Aminoflavone induces AhR mediated expression of CYP1A1, CYP1B1, and luciferase downstream of dioxin responsive download the handbook elements To confirm the finding that AF is capable of activating AhR signaling, 101 L hepatoma cells stably harboring three dioxin responsive elements upstream of a luciferase reporter were incubated with 0. 1% dimethyl sulfoxide or AF for 18 hours. Inhibitors,Modulators,Libraries After nor malizing raw luciferase units to the background levels seen in the DMSO control, we show that AF significantly increases luciferase expression in this system. However, compared with the positive control, B Naphthoflavone, it is evident that AF is a weak AhR agonist. This result is consistent with the previ ous finding that AF has agonistic effects on AhR.

Further, it was previously shown that AhR target genes CYP1A1, and to a lesser extent CYP1A2 and CYP1B1 are upregu lated in response to AF treatment, and may play role in the metabolism of AF itself. Inhibitors,Modulators,Libraries We went on to examine whether AF could induce AhR target genes in MDA MB 468 and Cal51. Cells were treated with a range of AF concentrations from 10nM to 10 uM, along with 1 uM of BNF as a positive control for AhR activation. While MDA MB 468 and Cal51 exhibit Inhibitors,Modulators,Libraries similar sensitiv ities to AF based on their GI50 values, we found that their ability to upregulate CYP1A1 and CYP1B1 expression after AF treatment was drastically different. AF strongly induced CYP1A1 and CYP1B1 expression in MDA MB 468, but to a much lesser extent in Cal51. Compared to MCF7, which has been shown to be responsive to AhR ligands, MDA MB 468 exhibits greater induction of CYP1A1 upon AhR activation.

Cal51 exhibits greater induction of CYP1A1 upon treatment with AhR activators as compared to MDA MB 231, which is AF resistant, but the induction is less than both MCF7 and MDA MB 468. SULT1A1 expression has also been linked to AF sensitivity. Inhibitors,Modulators,Libraries MDA MB 468 and Cal51 cells express SULT1A1 basally, but its expression is not induced by treatment with AF or BNF. Further, we have shown that knocking down AhR does not decrease basal SULT1A1 expression in MDA MB 468, and only minimally alters SULT1A1 expression in Cal51. Interestingly, Inhibitors,Modulators,Libraries direct knockdown of SULT1A1 in these cell lines results in signifi cantly increased resistance to AFs cytotoxic effects. Overall, these results suggest that cell populations with varying ability to induce AhR signaling may exhibit AF sensitivity.

Thus, active downstream AhR signaling may not be required to confer AF sensitivity. Endogenous selleckchem levels of AhR are not required for sensitivity to aminoflavone in MDA MB 468 and Cal51 human breast cancer cells It has been previously reported that AF sensitive MCF7 cells become resistant to AF upon attenuation of AhR sig naling. In addition, localization of AhR in the cellular cytoplasm has been shown to correlate with AF sensitivity.