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Objectives We have investigated whether KHG25855 (2-cyclohexylamino-1,3-thiazole hydrochloride) affected melanogenesis in B16 mouse melanoma cells, and the mechanisms involved. Methods Melanin content and tyrosinase activity were measured using an ELISA reader after cells were treated with KHG25855. KHG25855-induced signalling pathways were examined using Western blot analysis. Key findings KHG25855 decreased melanin production in a dose-dependent fashion, but KHG25855 did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and the related signal transduction pathways were also investigated. The effects of KHG25855 on the extracellular signal-regulated kinase and cAMP response element binding protein signalling pathways were determined, and KHG25855 was shown to have no effect on these signalling pathways. The Wnt signalling pathway is also deeply involved in melanogenesis, and so glycogen synthase kinase 3 β (GSK3 β) phosphorylation was assessed after KHG25855 treatment; KHG25855 caused GSK3 β phosphorylation (inactivation), but the level of β-catenin was not changed by KHG25855. Furthermore, α-melanocyte stimulating hormone-induced tyrosinase expression was downregulated by KHG25855. Conclusions We propose that KHG25855 showed hypopigmentary activity through tyrosinase downregulation via GSK3 β phosphorylation. [ABSTRACT FROM AUTHOR]

LGI family member 3 (LGI3) is a member of the LGI protein family. In our previous studies, LGI3 was determined to be expressed in adipose tissues, skin and the brain, where it served as a pleiotropic cytokine. The results indicated that LGI3 levels are increased in adipose tissues of obese individuals in comparison with control individuals and that LGI3 suppressed adipogenesis via its receptor, disintegrin and metalloproteinase domain-containing protein 23. Additionally, it was reported that LGI3 upregulates tumor necrosis factor-α and downregulated adiponectin and hypothesized that LGI3 may act as a proinflammatory adipokine involved in adipose tissue inflammation. In the present study, cytokine arrays were used to analyze cytokine levels in adipose tissues and plasma of LGI3-knockout mice and signaling protein arrays used to analyze the expression and phosphorylation of these proteins in LGI3-treated preadipocytes. The results suggested that expression levels of 129 gene products (24 cytokines and 105 signaling proteins) were altered in response to LGI3 deficiency or LGI3 treatment, respectively. Protein-protein interaction network analysis of LGI3-regulated gene products revealed that 94% of the gene products (21 cytokines and 100 signaling proteins) formed an interaction network cluster. Functional enrichment analysis for the LGI3-regulated gene products, including those from our previous studies, revealed an association with numerous biological processes, including inflammatory responses, cellular differentiation and development and metabolic regulation. Gene co-expression network analysis revealed that these LGI3-regulated gene products were involved in various biological processes in an overlapping and differential manner between subcutaneous and visceral adipose tissues. Notably, inflammatory responses were more strongly associated with the LGI3-regulated gene co-expression network in visceral adipose tissues than in subcutaneous adipose tissues. Analysis of expression quantitative trait loci identified four single nucleotide variants that affect expression of LGI3 in an adipose depot-specific manner. Taken together, the results suggested that LGI3 may serve depot-specific roles as an adipokine in adipose tissues. [ABSTRACT FROM AUTHOR]

Among G-proteins, Gh is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca2+-activated K+ (Maxi-K+) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPγS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPβS resulted in reversal of the GTPγS effect. When the Gαh7 antibody was applied, the GTPγS-stimulated channel activity was significantly inhibited to control level, suggesting that Gαh is involved in activation of the Maxi-K+ channel in smooth muscle cells. [ABSTRACT FROM AUTHOR]

We characterized the α1B-adrenoreceptor (α1B-AR)-mediated intracellular Ca2+ signaling involving Gαh (transglutaminase II, TGII) and phospholipase C (PLC)-δ1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca2+ concentration ([Ca2+]i) in response to activation of the α1B-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-δ1 substantially reduced both the peak and sustained levels of [Ca2+]i. Expression of TGII mutants lacking the interaction with PLC-δ1 resulted in a reduced capacitative Ca2+ entry. Reduced expression of PLC-δ1 displayed a transient elevation of [Ca2+]i and a reduction in capacitative Ca2+ entry. Expression of the C2-domain of PLC-δ1, which contains the TGII interaction site, resulted in reduction of the α1B-AR-evoked peak increase in [Ca2+]i, while the sustained elevation in [Ca2+]i and capacitative Ca2+ entry remained unchanged. These findings demonstrate that stimulation of PLC-δ1 via coupling of the α1B-AR with TGII evokes both Ca2+ release and capacitative Ca2+ entry and that capacitative Ca2+ entry is mediated by the interaction of TGII with PLC-δ1. [Copyright &y& Elsevier]

Objective: To investigate the efficacy of horse oil on lipopolysaccharide (LPS)-induced inflammation in human keratinocytes., Methods: Western blot analysis was performed to measure the expression of cyclooxygenase-2 (COX-2) and IκBα. ELISA was used to analyze prostaglandin E2 (PGE2) levels., Results: Horse oil decreased LPS-induced COX-2 and PGE2 levels in a dose-dependent manner. Nuclear factor-kappa B (NF-κB) plays a key role in the expression of inflammatory cytokines and mediators. Therefore, we investigated the influence of horse oil on the NF-κB signaling pathways. Horse oil inhibited translocation of NF-κB from the cytosol to the nucleus. Furthermore, LPS-induced degradation of IκBα was recovered by horse oil. The activation of p38 mitogen-activated protein kinase (MAPK) reportedly induces degradation of IκBα In agreement with this, LPS activated p38 MAPK and caused IκBα degradation. Conversely, horse oil inhibited LPS-induced p38 MAPK activation and IκBα degradation. In addition, a specific p38 MAPK inhibitor, SB203580, blocked IκBα degradation., Conclusion: Horse oil decreased COX-2 and PGE2 by inhibiting p38 MAPK activation, IκBα degradation, and the translocation of NF-κB.

Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3β (GSK3β) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation.

Methyl gallate (MG) was isolated from the bark of Acer barbinerve, which has traditionally been used in Oriental medicine. In the present study, we examined the effects of MG on melanin synthesis in Mel-Ab melanocyte cells. MG decreased melanin pigmentation in a concentration-dependent manner, but did not directly inhibit tyrosinase activity. Further analysis showed that MG had no effect on extracellular signal-regulated kinase (ERK) activation, but induced phosphorylation of glycogen synthase kinase (GSK)3β, which is known to increase β-catenin accumulation. Accordingly, the β-catenin level was increased by MG. However, a specific GSK3β inhibitor did not rescue the MG-induced inhibition of melanogenesis. Additionally, MG decreased the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, which regulate melanin synthesis. Based on these results, we conclude that MG inhibits melanogenesis by decreasing the expression of MITF and tyrosinase.

The purpose of this study was to investigate effects of myriocin, an inhibitor of serine palmitoyltransferase, on melanogenesis. It was found that myriocin increased melanin synthesis in a concentration-dependent manner. Moreover, myriocin up-regulated microphthalmia-associated transcription factor (MITF) and tyrosinase expression via phosphorylation of CREB, but it did not directly activate tyrosinase, a rate-limiting melanogenic enzyme. Furthermore, we demonstrated increased melanin synthesis with myriocin on a pigmented skin equivalent model established using Cervi cornus Colla (deer antler glue). One and 5 microM of myriocin darkened the color of the skin equivalent. These results suggest that myriocin may have potential effects for the treatment of hypopigmentary skin diseases like vitiligo or for sunless tanning.

We determined whether indole-3-carbinol (I3C) could affect DU145 human prostate carcinoma cell migration to prevent the development and progression of prostate cancer. Although previous studies have shown anticancer properties of I3C in various cancer cell lines, it has not been determined how I3C regulates epidermal growth factor (EGF)-induced migration and related signaling pathways. DU145 cells were treated with I3C (100 microM) in the absence or presence of EGF (10 ng/ml). Our results showed that I3C significantly inhibited DU145 cell migration with and without EGF stimulation. It has been reported that the beta-catenin signaling pathway controls androgen receptor (AR)-mediated prostate cancer progression, which plays a key role in the metastasis of prostate cancer. Western blot analysis demonstrated that I3C led to the phosphorylation of beta-catenin and subsequent degradation of beta-catenin in the absence and presence of EGF. In contrast, I3C did not have any effect on the expression of beta-catenin mRNA. From these results, we suggest that I3C inhibits EGF (dependent or independent)-induced DU145 cell migration through beta-catenin degradation.

Indole-3-acetic acid (IAA) has recently shown anticancer activity in combination with horseradish peroxidase. The current study demonstrated that IAA irradiated with ultraviolet B (IAA(UVB)) is able to generate free radicals and induce cell death in a time-dependent fashion in PC-3 prostate cancer cells, while PC-3 cells treated with IAA alone exhibited no toxic responses. It was also found through Western blot analysis that the cytotoxic effect of IAA(UVB) resulted from apoptosis. Treatment with IAA(UVB) for 24 hours showed a significant increase in phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, the stress signaling proteins. Furthermore, pro-caspases (-3, -8, and -9) were clearly down-regulated and poly(ADP-ribose) polymerase cleavages were demonstrated in the group treated with IAA(UVB). Flow cytometric analysis also demonstrated the induction of apoptosis by IAA(UVB) in PC-3 cells. In conclusion, this study demonstrated that IAA induced cell death in combination with UVB irradiation by increasing apoptosis in PC-3 cells.

Objectives: In the present study, we determined whether or not imidazole affects B16 murine melanoma cell migration to prevent melanoma metastasis., Methods: To determine the effects of imidazole on melanoma cell migration, B16 cells were treated with imidazole at various concentrations, and the migration was measured using a scratch migration assay., Key Findings: Imidazole did not exhibit cytotoxic effects on B16 cells at a concentration below 100 microm. The anti-migratory activity of imidazole was determined by the scratch migration assay. Our results showed that imidazole significantly inhibits B16 cell migration. It is known that the Wnt/beta-catenin signalling pathway regulates the progression of melanocytic tumours and determines the prognosis in cutaneous melanomas. Western blot analysis demonstrated that imidazole increases phosphorylation of beta-catenin and subsequent degradation of beta-catenin. Moreover, inhibition of melanoma cell migration by imidazole was restored by MG132, a proteasome inhibitor, via inhibition of beta-catenin degradation., Conclusions: Imidazole inhibits B16 cell migration through beta-catenin degradation, suggesting that imidazole is a potential candidate for the treatment of metastatic melanoma.

Indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) have emerged as a new strategy for cancer treatment. In the present study, we determined the effects of IAA/HRP treatment on TCCSUP human urinary bladder carcinoma cells. It was found that the IAA/HRP combination decreased cell viability of TCCSUP cells in a time- and dose-dependent manner, whereas IAA or HRP alone showed no such effect. In addition, the decreased cell viability was restored by pretreatment with ascorbic acid. To clarify the mechanism of death of TCCSUP cells by IAA/HRP, we investigated the signal transduction pathways related to the apoptosis. It was found that IAA/HRP activates p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). We further investigated the IAA/HRP-mediated apoptotic pathways and showed that IAA/HRP induces caspase-8 and caspase-9 activation, which results in caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. To further confirm whether IAA/HRP induces apoptotic cell death, we performed a DNA fragmentation assay after IAA/HRP treatment and found that IAA/HRP-treated cells showed typical apoptotic DNA ladder formation. From these results, we suggest that IAA/HRP induces apoptosis of TCCSUP human urinary bladder carcinoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.

Ultraviolet (UV) radiation is a major risk factor for the development of melanoma. Recent studies have reported that the intake of citrate-containing juices may reduce the risk of cancer. Thus, we investigated the effects of citrate on UVB-irradiated B16 murine melanoma cells. B16 cells had more evident apoptotic features with the combination of citrate/UVB than by citrate or UVB alone; cell death of HaCaT human keratinocytes was not observed with citrate/UVB. Western blot analysis demonstrated that citrate/UVB led to phosphorylation of the stress signaling proteins, such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Furthermore, citrate/UVB caused activation of caspase-9/-3 as well as cleavage of poly(ADP-ribose) polymerase (PARP). Correspondingly, cell cycle analysis showed that citrate/UVB clearly increased the sub-G0/G1 phase, which indicated apoptotic cell death of B16 cells. Therefore, our study has demonstrated that sub-lethal doses of citrate enhanced the apoptotic cell death of melanoma cells under UVB irradiation. From these results, we suggest that citrate might reduce the risk of developing melanoma induced by UVB.

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

Galphah (transglutaminase type II; tissue transglutaminase) is a bifunctional enzyme with transglutaminase (TGase) and guanosine triphosphatase (GTPase) activities. The GTPase function of Galphah is involved in hormonal signaling and cell growth while the TGase function plays an important role in apoptosis and in cross-linking extracellular and intracellular proteins. To analyze the regulation of these dual enzymatic activities we examined their calcium-dependence and thermal stability in enzymes from several cardiac sources (mouse heart, and normal, ischemic and dilated cardiomyopathic human hearts). The GTP binding activity of Galphah was markedly inhibited by Ca2+ whereas the TGase activity was strongly stimulated, suggesting that Ca2+ acts as a regulator, switching Galphah from a GTPase to a TGase. The TGase function of Galphah of both mouse and human hearts was more thermostable in the presence of Ca2+.

We characterized the alpha(1B)-adrenoreceptor (alpha(1B)-AR)-mediated intracellular Ca(2+) signaling involving G alpha(h) (transglutaminase II, TGII) and phospholipase C (PLC)-delta 1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to activation of the alpha(1B)-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-delta 1 substantially reduced both the peak and sustained levels of [Ca(2+)](i). Expression of TGII mutants lacking the interaction with PLC-delta 1 resulted in a reduced capacitative Ca(2+) entry. Reduced expression of PLC-delta 1 displayed a transient elevation of [Ca(2+)](i) and a reduction in capacitative Ca(2+) entry. Expression of the C2-domain of PLC-delta 1, which contains the TGII interaction site, resulted in reduction of the alpha(1B)-AR-evoked peak increase in [Ca(2+)](i), while the sustained elevation in [Ca(2+)](i) and capacitative Ca(2+) entry remained unchanged. These findings demonstrate that stimulation of PLC-delta 1 via coupling of the alpha(1B)-AR with TGII evokes both Ca(2+) release and capacitative Ca(2+) entry and that capacitative Ca(2+) entry is mediated by the interaction of TGII with PLC-delta 1.