Abstract 1363: Novel Nrf2 inhibitors and their application to overcome chemoresistance of anticancer drugs
Authors: Kumar Vineet , Foygel Kira , Paulmurugan Ramasamy , Malhotra Sanjay V. 
Published: July 2016

A large number of malignant tumors, for example, colonic, thyroid, endometrial, lung, ovarian, breast and pancreatic cancers, exhibit an increased Nrf2 expression and constitutive activation. This enhanced Nrf2 activation originates from rare gain-of-function mutations of Nrf2 itself, and from loss-of-function mutations, promoter hypermethylation or microRNAs targeting Nrf2 inhibitory protein Keap1/INRF. As a consequence of the enhanced Nrf2 activity, tumor cells acquire protection from apoptosis and are more capable of proliferation, both conditions favoring tumorigenesis on one hand and making tumor cells more refractory to chemo- and radiotherapy on the other hand. These findings suggest that Nrf2 exhibits profound protumorigenic activity, and can be a crucial target for anticancer therapy, particularly by overcoming chemoresistance.

In the current study, we have rationally created a library of about 150 flavonoid-based compounds and screened them for Nrf2 inhibition effect using a novel intact cell based bioluminescence imaging assay which directly measures the expression of NQO1, the downstream target gene of Nrf2. HeLa cells transfected with Luciferase reporter gene expressed under the promoter of NQO1 gene were treated with the library compounds at 10 μM concentration for 16 hours in a 96-well plate format. After treatment, the cells were imaged by the addition of 25 μg/ml of D-Luciferin using IVIS bioluminescence imaging system. The NQO1-ARE-luciferase signals measuring its expression in different wells were quantified. In parallel, the drug-associated toxicity was assessed by MTT assay, the results were used to normalize the bioluminescence signal, and the inhibitory effect was assessed by comparing the signal induced by each compound with the DMSO control. In this preliminary screening, 11 compounds that inhibited the expression of NQO1 by more than 3-fold, as compared to the DMSO control, were identified. Dose-response studies showed that these compounds inhibit Nrf2 in a linear dose-dependent pattern. Moreover, 3 of the hit compounds exhibited 6-10 fold of Nrf2 inhibition (compared to DMSO) at as low as 0.16 μM concentration. We also evaluated Hela cells treated with Doxorubicin (Dox, 1 μM) and four hit compounds (10 μM conc.) for induced apoptosis after 48 hours of treatment, to evaluate synergistic effect. Quantification studies by FACS assay using PI staining revealed that our hit inhibitors enhanced the apoptosis effect of Doxorubicin.

In conclusion, we have identified new potent inhibitors of Nrf2, which also enhanced the apoptotic effect of Doxorubicin. Further studies to probe the mechanism of action of these inhibitors and medicinal chemistry efforts to study the structure activity relationship are underway.

Citation Format: Vineet Kumar, Kira Foygel, Ramasamy Paulmurugan, Sanjay V. Malhotra. Novel Nrf2 inhibitors and their application to overcome chemoresistance of anticancer drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1363.