Ellagic Acid

This is an official report from the University of South Carolina about ellagic acid.
The best source of ellagic acid comes from Raspberries which are the raspberries used in the product "Ellagitannin"
Please click here to see our Report on Ellagitannin $43.95

Cervical Cancer Cells - HPV (Human Papilloma Virus) exposed to Ellagic Acid from Red Raspberries experienced apoptosis (normal cell death).

Ellagic Acid leads to G1 arrest of cancer cells, thus inhibiting and stopping mitosis (cancer cell division)

Ellagic Acid from Red Raspberries prevents destruction of the P53 gene by cancer cells. P53 is regarded as the safeguard of mutagenic activity in cervical cells.

The tests reveal similar results for breast, pancreas, esophageal, skin, colon, and prostate cancer cells. In addition, positive responses are observed in: periodontal disease, radiation induced chromosomal damage, slowing the degenerative process of aging, neurodegenerative diseases, diabetic retinopathy.

CANCER LETTERS ELSEVIER Cancer Letters 136 (1999) 215-221

p53/p2l(WAFl/ClPl) expression and its possible role in
GI arrest and apoptosis in ellagic acid treated cancer cells

Bhagavathi A. Narayanan, Otto Geoffroy, Mark C. Willingham, Gian G. Re Daniel W. Nixon

Cancer Prevention Program Hollings Cancer Center, Medical University of South Carolina, Charleston SC 29425, USA

Dept. of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA

Received 25 August 1998; received in revised form 15 October 1998, accepted 15 October 1998
Abstract

Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. 'Me effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. We found that ellagic acid at a concentration of 10-5 M induced GI arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p2l by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.
C) 1999 Published by Elsevier Science Ltd. All rights reserved.

1. Introduction

Ellagic acid has been demonstrated [1-5] in animal models to inhibit tumor growth induced by several chemical carcinogens, including polycyclic aromatic hydrocarbons, N-nitrosamines, aflatoxins and aromatic amines. Other studies [6,7] have demonstrated that ellagic acid, applied topically to mouse skin, could effectively inhibit TPA-induced omithine decarboxylase activity, hydroperoxide production and DNA synthesis. In addition to earlier reports, recent studies have shown that oral administration of ellagic acid significantly reduced the level of lipid peroxidase and liver dihydroxy proline in animal models [8,9]. The latter study [9] also indicated that oral administration of ellagic acid can circumvent carbon tetrachloride toxicity and subsequent lung fibrosis. Other than the few mentioned pharmacokinetic studies, the mechanisms of action of ellagic acid at the cellular and molecular level are still unclear. The focus of the present study was to understand the effect of ellagic acid on the cell cycle, DNA synthesis, apoptosis and overall cell proliferation inhibition. 'Me cervical carcinoma cell line (CaSki) which carries the HPV16 virus, whose E6 gene product complexes with wild type p53 causing loss of p53 function, was chosen to study the mechanisms of action of ellagic acid. To investigate whether the action of ellagic acid is exerted through modulation of the cell cycle and to test the functional state of p53, we analyzed the expression of p53 and p2l MRNA in ellagic acid treated and untreated CaSki cells in a time- and dose-dependent manner.

Ellagic acid purchased from Aldrich Chemicals was purified further by crystallization from ethanol until a purity of 99% was achieved using high-performance liquid chromatography (HPLC) (Figs. I and 2). The highest concentration (10-3 M) of ellagic acid was made in DMSO American Type Culture Collection(ATCC) andlowerconcentrations(10-'-10-'M) were made with DMSO. After confirming the purity of different dilutions of ellagic acid through HPLC, they were aliquoted, protected from light and stored at -200C.

experiments). The response of tumor cells treated with ellagic acid (in 1% DMSO) was compared with that of tumor cells treated with 1% DMSO alone. To determine the differential effect of ellagic acid with respect to dose and time on cell growth, 75% confluent cells were treated with different molar concentrations (10-5-10-9 M) and observed for 24, 48, 72 and 96 h. Trypsinized and PBS washed cells from the above experiments were counted using a Coulter particle counter at the specified time intervals. Cells harvested from similar sets of experiments were used for flow cytometry analysis.

CaSki cells treated with varying concentrations of ellagic acid and harvested at different time periods were trypsinized, washed in PBS, and fixed in 1% formaldehyde for 15 min on ice. After re-washing with PBS, the cells were fixed in 80% ethanol for 30 min. Appropriate volumes of the above cell suspension having 3 x cells were centrifuged and the pellets of cells were re-suspended with PBS and further treated with I mg/ml of RNAase (DNAase free) at room temperature for 30 min. Propidium iodide (I mg/ml,- final concentration in PBS) was added and flow cytometry analysis was performed after 30 min. Cell cycle analysis was performed using Epics xL MCL (Phoenix Flow System).

BrdUrd, an analogue of thymidine, is incorporated into DNA during S phase. The incubation of cells with BrdUrd (30 tLg/ml) was done at different time periods (0-96 h) with ellagic acid (10-8 M) exposure. Tle cells, pulsed with BrdUrd every 2 h, were harvested by trypsinization at several time intervals as mentioned earlier. The PBS washed cells fixed in 10% fonnalin and pretreated with 0.1% Triton XI 00, and 2 N HCI at 37'C for I 0 min were then treated with 0.1 M sodium borate for 5 min. Mouse monoclonal antibody (NCL-BrdUrd-Novocastra) followed by rhodamine conjugated with goat antimouse IgG was used to detect the incorporation of BrdUrd in the S phase cells.

2.5. DNA fragmentation
CaSki cells (I X 106) treated with and without ellagic acid (10-5 M) for 0, 24, 48 and 72 h were harvested by trypsinizing and suspended in I ml cell lysis buffer (5 mM Tris-HCI, 20 mM EDTA and 0.5% Triton X-100, pH 8.0) containing 100 jig/ml proteinase K and were incubated at 55'C for 4-6 h. Cells were again treated with RNAase (10 tLg/ml) for I h at 37'C. The supernatant was collected from the lysed cell extract and DNA extraction was carried out with an equal volume of Tris saturated phenol-chloroform. The phenol-chloroform extractions were repeated twice or more to get a clear aqueous phase. The purified samples were ethanol precipitated, and the pellet was air dried and re-suspended in 18 ttl of double distilled water. The final concentration of DNA was determined by UV absorbency at 260 nm. DNA (10 tLgAane) was electrophoresed on 1.8% agarose gels containing ethidium bromide (I iLg/rn].

Fig. 3. Cell cycle analysis (as described in Section 2) on CaSki cells exposed to ellagic acid at IO - 3 M concentration at different time intervals. (A) CaSki cells to exposed ellagic acid at 0 h. (B) CaSki cell exposed to ellagic acid for 24 h. (C) CaSki cells to exposed ellagic acid for 48 h. (D) CaSki cells exposed to ellagic acid for 72 h.

Whole cell RNA from CaSki cells was prepared by 2.7. Westem blot analysis the acid-guanidinium/phenol-chloroform extraction method of Chomczynski and Sacchi [10]. Formalde- Cells treated with ellagic acid (10-5 M) for 48 h hyde-agarose gel electrophoresis, transfer to nitrocel- were harvested by trypsinization. Cellular protein was lulose membranes, and hybridization were performed extracted and Western blotting for p2lwAF' protein as previously reported f I I ]. The probes for the consti- was carried out using p2l wAF' Western blotting kit tutively expressed gene glyceraidehyde phosphate with anti P21 wAF' monoclonal antibody (Calbiodehydrogenase (GAPDH) and p53 were a 0.5 kilobase chem-@09). Mouse monoclonal antibodies for (kbp) CDNA and a 2.7 kbp CDNA, respectively, from p53 (DOI) were purchased from Santa Cruz Biotechthe American Type Culture Collection (ATCC). The nology Inc. The reactive protein bands for p53 and probe for the p2l gene was a 1.05 kbp CDNA also). from ATCC. Radiolabeled probes were prepared by random primed labeling.

Cell cycle distribution analysis of ellagic acid treated (10-5 M) CaSki cells showed a minor GI arrest at 24 h as determined by flow cytometry. Cell cycle analysis after 48 h of exposure to ellagic acid, however, showed an enhanced GI peak. Such a cell cycle arrest was also observed at 48 h as a greatly educed number of cells in the S phase. A pre-G I apoptotic peak (31.8%) was very clear with an overlap of GI phase cells after 72 h (Figs. 3A-D). The percentage of cells in S phase was observed to be significantly reduced after 72 h.

The reduced mitotic rate (Fig. 4) which was observed in video time lapse microscopy [121, and the reduced rate of cell proliferation (Fig. 5) in ellagic acid treated cells was consistent with the observation of a significantly lower number of cells which showed incorporation of BrdUrd after treatment for 0-96 h. CaSki cells treated with 10-8 M ellagic acid in the presence of BrdUrd label showed a gradual decline in BrdUrd-labeled cells from 4 to 48 h. At 48 h 3% of treated cells contained the BrdUrd label compared to 33% in the control cells, indicating a significantly decreased rate of DNA synthesis (Fig. 6), which also reflected a similar pattern of a lower number of cells in the 'S' phase (in ellagic acid treated cells with 10-5 M) when analyzed using flow cytometry in cells treated for 48 h.

Intemucleosomal DNA cleavage, another indicator of 'apoptosis, was evaluated.by DNA fragmentation analysis. Apoptosis is usually associated with the activation of nucleases and intemucleosomal degradation of chromosomal DNA. To see a clear DNA fragmen tation, ellagic acid treated cells that showed more than 35% apoptosis (pre-GI peak) with flow cytometry analysis (Fig. 3D) were trypsinized and the DNA was extracted. DNA extracted from ellagic acid treated CaSki cells was run on a 1.8% agarose gel. Degradation of chromosomal DNA into small oligonucleosomal fragments was clearly observed in cells treated with ellagic acid (10-5 M) after 72 h of treatment (Fig. 7A).

Fig. 7. (A) DNA fragmentation analysis using agarosc gel (1.8%) electrophoresis. (B) RNA and Northern blot analysis (as described in Section 2) of CaSki cells exposed to ellagic acid (10-' M) for 48 h.

CaSki cells exposed to ellagic acid did not show any change in the level of p53 MRNA expression and its protein. However, ellagic acid did induce an increase in the expression of both p2l MRNA and protein (Figs. 7B and 8, respectively), an increase that remained significant after correction for GAPDH. Although a small increase in the MRNA level of p53 was observed, it was found to be consistent with the same level of increase in GAPDH. These results indicated that ellagic acid might induce GI arrest and apoptosis by increasing p2l via a p53-independent mechanism.

Plant-derived ellagic acid, a phenolic compound found in raspberries and other plant food, has previously been identified as a potent anticarcinogenic agent [1-9]. In this article we have shown an effect of ellagic acid on cell proliferation inhibition, cell cycle arrest and induction of apoptosis in a time- and dosedependent manner in cervical carcinoma cells (CaSki). CaSki cells were found to be sensitive to the effect of ellagic acid in inducing GI arrest within 24-48 h. Flow cytometry analysis of ellagic acid (IO -5 M) treated cells for 48 h revealed the accumulation of 82% of the cells at the GI peak, whereas the untreated cells showed only 30% of the cells in the GI peak. However, the mechanism of action of ellagic acid in inducing cell cycle arrest, decreasing DNA synthesis, with a lowered mitotic rate, and the induction of apoptosis is not clearly understood at this point. It is possible that all of these effects of ellagic acid are the consequences of a single primary event in which ellagic acid initiates a cascade of events leading to cell cycle arrest and reduced cell growth. A multitude of factors may modulate apoptosis, including growth factors, intracellular mediators of signal transduction and nuclear proteins regulating gene expression, DNA replication and cell cycle regulatory genes [13-17] in response to ellagic acid. The present study demonstrates that ellagic acid could induce apoptosis in a cell cycle dependent manner. Generally, the processes leading to DNA repair involve upregulation of p2l via p53-dependent mechanisms. In CaSki cells, ellagic acid could not induce a remarkable increase in the level of p53 MRNA and protein, although a greater elevation of p2l MRNA and protein was detected following treatment with ellagic acid. The activation of p2l by ellagic acid could be either through a p53independent mechanism or by a p53-dependent mechanism not involving the changes in the level of p53 protein. Recent studies [18,19] on cell death mechanisms have shown the possible involvement of caspases, death receptors, and mitochondrial activation in inducing cell death, which is independent of functional p53. However, the molecular events induced by ellagic acid which, in turn, upregulated p2i expression independent of p53, and the signaling pathways leading to p2l induction, are unclear at this point. The key findings from the present study provide evidence to support the potential effect of ellagic acid in cell proliferation inhibition via p2l-mediated GI arrest and cell death. Inhibition of tumor cell proliferation and induction of cell death by ellagic acid thus, may support a role for ellagic acid as a chemo preventive agent.

We would like to express our sincere appreciation to Dr. Clifford Schweinfest (CMSB, HCC) for critical review of the data. We would also like to thank Dr. Sally Self and Cary Wiggins for assistance with the flow cytometry analysis.

[1] E.C. Bate-Smith, Detection and determination of ellagitannins, Phytochemistry 11 (1972) 1153-1156.

121 J.P. Perchellet, H.U. Gaii. E.M. Perchellet. D.S. Klish, A.D. Armbrust, Antitumor promoting activities of tannin acid, ellagic acid, and several gailic acid derivatives in mou.-,e skin, Basic Life Sci. 59 (1992) 783-801.

[3) G.D. Stoner, A.M. Morse. lsothiocyanates and plant polyphcnois as inhibitors of lung and esophageal cancer, Cancer Lett. 114 (1997) 113-119.

[41 L.R. Dow, T.T. Chou, M.B. Bechle, C. Goddard. R.E. Identification of tricyclic analogs related to ellagic acid as potent/selective tyrosine protein kinase inhibitors. J. Med. Chem. 37 (1994) 224-223 1.

[51 D.H. Batch, L.M. Rundhaugen, G.D. Stoner. N.S. Pillay, W.A. Rosche, Structure-function relationships of the dietary anticarcinogen ellagic acid, Carcinogenesis 17 (19%) 265-. 269.

[6] M. Das, D.R. Bickers. H. Mukhtar, Effect of ellagic acid on hepatic and pulmonary xenobiotic metabolism in mice: Studies on the mechanism of its anticucinogenic action, Carcinogenesis 6 (1985) 140c)-1413.

[7] D. Ahn, D. Putt, L. Kresty, G.D. Stoner, D. Fromm, P.F. Holienberg, The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes p450 and Phase if enzymes, Carcinogenesis 17 (1996) 821428.

[8] R.W. Teel, Ellagic acid binding to DNA as a possible mechanism for its antimutagenic and anticucinogenic action, Cancer Lett. 30 (1986) 329-336.

[9) K.C. Ilresiamma, R. Kuttan, Inhibition of liver fibrosis by ellagic acid, Indian J. Physiol. Phannacol. 40 (1996) 363366.

[10] P Chomczynski, N. Sacchi, Single-step method of RNA isolation and acid guanidinium thiocyanate-phenoichlorofonn extraction, Ann. Biochem. 162 (1987) 156-159.

[111 G.G. Re, G.R. Antoun, T.F. Ziff, Modulation Of 2 constitutive transcriptional block at exon- I controls human c-myc expression, Oncogene 5 (1990) 1247-1250.

Willingham, Major DNA fragmentation is a late event in apoptosis, J. Histocbcm. Cytochem. 45 (1997) 923-934.

[13] E.D. Fisher, Apoptosis in cancer therapy: Crossing the threshold (mini review), Cell 78 (1994) 539-542.

[14] H.L. Hartwell, M.B. Kastan, Cell cycle control and cancer, Science 266 (1994) 1821-1828.

[15] B.C. Thompson, Apoptosis in the pathogenesis and treatment of disease, Science 267 (1995) 1457-1461.

[16) H. Stellar, Apoptosis: mechanisms mW genes of cellular suicide, Science 267 (1995) 1445-1449.

[181 Y.X. Zeng, S.W. El-Deiry, Regulation of p2i WAFI/CIPI expression by p53-independent pathways, Oncogene 12(5-8) (1996) 1557-1564.

[19] A. Ashkenazi, V.M. Dixit, Death receptors: Signaling and modulation, Science 281 (1998) 1305-1308.

Cervical carcinoma (CaSki) cells were grown at appropriate cell culture conditions at 370C in RPMI (Gibco) supplemented with L-glutamine, 10% fetal bovine serum (FBS) and antibiotics, in a water-saturated atmosphere of 5% C02. As mentioned earlier, different molar concentrations of ellagic acid were made with DMSO (ATCC grade for tissue culture experiments). The response of tumor cells treated with ellagic acid (in 1% DMSO) was compared with that of tumor cells treated with 1% DMSO alone. To determine the differential effect of ellagic acid with respect to dose and time on cell growth, 75% confluent cells were treated with different molar concentrations (10-5-10-9 M) and observed for 24, 48, 72 and 96 h. Trypsinized and PBS washed cells from the above experiments were counted using a Coulter particle counter at the specified time intervals. Cells harvested from similar sets of experiments were used for flow cytometry analysis.

CaSki cells (I X 106) treated with and without ellagic acid (10-5 M) for 0, 24, 48 and 72 h were harvested by trypsinizing and suspended in I ml cell lysis buffer (5 mM Tris-HCI, 20 mM EDTA and 0.5% Triton X-100, pH 8.0) containing 100 jig/ml proteinase K and were incubated at 55'C for 4-6 h. Cells were again treated with RNAase (10 tLg/ml) for I h at 37'C. The supernatant was collected from the lysed cell extract and DNA extraction was carried out with an equal volume of Tris saturated phenol-chloroform. The phenol-chloroform extractions were repeated twice or more to get a clear aqueous phase. The purified samples were ethanol precipitated, and the pellet was air dried and re-suspended in 18 ttl of double distilled water. The final concentration of DNA was determined by UV absorbency at 260 nm. DNA (10 tLgAane) was electrophoresed on 1.8% agarose gels containing ethidium bromide (I iLg/rn].

Fig. 8. Western blot analysis (as described in Section 2) of CaSki cells exposed to ellagic acid (10-5 M) for 48 h).

Acknowledgements We would like to express our sincere appreciation to Dr. Clifford Schweinfest (CMSB, HCC) for critical review of the data. We would also like to thank Dr. Sally Self and Cary Wiggins for assistance with the flow cytometry analysis.