Dataset Information

The mojastin mutant Moj-DM induces apoptosis of the human melanoma SK-Mel-28, but not the mutant Moj-NN nor the non-mutated recombinant Moj-WN.

ABSTRACT: In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC(50) of 160nM, while the GST-Moj-NN had an IC(50) of 493nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN inhibited the binding of SK-Mel-28 cells to fibronectin with an IC(50) of 11nM, followed by the GST-Moj-NN (IC(50) of 28nM), and the GST-Moj-DM (IC(50) of 46nM). The GST-Moj peptides' ability to induce apoptosis on SK-Mel-28 cells was determined using Annexin-V-FITC and nuclear fragmentation assays. Cells were incubated with 5muM GST-Moj peptides for 24h. At 5microM GST-Moj-DM peptide, 13.56%+/-2.08 of treated SK-Mel-28 cells were in early apoptosis. The GST-Moj-DM peptide also caused nuclear fragmentation as determined by fluorescent microscopy and Hoechst staining. The GST-Moj-WN and GST-Moj-NN peptides failed to induce apoptosis. We characterized the SK-Mel-28 integrin expression, as the first step in determining r-Moj binding specificity. Our results indicate that SK-Mel-28 cells express alphavbeta3, alphav, alpha6, beta1, and beta3 integrin receptors.

SUBMITTER: Seoane AI

PROVIDER: S-EPMC2930814 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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The mojastin mutant Moj-DM induces apoptosis of the human melanoma SK-Mel-28, but not the mutant Moj-NN nor the non-mutated recombinant Moj-WN.

Seoane Agustin I AI Tran Victoria L VL Sanchez Elda E EE White Stephanie A SA Choi Jason L JL Gaytán Brandon B Chavez Natalie N Reyes Steven R SR Ramos Carla J CJ Tran Luan H LH Lucena Sara E SE Sugarek Maria M Perez John C JC Mandal Stephanie A SA Ghorab Shervin S Rodriguez-Acosta Alexis A Fung Branden K BK Soto Julio G JG

Toxicon : official journal of the International Society on Toxinology 20100414 3

In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC(50) of 160nM, while the GST-Moj-NN had an IC(50) of 493nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN i ...[more]

PMID: 20398687

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Project description:Melanoma tumor antigen p97 or melanotransferrin (MTf) is an iron (Fe)-binding protein with high homology to serum transferrin. MTf is expressed at very low levels in normal tissues and in high amounts in melanoma cells. The over-expression of MTf in tumor cells was hypothesized to assist rapidly proliferating neoplastic cells with their increased Fe requirements. However, our recent characterization of the MTf knockout (MTf -/-) mouse demonstrated that MTf did not have an essential role in Fe metabolism. To understand the function of MTf, we utilized whole-genome microarray analysis to examine the gene expression profile of five models after modulating MTf expression. These models included two new stably transfected MTf hyper-expression models (SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type (SK-Mel-28 melanoma) where MTf was down-regulated by post-transcriptional gene silencing. These findings were compared to alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the gene array data were also assessed in a new model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased cellular proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and up-regulation, we identified three genes modulated by MTf expression. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and transcription factor 4 (Tcf4) were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and cell proliferation/survival, respectively. This study identifies novel molecular targets directly or indirectly regulated by MTf and potential pathways involved in its function. These molecular targets could be involved, at least in part, to the role of MTf in modulating proliferation. Experiment Overall Design: To prepare a construct capable of generating hairpin siRNA specific for human MTf mRNA, the expression vector, pSilencer™ 3.1-H1 neo (Ambion, Texas, USA) was used. The vector was used to clone transgene of 66-bp that transcribe 19-mer double-stranded hairpin RNAs of the target gene. The transgene were specifically targeted to positions 2031-2049-bp in the MTf gene (Genbank Accession: NM_005929). Transfections of pS-MTf transgenes or pS-scrambled vectors into human SK-Mel-28 melanoma cells were performed with LipofectamineTM 2000 reagent (Invitrogen, Melbourne, Australia). Total RNA was isolated from the cells using TRIzol Reagent® (Sigma-Aldrich) according to the manufacturer’s protocol. Total RNA from the stably-transfected SK-Mel-28 melanoma cell lines were prepared and hybridized onto Human Genome U133 Plus 2.0 Array. The human GeneChip® U133 Plus 2.0 consists of greater than 47,000 transcripts and variants from over 38,500 well characterized human genes (Affymetrix, Santa Clara, CA).

Dataset Information

The mojastin mutant Moj-DM induces apoptosis of the human melanoma SK-Mel-28, but not the mutant Moj-NN nor the non-mutated recombinant Moj-WN.

Publications

The mojastin mutant Moj-DM induces apoptosis of the human melanoma SK-Mel-28, but not the mutant Moj-NN nor the non-mutated recombinant Moj-WN.

Similar Datasets

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