG, Mitchell CA, Frey RS, Javaid K, Malik AB, Schurmans S, Tridandapani S, Marsh CB (2004) SHIP2 is recruited to the cell membrane upon macrophage colony-stimulating factor (M-CSF) Stimulation and regulates M-CSF-induced signaling. J Immunol 173:6820–6830
Fuhler GM, Brooks R, Toms B, Iyer S, Gengo EA, Park MY, Gumbleton M, Viernes DR, Chisholm JD, Kerr WG (2012) Therapeutic potential of SH2 domain-containing inositol-5′- phosphatase 1 (SHIP1) and SHIP2 inhibition in cancer. Mol Med 18:65–75
Zhou P, Kitaura H, Teitelbaum SL, Krystal G, Ross FP, Takeshita S (2006) SHIP1 negatively regulates proliferation of osteoclast precursors via akt-dependent alterations in D-type cyclins and p271. J Immunol 177:8777–8784
Conde C, Gloire G, Piette J (2011) Enzymatic and non-enzymatic activities of SHIP-1 in signal transduction and cancer. Biochem Pharmacol 82:1320–1334
Hamilton MJ, Ho VW, Kuroda E, Ruschmann J, Antignano F, Lam V, Krystal G (2011) Role of SHIP in cancer. Exp Hematol 39:2–13
Pilon-Thomas S, Nelson N, Vohra N, Jerald M, Pendleton L, Szekeres K, Ghansah T (2011) Murine pancreatic adenocarcinoma dampens SHIP-1 expression and alters MDSC homeostasis and function. PLoS one 6(11):1–10
Acknowledgments
We would like to thank Dr. Weston Porter Department Veterinary Integrated Bioscience, at Texas A&M University, College Station, and Texas for providing imaging equipment. Lastly we would like to thank Stefan Wypyszyk at Stiebs LLC (Kirkland, WA) for kindly supplying the pomegranate juice. Financial support for this research has been provided by the National Institutes of Health (KOIATOO 4597 to SMT).
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Banerjee, N., Talcott, S., Safe, S. et al. Cytotoxicity of pomegranate polyphenolics in breast cancer cells in vitro and vivo: potential role of miRNA-27a and miRNA-155 in cell survival and inflammation. Breast Cancer Res Treat 136, 21–34 (2012). https://doi.org/10.1007/s10549-012-2224-0
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DOI: https://doi.org/10.1007/s10549-012-2224-0