h-89 and Melanoma

Wnt/β-catenin signaling is important in development and differentiation of melanocytes.. The object of this study was to evaluate the effects of several Wnt/β-catenin signaling inhibitors on pigmentation using melanoma cells.. Melanoma cells were treated with Wnt/β-catenin signaling inhibitors, and then melanin content and tyrosinase activity were checked.. Although some inhibitors showed slight inhibition of pigmentation, we failed to observe potential inhibitory effect of those chemicals on pigmentation of HM3KO melanoma cells. Rather, one of powerful Wnt/β-catenin signaling inhibitors, ICG-001, increased the pigmentation of HM3KO melanoma cells. Pigmentation-enhancing effect of ICG-001 was reproducible in other melanoma cell line MNT-1. Consistent with these results. ICG-001 increased the expression of pigmentation-related genes, such as MITF, tyrosinase and TRP1. When ICG-001 was treated, the phosphorylation of CREB was significantly increased. In addition, ICG-001 treatment led to quick increase of intracellular cAMP level, suggesting that ICG-001 activated PKA signaling. The blockage of PKA signaling with pharmaceutical inhibitor H89 inhibited the ICG-001-induced pigmentation significantly.. These results suggest that PKA signaling is pivotal in pigmentation process itself, while the importance of Wnt/β-catenin signaling should be emphasized in the context of development and differentiation.

Topics: beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Line, Tumor; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Humans; Isoquinolines; Melanins; Melanocytes; Melanoma; Monophenol Monooxygenase; Pigmentation; Protein Kinases; Pyrimidinones; Sulfonamides; Tumor Cells, Cultured; Wnt Proteins; Wnt Signaling Pathway

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. In order to determine the effects of rosmarinic acid on melanogenesis and elucidate the molecular events of melanogenesis induced by rosmarinic acid, several experiments were performed in B16 melanoma cells. In this study, we showed that the melanin content and tyrosinase expression were increased by rosmarinic acid in a concentration-dependent manner. In addition, after the melanin content was increased by rosmarinic acid, it was reduced by H-89 and KT 5720, protein kinase A (PKA) inhibitors, but not by SB203580, a p38(mapk) inhibitor, or Ro-32-0432, a PKC inhibitor, which suggests the involvement of PKA in rosmarinic acid-induced melanogenesis. Consistent with this, rosmarinic acid induced the phosphorylation of CRE-binding protein (CREB), but had no effect on the phosphorylation of p38(mapk) or the inhibition of Akt phosphorylation. Additionally, rosmarinic acid induced the activation of cAMP response element (CRE) without having any effect on cAMP production, which suggests that rosmarinic acid-induced melanogenesis is mediated by PKA, which occurs downstream of cAMP production. This result was further confirmed by the fact that rosmarinic acid-induced phosphorylation of CREB was inhibited by H-89, but not by PD98059, a MEK1 inhibitor, or by LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Rosmarinic acid-induced expression of tyrosinase protein was attenuated by H-89. Based on these results, we report for the first time that rosmarinic acid induces melanogenesis through PKA activation signaling.

Topics: Animals; Carbazoles; Cell Line, Tumor; Cinnamates; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Depsides; Enzyme Activation; Indoles; Isoquinolines; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Oncogene Protein v-akt; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyrroles; Rosmarinic Acid; Signal Transduction; Sulfonamides

Interleukin-6 (IL-6) is a bifunctional growth factor in malignant melanoma; its expression increases during the malignant progression of the disease. Histamine, detected in large amounts in normal and pathological proliferating tissues, is an important paracrine and autocrine regulator of normal and tumour cell proliferation as well.. We investigated the presence and function of IL-6 and histamine in the WM35 primary human melanoma cell line with respect to their direct role in cell proliferation and their regulatory interactions.. IL-6 inhibited the proliferation of WM35 melanoma cells and increased significantly the expression of histidine decarboxylase as well as histamine production. It had dose-dependent effects on the proliferation: high concentration (10-5 M) was inhibitory through H1 histamine receptors while low histamine concentration acting on H2 receptors, with a simultaneous increase of cAMP, enhanced colony formation in the monolayer. Furthermore, IL-6 increased the H1- but decreased the H2-histamine receptor expression of the melanoma cells. On the other hand, histamine was locally synthesized by the WM35 melanoma cells.. We suggest that the growth arrest induced by IL-6 is in part mediated by its dual action on histamine: a shift toward H1 receptor predominance and an elevation of locally produced histamine with prevalent action on the inhibitory response triggered through the H1 receptor. These findings suggest a local cross-talk between histamine and IL-6 in the regulation of melanoma growth.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Bucladesine; Cell Division; Colforsin; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Gene Expression; Guanidines; Histamine; Histamine H1 Antagonists; Histamine H2 Antagonists; Humans; Imidazoles; Interleukin-6; Isoquinolines; Melanoma; Polymerase Chain Reaction; Receptors, Interleukin-6; Skin Neoplasms; Sulfonamides; Tumor Cells, Cultured

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).

Topics: Amino Acid Sequence; Cell Division; Collagen; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Isoquinolines; Melanoma; Molecular Sequence Data; Peptide Biosynthesis; Sulfonamides; Tumor Cells, Cultured

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Topics: Adenoviruses, Human; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Indole Alkaloids; Indoles; Isoflavones; Isoquinolines; Lactoferrin; Low Density Lipoprotein Receptor-Related Protein-1; Maleimides; Melanoma; Naphthalenes; Phenols; Protein Kinase C; Receptors, Immunologic; Signal Transduction; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator