Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells

A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in anin vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0.01 to 200 μg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 μg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C₂ ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.     

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 effects of whisky polyphenols on melanogenesis in mouse B16 melanoma cells

Whisky exerts an inhibitory effect on melanogenesis in B16 cells, the anti-melanogenic activity being positively correlated with the aging period and anti-oxidative activity of whisky. We examined the correlation between the inhibition of melanogenesis and the concentration of each compound in various whiskies to evaluate the importance of 11 different whisky polyphenols, including ellagic acid, gallic acid and lyoniresinol, in the anti-melanogenic activity of whisky. The concentration of all the compounds was positively correlated with the anti-melanogenic activity of whisky. Ellagic acid, gallic acid and lyoniresinol were the predominant polyphenols in the whiskies measured by HPLC. These three compounds also significantly inhibited the melanogenesis and tyrosinase activity in B16 cells. Ellagic acid, gallic acid and lyoniresinol were confirmed as the major participants in the anti-melanogenic activity of whisky.     

Involvement of extracellular signal-regulated kinase in nobiletin-induced melanogenesis in murine B16/F10 melanoma cells

Nobiletin contributes to pharmacological activities such as anti-cancer and anti-inflammatory effects, but little is known about its effect on melanogenesis. In this study, we found that nobiletin increased melanin content and tyrosinase activity in murine B16/F10 melanoma cells. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK) pathway with U0216 resulted in inhibition of nobiletin-induced melanin synthesis and tyrosinase expression.     

Inhibitory effect of ectoine on melanogenesis in B16-F0 and A2058 melanoma cell lines

Skin injuries, congenital lesions, melasma, Addison's disease and many pigment abnormalities prompt us to search for an effective whitening agent. Ideal whitening agent is a natural compound that can inhibit melanogenesis and has no cytotoxic effects. In a previous study, we have developed an optimum method for the production and characterization of ectoine from a halophilic bacterium isolated from a salt environment in Taiwan was identified as Marinococcus sp. In the present study, we screened the whitening properties of the biosynthesized ectoine using mouse and human melanoma cell lines, B16-F0 and A2058. Here, we examined the cell viabilities of melanoma cells after ectoine treatment at various concentrations up to 500 μM. Also, we addressed the melanin synthesis of melanoma cells after treatment with ectoine. The inhibitory effects of ectoine on tyrosinase activity were assessed in both mushroom tyrosinase and cellular tyrosinase. Furthermore, we investigated the type of inhibition of mushroom tyrosinase using Lineweaver–Burk enzyme kinetic. The melanogenesis-related gene expression (tyrosinase, TRP1, TRP2 and MITF) and their protein secretion were determined by the assays of quantitative real-time PCR and western blots, respectively. Our results demonstrated that ectoine is a safe and effective whitening agent, inhibited melanin synthesis, reduced both mushroom tyrosinase and cellular tyrosinase, and had various inhibitory effects on the expressions of melanogenesis-related genes and secretion of proteins in mouse and human melanoma cell lines. Thus, we suggest that ectoine can serve as a useful and safe new agent in cosmetic and clinical applications.     

Inhibitory effect of Boerhaavia diffusa on experimental metastasis by B16F10 melanoma in C57BL/6 mice

Administration of the aqueous methanol (3:7) extract of B.diffusa was found to be effective in reducing the metastases formation by B16F10 melanoma cells. Prophylactic administration of the extract (0.5 mg/dose) inhibited the metastases formation by about 95% as compared to untreated control animals. There was 87% of inhibition in the lung metastases formation in syngenic C57BL/6 mice, when the extract was administered simultaneously with tumour challenge. Biochemical parameters such as lung collagen hydroxyproline, hexosamines and uronic acid levels were also reduced significantly (P < 0.001) in the treated animals. Levels of serum sialic acids and gamma-glutamyltranspeptidase that are markers of neoplastic proliferation were also reduced in the tumour plus extract treated animals. More over treatment with the extract enhanced the survival of the animals more than double that of untreated control animals. When a non-toxic concentration of the extract was treated directly to the B16F10 cells in vitro, it inhibited the cell proliferation as estimated by the 3H - thymidine uptake assay. From the Zymogram analysis using culture supernatant from the extract treated cells it became evident that the components of the extract inhibited the expression or activity of gelatinases A and B (MMP-2 and MMP-9). Since the MMPs are intimately associated with cell invasion and angiogenesis, inhibition of these functions along with the anti-proliferative activity (cytostatic) may be contributing to the antimetastatic property shown by B. diffusa.     

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.     

Evaluation of apoptotic effect of cyclic imide derivatives on murine B16F10 melanoma cells

Cyclic imides are a large class of compounds obtained by organic synthesis including several sub-classes (succinimides maleimide, glutarimide, phthalimides naphtalimides, and its derivatives). Recently, some cyclic imide derivatives have shown important results as potential antitumor agents, as a Mitonafide and Amonafide. Based on this fact, we have studied antitumoral properties of nine cyclic imide derivatives, four of which are unpublished compounds, against Murine Melanoma Cells (B16F10). Initially, the MTT assay was used to select the compound with the best cytotoxic potential. After this selection, the compound 2-benzyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (4), which showed the best cytotoxic effects, was evaluated by flow cytometry, and a significant increase was observed in the proportion of cells in the subG0/G1, S and G2/M phases accompanied by a significant decrease in the G0/G1 phases. Then the mechanism involved on the death route (necrosis or apoptosis) was evaluated the by bromide and acridine orange method and by an Annexin V-FITC Apoptosis Detection kit. These results confirm that the percentage of B16F10 cells observed in the sub G0/G1 phase were undergoing apoptosis. The biological effects observed in the current study for the cyclic imide derivatives suggested promising applications, especially for the prototype compound 4.     

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.     

Differential distribution of B16F10 melanoma cells in the liver lobule

The distribution pattern and the number of tumor cells arrested in the liver were studied in mouse livers. Mice were perfused intravascularly with a suspension of B16F10 melanoma cells. The animals were sacrificed at 0, 1, 5, and 20 min after tumor cell perfusion. The pattern of tumor cell distribution was studied by morphological methods, and by a combined method of fluorescent-tumor cell labelling and histochemical succinate dehydrogenase activity on frozen sections, in order to define the localization of tumor cells arrested in the liver lobule. The results show that the tumor cells have an exclusive distribution in the periportal regions of the liver lobule (identified as the high succinate dehydrogenase activity areas), and that the cells are not arrested in the pericentral regions (identified as the low succinate dehydrogenase activity areas). In addition, indomethacin treatment (2 mg/kg/day) induced an increase in the number of melanoma cells arrested in the liver, but a different distribution with respect to controls was not observed. These results show that periportal regions of the liver lobule constitute a particular domain in which the B16F10 melanoma cells present a special retention ability that can be modulated by indomethacin treatment.     

Effect of serum withdrawal on the proliferation of B16F10 melanoma cells

B16F10 murine melanoma cell proliferation was inhibited after 48 h in medium with serum in the range 0.1 to 0.5% by volume. Cell viability was mostly retained, whereas cells completely deprived of serum died. Growth-arrested cultures showed serum-dependent suppression of DNA synthesis. The response was typically that of a 'cell cycle freeze', verified by flow cytometric distribution of cells. Consequently, serum deprivation did not lead to synchrony when serum was restored to arrested populations. Furthermore, there was no change in PCNA expression in arrested cells.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

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.