DR [Dermatology Reports 2011; 3:e36] [page 77] Ellagic acid inhibits melanoma growth in vitro J. Daniel Jensen,1 Jeffrey H. Dunn,1 Yuchun Luo,1 Weimin Liu,1 Mayumi Fujita,1,2 Robert P. Dellavalle1,2 1Department of Dermatology, School of Medicine, University of Colorado Denver, Aurora; 2Denver Veterans’ Affairs Medical Center, Denver, CO, USA Abstract Ellagic is a polyphenolic compound with anti-fibrotic and antioxidant properties, and exhibits antitumor properties against various cancer cells in vitro. There are few studies, however, which examine the effects of ellagic acid on melanoma. In the present study, we observe effects of ellagic acid on melanoma cells in vitro. Three metastatic melanoma cell lines (1205Lu, WM852c and A375) were exam- ined to determine the effects of ellagic acid on melanoma cell viability, cell-cycle, apoptosis, NF-κβ activity, and IL-1β & IL-8 secretion. Cell viability assays demonstrated that ellagic acid possesses an inhibitory effect on cell prolifera- tion at concentrations between 25 and 100 mM. In addition, ellagic acid promoted G1 cell cycle arrest, increased levels of apoptosis and decreased synthesis of IL-1β and IL-8 in melanoma cells. Ellagic acid also decreased NF-κβ activity, suggesting at least one poten- tial mechanism by which ellagic acid may exert its effects in melanoma cells. Our findings sup- port further investigation into prospective roles for ellagic acid as a therapeutic, adjuvant, or preventive agent for melanoma. Introduction Ellagic acid (EA) is a polyphenolic com- pound found in various types of fruit, including berries, pomegranates, and nuts, and is becoming a popular dietary supplement. It pos- sesses antifibrotic,1,2 antiproliferative,3,4 and antitumorigenic5,6 properties. EA is known to be protective against several types of cancer. EA has been shown to induce apoptosis in human melanoma cells in vitro.7 Few studies have investigated the effects of EA on melanoma, however, and the mechanis- tic action of EA in this setting is not well defined. In vitro studies have shown that EA decreases tyrosinase activity in mouse melanoma cells by chelating the copper tyrosi- nase cofactor.8 We are not aware of any other studies that have defined a mechanism by which EA exerts its effects in melanoma. The exact mechanism by which EA exerts its effects in other types of cancer is also unclear, but several potential mechanisms have been suggested. EA has been shown to modulate a variety of signaling pathways in cancer cells, including NF-κB, iNOS, and Wnt. Alternative mechanisms through which EA exerts anti- cancer effects have also been proposed. One group9 showed that EA prevented copper and catecholamine transmitter-mediated oxidative DNA damage, thus suggesting a protective role for EA in preventing reactive oxygen species production, lipid peroxidation, and DNA strand breaks. EA has also been shown to induce apoptosis via caspase pathways as well as potentiating trans-retinoic acid-mediated cell-differentiation on human leukemia cell lines.10 Another study showed that EA inhibits components of Wnt signaling pathways known to play a pivotal role in human colon carcino- genesis.11 Additionally, EA has been shown to reduce hepatic phase I CYP enzymes responsi- ble for converting estrogen to harmful metabo- lites implicated in mammary tumorigenesis.12 EA has been shown to downregulate iNOS, COX-2, TNF-a and IL-6 secretion by inhibiting nuclear factor-kappa β (NF-κβ) in colon and pancreatic cancers.13 Pomegranate fruit extracts (including EA) decrease NF-κβ expression in UVB-stimulated keratinocytes, decreasing skin tumorigenesis.14,15 Here we report on the effect of EA on melanoma cells, including inhibition of the NF-κB pathway. Materials and Methods Tissue culture Human metastatic melanoma cell lines (1205LU, WM852c and A375) were cultured in RPMI 1640 medium (GIBCO BRL, Gaithers- burg, Maryland, USA) supplemented with 10% fetal bovine serum (FBS, Gemini Bio-Products, Inc., Woodland, CA, USA) and antibiotics (penicillin (10,000 IU/mL), streptomycin (10,000 IU/mL), and amphotericin B (25 microg/mL, Cellgro, Manassas, VA, USA) and were incubated at 37°C and 5% CO2. Ellagic acid treatment EA was obtained from (MP Biochemicals, Solon, OH, USA) and dissolved in sterile DMSO (5 mM) and stored at -20°C. Separate, fresh 100 mL aliquots of EA were used for each experiment and excess reagent was disposed of according to protocol. Cell Titer 96 aqueous one solution cell proliferation assay (MTS assay) for the quantification of cell viability Experiments were performed according to the manufacturer’s instructions (Promega, Madison, WI, USA). Approximately 2.5¥103 cells were seeded in 96-well plates and incu- bated at 37C° and 5% CO2 for 1 day. EA was added to cells in 0, 25, 50 and 100 mM concen- trations and cells were incubated for 24, 48 and 72 h under previously described conditions. MTS reagent was added to the cells and incu- bated for 1 h, followed by spectophotometric analysis using an ELX808 Ultra Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) and KCjunior v. 1.10 software (BioTek Instruments). Annexin V apoptosis detection assay An annexin-FITC apoptosis detection kit (BD Biosciences Pharmingen, San Diego, CA, USA) was used, following the manufacturer’s instructions; 4¥105 cells were seeded in 10 cm plates and incubated for 24 h. Cells were treat- ed with ellagic acid in DMSO at 0, 25, 50 and 100 mM concentrations. Cells were incubated as previously described for 72 h and stained with Annexin V antibodies and PI. Samples were analyzed by the FACS core using a Beckman Coulter FC500 flow cytometer (Beckman Coulter). Apoptotic cells were defined as being positive for Annexin V. Cell cycle analysis 4¥105 cells were seeded on 10 cm tissue cul- ture dish and incubated overnight prior to treatment with EA. Cells were treated with 0 or 50 mM EA and incubated at 37°C for 48 h. Cells were then detached from the plate, stained with propidium iodide (PI) and allowed to incu- bate overnight at 4°C. Cells were then analyzed with by the University of Colorado Denver Fluorescent Activated Cell Sorting (FACS) core using a Beckman Coulter FC500 flow cytometer (Beckman Coulter Inc, Brea, CA, USA). Dermatology Reports 2011; volume 3:e36 Correspondence: Robert P. Dellavalle, Chief, Dermatology Service, Department of Veteran Affairs Medical Center, 1055 Clermont Street, Box 165 Denver, CO 80220, USA. Tel. +1.303.399.8020, ext. 2475 - Fax: +1.303.393.4686. E-mail: robert.dellavalle@ucdenver.edu Key words: ellagic acid, melanoma, NF-κB, IL-1β, IL-8. Received for publication: 27 August 2011. Accepted for publication: 31 August 2011. This work is licensed under a Creative Commons Attribution NonCommercial 3.0 License (CC BY- NC 3.0). ©Copyright J.D. Jensen et al., 2011 Licensee PAGEPress, Italy Dermatology Reports 2011; 3:e36 doi:10.4081/dr.2011.e36 No n- co mm er cia l u se on ly [page 78] [Dermatology Reports 2011; 3:e36] NF-κβ luciferase reporter assay 2.5x103 cells were seeded into 24-well plates and were transfected after 12 h of incubation. Transfection with pNFκβ-MetLuc2 Reporter Vector (Clontech, Mountain View, CA, USA) was performed according to protocol described by the Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA, USA). Cells were then treated with 0, 25, 50 and 100 mM EA and incubated for 24 or 48 h. Luminescence was then measured with a lumi- nometer (Promega, Madison, WI, USA). Measurement of gene expression (IL-1β, IL-8) 1205Lu metastatic melanoma cells were seed- ed and incubated for 24 h under previously described conditions. Cells were then treated with 0 or 50 mM EA for 48 h. Cells were detached from culture dishes and RNA was extracted from treated and untreated cells using the RNAqueous-Micro kit (Ambion, Austin, TX, USA), and subsequently reverse transcribed using random primers and MMLV reverse tran- scriptase (Promega, Madison, WI, USA). Real- time quantitative reserve transcription-PCR (qRT-PCR) was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on the MX3000P PCR sys- tem (Strategene, La Jolla, CA, USA). Primers were designed to generate a PCR product of 50 to 150 bp. Thermal cycling conditions were 95°C for 10 min followed by 42 cycles of 15 s at 95°C, 1 min at 60°C. GAPDH was used as a control to nor- malize the results. Statistical analysis The viabilities of each treated cell line were compared to control was evaluated using a Student’s t-test. Values of P <0.05 were consid- ered to be statistically significant. Results Melanoma cell viability is inhibited by Ellagic acid Cells were incubated with different concen- trations of EA for 24, 48, and 72 h and cell viabil- ity was measured using the MTS assay (Figure 1A, B). Inhibition of cell growth was detected as early as 24 h at 50 and 100 mM concentrations EA for 1205Lu cells (P<0.01 and P<0.05, respec- tively) and at 25, 50, and 100 µM concentrations EA for WM852c cells (P <0.05, P<0.05 and P <0.01, respectively). EA continued to inhibit cell growth for a 72 h period at 25, 50 and 100 mM concentrations (P <0.05, P<0.01 or P<0.001 for all data points measured). Ellagic acid induces apoptosis in melanoma cell lines Decreased viability is caused by cell death and/or decreased cell growth. In order to deter- mine if EA exerted a proapoptotic effect in metastatic melanoma, three cell lines (1205LU, WM852c, A375) were incubated in 25, 50 and 100 mM concentrations of EA for 72 h (Figure 2A). After staining for the apoptotic marker Annexin V, cell lines were sorted with flow cytometry. 1205LU cells demonstrated increased apoptosis when compared to controls at 50 and 100 mM (P<0.01 and 0<0.001, respec- tively). Furthermore, both WM852c and A375 cell lines had increased levels of apoptotic cells at 100 mM concentrations EA (P <0.05). In gen- eral, there was a trend toward increased apop- tosis with increasing concentrations of EA. Ellagic acid induced G1 cell cycle arrest In order to determine if EA inhibited cell proliferation via cell cycle arrest, WM852c cells were incubated for 48 h in 50 mM EA. After staining, cells were sorted according to the phase of their cell cycle (Figure 2B). Cell cycle analysis of WM852c cells in G1 phase suggest- ed disruption of cellular replication (P<0.01), suggesting that EA inhibited melanoma cell proliferation via G1 cell cycle arrest. Ellagic acid downregulates NF-κβ activity in metastatic melanoma cells Several studies reported that EA modulates the NF-κβpathway in cancer cells, therefore NF- κβ activity and expression in EA-treated cells was compared to untreated controls (Figure 3A, B). At 24 h, NF-κβ expression was decreased in both cell lines for all concentrations tested except for 25 mM in 1205Lu (for 1205Lu, P<0.001 for 50 and 100 mM concentrations; for WM852, P <0.01 for 50 mM, P <0.001 for 25 and 100 mM EA). At 48 h, there was decreased expression of NF-κβ with EA treatment at 25, 50 and 100 mM (for 1205Lu, P <0.01 for all concen- trations; for WM852, P <0.05, P<0.01 and P<0.01 for 25, 50 and 100 mM EA, respectively). Ellagic acid decreases synthesis of IL-1β and IL-8 in metastatic melanoma cells Downstream effectors of the NF-κβ pathway which control cell proliferation and apoptosis were measured using quantitative RT-PCR. As indicated in Figure 4, mRNA coding for IL-1β and IL-8 8 were significantly decreased (P<0.05) by treatment with EA. Discussion This study suggests that EA induces apopto- sis and G1 cell cycle arrest in melanoma cells at in vitro concentrations between 25 and 100 mM. Inhibition of the NF-κβ pathway was also shown to be a potential mechanism by which EA exerts its antiproliferative effects. The NF- κβ pathway frequently plays a central role in the pathogenesis of many types of cancer, and therapeutic modalities which target the NF-κβ pathway and its downstream intermediates are under investigation. Dysregulation of the NF-κβ pathway in the Article Figure 1. Cell growth over time with differ- ent concentrations of EA. A) WM852c and B) 1205Lu cells were incubated with dif- ferent concentrations of EA for 24, 48, and 72 h and cell viability was measured using MTS assay. Figure 2. A) Apoptotic cells at 72 h. Three cell lines (1205Lu, WM852c, A375) were incubated in 25, 50 and 100 mM concen- trations of EA for 72 h. After staining for the apoptotic marker Annexin V, cell lines were then sorted with flow cytometry. B) Cell cycle analysis. WM852c cells were incubated for 48 h in 50 mM EA. After staining, cells were sorted according to the phase of their cell cycle. A B A BNo n- co mm er cia l u se on ly [Dermatology Reports 2011; 3:e36] [page 79] setting of melanoma, a particularly treatment- resistant malignancy, is very common. Inhibition of NF-κβ has been explored as a potential therapeutic strategy against melanoma with positive results.16 For example, NF-κβ upregulation has been shown to pro- mote angiogenesis in melanoma,17 a key mechanism in tumor growth and mainte- nance. NF-κβ also increases the rate of melanoma metastasis.18 Conversely, suppres- sion of NF-κβ attenuates the invasive poten- tial of tumors.19 Further evidence of the impor- tance of the NF-κβ pathway in melanoma is that ablation of an upstream effector of the NF- κβ pathway, Ikappaβ kinase beta (IKΚβ), inhibits melanoma tumorigenesis20 and increased susceptibility to chemotherapy.21 Furthermore, p53 is inversely related to NF-κβ expression,22 and p53-mediated G1 cell cycle arrest may result from downregulation of NF- κβ in cancer cell.4 These findings support a potential role for EA as a therapeutic, adjuvant, or preventive agent for melanoma. Future work, however, should address the limitations of this study. While our data shows some evidence that EA treatment is associated with increased cell- cycle arrest and apoptosis, as well as decreased melanoma cell viability and NF-κβ activity, these results were not obtained in the pres- ence of a vehicle control. Follow up work is therefore indicated to determine if EA specifi- cally inhibits melanoma through the pathways observed in this study. Furthermore, the con- centrations of EA used in our study are higher than levels that are generally sustainable by normal consumption of EA-containing foods (typically ranging from 5-15 mM.23,24 While the use of higher doses of EA as an adjuvant to other therapies may prove to be of some bene- fit to patients it is not clear from these results if EA may exert therapeutic benefits at practi- cal dietary doses. References 1. Thresiamma KC, Kuttan R. Inhibition of liver fibrosis by ellagic acid. Indian J Physiol Pharmacol 1996;40:363-6. 2. Devipriya N, Sudheer AR, Srinivasan M, Menon VP. Effect of Ellagic acid, a plant polyphenol, on fibrotic markers (MMPs and TIMPs) during alcohol-induced hepa- totoxicity. Toxicol Mech Methods 2007;17:349-56. 3. Losso JN, Bansode RR, Trappey A 2nd, et al. In vitro anti-proliferative activities of ellagic acid. J Nutr Biochem 2004;15:672- 8. 4. Narayanan BA, Geoffroy O, Willingham MC, et al. p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apop- tosis in ellagic acid treated cancer cells. Cancer Lett 1999;136:215-21. 5. Mukhtar H, Das M, Khan WA, et al. Exceptional activity of tannic acid among naturally occurring plant phenols in pro- tecting against 7,12 dimethylbenz(a)- anthracene-, benzo(a)pyrene-, 3-methyl- cholanthrene-, and N-methyl-N-nitro- sourea-induced skin tumorigenesis in mice. Cancer Res 1988;48:2361-5. 6. Kowalczyk MC, Kowalczyk P, Tolstykh O, et al. Synergistic effects of combined phyto- chemicals and skin cancer prevention in SENCAR mice. Cancer Prev Res (Phila) 2010;3:170-8. 7. Kim S, Liu Y, Gaber MW, et al. Develop- ment of chitosan-ellagic acid films as a local drug delivery system to induce apop- totic death of human melanoma cells. J Biomed Mater Res B Appl Biomater 2009; 90:145-55. 8. Shimogaki H, Tanaka Y, Tamai H, Masuda M. In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition. Int J Cosmet Sci 2000;22:291-303. 9. Spencer WA, Jeyabalan J, Kichambre S, Gupta RC. Oxidatively generated DNA damage after Cu(II) catalysis of dopamine and related catecholamine neurotransmit- ters and neurotoxins: Role of reactive oxy- gen species. Free Radic Biol Med 2010; 50:139-47. 10. Hagiwara Y, Kasukabe T, Kaneko Y, et al. Ellagic acid, a natural polyphenolic com- pound, induces apoptosis and potentiates retinoic acid-induced differentiation of human leukemia HL-60 cells. Int J Hematol 2010;92:136-43. 11. Sharma M, Li L, Celver J, et al. Effects of fruit ellagitannin extracts, ellagic acid, and their colonic metabolite, urolithin A, on Wnt signaling. J Agric Food Chem 2009; 58:3965-9. 12. Aiyer H, Gupta RC. Berries and ellagic acid prevent estrogen-induced mammary tumorigenesis by modulating enzymes of estrogen metabolism. Cancer Prev Res (Phila) 2010;3:727-37. 13. Umesalma S, Sudhandiran G. Differential inhibitory effects of the polyphenol ellagic acid on inflammatory mediators NF- kappaB, iNOS, COX-2, TNF-alpha, and IL-6 in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. Basic Clin Pharma- col Toxicol 2010;107:650-5. 14. Afaq F, Malik A, Syed D, et al. Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated pro- tein kinases and activation of nuclear fac- tor kappa B in normal human epidermal keratinocytes paragraph sign. Photochem Photobiol 2005;81:38-45. 15. Afaq F, Saleem M, Krueger CG, et al. Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-kappaB pathways and inhibits skin tumorigenesis in CD-1 mice. Int J Cancer 2005;113:423-33. 16. Czyz M, Lesiak-Mieczkowska K, Kopro - wska K, et al. Cell context-dependent activ- Article Figure 3. Expression of NF-κβ after EA treatment. A plasmid containing a luciferase reporter gene under the control of NF-κβ was transfected into WM852c (A) and 1205LU cells (B) and measured after 24 hours incubation. Figure 4. Expression of IL-1β and IL-8 cytokines after EA treatment. Metastatic melanoma cells were seeded and incubated for 24 h. Cells were then treated with 0 or 50 mM EA for 48 h. RNA was extracted from treated and untreated cells and meas- ured by qRT-PCR. GAPDH was used as a control. *P <0.05 A B No n- co mm er cia l u se on ly [page 80] [Dermatology Reports 2011; 3:e36] ities of parthenolide in primary and meta- static melanoma cells. Br J Pharmacol 2010;160:1144-57. 17. Karst AM, Gao K, Nelson CC, Li G. Nuclear factor kappa B subunit p50 promotes melanoma angiogenesis by upregulating interleukin-6 expression. Int J Cancer 2009;124:494-501. 18. Wu FH, Yuan Y, Li D, et al. Endothelial cell- expressed Tim-3 facilitates metastasis of melanoma cells by activating the NF- kappaB pathway. Oncol Rep 2010;24:693-9. 19. Kim A, Kim MJ, Yang Y, et al. Suppression of NF-kappaB activity by NDRG2 expres- sion attenuates the invasive potential of highly malignant tumor cells. Carcino- genesis 2009;30:927-36. 20. Yang J, Splittgerber R, Yull FE, et al. Conditional ablation of Ikkb inhibits mela- noma tumor development in mice. J Clin Invest 2010;120:2563-74. 21. Amschler K, Schon MP, Pletz N, et al. NF- kappaB inhibition through proteasome inhibition or IKKbeta blockade increases the susceptibility of melanoma cells to cytostatic treatment through distinct pathways. J Invest Dermatol 2010;130: 1073-86. 22. Rasmussen MK, Iversen L, Johansen C, et al. IL-8 and p53 are inversely regulated through JNK, p38 and NF-kappaB p65 in HepG2 cells during an inflammatory response. Inflamm Res 2008;57:329-39. 23. Mertens-Talcott SU, Jilma-Stohlawetz P, Rios J, et al. Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum l.) polyphenols after ingestion of a standardized extract in healthy human volunteers. J Agric Food Chem 2006;54:8956-61. 24. Seeram NP, Lee R, Heber D. Bioavailability of ellagic acid in human plasma after con- sumption of ellagitannins from pomegran- ate (Punica granatum L.) juice. Clin Chim Acta 2004;348:63-8. Article No n- co mm er cia l u se on ly