Search

Analyzes the expression of genes for tyrosinase, tyrosinase-related protein 1, tyrosinase-related protein 2 and melanocortin-1 receptor using reverse transcription polymerase chain reaction. Result of the incubation of melanocyte-stimulating hormone/isobutylmethylxanthine; Correlation of the abundance and gel mobility pattern with changes in activity in hybrids and parental melanoma cells.

Background: Estrogen receptor (ER) and human epidermal growth factor receptor (HER) family receptors interact in breast cancer; co-targeting these receptors is of interest. We previously reported on a synergistic growth inhibition for the combination of trastuzumab plus tamoxifen in HER2+/ER+ BT474 cells, but no induction of apoptosis. Herein we describe the effects of co-targeting in models of differing HER2 overexpression status (MCF7 HER2-normal/ER+, BT474 HER2-overexpressing/ER+)., Materials and Methods: Assays of proliferation were carried-out using WST-1, cell cycle using flow cytometry, and apoptosis by determination of sub-G1 population and by annexin-V., Results: Combining a dual HER2/EGFR kinase inhibitor with anti-estrogens induces apoptosis of BT474 cells. Furthermore, in MCF7 cells, despite HER2-normal status and lack of response to single-agent HER2 inhibitors, addition of HER2 inhibitors or dual HER2/EGFR inhibitor to anti-estrogens augments the antiproliferative effect of anti-estrogens, and converts the drug effect from cytostatic to apoptosis-inducing., Conclusion: ER-HER co-targeting enhanced the antitumor effects and can bring about effects of targeting HER2 in models of HER2-normal breast cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

In previous studies, fusion of peritoneal macrophages or blood monocytes with mouse melanoma cells produced hybrids with upregulated expression of the glycosyltransferase beta1,6-N-acetylglucosaminyltransferase V (GnT-V) and its enzymatic product, beta1,6-branched oligosaccharides. This correlated with marked increases in motility, metastatic potential and, surprisingly, melanin content. This study was designed to establish direct roles for beta1,6-branched oligosaccharides in melanogenesis and motility. The levels of beta1,6-branched oligosaccharides were lowered by transfecting beta1,4-N-acetylglucosaminyltransferase III, a competitive inhibitor of GnT-V. beta1,4-N-acetylglucosaminyltransferase III transfection virtually eliminated melanin production and markedly decreased chemotactic motility. This implied that the metastatic and melanogenic phenotypes in hybrids were each upregulated by beta1,6-branched oligosaccharides. Although roles for beta1,6-branched oligosaccharides in motility and metastasis have been reported previously, this is the first study to directly implicate these structures in melanogenesis. Although drawn from experimental models, the findings might explain the well known hypermelanotic regions of human cutaneous malignant melanoma as hypermelanotic cutaneous malignant melanoma cells are rich in beta1,6-branched oligosaccharides. They might also explain why melanogenesis pathways differ between malignant and normal melanocytes as GnT-V is a myeloid-associated enzyme that is aberrantly expressed in melanoma cells but not in normal melanocytes.

It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed macrophage-specific glycosylation, especially increased GnT-V activity, beta1,6 branch formation in glycoproteins, accompanied by enhanced metastatic potential in vivo and motility in vitro. These hybrids also express upregulated melanocortin-1 receptor (MC1-R) activity and exhibit increased motility after melanocyte-stimulating hormone (MSH) treatment. In this report, we show that MSH-mediated stimulation of motility is mediated through enhanced expression of c-Met proto-oncogene. In metastatic hybrids c-Met expression is induced by MSH, and addition of c-Met neutralizing antibody to cells inhibits MSH-induced motility but not the basal motility of the cells. Furthermore, abrogation of the chemoattractant gradient concentration by addition of hepatocyte growth factor (HGF) recombinant protein, a cognate ligand of c-Met receptor, reduces the MSH-induced effect on motility. A similar result was also obtained by the addition of blocking anti-alphaHGF antibody in the chemoattractant chamber. Again, the metastatic hybrids, but not the nonmetastatic hybrids or parental melanoma cells, showed significant motile response to rHGF chemoattractant, and that motility is further induced when cells were stimulated with MSH/isobutylmethyl xanthine (IBMX). Synergistic stimulation on motility was also observed with those hybrids treated with MSH/IBMX and when rHGF and fibronectin (FN), in combination, were used as chemoattractants. These indicate that MSH/IBMX-induced motility might involve c-Met pathways as well as extracellular matrix (ECM)/integrin pathways in a cooperative fashion. Ets-1, a transcription factor involved in the expression of c-Met, is also found to be induced in metastatic hybrids after exposure to MSH/IBMX. Implication of the result is discussed in light of the role of c-Met and its interacting proteins in the development of metastatic phenotypes and its therapeutic intervention.