Mel-Ab Cells Research Articles

Depigmentation of Melanocytes by the Treatment of Extracts from Traditional Chinese Herbs: a Cell Culture Assay

To obtain potential skin whitening agents from traditional Chinese herbs, we tested changes of melanin content in melanocyte cell lines after treatment with extracts of 90 traditional Chinese herbs. Mouse melanocyte cell lines were used. Depigmentation activity of the herb extracts were first screened in Mel-Ab cells, and then re-evaluated in melan-a cells and co-culture of melan-a and SP-1 cells. Melanin content and cell viability were the two indications for evaluation. Tyrosinase activity and the expression of melanin synthesis related enzymes in cells treated with the herb extracts were also tested. Nine herb extracts were proved to have depigmentation activity similar to or better than that of arbutin and low cytotoxicity to melanocytes. Two of them were more effective in co-cultured melan-a cells. Most of the effective herb extracts inhibited tyrosinase activity and the expression of tyrosinase. Some of them also inhibited tyrosinase related protein-1 and/or tyrosinase related protein-2 in cultured cells. We have found 9 herb extracts to be promising skin whitening agents. Among them, water extract of Galla Chinensis and ethanol extract of Radix Clematidis exhibited higher depigmentation activity and caused lower tyrosinase activity in cell culture assays and are worthy to be further studied.

Cytoprotective effect of green tea extract and quercetin against hydrogen peroxide-induced oxidative stress

In this study, we evaluated the cytoprotective effects of antioxidative substances in hydrogen peroxide (H2O2) treated Mel-Ab melanocytes. Tested substances include selenium, quercetin, green tea (GT) extract, and several vitamins (ascorbic acid, Trolox, and folic acid). Of these, both quercetin and GT extract were found to have strong cytoprotective effects on H2O2-induced cell death. We also examined additive effects, but no combination of two of any of the above substances was found to act synergistically against oxidative damage in Mel-Ab cells. Nevertheless, a multi-combination of GT extract, quercetin, and folic acid appeared to prevent cellular damage in a synergistic manner, which suggests that combinations of antioxidants may be of importance, and that co-treatment with antioxidants offers a possible means of treating vitiligo, which is known to be related to melanocyte oxidative stress.

Terrein: a new melanogenesis inhibitor and its mechanism

Terrein is a bioactive fungal metabolite whose effects are almost unknown. In this study, we found for the first time that terrein has a strong hypopigmentary effect in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment of Mel-Ab cells with terrein (10-100 microM) for 4 days significantly reduced melanin levels in a dose-dependent manner. In addition, terrein at the same concentration also reduced tyrosinase activity. We then investigated whether terrein influences the extracellular signal-regulated protein kinase (ERK) pathway and the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. Terrein was found to induce sustained ERK activation and MITF down-regulation, and luciferase assays showed that terrein inhibits MITF promoter activity in a dose-dependent manner. To elucidate the correlation between ERK pathway activation and a decreased MITF transcriptional level, PD98059, a specific inhibitor of the ERK pathway, was applied before terrein treatment and found to abrogate the terrein-induced MITF attenuation. Terrein also reduced the tyrosinase protein level for at least 72 h. These results suggest that terrein reduces melanin synthesis by reducing tyrosinase production via ERK activation, and that this is followed by MITF down-regulation.

Effects of lysophosphatidic acid on melanogenesis

In this study, we investigated the effects of lysophosphatidic acid (LPA) on melanogenesis in Mel-Ab cells. We found that LPA significantly attenuates melanin synthesis, and reduces the activity of tyrosinase, the rate-limiting melanogenic enzyme. Interestingly, LPA was also found to induce the activation of a 90 kDa ribosomal S6 kinase (RSK-1), which is known to phosphorylate microphthalmia-associated transcription factor (MITF) at serine 409. Though it has been previously reported that the phosphorylation of MITF is followed by the degradation of MITF, we found that LPA significantly inhibited MITF promoter activity, and that this reduced MITF and tyrosinase protein production. Our results indicate that LPA contributes to reduced melanin synthesis via the down-regulation of MITF.

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.

Ceramide inhibits cell proliferation through Akt/PKB inactivation and decreases melanin synthesis in Mel-Ab cells.

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell-permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Our results show that C2-ceramide inhibits DNA synthesis in Mel-Ab cells and G361 human melanoma cells in a dose-dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C2-ceramide is able to influence extracellular signal-regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C2-ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C2-ceramide-induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel-Ab cells. Furthermore, we assessed the effects of C2-ceramide on the pigmentation of Mel-Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C2-ceramide at concentrations in the range of 1-10 microM, and that the pigmentation-inhibiting effect of C2-ceramide is much greater than that of kojic acid at 1-100 microM. In addition, we found that the activity of tyrosinase is reduced by C2-ceramide treatment. Our results demonstrate that C2-ceramide reduces the pigmentation of Mel-Ab cells by inhibiting tyrosinase activity.

Phorbol 12‐myristate 13‐acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16‐Bl6 melanoma variants

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential. © 1996 Wiley-Liss, Inc.

Phorbol 12-myristate 13-acetate enhances nm23 gene expression in murine melanocytes but not in syngeneic B16-Bl6 melanoma variants

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

Differences in expression of metalloproteinases and plasminogen activators in murine melanocytes and B16 melanoma variants: Lack of association with In vitro invasion

We investigated in vitro chemotactic responses to fibronectin and laminin, invasion through reconstituted basement membrane (Matrigel) and secretion of matrix metalloproteinases and plasminogen activators by non-tumorigenic Mel-ab melanocytes; B16 melanoma; and the metastatic sublines, B16F1, B16F10 and B16BL6. In vitro chemotactic and invasive ability were not associated with in vivo metastatic potential. Secretion of various matrix-degrading enzymes was not related to in vitro invasion. Conditioned media from all B16 melanoma sublines, but not from Mel-ab cells, contained the M(r) 92,000 progelatinase. The activated M(r) 85,000 species was present only in conditioned media from Mel-ab, B16 and B16F1 cells. Mel-ab cells secreted copious amounts of the M(r) 72,000 progelatinase, and the M(r) 66,000 active form was also present in conditioned media. Secretion of the M(r) 72,000 progelatinase by B16 melanoma sublines was markedly lower, and only conditioned media from B16 cells contained the activated M(r) 66,000 form. Furthermore, cell lysates of Mel-ab cells contained a M(r) 67,000 metalloproteinase which was absent in the tumor cells. All cells secreted tissue plasminogen activator; however, the metastatic B16F1, B16F10 and B16-BL6 cells also secreted urokinase plasminogen activator. Our results indicate that matrix metalloproteinase secretion by itself is not associated with tumorigenicity or metastatic potential. Secretion of urokinase plasminogen activator, and not tissue plasminogen activator, reflected the metastatic characteristics of the B16 melanoma tumor sublines.

Growth of melanocytic cells is associated with down-regulation of protein kinase C alpha, delta, and epsilon isoforms. Possible role of diacylglycerol.

Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.

Differential induction of 12-O-tetradecanoylphorbol-13-acetate sequence gene expression in murine melanocytes and melanoma cells.

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281-3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of TIS gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.

Induction of glutathione content in murine melanocytes after transformation with c-H-ras oncogene

Malignant melanoma tumors are inherently resistant to anticancer drugs, yet the mechanism of this resistance is not understood. B16 melanoma, a spontaneous tumor which arose in the C57BL/6 mouse; BL6 melanoma, a highly invasive variant; and Mel-ab melanocytes, isolated from C57BL/6 mouse skin, were examined for intracellular glutathione (GSH) content. GSH was higher in BL6 and B16 cells than in Mel-ab cells, with the highest concentration in BL6 cells. Since GSH is thought to be involved in the resistance of many cells, including melanoma, to cytotoxic drugs, we determined whether intracellular GSH content was altered during transformation of Mel-ab cells. After transfection with pHO6T1 plasmid DNA, containing an activated c-H-ras oncogene flanked by transcriptional enhancers, 1.3% of successfully transfected Mel-ab melanocytes formed distinct colonies in soft agar, compared to 0.2% of cells transfected with control pHO6 plasmid without H-ras. Approximately 53% of the pHO6T1-transfected colonies isolated from soft agar grew in 5% CO2 in the absence of phorbol-12-myristate-13-acetate, a requirement for the extended growth of Mel-ab cells. Cells transfected with control pHO6 plasmid and non-transfected Mel-ab cells did not survive under these conditions. All of the isolated pHO6T1 transfected cells formed tumors when inoculated into C57BL/6 mice. Transformed cells had higher GSH content than non-transfected Mel-ab cells, whether expressed on a cellular or cell volume basis. Although the amount of oxidized glutathione was greater in the tumorigenic cells, this could not account for the overall increase in GSH. Neither glutathione S-transferase nor gamma-glutamyl transpeptidase activities were increased in the H-ras-transfected cells. Northern blot analysis confirmed H-ras-specific RNA in pHO6T1-transformed cells.