Cell Cycle Arrest and Apoptosis of Melanoma Cells by Docosahexaenoic Acid : Association with Decreased pRb Phosphorylation 1

The incidence of cutaneous malignant melanoma is undergoing a dramatic increase in persons with light-color skin in all parts of the world. The prognosis for individuals with advanced disease is dismal due to the lack of effective treatment options. Thus, there is a need for new approaches to control tumor progression. Epidemiological, experimental, and mechanistic data implicatev-6 polyunsaturated fatty acids (PUFAs) as stimulators and long-chainv-3 PUFAs as inhibitors of development and progression of a range of human cancers, including melanoma. The aim of this study was to assess the mechanisms by which docosahexaenoic acid (DHA), an v-3 PUFA, affects human melanoma cells. Exponentially growing melanoma cell lines were exposedin vitro to DHA and then assessed for (a) inhibition of cell growth; (b) expression of cyclins and cyclin-dependent kinase inhibitors in individual cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to cyclin D1, cyclin E, p21, or p27; and (c) expression of total pRb independent of phosphorylation state and hypophosphorylated pRb in fixed cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to pRb or pRb, respectively. After treatment with increasing concentrations of DHA, cell growth in a majority of melanoma cell lines (7 of 12) was inhibited, whereas in 5 of 12 cell lines, cell growth was minimally affected. Two melanoma cell lines were examined in detail, one resistant (SK-Mel-29) and one sensitive (SK-Mel110) to the inhibitory activity of DHA. SK-Mel-29 cells were unaffected by treatment with up to 2 mg/ml DHA whether grown in the absence or presence of 1% fetal bovine serum (FBS). No appreciable change was observed in cell growth, cell cycle distribution, the status of pRb phosphorylation, cyclin D1 expression, or the levels of the cyclin-dependent kinase inhibitors p21 and p27. In contrast, SK-Mel-110 cell growth was inhibited by DHA with the cells accumulating either in G1 or S phase: 0% in SK-Mel-29 versus 13.3 or 41.2% in SK-Mel-110 in the absence or presence of FBS, respectively. In the absence of serum, considerable death occurred by apoptosis. In addition, DHA treatment resulted in increasing numbers of SK-Mel-110 cells (from 12 to>40%) expressing hypophosphorylated pRb, whereas the levels of cyclin D1 and p21 changed little. Expression of p27 in these cells increased >2.5 times when grown in the absence of FBS but not in the presence of 1% FBS. Thus, we show for the first time that DHA inhibits the growth of cultured metastatic melanoma cells. Furthermore, growth inhibition correlates with a quantitative increase in hypophosphorylated pRb in the representative sensitive melanoma cell line SK-Mel-110. Although multiple factors influence pRb phosphorylation, it appears that both cyclin D1 and p21 expression do not change in the presence of DHA, although p27 was strikingly increased in SK-Mel-110 cells in the absence of FBS. The fact that pRb became hypophosphorylated after exposure to DHA suggests a cross-talk mechanism between fatty acid metabolism and the pRb pathway. Determining the mechanism by which PUFAs can inhibit melanoma growth will be an important first step in the rational use of PUFAs as antitumor agents. INTRODUCTION Epidemiological studies predict that, given the present rate of increase, 1 in 75 Americans born today will develop cutaneous malignant melanoma sometime in their lifetime (1). Moreover, although early stage melanoma that is confined to the epidermis or superficial dermis is curable, the prognosis for individuals with deep invasion of the dermis and metastases is dismal, with a 5-year survival rate of only ;10%. This poor outcome is due to the lack of effective treatment options (2–4). One potential and innovative adjuvant therapeutic modality is a dietary intervention designed to modify the relative consumption of the essential PUFAs 3 (3). There is a growing body of data indicating that dietary fat influences the development and progression of many cancers (5–8), including malignant melanoma (9–14), with the polyunsaturatedv-6 fatty acids (n-6 PUFAs) exerting stimulatory effects (9, 10) andv-3 fatty acids (n-3 PUFAs) having suppressive activity (11). These epidemiological observations are supported by experimental studies, which demonstrated enhancing effects of high-fat diets on UV-induced skin carcinogenesis (15–18) and inhibition by feeding dietary n-3 PUFA (17). Previously published studies have shown that n-6 PUFA stimulates the growth of human breast (19, 20) and prostate (21) cancer cell lines in vitro, whereas DHA and EPA, two long-chain n-3 PUFAs that are present at high concentrations in some fish oils, inhibit growth (19, 21–23). We report here that DHA inhibited the growth of . 50% of human melanoma cell lines studied by us in vitro. Furthermore, in a representative sensitive melanoma cell line, this inhibition was accompanied by alterations in the phosphorylation status of the pRb gene product, the primary role of which is to control the commitment of cells to enter S phase (24–26). At present, little is known about the effects of fatty acids on cell cycle control. Thus, the present study provides information about potential mechanistic interactions of fatty acids with specific components of the cell cycle machinery. MATERIALS AND METHODS Melanoma Cell Lines. Twelve human metastatic melanoma (SK-Mel) cultures were established as described previously (27, 28) and routinely passaged in DMEM (BioWhittaker, Walkersville, MD) containing 7.5% FBS (Gemini Bioproducts, Calabasas, CA), penicillin (100 U/ml), and streptomycin (100mg/ml). All cell lines were confirmed to be free of Mycoplasmabefore use. Calculation of Cell Doubling Times. Cells were plated in DMEM containing 7.5% FBS at a density of 2000 cells per well of a 12-well tissue culture plate and incubated at 37°C. After 24 h, the cells were washed twice with PBS and refed with DMEM in the presence or absence of 1.0% FBS. Every 2 days thereafter, the cells were harvested by trypsinization and cell number was determined using a Coulter Counter (Coulter Electronics, Hialeah, FL). The cultures were refed every 3 days of the experiment. After 8 days of growth, the Received 11/2/99; accepted 5/30/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the American Health Foundation. 2 To whom requests for reprints should be addressed, at The American Health Foundation, One Dana Road, Valhalla, NY 10595. Phone: (914) 789-7158; Fax: (914) 5926317; E-mail: talbino@aol.com. 3 The abbreviations used are: PUFA, polyunsaturated fatty acid; DHA, docosahexaenoic acid; Rb, retinoblastoma; CKI, cyclin dependent kinase inhibitor; mAb, monoclonal antibody; EPA, eicosapentaenoic acid; CDK, cyclin dependent kinase; FBS, fetal bovine serum; DAPI, 49,6-diamidino-2-phenylindole.