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Eugenol Causes Melanoma Growth Suppression through Inhibition of E2F1 Transcriptional Activity

2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês

10.1074/jbc.m411429200

1083-351X

Rita Ghosh, Nagalakshmi Nadiminty, James E. Fitzpatrick, William L. Alworth, Thomas J. Slaga, Addanki P. Kumar,

Tannin, Tannase and Anticancer Activities

Metastatic malignant melanoma is an extremely aggressive cancer, with no currently viable therapy. 4-Allyl-2-methoxyphenol (eugenol) was tested for its ability to inhibit proliferation of melanoma cells. Eugenol but not its isomer, isoeugenol (2-methoxy-4-propenylphenol), was found to be a potent inhibitor of melanoma cell proliferation. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point. More significantly, 50% of the animals in the control group died from metastatic growth, whereas none in the treatment group showed any signs of invasion or metastasis. Eugenol was well tolerated as determined by measurement of bodyweights. Examination of the mechanism of the antiproliferative action of eugenol in the human malignant melanoma cell line, WM1205Lu, showed that it arrests cells in the S phase of the cell cycle. Flow cytometry coupled with biochemical analyses demonstrated that eugenol induced apoptosis. cDNA array analysis showed that eugenol caused deregulation of the E2F family of transcription factors. Transient transfection assays and electrophoretic mobility shift assays showed that eugenol inhibits the transcriptional activity of E2F1. Overexpression of E2F1 restored about 75% of proliferation ability in cultures. These results indicate that deregulation of E2F1 may be a key factor in eugenol-mediated melanoma growth inhibition both in vitro and in vivo. Since the E2F transcription factors provide growth impetus for the continuous proliferation of melanoma cells, these results suggest that eugenol could be developed as an E2F-targeted agent for melanoma treatment. Metastatic malignant melanoma is an extremely aggressive cancer, with no currently viable therapy. 4-Allyl-2-methoxyphenol (eugenol) was tested for its ability to inhibit proliferation of melanoma cells. Eugenol but not its isomer, isoeugenol (2-methoxy-4-propenylphenol), was found to be a potent inhibitor of melanoma cell proliferation. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point. More significantly, 50% of the animals in the control group died from metastatic growth, whereas none in the treatment group showed any signs of invasion or metastasis. Eugenol was well tolerated as determined by measurement of bodyweights. Examination of the mechanism of the antiproliferative action of eugenol in the human malignant melanoma cell line, WM1205Lu, showed that it arrests cells in the S phase of the cell cycle. Flow cytometry coupled with biochemical analyses demonstrated that eugenol induced apoptosis. cDNA array analysis showed that eugenol caused deregulation of the E2F family of transcription factors. Transient transfection assays and electrophoretic mobility shift assays showed that eugenol inhibits the transcriptional activity of E2F1. Overexpression of E2F1 restored about 75% of proliferation ability in cultures. These results indicate that deregulation of E2F1 may be a key factor in eugenol-mediated melanoma growth inhibition both in vitro and in vivo. Since the E2F transcription factors provide growth impetus for the continuous proliferation of melanoma cells, these results suggest that eugenol could be developed as an E2F-targeted agent for melanoma treatment. Melanoma is one of the fastest growing cancers in the developing world with the incidence having tripled in the last three decades (1Dennis L.K. Arch. Dermatol. 1999; 135: 275-280Crossref PubMed Google Scholar). Chemotherapy, immunotherapy, and vaccines have all produced limited benefits especially since the responses are typically short-lived, with no significant effect on overall survival. As a first step toward developing new compounds for effective melanoma management, we screened a panel of naturally occurring compounds for antiproliferative activity toward melanoma cells. From this screen, we have identified 4-allyl-2-methoxyphenol (eugenol) 1The abbreviations and trivial names used are: eugenol, 4-allyl-2-methoxyphenol; isoeugenol, 2-methoxy-4-propenylphenol; CDK, cyclin-dependent kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; RT, reverse transcription; pRb, retinoblastoma tumor suppressor protein.1The abbreviations and trivial names used are: eugenol, 4-allyl-2-methoxyphenol; isoeugenol, 2-methoxy-4-propenylphenol; CDK, cyclin-dependent kinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; RT, reverse transcription; pRb, retinoblastoma tumor suppressor protein. as a potent inhibitor of both anchorage-dependent and anchorage-independent growth of melanoma cells representing the different stages of melanoma progression. The structures of eugenol and the isomeric isoeugenol are shown in Fig. 1. Eugenol is found in reasonable quantities in the essential oils of different spices such as Syzgium aromaticum (clove), Pimenta racemosa (bay leaves), and Cinnamomum verum (cinnamon leaf). Eugenol has been used as an antiseptic, antibacterial, analgesic agent in traditional medical practices in Asia as well as in dentistry in cavity-filling procedures. Eugenol has been demonstrated to inhibit prostaglandin biosynthesis (2Rasheed A. Laekeman G. Totte J. Vietinch A. Herman A.G. N. Engl. J. Med. 1984; 310: 50-51PubMed Google Scholar) and to block COX-2 activity with an IC50 value of 129 μmol (3Huss U. Ringbom T. Perera P. Bohlin L. Vasange M.J. J. Nat. Prod. (Lloydia). 2002; 65: 1517-1521Crossref PubMed Scopus (79) Google Scholar). In long term carcinogenicity experiments by various groups in CD-1 mice and F344 rats, eugenol was not associated with tumor formation (4Miller J.A. Swanson A.B. Miller E.C. Miller E.C. Naturally Occurring Carcinogens: Mutagens and Modulators of Carcinogenesis. Tokyo Japan Science Society Press/Baltimore University Park Press, Tokyo/Baltimore, MD1979: 111-125Google Scholar). Based on numerous long term carcinogenicity studies, the National Toxicology Program concluded that eugenol was not carcinogenic to rats and that there was no evidence that unequivocally proved the carcinogenic nature of eugenol in mice (National Toxicology Program). More recently, in a skin carcinogenesis study using the initiating agent 7,12-dimethylbenz(a)anthracene followed by three times weekly cutaneous applications of eugenol for 63 weeks in a group of female ICR/HA Swiss mice, no carcinomas were found (5Van Duuren B.L. Sivak A. Segal A. Orris L. Langseth L. J. Natl. Cancer Inst. 1966; 37: 519-526PubMed Google Scholar). In a skin painting study by Van Duuren and Goldschmidt (6Van Duuren B.L. Goldschmidt B.M. J. Natl. Cancer Inst. 1976; 56: 1237-1242Crossref PubMed Scopus (289) Google Scholar), eugenol was reported as being partially effective in inhibiting benzo(a)pyrene-induced skin carcinomas. Eugenol was shown to inhibit DMBA-croton oil-induced papillomas by about 84% (7Sukumaran K. Unnikrishnan M.C. Kuttan R. Indian J. Physiol. Pharmacol. 1994; 38: 306-308PubMed Google Scholar). Eugenol is not mutagenic, although the incidence of sister chromatid exchange was found to increase in Chinese hamster ovary cells (8Stich H.F. Stich W. Lam P.P. Mutat. Res. 1981; 90: 355-363Crossref PubMed Scopus (68) Google Scholar). Eugenol has neither been previously reported to be effective against melanoma nor been systematically tested in other common cancers. The E2F proteins are a family of transcription factors with an important role in regulating cell cycle progression (9Cam H. Dynlacht B.D. Cancer Cell. 2003; 3: 311-316Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). It has been shown that deregulated transcriptional activity of the E2F family in autonomously growing melanoma cells provides the impetus for continuous proliferation of melanoma cells. Specifically, E2F2 and E2F4 are predominant in actively proliferating melanocytes, melanoma cells, and freshly isolated melanoma tumors. The up-regulated E2F activity in melanoma cells is dependent on persistent cyclin-dependent kinase (CDK) activity and inactivation of the pocket proteins (10Halaban R. Cheng E. Smicum Y. Germino J. J. Exp. Med. 2000; 191: 1005-1016Crossref PubMed Scopus (69) Google Scholar). It has also been shown that the members of the E2F family known to cause growth arrest and apoptosis are either absent or expressed at low levels in melanoma cells, therefore providing a growth advantage to melanoma cells (10Halaban R. Cheng E. Smicum Y. Germino J. J. Exp. Med. 2000; 191: 1005-1016Crossref PubMed Scopus (69) Google Scholar). A clinical agent that can target the continuous cycling of melanoma cells would be an attractive tool for effective inhibition of melanoma cell growth. Our results show that eugenol is a potent inhibitor of both anchorage-dependent and anchorage-independent growth of melanoma cells in culture, causes significant tumor growth delay (p = 0.0057), decreases size of tumors, and inhibits melanoma invasion and metastasis in B16 xenograft animals. Eugenol also arrests cells in the S phase of cell cycle, induces apoptosis, and inhibits E2F1 transcriptional activity. The growth inhibitory effect of eugenol is partially abrogated by overexpression of E2F1. These results suggest a potential role for E2F1 in eugenol-mediated melanoma management. Materials—Eugenol was purchased from Sigma; all the polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CellTiter96 Aqueous One solution, DeadEnd colorimetric apoptosis detection system, and Dual-Luciferase assay system were from Promega Corp. (Madison, WI). All other reagents were molecular biology grade from Sigma. The E2F1 plasmids were a gift from Dr. David G. Johnson at University of Texas M. D. Anderson Cancer Center, Smithville, TX. Cell Lines—The human melanoma cells that represent disease progression (Sbcl2-primary melanoma; WM3211-radial growth phase; primary RGP, WM98-1-radial and vertical growth phase; primary RGP and VGP, WM1205Lu-metastatic melanoma) were a gift from Dr. Meenhard Herlyn at the Wistar Institute in Philadelphia. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 0.5% insulin. All melanoma cell lines were maintained in a humidified incubator with 5% CO2 at 37 °C. Animal Study—Female B6D2F1 mice bearing established B16 melanomas (∼50 mm3) were randomized into a control and a treatment group of eight animals each at Piedmont Research Center in Morrisville, NC. The control group received corn oil (vehicle), and the treatment group received 125 mg of eugenol/kg of body weight twice a week intraperitoneally for the duration of the study. The dose was administered in a volume of 0.2 ml/20 g of bodyweight and was adjusted for the body weight of the animal. The choice of 125 mg/kg of bodyweight of eugenol was based on an earlier maximum tolerated dose study using three different doses. Animals were euthanized when their tumors reached the end point volume of 2,000 mm3, and the time to end point was calculated for each mouse. The percentage of tumor growth delay was used as treatment outcome (defined as the percentage of increase in median time to end point of treated versus control mice). Significance of efficacy was calculated using log rank analysis. All animal procedures were conducted in strict adherence to recommendations of the Guide for Care and Use of Laboratory Animals. Proliferation Assay—Actively growing human melanoma cells were plated in 96-well plates at a density of 4 × 103 cells/well in triplicates. After 24 h at 37 °C with 5% CO2, cells were treated with different concentrations of eugenol (0.5-2.5 μm), isoeugenol (0.5-5.0 μm), or the solvent (ethanol). Cell proliferation following treatment was carried out with the CellTiter96 Aqueous One solution assay (Promega Corp.) as described elsewhere (11Ghosh R. Ott A.M. Seetharam D. Slaga T.J. Kumar A.P. Melanoma Res. 2003; 13: 119-127Crossref PubMed Scopus (35) Google Scholar). Briefly, plated cells were incubated with the dye solution containing tetrazolium at 37 °C for 4 h. The reaction was terminated with a stop solution that solubilizes the formazan product formed. Absorbance at 570 nm was recorded using a SpectraMaxPlus plate reader (Molecular Devices). Proliferation assays were performed five times in triplicate wells. The trypan blue exclusion assay was initially used to measure cell viability. Colony Formation Assay—A colony-forming assay as described by Kumar et al. (12Kumar A.P. Garcia G.E. Ghosh R. Rajnarayanan R.V. Alworth W.L. Slaga T.J. Neoplasia. 2003; 5: 255-266Crossref PubMed Google Scholar) was used. Logarithmically growing melanoma cells were trypsinized and plated at a density of 8,000 cells/ml in 0.5% agarose plates in duplicate. After incubating for 14 days, colonies were stained with 0.02% p-iodonitrotetrazolium. After 6 h, colonies containing more than 50 cells and stained dark pink were counted in eight different fields. The experiment was repeated twice in duplicate. Flow Cytometric Analysis—Actively growing cells were plated at a density of 106 cells in 100-mm dishes. Cells at ∼70% confluency were treated with either 0.5 μm eugenol in ethanol or solvent (ethanol) for 20 and 36 h. Cells were harvested and resuspended in 1 ml of Krishan stain containing 1.1 mg/ml sodium citrate, 46 μg/ml propidium iodide, 0.01% Nonidet P-40, and 10 μg/ml RNase (13Krishan A. J. Cell Biol. 1975; 66: 188-193Crossref PubMed Scopus (1469) Google Scholar). These cells were subject to flow cytometric analysis on a Beckman Coulter XL flow cytometer (Beckman Coulter) at the University of Colorado Comprehensive Cancer Center Flow Cytometry core facility. Data analysis was carried out with the Modfit LT software (Verity Software House, Topsham, ME). Three independent flow cytometry analyses were carried out. Apoptosis Detection—Melanoma cells treated with ethanol or 0.5, 1, and 2.5 μm eugenol in ethanol for 18 h and observed by phase contrast microscopy. From this initial experiment, we chose to demonstrate the induction of apoptosis at the biochemical level using the malignant melanoma cell line WM1205Lu. The DeadEnd colorimetric apoptosis detection system (Promega Corp.) was used to detect apoptosis as described previously (11Ghosh R. Ott A.M. Seetharam D. Slaga T.J. Kumar A.P. Melanoma Res. 2003; 13: 119-127Crossref PubMed Scopus (35) Google Scholar). This assay uses a modified terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method to detect apoptotic cells in situ. Cells were grown and treated as described above. Cells were washed and fixed on poly-l-lysine-coated slides in 2% formaldehyde solution. After permeabilizing cells with Triton X-100, double-stranded breaks were labeled with the biotinylated nucleotide mix and the terminal deoxynucleotidyltransferase enzyme. After the reactions were terminated, apoptosis was detected as the dark brown color of the horseradish peroxidase-labeled streptavidin bound to the biotinylated nucleotides. Two independent TUNEL assays were carried out with the DNase I-treated cells as positive control and a negative control without the terminal deoxynucleotidyltransferase enzyme. We also performed DNA fragmentation analysis to assess apoptosis induction by eugenol. Whole Cell Extracts and Western Blotting—Cells were either treated with 0.5 μm eugenol or left untreated for 18 h. Cells were harvested and lysed in a buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF, 1 mm NaVO4, 1 mm phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, 25 μg/ml aprotinin, 25 μg/ml pepstatin, and 1 mm dithiothreitol. Lysed cells were passed through a 25-gauge needle, and the released material was centrifuged at 12,000 rpm for 30 min. Protein content in the supernatant was determined by the method of Bradford (14Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (208724) Google Scholar). Western blotting was carried out as described elsewhere (11Ghosh R. Ott A.M. Seetharam D. Slaga T.J. Kumar A.P. Melanoma Res. 2003; 13: 119-127Crossref PubMed Scopus (35) Google Scholar). Briefly, equal amounts of whole cell extracts were fractionated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Antibodies used were as follows: polyclonal anti-E2F1, -E2F2, -E2F3, -E2F4, -E2F5, and -E2F6. We used the Western Lightning chemiluminescence reagent Plus for detection according to the manufacturer's instructions (PerkinElmer Life Sciences). All the blots were probed with β-actin to normalize for loading differences. Western blotting was repeated twice with different batches of extracts. Transfection and Transient Expression Assay—Transient transfection assays were performed in WM1205Lu cells using a Lipofectamine reagent (Invitrogen) as published previously (15Ghosh R. Tummala R. Mitchell D.L. FEBS Lett. 2003; 554: 427-432Crossref PubMed Scopus (7) Google Scholar). Wild type or mutant E2F1-luciferase reporter plasmids (1 μg/well) and pRL-TK plasmid (50 ng/well) were incubated with Lipofectamine for 30 min and then added to the cells. 24 h after transfection, the cells were treated with 1 μm eugenol in ethanol for 4 h. Following treatment, luciferase reporter assay was performed with the Dual-Luciferase reporter assay system from Promega Corp. As per the manufacturer's recommendation, cells were harvested and then lysed in passive lysis buffer. Cell lysate was cleared from debris by centrifugation at 10,000 rpm for 5 min at 4 °C. Luciferase activity was assessed in duplicate samples containing equal amounts of protein. The assay contained 20 μl of cell lysate and 50 μl of firefly luciferase buffer (luciferase assay reagent II). Firefly luciferase was measured on the Victor plate reader. 50 μl of Renilla luciferase (stop and glow) buffer was added, and Renilla luciferase activity was measured. Renilla luciferase activity was used to normalize transfection efficiency. Results are expressed as the ratio of firefly luciferase/Renilla luciferase at equal amounts of protein. For overexpression of E2F1, WM1205Lu cells in triplicates were transfected with 5 and 10 μg of pCDNA3E2F1-overexpressing plasmid or pCDNA3 (control) plasmid. 24 h after transfection, the cells were treated with eugenol for 4 h. The number of live cells was counted in duplicates using the trypan blue exclusion assay. All transfection experiments were carried out twice each with two different plasmid preparations. Gene Expression Analysis—WM1205Lu cells at 80% confluency were treated with 0.5 μm eugenol in ethanol for 18 h. Total RNA was isolated with the TRIzol reagent (Ambion, Austin, TX) according to vendor instructions. To determine changes in gene expression following eugenol treatment, we used the cell cycle pathway-specific gene expression profiling system from SuperArray Bioscience Corp. (Frederick, MD). We made biotinylated cDNA from total RNA by RT-PCR according to instructions provided by SuperArray Bioscience Corp. The denatured probe was hybridized to the membranes containing 96 cDNA fragments involved in cell cycle regulation. Data were extracted from the raw image and analyzed with the GEArray analyzer software (Super Array Bioscience Corp. (Frederick, MD)). All raw signal intensities were corrected for background by subtracting the signal intensity of pUC18 (negative control) and normalized to Homo sapiens peptidylprolyl isomerase A; PPIA (housekeeping gene). The corrected and normalized signal was used to estimate the relative abundance of transcripts. The array results were verified by quantitative RT-PCR. Preparation of Nuclear Extracts—Nuclear extracts were prepared as described elsewhere (16Ghosh R. Peng C.-H. Mitchell D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6918-6923Crossref PubMed Scopus (11) Google Scholar). Eugenol-treated (0.5 μm) and control cells were harvested and homogenized in a buffer containing 10 mm Hepes, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1.0 mm dithiothreitol, 1.0 mm phenylmethylsulfonyl fluoride, and protease inhibitors including 1 mm phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, 25 μg/ml aprotinin, 25 μg/ml pepstatin. Nuclei were separated from cell debris and lysed in a buffer containing 20 mm Hepes, pH 7.9, 25% (v/v) glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1.0 mm dithiothreitol, and the protease inhibitors. Nuclear protein was collected by centrifugation at 15,000 × g for 45 min. Protein content in the supernatant was determined by the method of Bradford (14Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (208724) Google Scholar). Immunocytochemistry and Immunohistochemistry—Untreated or 1 μm eugenol-treated cells were fixed in paraformaldehyde on poly-l-lysine-coated slides. For immunocytochemical detection of E2F1, we used 1:200 dilution of the primary antibody and followed the manufacturer's recommendations for the Histostain Plus kit (Zymed Laboratories Inc., San Francisco, CA). For immunohistochemical detection of E2F1 in melanoma tumor tissues, we deparaffinized tissue sections with xylene and rehydrated in a graded series of ethanol. After the appropriate washes in phosphate-buffered saline, immunochemical detection was carried out in the same way as cytochemical detection of E2F1. We used an E2F1 blocking antibody as a negative control for immunocytochemical as well as immunohistochemical detection of E2F1. The immunocytochemical assay was performed twice with cells from different passages. A Coolpix digital camera attached to a Nikon T1-SM or Zeiss microscope was used to obtain photographs of the cells and tissues. Eugenol Inhibits Anchorage-dependent and -independent Growth of Melanoma Cells in Culture—The CellTiter96 proliferation assay was used to measure proliferation of the cells for up to 72 h following the addition of increasing concentrations of eugenol. As shown in Fig. 2, A-D, eugenol addition inhibited the growth of all the human melanoma cell lines tested. The Sbcl2 and WM3211 cells showed 50% growth inhibition in 0.5 μm eugenol after 24 h, whereas the WM98-1 and WM1205Lu cells needed twice as much time for 50% growth inhibition at this concentration of eugenol. At 72 h, however, there was no difference in response between the different cell lines using 0.5 μm eugenol. Fig. 2E shows that isoeugenol, an isomer of eugenol, did not inhibit the growth of any of the human melanoma cell lines at concentrations up to 5 μm. Since anchorage-independent growth is a hallmark of cancer cells, we tested to see whether eugenol could inhibit colony formation on soft agar. Data presented in Fig. 2F show that eugenol inhibited colony formation in all the human melanoma cells. The metastatic melanoma cell line WM1205Lu showed the lowest inhibition in colony formation as compared with the other melanoma cells at 0.5 μm eugenol. Taken together, these results show that eugenol inhibits both anchorage-dependent and anchorage-independent growth of melanoma cells. Eugenol Causes Significant Tumor Growth Delay, Decreases Tumor Size, and Prevents Tumor Metastasis in B16F10 Xenograft Mice—We tested the effect of eugenol in vivo in the B16 melanoma xenograft model system as shown in Table I. As shown in Table II tumors in the vehicle group grew rapidly to the end point volume, and four mice died due to metastasis between days 8 and 14. The median time for the tumor to grow to end point for the control group was 12.6 days. There was a highly significant 19% tumor growth delay (p = 0.0057) in the group treated with eugenol. The size of tumors in the treatment group was about 62% of that of the tumors of control animals on day 15. Very significantly, 50% of the animals in the control group developed non-treatment-related metastases, whereas none of the animals in the treatment group showed any signs of invasion or metastasis. Daily examination and body weight measurements of the animals showed no difference in body weight between control and treatment group animals, indicating that eugenol was well tolerated.Table ITreatment protocolGroupTreatment RegimenScheduleNumberAgentWeightRoutenmg/kg18Corn oil0Intraperitoneal2×/week28Eugenol125Intraperitoneal2×/week Open table in a new tab Table IISummary of eugenol treatment responseGroupMedianTTET - C%TGDLog rank sigMean tumor sizeNumber of NTRmn112.600None3417.3 ± 107.1 mg42152.419%**, p value = 0.0057a, p value = 0.00572135.8 ± 435.9 mg0a ** , p value = 0.0057 Open table in a new tab Tumor tissue (Fig. 3) from control group animals stained with hematoxylin and eosin (Fig. 3A) demonstrates numerous confluent collections of atypical cells with large pleomorphic nuclei, clumped chromatin, and atypical mitotic figures. Interspersed between the melanoma cells are numerous melanin-containing melanophages. Following treatment with eugenol (panel ii), diffuse areas of tumor necrosis intermixed with occasional small aggregates of damaged melanocytes that demonstrate nuclear pyknosis and condensation of the cytoplasm are seen. Diffuse areas of tumor necrosis with no viable-appearing melanoma cells are also seen. We used the modified TUNEL assay to determine whether the tumors in the treatment group were undergoing apoptosis. In the modified TUNEL assay, streptavidin-labeled dUTP is incorporated into the 3′-OH ends of apoptotic DNA by the enzyme terminal deoxynucleotidyltransferase to produce brown staining in cells undergoing apoptosis. As shown in Fig. 3B (panel i), tumor sections of the control animals showed negligible brown staining. On the other hand, in Fig. 3B, panel ii, intense brown staining is visible, indicating that eugenol treatment induces apoptosis in melanoma tumors. Eugenol Blocks Cell Cycle Progression and Induces Apoptosis—Logarithmically growing WM1205Lu cells were treated with eugenol and subjected to flow cytometry analysis as described under “Experimental Procedures.” Results in Fig. 4A show that there was a 40% increase in cells in the S phase accompanied by a decrease in the G1 phase cells with no significant change in the G2/M phase cells following eugenol treatment. Further these data also show that the cells remain blocked in the S phase up to 36 h. Other human melanoma cells (Sbcl2, WM3211, and WM98-1) also showed a similar pattern of S phase block upon eugenol treatment (data not shown). Further we found the consistent appearance of a sub-G1 peak during flow cytometry analysis that is indicative of apoptotic cells (data not shown). We treated melanoma cells with 0.5, 1, and 2.5 μm eugenol for 18 h and then observed the cells under a phase contrast microscope. Fig. 4B shows the morphological changes that occur in a representative cell line WM1205Lu following eugenol treatment. As shown in Fig. 4B, panels ii-iv, all the cells treated with eugenol showed blebbing of membranes, shrinkage of cytoplasm, and condensation of nuclear material, as well as gradually lifting off the dishes. These characteristic features of apoptosis occurred in a dose-dependent manner and was absent in untreated cells (Fig. 4B, panel i). To confirm the induction of apoptosis, we performed the modified TUNEL assay to detect apoptotic cells in situ using the metastatic melanoma cell line, WM1205Lu. As shown in Fig. 4C, panels ii-iv, increasing the concentration of eugenol from 0.5 to 2.5 μm produced an increase in the number of brown stained cells, indicating that these cells were undergoing apoptosis. In Fig. 4C, panel i, the untreated control showed no staining. Cells treated with DNase I were used as a positive control (data not shown). A characteristic feature of apoptotic cells is the activation of endonucleases that attack internucleosomal DNA resulting in DNA fragments that are 180-200 bp. We found that DNA isolated from eugenol-treated cells, but not from control cells, showed the laddering effect (data not shown). Taken together, the data presented in Fig. 4 clearly show that eugenol blocks cells in the S phase of the cell cycle and induces apoptosis in the human melanoma cells, WM1205Lu. It is not known, however, whether the S phase block is essential for induction of apoptosis. Eugenol Modulates Expression of E2F Family Members— Since we found that eugenol blocks cells in the replication phase of the cell cycle, we examined the cell cycle regulatory genes involved in the eugenol response using the pathway-specific gene expression system as described under “Experimental Procedures.” The raw data shown in Fig. 5A were corrected for background by subtracting the signal intensity of pUC18 (negative control) and normalizing to peptidylprolyl isomerase A (PPIA; housekeeping gene), and the relative abundance of transcripts between control and eugenol-treated samples was determined (Fig. 5B). To determine genes that are experimentally and biologically relevant, we filtered the data for those genes whose expression level increased or decreased at least 2-fold. Based on this analysis, we determined that members of the E2F family of transcription factors are modulated by eugenol treatment. Quantitative-RT-PCR with glycer-aldehyde-3-phosphate dehydrogenase as an internal control was used to validate the array results. Results presented in Fig. 5C show the amplification products of the E2F family (E2F1-E2F6) along with glyceraldehyde-3-phosphate dehydrogenase (bottom band). Transcript abundance was calculated as the ratio of the target transcript to that of its internal control. A graphical representation of the relative abundance of transcripts in eugenol-treated and untreated cells is shown in Fig. 5D. We found that E2F1, E2F2, and E2F3 were all down-regulated 2-fold or more following treatment wi