Figure 6 11a induces G1/S phase cell cycle arrest in a time dependent selleck bio manner in HCT116 isogenic cell lines. Figure 7 11a induces G1/S phase cell cycle arrest in a dose dependent manner in HCT116 isogenic cell lines. Discussion In this study, we evaluated the dependency of 11a cytotoxicity on PNR expression and further investigated the mechanism of 11a cellular cytotoxicity using various cell-based assays. Although 11a was originally reported as a synthetic agonist for PNR, it was found not to be a direct PNR agonist in the recently developed TR-FRET assay which measured PNR-RetCoR dissociation [32]. Consistent with this finding, our cell-based assays similarly showed that 11a was unlikely to be a direct agonist for PNR.

We employed luciferase reporter assays to compare the ability of 11a to activate different subfamily II nuclear receptors, of which PNR is a member [36]. However, our results showed that 11a is least potent in PNR activation, whereas 11a could weakly activate other members of subfamily II such as TLX, COUP-TFI and COUP-TFII at the different concentrations tested. Further studies using stable cell lines overexpressing PNR confirmed that the 11a-induced cytotoxic effects are PNR independent. Together, our results indicate that 11a may have cellular targets other than PNR to confer cytotoxic effects. Although the cellular targets of 11a remain to be determined, 11a was found to activate COUP-TFII in DR2-luciferase reporter assay and COUP-TFII target genes in two breast cancer cell lines. The only other known weak agonist for COUP-TFII is atRA [44].

atRA was shown to activate COUP-TFII on the NGFI-A promoter in the luciferase reporter assay [44]. Induction of RARB2 by atRA causes growth inhibition and apoptosis in cancer cells and this process requires the orphan nuclear receptor COUP-TFII [42]. Since 11a activated COUP-TFII in the DR2 luciferase assay (Figure 1B) and induced RARB2 and NGFI-A gene expression to a comparable level as atRA (Figure S3), it is possible that 11a could serve as an agonist for COUP-TFII and substitute atRA in some cancer treatment. For example, all-trans retinoic acid has long been used for the treatment of acute promyelocytic leukemia (APL) and were shown to inhibit solid tumor growth [55], however, the strong cytotoxicity prevents its wide use in cancer treatment. The concentrations of RAs required for activation of COUP-TFII are 10-100 times higher than the physiological levels [44], on the contrary, 11a at 10-time lower concentration could manifest the same effect in the COUP-TFII target gene activation (Figure 3). Whether 11a-induced cytotoxicity is at least in part mediated through COUP-TFII Anacetrapib is worth further investigation.