Abstract B160: Esculetin induces antiproliferative and apoptotic response in PANC-1 cells by directly binding to Keap1
Authors: Arora Rashi , Sawney Sharad , Saini Vikas , Tiwari Manisha , Saluja Daman 
Published: May 2016

Abstract
Esculetin, a coumarine derivative, has been reported to inhibit growth of various human cancer cells. However, the targets to which esculetin binds and the possible mechanism for its cytotoxicity in cancer cells remain to be elucidated. Herein the effect of esculetin on PANC-1 cells and its mechanism of action were evaluated.
Treatment with 100 μM esculetin resulted in significant growth inhibition in PANC-1 cells with G1-phase cell cycle arrest within 12 hour. Induction of apoptosis was confirmed in cells on 24 hour treatment with 100μM esculetin by flow cytometric analysis of apoptotic marker annexin V and temporal increase in the active form of caspase 3, 8 and 9. Apoptosis was found to be associated with loss of mitochondial membrane potential analyzed through measurement of fluorescence by JC-1 dye and increase in cytosolic cytochrome-C levels.
A notable decrease in intracellular ROS observed in PANC-1 cells on esculetin treatment, propelled focus on regulation of Nrf2, a major antioxidant response regulator. Keap1 protein sequesters Nrf2 transcription factor in the cytoplasm and promotes its degradation. Direct inhibition of Nrf2-Keap1 interaction has recently emerged as an attractive strategy for cancer treatment. Co-immunoprecipation assays carried out on both Nrf2 and Keap1 revealed a loss of interaction between the two on esculetin treatment. Nuclear accumulation of Nrf2 observed with confocal microsopy and western blot and upregulation of expression of Nrf2 regulated gene NQO1 observed with qPCR further supported the activation of Nrf2. To account for the loss of Nrf2-Keap1 interaction on esculetin treatment, direct binding potential between esculetin and keap1 was checked. Molecular docking studies predicted that esculetin displayed significant interactions in terms of hydrogen bond, π-cationic and π-π aromatic interactions with the subpocket P1 and P3 of the Keap1 receptor to provide inhibitory profile. The direct binding of esculetin with keap1 was then confirmed by pull down assay using esculetin conjugated sepharose beads. A recently established implication of Nrf2 activation is its antagonistic effect on NF-κB, an anti-apoptotic transcription factor that promotes cell cycle progression. Here too, p65-NF-κB protein levels were found to decrease on Nrf2 activation in a temporal fashion on esculetin treatment on PANC-1 cells.
Our findings thus suggest that esculetin binds to Keap1 and thereby promotes nuclear accumulation of Nrf2 in PANC-1 cells that possibly induces antiproliferative and apoptotic response by attenuating NF-κB.
Citation Format: Rashi Arora, Sharad Sawney, Vikas Saini, Manisha Tiwari, Daman Saluja. Esculetin induces antiproliferative and apoptotic response in PANC-1 cells by directly binding to Keap1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B160.