Enzalutamide is an androgen receptor inhibitor

by Selleckchem | Feb 27 2013

Little is known of pathways involved in upstream control of Notch1 gene expression and activity in keratinocytes, and mammalian cells in general. To address this issue we undertook a chemical genetics approach. Rather than screening a large collection of unknown chemicals, enzalutamide we chose a library of 489 compounds, approved by the Food and Drug Administration and of established target selectivity, using a Luciferase Notch/CSL-responsive reporter as a read-out. The negative regulators of Notch signaling identified by this screen included inhibitors of metalloproteases and secretase, which are required for endogenous Notch activation4, confirming the validity of the assay . Statistical analysis of the results pointed to a number of other candidate pathways.
In particular, the most significant compounds to induce Notch activity were kinase inhibitors Topotecan that target components of signaling networks connected with EGFR signaling, which was of special interest, given the relevance of this pathway in keratinocytes and cancer To validate the findings of our screen, we compared the effects of EGFR inhibition and stimulation on endogenous Notch signaling in human primary keratinocytes. A dose-response of human primary keratinocytes to the EGFR inhibitor AG1478 was determined, on the basis of decreased phosphorylation of the EGFR, ERK1/2, c-Jun and Elk proteins, as well as Tyrphostin AG-1478 downmodulation of c-Fos, which is controlled by growth factors more indirectly, through SRF and TCF-dependent transcription, at the level of gene transcription .
At the same doses, there was induction of the canonical Notch target genes Hes1, Hes5 and Herp1, while, conversely, EGF treatment suppressed expression of these genes . In parallel with this effect, Notch1 mRNA levels were increased by EGFR inhibition, while they were down-regulated by EGF treatment.Consistent with a GS-1101 transcriptional mechanism, no increase of Notch1 mRNA stability was observed in EGFR inhibitor-treated cells after Actinomycin D treatment . The results were confirmed at the protein level, by immunoblotting of AG1478- and EGF-treated keratinocytes with antibodies against total and cytoplasmic activated forms of Notch1 as well as Hes1 . Effects similar to those of AG1478 were also elicited by Tarceva, an EGFR inhibitor approved for clinical use13.
Besides chemical inhibition, up-regulation of Notch1 activity and expression were also observed after knockdown of EGFR expression by transfection of keratinocytes with Adriamycin specific siRNAs . Unlike Notch1, Notch2 expression was modulated by EGFR signaling at the mRNA but not protein level , while no consistent changes were found in expression of the Notch ligands Jagged 1 and Delta like 1 EGFR suppression is expected to cause growth inhibition and increased apoptosis7, 14, a fact that we experimentally confirmed, raising the possibility that the induction of Notch1 expression is only an indirect consequence of these events. However, treatment of keratinocytes with TNF at pro-apoptotic concentrations had no effects on levels of Notch1 expression, which was also not affected by suppression of keratinocyte growth by TGF treatment .