1-oleoyl-2-acetylglycerol and Melanoma

In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Diglycerides; HSP70 Heat-Shock Proteins; Humans; Hyperpigmentation; Keloid; Melanins; Melanocytes; Melanoma; Mitochondria; Mitochondrial Proteins; RNA Interference; RNA, Small Interfering; Skin Pigmentation

Tyrosinase is a key enzyme for melanin biosynthesis, and hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be improved by treatment with depigmenting agents. In the present study, piperlonguminine from Piper longum was discovered to inhibit melanin production in melanoma B16 cells stimulated with alpha-melanocyte stimulating hormone (alpha-MSH), 3-isobutyl-1-methylxanthine or protoporphyrin IX, where the compound exhibited stronger depigmenting efficacy than kojic acid. However, piperlonguminine did not affect 1-oleoyl-2-acetyl-sn-glycerol-induced melanogenesis and did not affect protein kinase C-mediated melanin production. Surprisingly, piperlonguminine did not inhibit the catalytic activity of cell-free tyrosinase from melanoma B16 cells but rather suppressed tyrosinase mRNA expression. This effect was attributed to the inhibitory action of piperlonguminine on alpha-MSH-induced signaling through cAMP to the cAMP responsive element binding protein that in turn regulates the expression of the microphthalmia-associated transcription factor, a key activator of the tyrosinase promoter. This study demonstrates that piperlonguminine is an efficient depigmenting agent with a novel mechanism of action.

Topics: 1-Methyl-3-isobutylxanthine; alpha-MSH; Animals; Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Diglycerides; Dioxolanes; Down-Regulation; Humans; Melanins; Melanoma; Microphthalmia-Associated Transcription Factor; Molecular Structure; Monophenol Monooxygenase; Phosphodiesterase Inhibitors; Piper; Plant Extracts; Protoporphyrins; RNA, Messenger

Epidermal melanocytes have a higher sensitivity to sulphur mustard (HD) compared with other skin cell types. This may be due to the enzymatic production of melanin precursors exerting an additional cytotoxic effect following HD depletion of the cellular protectant, GSH. Stimulation of the protein kinase C pathway in melanocytes is known to increase melanin production in melanocytes and melanoma cell lines. In order to investigate the role of pigment synthesis in HD toxicology, cultures of an unpigmented melanoma cell line (C32) and of a pigmented melanoma line (G361) were treated with the potent diacyl glycerol analogue, oleoyl acetyl glycerol (OAG), in order to determine if protein kinase C-mediated increases in pigment production could increase sensitivity to subsequent HD exposure. Stimulation of C32 cells with OAG exerted a significant protective effect against the cytotoxic effects of HD. However, this was not due to increased melanin synthesis because this cell line cannot synthesize melanin pigments. The protective action observed is postulated to be due to modulation of protein kinase C activity. In contrast, stimulation of G361 melanoma cells with OAG resulted in an increased level of cytotoxicity upon subsequent exposure to HD. Protein kinase C controls several cellular pathways including checkpoints in the cell cycle, stalling the cell in G and promoting transition through the G2/M boundary. Given the genotoxic properties of HD, these two points in the cell cycle are important in determining the overall cytotoxic effect of HD. Control of the cell cycle by protein kinase C modulation and manipulation of melanin synthetic pathways may have therapeutic benefits.

Topics: Cell Division; Dermatologic Agents; Diglycerides; Humans; Melanins; Melanoma; Mustard Gas; Protein Kinase C; Skin Neoplasms; Tumor Cells, Cultured

The mechanism by which ultraviolet radiation induces melanogenesis in epidermal melanocytes is unknown. Previous observations that in cultured human melanocytes 1-oleoyl-2-acetylglycerol augmented both basal and ultraviolet radiation-induced melanogenesis, suggested that the responses were mediated via protein kinase C. However, paradoxically the phorbol ester TPA was without effect. Therefore, the present study has examined the involvement of protein kinase C in melanogenesis. Analysis of the isozyme profile of human melanocytes revealed the presence of protein kinase C alpha, beta I, epsilon and zeta but not the isozyme eta. Following exposure to 500 nM TPA for 24 hours, isozymes alpha, beta I and epsilon were downregulated, but zeta was unaffected. Similar isozyme profiles were observed in S91 and SKMEL3 melanoma cells. The melanogenic responses to 1-oleoyl-2-acetylglycerol and ultraviolet radiation were unaffected by inhibition of protein kinase C with Ro31-8220, or ablation by downregulation with 500 nM TPA, in human melanocytes and melanoma cells. 1-Oleoyl-2-acetylglycerol had no effect on protein kinase C activity in human melanocytes, as measured by rapid phosphorylation of the 80 kDa protein myristoylated alanine-rich C kinase substrate (MARCKS). Ultraviolet radiation induced a small increase in MARCKS protein phosphorylation but this effect was inhibited by pretreatment for 24 hours with 500 nM TPA, which had no effect on ultraviolet-induced melanogenesis. Overall, these findings indicate that 1-oleoyl-2-acetylglycerol and ultraviolet radiation activate melanogenesis via protein kinase C-independent pathways.

Topics: 1-Methyl-3-isobutylxanthine; 3T3 Cells; Animals; Diglycerides; Enzyme Induction; Humans; Indoles; Intracellular Signaling Peptides and Proteins; Isoenzymes; Melanins; Melanocytes; Melanoma; Melanoma, Experimental; Membrane Proteins; Mice; Myristoylated Alanine-Rich C Kinase Substrate; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Proteins; Signal Transduction; Skin Neoplasms; Taurodeoxycholic Acid; Tetradecanoylphorbol Acetate; Ultraviolet Rays

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.

Topics: Biological Transport; Carcinoma, Squamous Cell; Cytoplasmic Granules; Diglycerides; Flow Cytometry; Humans; Keratinocytes; Lectins; Melanins; Melanoma; Microscopy, Electron; Tumor Cells, Cultured