Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation.
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 implicate omega-6 polyunsaturated fatty acids (PUFAs) as stimulators and long-chain omega-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 omega-3 PUFA, affects human melanoma cells. Exponentially growing melanoma cell lines were exposed in 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, p21WAF1/CIP1, or p27(KIP1); and (c) expression of total pRb(T) independent of phosphorylation state and hypophosphorylated pRb(P-) in fixed cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to pRb(T) or pRb(P-), 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-Mel-110) to the inhibitory activity of DHA. SK-Mel-29 cells were unaffected by treatment with up to 2 microg/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.
Albino
A P
AP
The American Health Foundation, Valhalla, New York 10595, USA. talbino@aol.com
Juan
G
G
Traganos
F
F
Reinhart
L
L
Connolly
J
J
Rose
D P
DP
Darzynkiewicz
Z
Z
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D016895:Culture Media, Serum-Free; D019938:Cyclin D1 / Q000096:biosynthesis; D019927:Cyclin E / Q000096:biosynthesis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000096:biosynthesis; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D005434:Flow Cytometry; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D010766:Phosphorylation / Q000187:drug effects; D016160:Retinoblastoma Protein / Q000378:metabolism*; D012878:Skin Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D016895:Culture Media, Serum-Free; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D006131:Growth Inhibitors; D008869:Microtubule-Associated Proteins; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004281:Docosahexaenoic Acids