Treatment of mouse melanoma cells with phorbol 12-myristate 13-acetate counteracts mannosylerythritol lipid-induced growth arrest and apoptosis

Mannosylerythritol lipid (MEL), an extracellularglycolipid from yeast, induces the differentiation ofHL-60 promyelocytic leukemia cells towardsgranulocytes. We show here that MEL is also a potentinhibitor of the proliferation of mouse melanoma B16cells. Flow-cytometric analysis of the cell cycle ofMEL-treated B16 cells revealed the accumulation ofcells in the sub-G₀/G₁ phase, which is a hallmark ofcells undergoing apoptosis. Treatment of B16 cellsfor 24 h with phorbol 12-myristate 13-acetate (PMA),an activator of protein kinase C (PKC), did notinterfere with the growth and survival of the cells,but it effectively counteracted the MEL-induced growtharrest and apoptosis. The activity of PKC was reducedin B16 cells treated with MEL at a concentration atwhich MEL induced apoptosis. However, incubation withPMA in addition to MEL reversed this reduction in theactivity of PKC. These results suggest thatconverging signaling pathways are triggeredindependently by MEL and PMA and that the signalsmight both be mediated by PKC.     

Involvement of phospholipase D1 in melanogenesis of mouse B16 melanoma cells

In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.     

Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma

Melanogenesis is one of the characteristic functional activities of melanocyte/melanoma and is regulated via mitogen-activated protein kinase (MAPK) and Akt/protein kinase B (PKB) pathways. Placental total lipid fraction (PTLF), prepared from a hydroalcoholic extract of fresh term human placenta contains sphingolipids and was recently shown to stimulate melanogenesis via up-regulation of the key enzyme tyrosinase in B16F10 mouse melanoma cells. How such lipids mediate their effects on pigmentation and tyrosinase expression is a particularly important aspect of melanogenesis. To study the signaling that leads to tyrosinase expression, we have investigated the roles of the MAPK and Akt/PKB pathways in B16F10 melanoma cells in melanogenesis in response to PTLF. Treatment of cells with PTLF led to the time dependent phosphorylation of p38 MAPK. SB203580, a p38 MAPK inhibitor, completely blocked the PTLF-induced melanogenesis by inhibiting promoter activity and subsequent expression of tyrosinase. Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 a blocker of the Akt signaling pathway, or an inhibitor of MEK (MAPK/ERK Kinase), PD98059 when included along with PTLF was found to potentiate PTLF-induced phosphorylation of p38 MAPK together with tyrosinase expression and melanogenesis. The results suggest that the activation of p38 MAPK plays a crucial role in PTLF-induced B16F10 melanogenesis by up-regulating tyrosinase expression.     

Differentiation-inducing activity of lupane triterpenes on a mouse melanoma cell line

Lupane triterpenes were found to promote melanogenesis, a hallmark of B16 2F2 mouse melanoma cell differentiation. Studies of the structure-activity relationships demonstrated that the keto function at C-3 of the lupane skeleton played important roles in the melanogenic activities of lupane triterpenes on melanoma cells. The carbonyl group at C-17 of lupane triterpenes was essential against their apoptosis-inducing activity against human cancer cells via the inhibition of topoisomerase I. We investigated whether signaling mechanisms were involved in the stimulative effects of lupane triterpenes on the melanogenesis of B16 2F2 cells. In experiments using selective inhibitors against various signal transduction molecules and Western blotting analysis, it was suggested that p38 MAPK was involved in melanoma cell differentiation as a downstream effector of PKA. Lupeol (compound 1), a lupane triterpene, induced dendrite formations, a morphological hallmark of B16 2F2 cell differentiation by rearrangement of the actin cytoskeleton. The activation of cofilin, an actin depolymerizing factor, by compound 1 caused actin fiber disassembly in B16 2F2 cells. Furthermore, compound 1 was shown to inhibit the cell motilities of human melanoma and neuroblastoma in vitro.     

Protein kinase C and linoleic acid-induced inhibition of melanogenesis

Linoleic acid has been shown to inhibit melanogenesis in cultured B16 mouse melanoma cells. We report here the possible involvement of protein kinase C (PKC) in linoleic acid-induced inhibition of melanogenesis in B16 cells. A single PKC subspecies (alpha-PKC) was detected in B16 cells. The enzyme was activated by linoleic acid in vitro. The effective concentrations at which PKC was activated (25 microM; maximum response) were consistent with those for the inhibition of melanogenesis in cell culture system. In addition, the permeable diacylglycerol 1-oleoyl-2-acetyl glycerol that activates PKC also inhibits melanogenesis at 100 microM. These results suggest that activation of PKC plays a pivotal role in the linoleic acid-induced inhibition of melanogenesis in B16 cells.     

Regulation of ornithine decarboxylase in B16 mouse melanoma cells: synergistic activation of melanogenesis by alphaMSH and ornithine decarboxylase inhibition

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationic compounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression of genes regulating cell growth and/or differentiation can be controlled by alpha-melanocyte-stimulating hormone (alphaMSH) and other melanogenesis modulating agents. In the B16 mouse melanoma model, alphaMSH stimulates melanogenesis by upmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibits melanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenic pathway. Treatment of B16 cells with TPA or alphaMSH rapidly stimulated ODC activity. The effect was stronger for TPA and appeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor alpha-difluoromethylornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMO stimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatment with alphaMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic response of B16 cells to alphaMSH. Rather, increased intracellular concentrations of polyamines following ODC induction might constitute a feedback mechanism to limit melanogenesis activation by alphaMSH.     

Specific protein production during melanogenesis in B16/C3 melanoma cells

The mouse melanoma cell line B16/C3 offers an excellent in vitro model for studying melanocyte differentiation. Melanogenesis can be induced by serum, a hormone-supplemented serum-free medium, melanocyte stimulating hormone, and dibutyryl cAMP. The tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, 5-bromodeoxyuridine, and acidic pH inhibit this process. Using two-dimensional polyacrylamide gel electrophoresis, we have identified four cellular proteins whose production is modulated during melanogenesis, a process which includes concomitant increases in levels of tyrosinase, the rate limiting enzyme for melanin biosynthesis, melanization, and ultimately, cell death. The production of these proteins are coordinately expressed or inhibited in response to the diverse inducers and inhibitors of melanogenesis. We conclude from these studies that these specific proteins are intimately involved in the differentiation of B16/C3 melanoma cells.     

Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells

Melanogenesis is a principal parameter of differentiation in melanocytes and melanoma cells. Our recent study has demonstrated that phospholipase D1 (PLD1) regulates the melanogenic signaling through modulating the expression of tyrosinase, the rate-limiting step enzyme in the melanin biosynthesis. The current study was designed to gain more insight into the involvement of PLD1 in the regulation of melanogenesis. To investigate the role of PLD1, we examined the effect of knockdown of endogenous PLD1 by small interference RNA (siRNA) on melanogenesis in B16 melanoma cells. It was shown that the melanin synthesis was induced in PLD1-knockdowned cells, and also that the level of melanin synthesis was well correlated with increases in expression level of tyrosinase and its related proteins (Tyrp1 and Dct). Furthermore, the reduction of expression levels of PLD1 by siRNA transfection was accompanied by diminution of ribosomal S6 kinase 1 (S6K1) phosphorylation. The activity of mammalian target of rapamycin (mTOR) is essential for phosphorylation of S6K1 and the treatment malanoma cells with rapamycin, a potent inhibitor of mTOR effectively induced melanogenesis. The results obtained here provide possible evidence that PLD1 exerts a negative regulatory role in the melanogenic process through mTOR/S6K1 signaling.     

Stimulation of melanin synthesis of B16-F10 mouse melanoma cells by bufalin.

Bufalin, which is one of prominent components of Chinese toad venom, was found to decrease the rate of cell proliferation of mouse melanoma clone B16-F10 cells and a concomitant stimulation of expression of its melanotic phenotype. The effect of bufalin on melanogenesis included stimulation of tyrosinase activity and increase of cellular melanin content. These effects became apparent after 48 hr exposure to 10(-4) M bufalin and increased thereafter. Other cardiotonic steroids, such as cinobufagin and ouabain, at the concentration of 10(-4) M for 6 days, also showed the stimulatory effect on melanin synthesis of B16-F10 cells, but not digitoxigenin.     

Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A.

Clostridium difficile toxin A increases paracellular permeability in colonic epithelial T84 cells by mechanisms involving RhoA glucosylation and actin depolymerization. However, we previously observed that toxin A-mediated decline in transepithelial electrical resistance preceded changes in cell morphology and tight junction ultrastructure (Hecht, G., Pothoulakis, C., LaMont, J. T., and Madara, J. L. (1988) J. Clin. Invest. 82, 1516-1524). Recent studies also showed that C. difficile toxins induce early cellular responses, including activation of mitogen-activated protein kinases, generation of reactive oxygen metabolites, and calcium influx. The aim of this study was to investigate whether toxin A-induced early cellular responses contribute to the permeability changes. We found that toxin A stimulated the activities of membrane and cytosolic protein kinase Calpha (PKCalpha) and cytosolic PKCbeta. A specific PKCalpha/beta antagonist (myristoylated PKCalpha/beta peptide) blocked toxin A-mediated RhoA glucosylation. Furthermore, decreased transepithelial electrical resistance and increased translocation of ZO-1 from tight junction occurred within 2-3 h of toxin A exposure and were also inhibited by PKCalpha/beta antagonist. During this time period, toxin exposure did not induce translocation of ZO-2, dephosphorylation or translocation of occludin, or cell rounding. Our data indicate that PKC signaling regulates toxin A-mediated paracellular permeability changes and ZO-1 translocation.     

In vitro and in vivo melanogenesis inhibition by biochanin A from Trifolium pratense

Our previous study showed that a methanol extract from Trifolium pratense exerted potent inhibitory activity on melanogenesis in mouse B16 melanoma cells. In the present study, the active compound in this Chinese herb extract was isolated and identified as biochanin A by mass spectrum, (1)H-NMR, and (13)C-NMR analysis. The inhibitory effects of biochanin A on melanogenesis were investigated in vitro in cultured melanoma cells and in vivo in zebrafish and mice. Biochanin A dose-dependently inhibited both melanogenesis and cellular tyrosinase activity in B16 cells and in zebrafish embryos. Application of a cream containing 2% biochanin A twice daily to the skin of mice also increased the skin-whitening index value after 1 week of treatment, and the increase continued for another 2 weeks. Biochanin A was confirmed as a good candidate for use as a skin-whitening agent in the treatment of skin hyperpigmentation disorders.     

In vitro and in vivo effects of an anti-mouse endoglin (CD105)–immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC₅₀ values of 4.2 × 10^?9 M for B16MEL4A5 and 7.7 × 10^?11 M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.     

Inhibitory effects of whisky congeners on melanogenesis in mouse B16 melanoma cells

We examined the effect of whisky congeners, substances other than ethanol in whisky, on melanogenesis in mouse B16 melanoma cells. Treatment with whisky congeners significantly blocked melanogenesis. Our results indicate that the inhibitory effects of whisky congeners on melanogenesis is due to direct inhibition of tyrosinase activity and to suppression of tyrosinase protein levels.     

Inhibitory effect of rose hip (Rosa canina L.) on melanogenesis in mouse melanoma cells and on pigmentation in brown guinea pigs

The compounds present in rose hips exerting an inhibitory action against melanogenesis in B16 mouse melanoma cells were investigated by dividing an aqueous extract of rose hips (RE) into four fractions. The 50% ethanol eluate from a DIAION HP-20 column significantly reduced the production of melanin and was mainly composed of procyanidin glycosides. We also found that this 50% ethanol eluate reduced the intracellular tyrosinase activity and also had a direct inhibitory effect on tyrosinase obtained as a protein mixture from the melanoma cell lysate. We also investigated the effect of orally administering RE on skin pigmentation in brown guinea pigs, and found that the pigmentation was inhibited together with the tyrosinase activity in the skin. These data collectively suggest that proanthocyanidins from RE inhibited melanogenesis in mouse melanoma cells and guinea pig skin, and could be useful as a skin-whitening agent when taken orally.