Eugenol Causes Melanoma Growth Suppression through Inhibition of E2F1 Transcriptional Activity

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

doi:10.1074/jbc.m411429200

Abstract

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.

Highlights

Metastatic malignant melanoma is an extremely aggressive cancer, with no currently viable therapy. 4-Allyl-2methoxyphenol was tested for its ability to inhibit proliferation of melanoma cells
These results show that eugenol inhibits both anchorage-dependent and anchorage-independent growth of melanoma cells
In an effort to develop molecular mechanismbased treatment strategies, we have identified eugenol as a compound with the ability to inhibit the growth of melanoma cells in culture

Summary

EXPERIMENTAL PROCEDURES

Materials—Eugenol was purchased from Sigma; all the polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell proliferation following treatment was carried out with the CellTiter Aqueous One solution assay (Promega Corp.) as described elsewhere [11]. Apoptosis Detection—Melanoma cells treated with ethanol or 0.5, 1, and 2.5 ␮M 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 [11]. Eugenol-treated (0.5 ␮M) 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 ␮g/ml leupeptin, 25 ␮g/ml aprotinin, 25 ␮g/ml pepstatin. A Coolpix digital camera attached to a Nikon T1-SM or Zeiss microscope was used to obtain photographs of the cells and tissues

RESULTS

Corn oil

DISCUSSION