Even though PNR is not the cellular target of 11a, 11a make it clear might still have cytotoxic effects and thus can be explored as an anti-cancer drug. To test this possibility, we performed systematic cytotoxicity studies using the NCI-60 cell lines. Our results revealed that 11a could induce cytotoxicity in a broad range of cell lines, which is contradictory to a previous report showing that 11a was non-toxic in CHO cells [31]. The cytotoxicity of 11a was evaluated in a NCI-60 cell line screen using the SRB cell viability assay, with the advantage of differentiating cell killing (LC50, 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) from growth inhibition (GI50, 50% reduction in the net protein increase in control cells during the drug incubation) [47,56-58].

The NCI-60 cell line study screens classical and newly synthetic compounds with unique structures and functional groups to assess the mechanism of cytotoxicity and characterize selected cytotoxic effects towards certain cancer types [47], with the aim of finding novel drugs for cancer research and treatment. The sensitivity to 11a is strongly correlated with p53 status, not only in the NCI-60 screen (Figure 3) but also with the HCT116 isogenic cell lines (Figure 5). Because 11a did not strongly induce apoptosis in various cell lines, we focused on G1/S cell cycle arrest in isogenic HCT116 cell lines with null or wild type p53. Although p53+/+ and p53-/- HCT116 cell lines both underwent G1/S phase cell cycle arrest, the p53 wild type cells exhibited higher sensitivity towards 11a as compared with the p53-/- cells, and the checkpoint could not be recovered even after 24 hours treatment.

We concluded that the differential sensitivity was at least partially due to p53 function in HCT116 cell lines. The cell cycle arrest in the p53 null cell line may have been caused by p21 induction (Figures 6A, ,7A)7A) and the activity of other players like Cdk2 and Rb, which regulate the G1/S phase transition [59-61]. PNR was proposed as a putative therapeutic target for various diseases including p53-positive and ER��-positive cancers. Because endogenous ligands have not been identified for PNR, efforts have been made to identify synthetic PNR agonists. The lack of highly sensitive assays and a crystal structure of PNR greatly limit the discovery of synthetic PNR agonists.

13-cis-retinoic acid, the natural retinal pigment, could only confer agonistic activity towards PNR at non-physiological concentrations [31]. Thus far, only 11a was described as a potent PNR agonist by one study [31], yet this is challenged by a recent study [32]. Our results agree with the latter study, demonstrating that 11a Batimastat cytotoxicity is independent of PNR. Rather, we show that 11a exhibits differential cytotoxicity in various cancer cell lines, and this cytotoxicity correlates with p53 mutation status.