Phytochemical potential of Daphne gnidium in inhibiting growth of melanoma cells and enhancing melanogenesis of B16-F0 melanoma

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16-F0 and B16-F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells B16-F0 and B16-F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2-M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16-F0 melanoma cells. Copyright © 2012 John Wiley & Sons, Ltd.     

A New Method for Estimation of the Mycelial Weight in Koji

A New method was devised for the estimation of the mycelial weight in rice-koji. In this method, the content of glucosamine in koji was used for the calculation of mycelial weight. The content of glucosamine in the mycelia of Aspergillus oryzae, koji, and rice was determined by a colorimetry after hydrolysis of these materials with sulfuric acid and purification of glucosamine fraction with a Dowex 50 W column. And the values of glucosamine were 114.5 mg/g in mycelia, 3.03 in the koji for amazake,* 1.34 in the koji for sake, and 0.0 in rice. The mycelial contents calculated from these data were 2.6% dry weight in amazake-koji and 1.2% in sake-koji.     

Purification and characterization of invertase isozymes from Fusarium oxysporum

Fusarium invertase appears to exist in two forms, mycelial (P-1) and conidial (P-2) types. They were purified and partially characterized, but their specific activities were too low when compared with yeast and Neurospora invertases. Present studies describe a method for isolation of highly purified enzymes and their properties. The enzymes are homogeneous by several criteria. Estimation of molecular weights revealed the subunit structure of each invertase, and the association-dissociation of subunits seem to occur as temperature varies. Amino acid compositions and other properties have been studied. The two invertases are glycoproteins which contain 36% (P-1) and 23% (P-2) carbohydrates (predominantly mannose with smaller percentages of glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine). Comparison with the properties of the previous preparations is also described.     

Photodynamic therapy-induced killing is enhanced in depigmented metastatic melanoma cells

The resistance of pigmented human melanomas over their unpigmented counterparts to a number of therapies has suggested that the presence of intracellular melanin plays a role in rendering these cells less susceptible to cell death, probably through the ability of this pigment to act as an intracellular antioxidant, thus neutralizing chemotherapeutic-induced ROS (reactive oxygen species). PDT (photodynamic therapy) was recently suggested as an attractive, adjunctive therapy owing to its cellular specificity and limited side effects. In the present study, we propose that first depigmenting melanomas with a reversible TYR (tyrosinase) inhibitor such as PTU (phenylthiourea) increases their susceptibility to HYP-PDT (hypericin-mediated PDT). Pigmented [UCT Mel-1 (University of Cape Town melanoma cell line 1)] and unpigmented (A375) melanomas were first characterized with respect to their TYR activities and melanin quantities and then treated with a TYR inhibitor for 48 h. Cell viability assays after treatment with 3 μM HYP-PDT showed a significant increase in cell death in depigmented melanomas compared with untreated melanomas that returned to the level of untreated melanoma cells on removing the TYR inhibitor. The present study supports the hypothesis that combining the inhibition of melanogenesis with PDT should be explored as a valid therapeutic target for the management of advanced melanoma.     

In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

驱虫斑鸠菊对B-16黑素瘤细胞酪氨酸酶活性及黑素生成影响

目的 :探讨驱虫斑鸠菊体外对酪氨酸酶活性影响 ,以及对小鼠B - 16黑素瘤细胞株细胞增殖、黑素合成以及细胞内酪氨酸酶的作用。方法 :利用四甲基偶氮唑蓝 (MTT)比色法测定药物对细胞增殖的影响 ;采用酶学方法研究药物对酪氨酸酶活性的影响 ;470nm比色法测定黑素含量。结果驱虫斑鸠菊体外可激活酪氨酸酶活性 ,增强B - 16鼠黑素瘤细胞增殖 ,提高酪氨酸酶和黑色素合成能力 ;对整体动物黑素细胞具有促进合成和分泌作用。结论在白癜风的治疗中 ,驱虫斑鸠菊可增强酪氨酸酶活性 ,进而促进黑素合成     

Changes in Glycosyldiacylglycerols during Germination of Black Mung Beans (Matpe)

The physico-chemical characteristics of purified arginine kinases from prawn and swimming crab were examined. The molecular weights of prawn and swimming crab enzymes were 40,500 and 40,000, respectively. Amino acid analysis indicated that there were some differences in the contents of proline, glycine, methionine, and lysine. The other amino acid compositions of these enzymes resembled each other.

Both enzymes were stable up to 20°C when they were treated for 10 min at various temperature levels. The enzymes lost their activities at temperatures higher than 25°C. They were more stable at pH 8.0 than pH 7.0. The optimum temperature for the enzyme of prawn was about 42°C and that for swimming crab was about 40°C. The pH optima for the activity of arginine kinase of prawn in the forward and in the reverse reactions were found to be 9.0 and 6.1, respectively. For the swimming crab, the similar optimum pHs at 9.2 in the forward reaction and 5.8 in the reverse reaction were observed. Both enzymes were activated most strongly with Mg^{2 +} and Mn^{2 +} followed by Ca^{2 +}, Co^{2 +}, and Fe^{2 +}. The enzymes were not activated by Sr^{2 +}, Cu^{2 +}, or Zn^{2 +}.

The optimum molar ratio of Mg^{2 +}: ATP in the forward reaction of prawn and swimming crab was found to be 1:1, and the molar ratio of Mg^{2 +} : ADP in the reverse reaction was 4:1 in both cases. Kinetic studies indicated that dissociation constants were rather different. In the prawn, dissociation constants for arginine, ATP, AP, and ADP were 0.19,0.31, 0.67, and 0.29 mM, respectively, but in the swimming crab, they were 0.10, 0.18, 0.22, and 0.11 mM, respectively.     

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata)

The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl₂. It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4°C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.     

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.     

A melanocyte-keratinocyte coculture model to assess regulators of pigmentation in vitro

Many melanocyte or skin equivalent models have been used to evaluate the potential efficacy of melanogenic compounds to regulate pigmentation, but there has been great variation in results, partially stemming from the use of different cell lines and diverse conditions for the melanogenic assays. In an earlier report, we optimized a microtiter format assay system to screen potential bioactive compounds using immortalized melan-a melanocytes. That assay system, termed the STOPR protocol, allowed effects on melanocyte proliferation and differentiation to be assessed in a highly sensitive, reproducible, and cost-effective manner. However, in the skin and hair, melanocytes interact with keratinocytes, fibroblasts, and other cell types, and testing of putative bioactive compounds on melanocytes alone in culture does not allow one to observe the interactions with those other cell types, such as would occur in vivo. Therefore, we developed a melanocyte-keratinocyte coculture protocol that allows testing of compounds for potential effects on pigmentation in a more physiologically relevant context. It is a sensitive, reproducible, and reliable model for testing melanogenic regulators, and we have standardized it with known melanogenic inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and stimulators (alpha-melanocyte-stimulating hormone, 8-methoxypsoralen, and 3,4-dihydroxyphenylalanine). This coculture system allows for large-scale screening of candidate compounds in conjunction with the STOPR protocol and provides a more physiologically relevant system to study melanocyte-keratinocyte interactions and to elucidate the regulatory mechanisms of melanogenic compounds.     

Modification of membrane fluidity in melanin-containing cells by low-level microwave radiation.

The treatment of a B16 melanoma cell line with 2.45-GHz pulsed microwaves (10 mW/cm2, 10-microseconds pulses at 100 pps, 1-h exposure; SAR, 0.2 W/kg) resulted in changes of membrane ordering as measured by EPR (electron paramagnetic resonance) reporter techniques. The changes reflected a shift from a more fluid-like phase to a more solid (ordered) state of the cell membrane. Exposure of artificially prepared liposomes that were reconstituted with melanin produced similar results. In contrast, neither B16 melanoma cells treated with 5-Bromo-2-Deoxyuridine (3 micrograms/day x 7 days) to render them amelanotic, nor liposomes prepared without melanin, exhibited the microwave-facilitated increase of ordering. Inhibition of the ordering was achieved by the use of superoxide dismutase (SOD), which strongly implicates oxygen radicals as a cause of the membrane changes. The data indicate that a significant, specific alteration of cell-membrane ordering followed microwave exposure. This alteration was unique to melanotic membranes and was due, at least in part, to the generation of oxygen radicals.     

水稻巯基蛋白酶抑制剂的分子修饰与其对稻瘟病菌的抑制作用

水稻巯基蛋白酶抑制剂（ＣＰＩ）经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用Ｎ－乙基顺丁烯二酰亚胺与ＣＰＩ反应,可以测出ＣＰＩ分子内有１９个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻ＣＰＩ的抑制活性不需要巯基参与;应用Ｎ－溴代丁二酰亚胺与ＣＰＩ反应,可测出ＣＰＩ分子内有２个Ｔｒｐ被修饰,修饰后,抑制活性全部丧失,表明Ｔｒｐ是保持抑制活性所必需的基团。水稻巯基蛋白酶抑制剂和丝氨酸蛋白酶抑制剂对稻瘟病菌丝体的生长均有抑制作用,但后者的抑制作用比前者更强,若将两种抑制剂混合使用,则对稻瘟病菌丝体的抑制作用非常强烈;当抑制剂加入量达７２μｇ时,即可产生明显的抑制作用。     

Activation of tyrosinase in mouse melanoma cell cultures

Summary Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37°C for a minimum of 8 h. Enzyme activity continued to increase for 48h at which time the maximal level of activation was observed. Activation did not occur at 4°C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation process increased the V _Max of tyrosinase 10-fold and lowered the K _M by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole. The “self-activation” response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells. This investigation was supported by Public Health Service grants CA41425 and CA30393 awarded by the National Cancer Institute, Bethesda, MD and by a research grant from the Proctor and Gamble Company.     

Purification of Ribonuclease L from Aspergillus sp. by Affinity Chromatography

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3′-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

Selection of malignant melanoma variant cell lines for ovary colonization

Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16–010 cells grew in circular in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melainin pigment (B16-010 < B16-05 < B16-01 ? B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.     

miR-146a抑制酪氨酸酶基因家族在小鼠黑色素细胞中表达

miRNA是在许多生物过程中都起着至关重要作用的一类内源性非编码的小RNA,与癌症、肿瘤的发生有关。现发现很多miRNA在黑色素生成中都有重要的调控作用,但miR-146a是否对黑色素的生成具有影响未见报道。本研究发现miR-146a通过靶向抑制酪氨酸酶相关蛋白1(tyrosinase related protein 1,TYRP1)的表达而使黑色素生成降低。在小鼠黑色素细胞中分别转染miR-146a mimic和miR-146a 抑制剂,通过qRT-PCR与Western印迹分析比较各实验组中TYRP1基因与酪氨酸家族相关基因酪氨酸酶(tyrosinase, TYR)、酪氨酸酶相关蛋白2(tyrosinase related protein 2, TYRP2)的表达差异。双荧光报告实验验证TYRP1与miR-146a的靶向关系,双荧光酶活性结果显示,实验组相比对照组,荧光素酶活性明显降低,说明TYRP1是miR-146a的靶基因之一;qRT-PCR和Western印迹结果显示实验组TYR、TYRP1及TYRP2 在mRNA水平和蛋白质水平表达均显著降低;紫外分光光度法检测黑色素含量,结果显示miR-146a mimic转染组黑色素含量明显下降,而抑制组的黑色素含量呈上升趋势。综上所述,miR-146a通过靶向抑制TYRP1基因的表达,而影响TYR家族成员的表达,调控黑色素的生物合成。     

Tyrosinase inhibitory effect and inhibitory mechanism of tiliroside from raspberry

Tiliroside was found to inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag time of tyrosine oxidation catalyzed by mushroom tyrosinase was obviously lengthened; 0.337?mM of tiliroside resulted in the lag time extension from 46.7?s to 435.1?s. A kinetic analysis shown that tiliroside was a competitive inhibitor for monophenolase and diphenolase with K_i values of 0.052?mM and 0.26?mM, respectively. Furthermore, tiliroside showed 34.5% (p?<?0.05) inhibition of intracellular tyrosinase activity and 54.1% (p?<?0.05) inhibition of melanin production with low cytotoxicity on B16 mouse melanoma cells at 0.168?mM. In contrast, arbutin displayed 9.1% inhibition of cellular tyrosinase activity and 29.5% inhibition of melanin production at the same concentration. These results suggested that tiliroside was a potent tyrosinase inhibitor and might be used as a skin-whitening agent and pigmentation medicine.     

Hot-Water Extracts from Adzuki Beans (Vigna angularis) Stimulate Not Only Melanogenesis in Cultured Mouse B16 Melanoma Cells but Also Pigmentation of Hair Color in C3H Mice

A hot-water extract of adzuki was obtained by boiling beans of adzuki (Vigna angularis). This hot-water extract was fractionated using HP-20 column chromatography. Its distilled water fraction (WEx) was found to stimulate tyrosinase activity in cultured mouse B16 melanoma cells and hair color pigmentation in C3H mice. At concentrations of 1–3 mg/ml, WEx stimulated melanogenesis without inhibiting cell growth. During this effect, WEx activated tyrosinase-inducing activity in the cells, but did not activate tyrosinase, which exists at an intracellular level. In this study, WEx increased cyclic adenosine-3′,5′-monophospate (cAMP) content in the cells and protein kinase A (PKA) activity, and stimulated translocation of cytosolic protein kinase C (PKC) to the membrane-bound PKC. These results suggest that the addition of WEx activates the adenylcyclase and protein kinase pathways and, as a result, stimulates melanogenesis. WEx was found to have pigmentation activity on hair color in C3H mice. It might be useful in anti-graying, protecting human skin from irradiation.     

水稻巯基蛋白酶抑制剂研究进展

综述水稻巯基蛋白酶抑制剂 (Oryzacystatin ,OC)近年的研究进展。巯基蛋白酶抑制剂 (CPI)统称为胱蛋白超家族。OCI含有CPI家族的典型保守序列Glu -Val-Val-Ala -Gly ,这是其抑制活性不可缺少的区段。利用水稻cDNA文库还克隆得到OCII。OCI与OCII之间在序列上有高度的同源性 ,但在对蛋白酶的抑制作用上有显著差异。OC对鞘翅目昆虫有较强的抗虫性。在抗病方面 ,OC可抑制稻瘟病菌丝体的生长     

Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)