Inhibitory effect of Cyrtomium falcatum on melanogenesis in α-MSH-stimulated B16F10 murine melanoma cells

Objective:To explore the anti-melanogenic potential of Cyrtomium falcatum.Methods:The effects of Cyrtomium falcatum crude extract and its solvent fractions on tyrosinase activity,melanin content,and the expressions of melanogenesis-related genes and proteins were analyzed inα-melanocyte-stimulating hormone(α-MSH)-stimulated B16F10 cells.Results:α-MSH treatment significantly increased tyrosinase activity,and extracellular and intracellular melanin content,as well as the expression levels of tyrosinase,microphthalmia-associated transcription factor(MITF),tyrosinase-related protein(TRP)-1,and TRP-2 in B16F10 cells.Treatment with Cyrtomium falcatum crude extract and its solvent fractions reduced tyrosinase activity and extracellular and intracellular melanin content and downregulated the expression levels of tyrosinase,MITF,TRP-1,and TRP-2 in a dose-dependent manner.Conclusions:Cyrtomium falcatum has potential anti-melanogenesis effects and can be used as a potential source material in cosmeceutical industry for the research and development of novel lead molecules with whitening properties.

Different effects of the inhibition of Src activity on Akt/PKB in melanoma cells with wild BRAF and mutated BRAF V600E

Src regulates cell adhesion, invasiveness, motility and growth in cancer cells. In melanoma, accumulating data show that Src inhibition can be effective and may enhance the effects of other agents. Increased Src expression and activity thus has recently become a target for drug therapy. Several melanoma cell lines were exposed to inhibitors of Src activity despite their broad specificity. To examine the particular activity of Src in human melanoma cells, we used SU6656, the selective inhibitor of Src family protein kinases. The activity of Src and cell proliferation were suppressed in HBL human cells, wild type melanoma cells and in SK-MEL-5 human melanoma cells harboring mutant BRAF V600E, upon their treatment with SU6656. The suppression of Src kinase activity had not inhibitory effects on Akt/PKB activity in SK-MEL-5 cells, which we have previously found in HBL cells. This may indicate that changes of Src involvement in the control of Akt/PKB activity and its downstream signaling could be induced by BRAF V600E mutation in SK-MEL-5 cells.

Effect of luteolin on apoptosis and vascular endothelial growth factor in human choroidal melanoma cells

AIM:To investigate the effects of luteolin on apoptosis,the cell cycle,and the expression and secretion of vascular endothelial growth factor(VEGF)in human choroidal melanoma cells(C918 and OCM-1).METHODS:C918 and OCM-1 cells cultured in vitro were treated with various concentrations of luteolin(0,5,10,15μmol/L).Cell growth was observed with an inverted microscope,and cell cycle arrest was detected by propidium iodide(PI)staining using flow cytometry.Apoptosis was detected by Hoechst33342 staining,and apoptosis rate was determined by Annexin V-FITC/PI experiments using flow cytometry.The expression of apoptosis-related proteins Bcl-2,Bax and VEGF was analyzed using Western blots.The levels of VEGF secreted by the cells into the supernatant was analyzed using ELISA.RESULTS:After treating with 5 to 15μmol/L luteolin for 48 h,the fusion degree of C918 and OCM-1 cells decreased,and more floating apoptotic cells appeared.Luteolin treatment increased the G0-G1 phase ratio of the C918 and OCM-1 cells,blocked cell cycle progression,and increased the apoptosis rate of the C918 and OCM-1 cells.Western blot showed that luteolin decreased the expression of Bcl-2 and VEGF in the C918 and OCM-1 cells and increased the expression of Bax protein.The ELISA results showed that 10 to 15μmol/L luteolin decreased the cell secretion of VEGF.CONCLUSION:Luteolin may induce apoptosis by regulating the levels of apoptosis-related proteins in C918 and OCM-1 cells.Luteolin can induce cell cycle arrest,decrease the expression of VEGF.

Inactivated Sendai Virus Induces Apoptosis in Murine Melanoma Cells by IGF-1R Down-regulation

The mortality of cancer patients has considerably improved due to progress in surgery, chemotherapy and radiotherapy. However, some types of cancers, such as melanoma, remain refractory to conventional strategies. Although melanoma accounts for only 4% of all dermatological malignancies, it is responsible for 80% of mortalities from skin tumors[11. The reported survival rate of melanoma over 5 years is not yet encouraging due to its chemo-resistance and rapid metastasis. Therefore, it is necessary to develop new drugs with potent activity and weak side-effect against melanoma.

Proteomic analysis of energy metabolism and signal transduction in irradiated melanoma cells

AIM: To analyze proteomic and signal transduction alterations in irradiated melanoma cells. METHODS: We combined stable isotope labeling with amino acids in cell culture (SILAC) with highly sensitive shotgun tandem mass spectrometry (MS) to create an efficient approach for protein quantification. Protein protein interaction was used to analyze relationships among proteins. RESULTS: Energy metabolism protein levels were significantly different in glycolysis and not significantly different in oxidative phosphorylation after irradiation. Conversely, tumor suppressor proteins related to cell growth and development were downregulated, and those related to cell death and cell cycle were upregulated in irradiated cells. CONCLUSION: Our results indicate that irradiation induces differential expression of the 29 identified proteins closely related to cell survival, cell cycle arrest, and growth inhibition. The data may provide new insights into the pathogenesis of uveal melanoma and guide appropriate radiotherapy.

Inhibition of HOXB7 Gene Expression in Melanoma Cells by Small Interfering RNA

Objective： HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma （MM） cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods： Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results： The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion： Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited.

RNAi-mediated Human Nestin Silence Inhibits Proliferation and Migration of Malignant Melanoma Cells by G1/S Arrest via Akt-GSK3β-Rb Pathway

Human Nestin（hNestin） has been found to express in melanoma, and its expression is positively correlated with the advanced stage of melanoma. However, the precise role of hNestin in the development of melanoma has not been fully understood. The present study aimed to explore the role of hNestin in the proliferation and invasion of melanoma cells. The lentivirus vector carrying a short hairpin RNAs（shRNAs） targeting hNestin（hNestin-sh RNA-LV） was stably infected into human melanoma cells UACC903, which expressed high levels of hNestin. The effects of hNestin knockdown on the proliferation, apoptosis, migration of melanoma cells and the related signaling pathways were investigated by immunofluorence, Western blotting and reverse transcription polymerase chain reaction（RT-PCR）, respectively. The results showed that hNestin was expressed in most melanoma specimens and the melanoma cells studied. Knockdown of hNestin expression significantly inhibited the proliferation of melanoma cells, blocked the formation of cell colony, arrested cell cycle at G1/S stage and suppressed the activation of Akt and GSK3β. hNestin-silent cells also showed a sheet-like appearance with tight cell-cell adhesion, decreased membrane expression of N-cadherin and β-catenin, and attenuated migration. Furthermore, hNestin silence resulted in the inhibition of tumor growth in vivo. Our study indicates that hNestin knockdown suppresses the proliferation of melanoma cells, which might be through affecting Akt-GSK3β-Rb pathway-mediated G1/S arrest, and hNestin silence inhibits the migration by selectively modulating the expression of cell adhesion molecules in the process of epithelial-mesenchymal transition.

Effect of Trichosanthin on Cell Cycle and Apoptosis of Murine Melanoma Cells

Objective:To study thei nhibitory effect of purified trichosanthin component on the proliferation of malignant melanoma.Methods:The effect of purified trichosanthin componenton the DNA synthesis,cell cycle and cell apoptos is of murinemelanomacellsweredetected by flow cytometry when culture dinvitro.Results:The significantG0/G1phasearrest was revealed by the inc rease of cellsinG0/G1 phase and decrease of cell sinS phase.The obviousapoptosis of melanoma cells was in duced bypuri fied trichos anthin component.G0/G1phase arrest was high lycorrelated withapop to sis(r=0.8705).Conclusion:The purified trichos anthin component can markedly inhibit melanoma cells by the suppression of DNA syn the sisin S phaseand cell mitosisas well as induction of cell apoptosis.

Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells

Background Melanoma has the highest mortality among all superficial malignant tumors.The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets.As a molecular switch that controls tumor metastasis,Ras homology C （RhoC） has been correlated with tumor progression,especially tumor invasion and metastasis.However,little research has been done about the effects of RNA interference （RNAi） targeting RhoC on the invasion and metastasis of melanoma.In this study,we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.Methods Based on the RhoC gene encoding information,three pGPU6/GFP/Neo-short hairpin （shRNA） plasmids were constructed.After detecting their silencing effects on the RhoC gene of A375 cells,the most effective pGPU6/GFP/ Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC.The lentivirus vector was used to infect A375 cells,and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.Results The plasmids pGPU6/GFP/Neo-shRNA 336,pGPU6/GFP/Neo-shRNA 453,and pGPU6/GFP/Neo-shRNA 680 were constructed.After they were transfected into A375 cells,the expressions of RhoC mRNA and protein were 1.47±0.26,1.13±0.16,1.39±0.11 and 70.98±9.21,50.67±6.06,65.77±4.06,respectively.pGPU6/GFP/Neo-shRNA 453 was the most effective sequence,and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC.pLenti6.3-EGFP-453 was used to infect A375 cells.The expression of RhoC mRNA and protein were 1.05±0.05 and 62.04±15.86 in the lentivirus group,4.21±0.24 and 220.86±24.07 in the negative lentivirus control group,and 4.63±0.32 and 257.39±12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant （P ＜0.05）.Conclusion The successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro,and significantly inhibit the RhoC mRNA and protein expression.

Components in Melanoma Cytoplasm Might Induce Murine BMSCs Transformation and Expression of Melan-A

This study explored the possibility that the components in melanoma cytoplasm induce murine BMSCs transformation and expression of Melan-A by morphologically observing the changes of BMSCs and immunocytochemically detecting Melan-A in the cells after culturing BMSCs in me-dium containing melanoma cytoplasm components （MCC）. MCC of B16 melanoma cells was prepared and BMSCs were cultured and induced by adding the MCC into culture medium. The cells were morphologically observed and Melan-A was immunohistochemically detected to confirm BMSCs transformation. MCC-induced BMSCs underwent morphological changes. A number of melanin granules appeared in the cytoplasm of the cells and some were released into surrounding areas. Several cells that might come from one cell formed a cluster, and their granules, together with those secreted by other induced BMSCs, formed a so-called ＂sphere-formed structure＂. The induced BMSCs expressed Melan-A. We are led to conclude that there might be some factors in the cytoplasm of melanoma cells that might induce BMSCs transformation toward melanogenic cell, or even melanoma.

AstragalosideⅣimproves melanocyte differentiation from mouse bone marrow mesenchymal stem cells

Vitiligo results in an autoimmune disorder destructing skin pigment cells,melanocytes(Mcs).This study aimed to investigate whether Astragaloside IV(AIV)could efficiently induce differentiation of bone marrow mesenchymal stem cells(BMMSCs)into Mcs.BMMSCs were induced and differentiated into Mcs with 0.1,0.2,and 0.4 mg/L AIV during 150-day.Morphologic changes of differentiated cells were observed.Levels of some melanocytic specific genes(TRP-1,TRP-2,MART-1,Mitf)were measured with quantitative polymerase chain reaction(qPCR)at 90,120,and 150 days of induction.After 90-day induction,the differentiated cells with 0.4 mg/L AIV demonstrated the typical morphology of Mcs,positive 3,4 dihydroxyphenylalanine staining,and positive staining of TRP-1,TRP-2,MART-1,and Mitf.After 90-and 120-days’induction with 0.4 mg/L AIV,TRP-1 expression was significantly elevated(p<0.01),and TRP-2 expression was significantly increased in 0.4 mg/L AIV-treated group compared to negative control(p<0.01),0.1 mg/L(p<0.01),and 0.2 mg/L(p<0.01)AIV-treated groups.Moreover,MART-1 expression was significantly up-regulated in 0.4 mg/L AIV-treated group compared to negative control,but without difference compared to 0.1 mg/L(p>0.05)and 0.2 mg/L(p>0.05)AIV-treated groups.During 90 to 150-day induction,there were no significant differences for Mitf levels between AIV-treated groups and negative control(p>0.05).In conclusion,90-day induction with 0.4 mg/L AIV up-regulated TRP-1,TRP-2,and MART-1 expression,indicating that AIV can efficiently induce Mcs differentiation from BMMSCs.These results provide experimental and theoretic evidence for AIV application in clinical vitiligo repigmentation treatment.

In-Vitro Inhibition of Human Melanoma (BLM) Cell Growth by Progesterone Receptor Antagonist RU-486 (Mifepristone)

RU-486 is an abortifacient which is used to terminate early pregnancy. It acts by blocking progesterone receptor. In our earlier study with progesterone, RU-486 was used as a progesterone receptor antagonist to find out the mechanism of progesterone action on melanoma cells. Results indicated that the effect of progesterone was not mediated through progesterone receptor. In the course of experiments, it was observed that RU-486 by itself inhibited mouse melanoma cell growth. Further research work with RU-486 showed a dose dependent inhibition of human melanoma cell growth. The mechanism of inhibition of cell growth was due to apoptosis and this effect of RU-486 was neither mediated through progesterone receptor nor glucocorticoid receptor. This in-vitro study suggested that melanoma also could be a target for RU-486 action, apart from breast, ovary and prostate cancers.

Anti-Melanogenesis Activity of Supercritical Carbon Dioxide Extract from Perilla frutescens Seeds

Perilla frutescens seed (PFS) oil is reported to inhibit skin photoaging;however, its effect on melanogenesis has not yet been investigated. Herein, we tested the anti-melanogenesis activity of an oil-based extract from PFS with supercritical carbon dioxide (scCO2). In a cell culture system, B16 mouse melanoma cells were treated with the PFS scCO2 extract and other samples. The PFS scCO2 extract decreased melanin production by approximately 90% in B16 mouse melanoma cells without cytotoxicity at 100 μg/mL. This effect was greater than that of the well-known melanogenesis inhibitor, kojic acid. Although a hexane-extracted PFS oil and a squeezed PFS oil also decreased melanin production in the B16 cells, the inhibitory effect of the PFS scCO2 extract was higher than both of these. Chemical analysis of the PFS scCO2 extract and squeezed PFS oil showed that almost 90% of the components of both oils were α-linolenic acid, linoleic acid, and oleic acid. Furthermore, the ratio of those three fatty acids across both samples was almost the same. When the three fatty acids were mixed in the same ratio as in the PFS scCO2 extract, the IC50 of the mixture for melanin production in B16 melanoma cells was identical to that of the PFS scCO2 extract. However, the IC50 of the squeezed PFS oil was approximately 6.6 times higher than that of the mixture. Although those fatty acids are the main inhibitory ingredients against melanin production in all of the extracts, some factor(s) in the squeezed PFS reduce their affinity with the cells. These results indicated that the PFS scCO2 extract could be a superior melanogenesis inhibitor. Although its main ingredients are probably the same as those of the squeezed PFS oil, it is necessary to extract with scCO2 for stronger anti-melanogenesis activity.

Effects of Genistein on Cell Cycle and Apoptosis of Two Murine Melanoma Cell Lines

The effects of genistein on several tumor cell lines were investigated to study the effects of genistein on cell growth, cell cycle, and apoptosis of two murine melanoma cell lines, B16 and K1735M2. These two closely related murine melanoma cell lines, however, have different responses to the genistein treatment. Genistein inhibits the growth of both the B16 and K1735M2 cell lines and arrests the growth at the G2/M phase. After treatment with 60 μmol/L genistein for 72 h, apoptosis and caspase activities were detected in B16 cells, while such effects were not found treatment the protein content and mRNA levels of n K1735M2. Further tests showed that after genistein p53 increased in B16, but remained the same in K1735M2. The protein content and mRNA levels of p21^WAF1/CIP1 increased in both cell lines after treatment. The results show that genistein might induce apoptosis in B16 cells by damaging the DNA, inhibiting topoisomerase Ⅱ, increasing p53 expression, releasing cytochrome c from the mitochondria, and activating the caspases which will lead to apoptosis.

Celecoxib in combination with retinoid CD437 inhibits melanoma A375 cell in vitro

This study aimed to investigate the effects of celecoxib,synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylicacid(CD437)and the combination of the two on cell proliferation,apoptosis,and cycle arrest of human malignant mela-noma A375 cells.3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazoliumbromide assay(MTT assay)was applied to determine the anti-proliferative effects of the drugs on human malignant melanoma A375 cells.Flow cytometry was performed to investigate the influence of the drugs on cell cycle and cell apoptosis.Both celecoxib and CD437 could inhibit the growth of human malignant melanoma A375 cells in a dose-dependent manner.Celecoxib at 80μmol/L inhibited proliferation,induced apoptosis and G2/M cell cycle arrest of human malignant melanoma A375 cells after treatment for 24 h[proliferation inhibiting rate:(50.2±2.51)%,apoptosis rate:(35.91±1.80)%].CD437 at 10μmol/L inhibited proliferation,induced apoptosis and G0/G1 cell cycle arrest of human malignant melanoma A375 cells after treatment for 24 h[proliferation inhibiting rate:(58.6±2.38)%,apoptosis rate:(28.03±0.77)%].Celecoxib in combination with CD437 could significantly enhance the effects of inhibiting proliferation and inducing apoptosis of human malignant melanoma A375 cells 24 h after treatment compared with the drug alone[proliferation inhibiting rate:(68.92±1.72)%,apop-tosis rate:(42.09±1.05)%,both P<0.05]and decrease the proportion of the S phase in the cell cycle.Celecoxib could inhibit the growth of human malignant melanoma A375 cells by inducing apoptosis and G2/M cycle arrest.CD437 could inhibit the growth of human malignant melanoma A375 cells by inducing apoptosis and G0/G1 cycle arrest.Celecoxib exhibited additive effects with CD437 on retarding the growth and inducing apoptosis of human malignant melanoma A375 cells.Celecoxib in combination with CD437 may become an effective method for prevention and treatment of human melanoma.

Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol

2-Hydroxytyrosol(2-HT),originally reported as a synthetic compound,was isolated for the first time as a fungal metabolite.2-HT was found to inhibit mushroom tyrosinase with an IC_(50) value of 13.0 mmol/L.Furthermore,2-HT dose-dependently inhibited tyrosinase activity(IC_(50),32.5 mmol/L)in the cell-free extract of B16 melanoma cells andα-melanocyte stimulating hormone(α-MSH)-stimulated melanin formation in intact B16 melanoma cells.

Super-resolution visible photoactivated atomic force microscopy

Imaging the intrinsic optical absorption properties of nanomaterials with optical microscopy(OM)is hindered by the optical diffraction limit and intrinsically poor sensitivity.Thus,expensive and destructive electron microscopy(EM)has been commonly used to examine the morphologies of nanostructures.Further,while nanoscale fluorescence OM has become crucial for investigating the morphologies and functions of intracellular specimens,this modality is not suitable for imaging optical absorption and requires the use of possibly undesirable exogenous fluorescent molecules for biological samples.Here we demonstrate super-resolution visible photoactivated atomic force microscopy(pAFM),which can sense intrinsic optical absorption with~8 nm resolution.Thus,the resolution can be improved down to~8 nm.This system can detect not only the first harmonic response,but also the higher harmonic response using the nonlinear effect.The thermoelastic effects induced by pulsed laser irradiation allow us to obtain visible pAFM images of single gold nanospheres,various nanowires,and biological cells,all with nanoscale resolution.Unlike expensive EM,the visible pAFM system can be simply implemented by adding an optical excitation sub-system to a commercial atomic force microscope.