Abstract
Sensitize melanomas to apoptosis and inhibit their growth and metastatic potential by compounds that mimic the activities of activating transcription factor 2 (ATF2)-driven peptides. Small-molecule chemical library consisting of 3,280 compounds was screened to identify compounds that elicit properties identified for ATF2 peptide, including (a) sensitization of melanoma cells to apoptosis, (b) inhibition of ATF2 transcriptional activity, (c) activation of c-Jun NH(2)-terminal kinase (JNK) and c-Jun transcriptional activity, and (d) inhibition of melanoma growth and metastasis in mouse models. Two compounds, celastrol (CSL) and acetyl isogambogic acid, could, within a low micromolar range, efficiently elicit cell death in melanoma cells. Both compounds efficiently inhibit ATF2 transcriptional activities, activate JNK, and increase c-Jun transcriptional activities. Similar to the ATF2 peptide, both compounds require JNK activity for their ability to inhibit melanoma cell viability. Derivatives of CSL were identified as potent inducers of cell death in mouse and human melanomas. CSL and a derivative (CA19) could also efficiently inhibit growth of human and mouse melanoma tumors and reduce the number of lung metastases in syngeneic and xenograft mouse models. These studies show for the first time the effect of CSL and acetyl isogambogic acid on melanoma. These compounds elicit activities that resemble the well-characterized ATF2 peptide and may therefore offer new approaches for the treatment of this tumor type.
Highlights
MethodsMouse melanoma (SW1) and human melanoma (LU1205) cells were maintained in DMEM supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics
Experimental Design—Small-molecule chemical library consisting of 3,280 compounds was screened to identify compounds that elicit properties identified for activating transcription factor 2 (ATF2) peptide, including (a) sensitization of melanoma cells to apoptosis, (b) inhibition of ATF2 transcriptional activity, (c) activation of c-Jun NH2-terminal kinase (JNK) and c-Jun transcriptional activity, and (d) inhibition of melanoma growth and metastasis in mouse models
A growing number of genetic and epigenetic changes in melanomas affect genes associated with melanocyte development and maintenance, cell cycle control, resistance to apoptosis, and angiogenic and metastatic capacity
Summary
Mouse melanoma (SW1) and human melanoma (LU1205) cells were maintained in DMEM supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics. The human melanoma cell lines MEWO and WM115 were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. Primary mouse melanocytes were cultured in F-12 medium supplemented with 10% fetal bovine serum, antibiotics, isobutylmethylxanthine, bovine pituitary extract, and 12-O-tetradecanoylphorbol-13-acetate. An ATF2 peptide (amino acids 51–100) was cloned into HA-tagged pcDNA3 vector as described previously [30]. 5xJun2tk-Luc (marker for ATF2 transcriptional activities), 5xTREtk-Luc (marker for AP1/c-Jun transcriptional activities), and 2xNF-κB-Luc were described previously [31]. Pharmacologic inhibitor of JNK (SP600125) was purchased (EMD Biosciences) and added (5 μmol/L) to cultured cells as indicated in Results
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