Anti-Hyperpigmentation-Related Potential Activities in B16BL6 Cells and Chemical Composition of Essential Oil from Chamaecyparis pisifera Leaves

Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes

Ascorbic acid, a potential antioxidant, is known to inhibit melanogenesis. However, there are conflicting findings that ascorbic acid has very low stability and acts as a pro-oxidant, eventually increasing proliferation and melanin content in melanoma cells. In the present study, we explored the effects of ascorbic acid on the activity and expression of tyrosinase and melanin pigmentation in the presence and absence of α-melanocyte-stimulating hormone (α-MSH) using B16F10 melanoma cells. The mechanism by which ascorbic acid stimulated the expression of tyrosinase was also investigated. No inhibitory effect on melanin content was observed in ascorbic acid-treated cells, regardless of the presence of α-MSH. Ascorbic acid stimulated the activity and expression of tyrosinase and increased the expression of melanogenic regulatory factors, such as tyrosinase-related protein-1 (TRP-1), dihydroxyphenylalaminechrome tautomerase (TRP-2), and microphthalmia-associated transcription factor (MITF). Ascorbic acid also induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). The inhibition of p38 MAPK pathway by SB203580 led to the suppression of tyrosinase, TRP-1, and TRP-2 expression in cells treated with ascorbic acid. Combined treatment with N-acetyl-L: -cysteine and/or desferrioxamine mesylate attenuated the stimulating effect of ascorbic acid on tyrosinase activation in the cells. Collectively, ascorbic acid stimulates tyrosinase activity and expression in B16F10 cells via activation of p38 MAPK signaling and subsequent up-regulation of MITF, tyrosinase, and TRP expression.

A variant of B-16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition of MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F1 cells. MSH induced a two fold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.

Cloudman S91 mouse melanoma cells lose their ability to demonstrate an MSH-induced increase in tyrosinase activity as cell density increases. This loss in hormone responsiveness occurs before confluency is reached and cannot be reversed by exposure of cells to increasing concentrations of MSH. The failure of high-density cultures to respond to MSH is apparently not the result of an inability of MSH to stimulate cAMP production, since either low- or high-density cultures exposed to MSH demonstrate equivalent increases in intracellular levels of cAMP. Further, neither theophylline (1mM), dibutyryl cyclic AMP (10(-4)M), or prostaglandin E1 (10(-6)M) is effective in stimulating tyrosinase activity in melanoma cells cultured at densities exceeding 6 X 10(4) cells/cm2. This finding suggests that the decay of hormone responsiveness occurs at a cellular site distal to cAMP production. The decrease in tyrosinase stimulation by MSH as cell density increases is also apparently not the result of an increase in activity of any soluble inhibitor of the enzyme, for cytosol preparations from high-density cultures (10(5) cells/cm2) fail to inhibit tyrosinase activity in cell homogenates from low-density cultures treated with MSH.

In a previous investigation of tyrosinase activity in the cells of the nucleus substantiae nigrae of adult cats and monkeys, diphenolase, but not monophenolase activity, was detected. This is possibly due to inhibition of monophenolase. When the copper radicle in tyrosinase is oxidized, monophenolase cannot be detected, but reducing agents activate and unmask this component of tyrosinase. The effects of reducing agents in activating monophenolase have been investigated. Tyrosine is converted to melanin by monophenolase in adult nigra cells of the cat and monkey when the enzyme is activated by the reducing agents L-dopa, L-ascorbic acid, and L-adrenaline. Variations in physical factors influence the intensity of the activated monophenolase reaction resulting from incubation of sections in tyrosine-dopa mixtures; the reaction is inhibited by general enzyme inhibitors and specific tyrosinase inhibitors. The significance of the results in relation to tyrosinase activity in adult nigra cells is discussed.

Siegesbeckia glabrescens Makino (SGM) has been traditionally used to treat many disorders, including rheumatoid arthritis, hypertension, and acute hepatitis. However, the biological activities of SGM in skin remain unclear. The present study explored the effects of SGM flower absolute (SGMFAb) on skin-whitening-linked biological activities in B16BL6 cells. SGMFAb was extracted using hexane, and its composition was analyzed through gas chromatography/mass spectrometry analysis. The biological effects of SGMFAb on B16BL6 melanoma cells were detected via WST and BrdU incorporation assays, ELISA, and immunoblotting. SGMFAb contained 14 compounds. In addition, SGMFAb was noncytotoxic, attenuated the serum-induced proliferation of, and inhibited melanin synthesis and tyrosinase activity in α-MSH-exposed B16BL6 cells. SGMFAb also reduced the expressions of MITF (microphthalmia-associated transcription factor), tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 in α-MSH-exposed B16BL6 cells. Moreover, SGMFAb downregulated the activation of p38 MAPK, ERK1/2, and JNK in α-MSH-stimulated B16BL6 cells. In addition, SGMFAb reduced the expressions of three melanosome-transport-participating proteins (myosin Va, melanophilin, and Rab27a) in α-MSH-stimulated B16BL6 cells. These results indicate that SGMFAb positively influences skin whitening activities by inhibiting melanogenesis and melanosome-transport-related events in B16BL6 cells, and suggest that SGMFAb is a promising material for developing functional skin whitening agents.

Impatiens textori Miq. (ITM; family Balsaminaceae) is a traditional medicinal plant with many biological activities, which include anti-allergic, anti-inflammatory, and anti-pruritic properties. However, it remains to be determined whether ITM affects biological activities in the skin. Thus, we investigated the effects of ITM flower absolute (ITMFAb) extract on the biological activities of skin, especially those related to skin wound repair and whitening. ITMFAb was extracted with hexane, and its composition was determined through GC/MS. The biological activities of ITMFAb on HaCaT keratinocytes and B16BL6 melanoma cells were analyzed using a water-soluble tetrazolium salt, 5-bromo-2′-deoxyuridine incorporation, a Boyden chamber, an ELISA, a sprouting assay, and by immunoblotting. These analyses were performed in a range of ITMFAb concentrations that did not inhibit the viability of the cells (HaCaT, ≤400 µg/mL; B16BL6, ≤200 µg/m). Forty components were identified in ITMFAb. ITMFAb stimulated proliferation, migration, sprout outgrowth, and type I and IV collagen synthesis and upregulated the activations of ERK1/2, JNK, p38 MAPK, and AKT in HaCaT cells. In addition, ITMFAb attenuated the serum-induced proliferation of B16BL6 cells. ITMFAb inhibited melanin synthesis, tyrosinase activity, and expressions of MITF and tyrosinase in α-MSH-exposed B16BL6 cells. These findings indicate that ITMFAb has beneficial effects on wound repairing and whitening-linked responses in the skin and suggest the potential use of ITMFAb as a natural material for the development of skin wound repair and whitening agents.

Stimulation by Ionophores of Tyrosinase Activity of Mouse Melanoma Cells in Culture

Aralia elata (Miq.) Seem. (AES; family Araliaceae) is a medicinal plant and has been reported to have various bioactivities, including anticancer and hepatotoxicity protective activities. However, no studies have investigated the biological activities of AES or its extracts on skin. To address this, we aimed to explore the effect of AES-flower-derived absolute-type essential oil (AESFEO) on skin-related biological activities, especially skin wound healing and whitening-related responses in skin cells (human-derived keratinocytes [HaCaT cells] and melanocytes [B16BL6 cells]) and to identify the components of AESFEO. Cell biological activities were analyzed using WST and BrdU incorporation assays, ELISA, or by immunoblotting. In HaCaT cells, AESFEO promoted proliferation, type IV collagen production, and enhanced the phosphorylations of Erk1/2, p38 MAPK, JNK, and Akt. In B16BL6 cells, AESFEO reduced serum-induced proliferation, α-MSH-stimulated increases in melanin synthesis and tyrosinase activity, and α-MSH-induced increases in MITF, tyrosinase, TRP-1, and TRP-2 expressions. In addition, AESFEO inhibited the phosphorylation of Erk1/2, p38 MAPK, and JNK in α-MSH-stimulated B16BL6 cells. Eighteen compounds were identified in AESFEO by GC/MS. These results suggest that AESFEO has beneficial effects on keratinocyte activities related to skin wound healing and melanocyte activities related to inhibition of skin pigmentation. AESFEO may serve as a useful natural substance for developing agents that facilitate skin wound healing and inhibit melanogenesis.

Increased Melanoma Growth and Metastasis Spreading in Mice Overexpressing Placenta Growth Factor

In the present study, the chemical compositions and skin whitening-related antioxidant and anti-melanogenic effect of essential oils (EOs) extracted from Chrysanthemum borealeMakino (CBM) (CBMEOs) at vegetative, pre-flowering and full-flowering are investigated and contrasted among the three stages. The yields and components of the CBMEOs were different at each stage. The CBMEOs increased DPPH and ABTs scavenging activities and attenuated the α-melanocyte stimulating hormone (α-MSH)-induced tyrosinase activity and melanin synthesis in B16BL6 cells. Among CBMEO components, eugenol had the highest DPPH and ABTs scavenging activities and cuminaldehyde was the strongest inhibitor of α-MSH-induced tyrosinase activity and melanin synthesis. The CBMEOs in each stage showed the different levels of phosphorylation of extracellular signal-regulated kinase1/2 and p38 MAPK. These findings demonstrate that the CBMEOs have antioxidative and anti-melanogenic activities in all the CBM harvesting stages, resulting in skin-whitening biological activities and that the levels of their component contents and bioactivities differ among the CBM harvesting stages. The CBMEOs may have the potential for use in cosmetics and alternative medicine.

This study was performed to assess the antioxidant activities and whitening effects of Agrimonia pilosa Ledeb on melanin synthesis. The whitening effects of Agrimonia pilosa Ledeb water extracts were examined by in vitro mushroom tyrosinase assay and B16BL6 melanoma cells. We assessed inhibitory effect of Agrimonia pilosa Ledeb water extract on expression of melanogenic enzyme proteins including tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinaserelated protein 2 (TRP-2) in B16BL6 cells. Inhibitory effect of Agrimonia pilosa Ledeb onto free radical generation was determined by measuring DPPH and hydroxyl radical scavenging activitie. Our results indicated that Agrimonia pilosa Ledeb water extract effectively inhibited free radical generation. In DPPH and hydroxy radical scavenging activity, Agrimonia pilosa Ledeb water extract had a potent anti-oxidant activity in a dose-dependent manner. They significantly inhibited tyrosinase activity in vitro and in B16BL6 melanoma cells. Also, Agrimonia pilosa Ledeb suppressed the expression of tyrosinase in B16BL6 melanoma cells. These results show that Agrimonia pilosa Ledeb inhibited melanin production on the melanogenesis. The underlying mechanism of Agrimonia pilosa Ledeb on whitening activity may be due to the inhibition of tyrosinase activity. We suggest that Agrimonia pilosa Ledeb may be useful as new natural active ingredients for antioxidant and whitening cosmetics.

본 연구에서는 정공피 물 추출물의 미백 효과를 확인하기 위하여 항산화 효능을 검증하였고, 멜라닌 생성의 첫 단계인 tyrosinase 활성, mouse melanoma B16BL6 세포 생존율 및 TRP-1, TRP-2 mRNA 발현 정도를 측정하였다. 정공피 물 추출물은 DPPH 및 hydroxy 라디칼 소거 작용을 통하여 높은 항산화 효능을 나타내었고, tyrosinase, TRP-1, TRP-2의 활성을 효과적으로 억제하는 것을 확인하였다. 이는 정공피 물 추출물이 직접적으로 B16BL6 melanoma 세포의 tyrosinase 단백질의 활성을 저해시킴으로써 멜라닌 생성을 억제하는데 효과적임을 의미한다. 결론적으로 정공피 추출물은 항산화 활성과 미백 효과가 우수하여 기능성 화장품의 천연 소재로서 활용 가능성이 매우 높은 것으로 판단된다.

본 연구에서는 효소처리 한 대두추출물의 항산화 및 미백효과를 확인하기 위하여 DPPH 및 hydroxyl radical 포착활성을 측정하였고, 멜라닌 생성의 첫 단계인 tyrosinase 활성, mouse melanoma B16BL6 세포 생존율 및 TRP-1, TRP-2 발현 저해활성을 측정하였다. 또한 기존의 기계적 마쇄 가공한 대두추출물과 효소처리 가공한 대두추출물의 항산화 및 미백효과를 비교 조사하였다. 효소처리 한 대두추출물의 DPPH radical 소거능과 hydroxyl radical 소거능이 마쇄처리 한 추출물보다 높았으며, 특히 효소처리군이 마쇄군에 비하여 20% 이상 높은 항산화력을 나타내었다. 또한 마쇄군에 비하여 효소처리군이 tyrosinase와 TRP-1, TRP-2의 더 높은 활성 억제능을 보였다. 이는 대두가 B16BL6 melanoma 세포의 tyrosinase 단백질의 활성을 저해시킴으로써 멜라닌 생성을 억제하는데 효과적임을 의미하는 동시에 효소처리에 의하여 대두의 영양소의 손실이 더 적었음을 나타낸다. 결론적으로 효소처리에 의한 대두 추출물은 항산화 활성과 미백 효과가 우수하여 기능성 화장품의 천연 소재로서 활용가능성이 매우 높은 것으로 사료된다.

Several studies have indicated that extracellular matrix (ECM) proteins can influence melanocyte behaviour in vitro. However, the choice of medium is known to have a profound effect on melanocyte behaviour and it is currently difficult to ascribe which reported effects are due to ECM proteins and those which are attributable to the medium used in these different studies. The purpose of this study was to learn more about the influence of ECM proteins on melanocyte function by examining a range of cell-derived and individual ECM proteins for their impact on melanocyte tyrosinase activity in cells cultured under conditions of varying mitogenic drive. We found that ECM derived from human dermal fibroblasts, bovine endothelial cells and a human endothelial cell line as well as collagen I, collagen IV, fibronectin, and to a lesser extent laminin, were all capable of increasing tyrosinase activity in cultures of normal melanocytes. Effects of these ECM were seen most consistently in media with relatively few mitogens, for example ECM proteins influenced melanocyte morphology and this was seen most readily in cells cultured in medium without any mitogens (which ordinarily fails to support melanocyte survival). This study illustrates that ECM proteins can influence melanocyte morphology, proliferation, and tyrosinase activity in vitro and supports a possible role of ECM proteins in the regulation of melanocyte function in vivo.