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.

Effects of histamine on growth and apoptosis of human melanoma cells A375

Objective: To investigate the effects of histamine on growth and apoptosis of human melanoma cells A375. Methods: The effect of histamine on growth of A375 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis by double staining with Annexin V-FITC and PI, and active caspase-3 analysis by staining FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody were made by flow cytometer. StreptAvidin-Biotin Complex (SABC) immunocytochemical assays were adopted to detect Bax/Bcl-2 protein expressions.Results: Histamine inhibited proliferation of A375 cells in a dose- and time-dependent manner, and altered cell cycle distribution of A375 cells revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. Histamine induced apoptosis of A375 cells (P<0.05), elevated the cells population with detectable active caspase-3 (P<0.05), increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly (P<0.05). Conclusion: That histamine inhibits cell cycle progress of A375 cells is one of the possible mechanisms of proliferation arrest of A375 cells elicited by histamine. Histamine mediates apoptosis in A375 cells that may be caspase-dependent through mitochondria routine. Histamine with high concentration inhibits growth of A375 cells in vitro by interfering proliferation and inducing apoptosis of cells.

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.

Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells

Objective; Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta.Apart from its expression in placenta,brain and testis,syncytin has also been found in many cancers.Although syncytin has been proposed to serve as a positive prognostic marker in some cancers,the underlying mechanism is unclear.The aim of this study is to evaluate the effects of syncytin expression on the invasive phenotype of melanoma cells.Methods：The eukaryotic expression plasmid for syncytin-EGFP was constructed and transfected into B16F10 melanoma cells.The effect of syncytin on the invasion potential of rumor cells was evaluated in B16F10 subline cells that stably expressed syncytin-EGFP fusion protein or EGFP alone.Results：The B16F10 sublines that stably expressed syncytin-EGFP or EGFP alone were established respectively and confirmed by immunofluorescent and immtmoblotting assay.Syncytin expression in B16F10 cells was associated with decreased cell proliferation,migration and invasion.Multinucleated giant cells that contained as many as five nuclei were induced in syncytin-expressing cells.In addition,syncytin expression did not alter the sensitivity of B16F10 cells to trichosanthin,a toxin that damages syncytiotrophoblasts more efficiently than other tissues.Conclusions：These results suggest that syncytin expression in some cancers may confine their invasion potential and thus serve as a positive prognostic factor.

A study on melanoma treatment using dendritic cells loaded with antigens purified from melanoma cell lines

Objective The aim of this study was to purify effective tumor peptide complexes from human melanoma cell lines to enhance the treatment effects on melanoma.Methods We purified heat shock protein 70(HSP70)-peptide complexes(PCs)from human melanoma cell lines A375,A875,M21,M14,WM-35,and SK-HEL-1.We named the purified product as M-HSP70-PCs and determined its immunological activities.Autologous HSP70-PCs purified from primary tumor cells of melanoma patients(9 cases)were used as controls.These two tumor antigenic complexes were loaded into dendritic cells(DCs)and used to stimulate an antitumor response against tumor cells in the corresponding patients.Results Mature DCs pulsed with M-HSP70-PCs stimulated autologous T cells to secrete the same levels of type I cytokines as the autologous HSP70-PCs.Moreover,DCs pulsed with M-HSP70-PCs endued CIK cells with an equal ability as autologous HSP70-PCs to kill melanoma cells in the patients.Conclusion M-HSP70-PCs may be used as an efficient and generalized tumor antigen in the treatment of DC-based malignant melanoma.

<i>In-Vitro</i>Effect of Steroids on Melanoma Cell Growth—A Prelude to Melanoma Treatment?

Skin is not only a target organ for various sex steroids and hormones, but also an endocrine organ, which produces sex steroids. It has been suggested by Nikolakis et al. that impairment in skin steroidogenesis may result in inflammatory or autoimmune or other skin disorders. Melanoma is one such skin disease or disorder, which is believed to be caused by UV rays. But, epidemiological, clinical, in-vivo and in-vitro studies suggested the involvement of steroids in the regulation of melanoma growth. However, these studies either did not identify the steroid involved or did not relate to the protective function of the steroid in menstruating females in melanoma, as reported by the clinical studies. In this context, our studies with mouse and human melanoma cell lines showed that female sex steroid progesterone not only inhibited melanoma cell growth, but also affected adhesion and migration functions. In addition, our studies also showed that the effect of progesterone was not a toxic or spurious, but a specific effect on melanoma cells. Hence, our in-vitro studies along with previous other studies subscribed to the idea proposed earlier by Slominski et al. that modulation of local steroids could be a new therapeutic approach for treatment of skin disease or disorder, melanoma.

Potential mechanism for the effects of dexamethasone on growth of human melanoma cells in vitro

This study deals with the inhibitory effects of dexamethsone on the proliferation of a human melanoma cell line in vitro. A retarded cell proliferation was observed in M-5A cells with the presence of specific high affinity glucocorticoid steroid receptors. There was a correlation between the number of glucococrticoid binding sites and the reduction of cell proliferation in term of reduced plating efficiency. Arrest or accumula- tion of M-5A cells in G1 phase or both in G1 and G2 phase appeared to be involved in the growth inhibitory effect by dexamethsone. The magnitude and duration of cell cycle arrest up to 72 hours were dose dependent. There was a correlation between the duration of the disturbance of the cell cycle progression in M-5A cells after dexamethsone treatment and the inhibition of cell proliferation. Synthesis of receptor protein was not specifically stimulated or inhibited relative to the effect on cellular protein content in general. This may conclude that in the melanoma M-5A cell, death after glucocorticoid treatment is somehow related to the glucocorticoid receptor content and to the disturbance of cell cycle distribution.

Cytotoxic Effect of Dexamethasone Restricted to Noncycling, Early G1 Phase of Melanoma Cells

This study deals with the inhibitory effects of dexamethsone on the proliferation of a human melanoma cells after separation into different fractions according to their position in the cell cycle using a gravity sedimentation chamber. Fractions with high percentage of G1 cells were more susceptible to the action of dexamethsone than those with a lower percentage of G1 cells. Dexamethsone stimulated the rate of incorporation of radioactive precursors into acid-precipitable materials of melanoma M-5A cells starting 24 hours after treatment for one hour. Moreover, dexamethsone treatment markedly increased the volume of the M-5A cells with increasing the possibility of stimulating the transcriptional/translational activity in the cells. This study may hopefully stimulate the development of new approaches in systemic and/or regional chemotherapy with malignant melanoma using dexamethsone with other alkylating agents to get synergestic interaction.

Comparative Analysis of Protein Expression Concomitant with DNA Methyltransferase 3A Depletion in a Melanoma Cell Line

DNA methyltransferase 3A (Dnmt3a), a de novo methyltransferase, has attracted a great deal of attention for its important role played in tumorigenesis. We have previously demonstrated that melanoma is unable to grow in-vivo in conditions of Dnmt3a depletion in a mouse model. In this study, we cultured the Dnmt3a depletion B16 melanoma (Dnmt3a-D) cell line to conduct a comparative analysis of protein expression con-comitant with Dnmt3a depletion in a melanoma cell line. After two-dimensional separation, by gel electro-phoresis and liquid chromatography, combined with mass spectrometry analysis (1DE-LC-MS/MS), the re-sults demonstrated that 467 proteins were up-regulated and 535 proteins were down-regulated in the Dnmt3a-D cell line compared to the negative control (NC) cell line. The Genome Ontology (GO) and KEGG pathway were used to further analyze the altered proteins. KEGG pathway analysis indicated that the MAPK signaling pathway exhibited a greater alteration in proteins, an interesting finding due to the close relation-ship with tumorigenesis. The results strongly suggested that Dnmt3a potentially controls the process of tu-morigenesis through the regulation of the proteins (JNK1, p38α, ERK1, ERK2, and BRAF) involved in tu-mor-related pathways, such as the MAPK signaling pathway and melanoma pathway.

Polydopamine Particles Effect on Melanoma Cells Proliferation and Melanin Secretion

Melanin is a biopolymer implicated in the protection of cellular membranes and DNA produced by melanocytes. This pigment has a dual role and should be considered as a photo-protector and as a photosensitizer due to its interaction with UV. The design of multifunctional and biologically responsive coatings is of major interest in modern biomaterials science. The aim of this study is not only to characterize the deposition of multilayered polyelectrolytes films made from polydopamine particles and polyamines like poly-(L-lysine hydrobromide) (PLL), but also to evaluate melanoma cells activity in terms of proliferation and their capacity to stimulate melanin secretion. One could expect that the presence of a melanin like material in the film may have a positive or a negative feedback on the melanin biosynthesis and consequently on melanoma development. Some comparisons are also done with pure polydopamine grains in suspension in the cell culture medium, to investigate if the immobilization of the polydopamine grains has an influence on their bioactivity.

Experimentally-Induced Inhibition of Growth in Melanoma Cell Cultures Separated by ~2 Kilometers When Both Share Excess Correlation Magnetic Fields: Macroscopic Evidence of Free-Space Quantum Teleportation?

In multiple experiments plates of melanoma cells separated by either 3 m or 1.7 km were placed in the centers of toroids. A specific protocol of changing, angular velocity, pulsed magnetic fields that has been shown to produce excess correlation in photon durations and shift in proton concentrations (pH) in spring water were generated around both plates of cells. Serial injections of 50 μL of standard concentrations of hydrogen peroxide into the “local” plates of cells during the 12 min of field activation produced conspicuous cell death (reduction of viable cells by about 50%) with comparable diminishments of cell numbers in the non-local plates of cells within 24 hr but only if both loci separated by either 3 m or 1.7 km had shared the “excess correlation” magnetic field sequence. The non-local effect did not occur if the magnetic fields had not been present. Higher or lower concentrations of peroxide or concentrations that eliminated all of the cells or very few cells in the local dishes were associated with no significant diminishment of non-local cell growth. The data indicate that there must be a critical number of cells remaining viable following the local chemical reaction for the excess correlation to be manifested in the non-local cells. We suggest that this specific spatial-temporal pattern of fields generated within the paired toroidal geometries promotes transposition of virtual chemical reactions as an information field. Calculations of the energy available per cell and per volume of the quantity of reactants injected into the local space from the intensity of the changing velocity toroidal magnetic field support previous measurements and derivations that the units of information transposition may involve discrete quantities that represent equivalents of photons, electrons and protons.

Calpastatin participates in the regulation of cell migration in BAP1-deficient uveal melanoma cells

AIM: To detect how BRCA-associated protein 1(BAP1) regulates cell migration in uveal melanoma(UM) cells. METHODS: Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, immunoprecipitations and surface plasmon resonance analyses were applied to identify BAP1 protein partners. Western blot and calpain activity assays were used to test the expression and function of calpastatin(CAST). RESULTS: CAST protein was confirmed as a new BAP1 protein partner, and loss of BAP1 reduced the expression and function of CAST in UM cells. The overexpression of CAST rescued the cell migration phenotype caused by BAP1 loss.CONCLUSION: BAP1 interacts with CAST in UM cells, and CAST and its subsequent calpain pathway may mediate BAP1-related cell migration regulation.

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.

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.

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.