Dataset Information

Impairment of SK-MEL-28 Development-A Human Melanoma Cell Line-By the Crataeva tapia Bark Lectin and Its Sequence-Derived Peptides.

ABSTRACT: Melanoma is difficult to treat with chemotherapy, prompting the need for new treatments. Protease inhibitors have emerged as promising candidates as tumor cell proteases promote metastasis. Researchers have developed a chimeric form of the Bauhinia bauhinioides kallikrein inhibitor, rBbKIm, which has shown negative effects on prostate tumor cell lines DU145 and PC3. Crataeva tapia bark lectin, CrataBL, targets sulfated oligosaccharides in glycosylated proteins and has also demonstrated deleterious effects on prostate and glioblastoma tumor cells. However, neither rBbKIm nor its derived peptides affected the viability of SK-MEL-28, a melanoma cell line, while CrataBL decreased viability by over 60%. Two peptides, Pep. 26 (Ac-Q-N-S-S-L-K-V-V-P-L-NH2) and Pep. 27 (Ac-L-P-V-V-K-L-S-S-N-Q-NH2), were also tested. Pep. 27 suppressed cell migration and induced apoptosis when combined with vemurafenib, while Pep. 26 inhibited cell migration and reduced nitric oxide and the number of viable cells. Vemurafenib, a chemotherapy drug used to treat melanoma, was found to decrease the release of interleukin 8 and PDGF-AB/BB cytokines and potentiated the effects of proteins and peptides in reducing these cytokines. These findings suggest that protease inhibitors may be effective in blocking melanoma cells and highlight the potential of CrataBL and its derived peptides.

SUBMITTER: Lie KCM

PROVIDER: S-EPMC10342017 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Impairment of SK-MEL-28 Development-A Human Melanoma Cell Line-By the Crataeva tapia Bark Lectin and Its Sequence-Derived Peptides.

Lie Kathleen Chwen Ming KCM Bonturi Camila Ramalho CR Salu Bruno Ramos BR de Oliveira Juliana Rodrigues JR Bonini Galo Márcia M Paiva Patrícia Maria Guedes PMG Correia Maria Tereza Dos Santos MTDS Oliva Maria Luiza Vilela MLV

International journal of molecular sciences 20230625 13

Melanoma is difficult to treat with chemotherapy, prompting the need for new treatments. Protease inhibitors have emerged as promising candidates as tumor cell proteases promote metastasis. Researchers have developed a chimeric form of the Bauhinia bauhinioides kallikrein inhibitor, rBbKIm, which has shown negative effects on prostate tumor cell lines DU145 and PC3. Crataeva tapia bark lectin, CrataBL, targets sulfated oligosaccharides in glycosylated proteins and has also demonstr ...[more]

PMID: 37445794

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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).

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, pSilencerâ¢ 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 ReagentÂ® (Sigma-Aldrich) according to the manufacturerâs 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 GeneChipÂ® 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

Impairment of SK-MEL-28 Development-A Human Melanoma Cell Line-By the Crataeva tapia Bark Lectin and Its Sequence-Derived Peptides.

Publications

Impairment of SK-MEL-28 Development-A Human Melanoma Cell Line-By the <i>Crataeva tapia</i> Bark Lectin and Its Sequence-Derived Peptides.

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