Esculetin induces antiproliferative and apoptotic response in pancreatic cancer cells by directly binding to KEAP1

Abstract

Background: A handful of studies have exploited antitumor potential of esculetin, a dihydroxy coumarine derivative; the targets to which it binds and the possible downstream mechanism for its cytotoxicity in cancer cells remain to be elucidated. Using pancreatic cancer cell lines as a model system, herein the study was initiated to check the efficacy of esculetin in inhibiting growth of these cancer cells, to decipher mechanism of its action and to predict its direct binding target protein.

Methods: The cytotoxicity of esculetin was determined in PANC-1, MIA PaCa-2 and AsPC-1 cell lines; followed by an inspection of intracellular levels of ROS and its associated transcription factor, p65-NF-κB. The interaction between transcription factor, Nrf2 and its regulator KEAP1 was studied in the presence and absence of esculetin. The effect of Nrf2 on gene expression of antioxidant response element pathway was monitored by real time PCR. Thereafter, potential binding target of esculetin was predicted through molecular docking and then confirmed in vitro.

Results: Esculetin treatment in all three pancreatic cancer cell lines resulted in significant growth inhibition with G1-phase cell cycle arrest and induction of mitochondrial dependent apoptosis through activation of caspases 3, 8 and 9. A notable decrease was observed in intracellular ROS and protein levels of p65-NF-κB in PANC-1 cells on esculetin treatment. Antioxidant response regulator Nrf2 has been reportedly involved in crosstalk with NF-κB. Interaction between Nrf2 and KEAP1 was found to be lost upon esculetin treatment in PANC-1 and MIA Paca-2 cells. Nuclear accumulation of Nrf2 and an upregulation of expression of Nrf2 regulated gene NQO1, observed on esculetin treatment in PANC-1 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 depicted in silico using molecular docking studies. Pull down assay using esculetin conjugated sepharose beads confirmed the binding between esculetin and KEAP1.

Conclusions: We propose that esculetin binds to KEAP1 and inhibits its interaction with Nrf2 in pancreatic cancer cells. This thereby promotes nuclear accumulation of Nrf2 in PANC-1 cells that induces antiproliferative and apoptotic response possibly by attenuating NF-κB.

Keywords: ARE pathway; Anticancer compound; Coumarins; Esculetin; KEAP1; Molecular target; NF-κB; Nrf2; Pancreatic cancer.

Fig. 1

Effect of esculetin on pancreatic cancer cells: A Effect of different concentrations of esculetin on cell viability using MTT assay in PANC-1, AsPc-1 and MIA PaCa-2 cell lines. B Cell cycle analysis of different cell lines ((I) PANC-1 (II) AsPc-1 and (III) MIA PaCa-2) in the absence and presence of esculetin (100 μM) using flow cytometric analysis of DNA content showing cell cycle arrest in G1 phase (a-synchronized population, b-vehicle control, c- esculetin treated cells for 12 h in I and II and 18 h in III). C Percent distribution of PANC-1 cells in different phases of cell cycle upon 100 μM esculetin treatment. (V stands for Vehicle control for indicated time, E stands for esculetin treated sample for indicated time). Data represents the mean ± SD of three independent experiments. The significance was determined using ANOVA (Bonferroni’s test). Key:*p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001)

Fig. 2

Esculetin induces apoptosis in pancreatic cancer cells: a Flow cytometric analysis of PANC-1 cells treated with 100 μM esculetin for indicated time showed temporal increase in surface expression of apoptotic marker- Annexin V indicating increased population of cells in apoptotic phase. b Percentage of PANC-1 cells exhibiting fluorescence in all four panels (healthy, early apoptosis, late apoptosis and necrosis) showing time dependent increase in apoptosis in Ecsuletin treated cells. c Percentage of cells with active APO BrdU indicating apoptosis in the absence and presence of esculetin (100 μM) as determined using TUNEL assay. d Western blot analysis showing an increase in expression of pro and active form of caspases (VC stands for vehicle control, E stands for esculetin treated sample for indicated time, CF stands for cleaved form, numerals represent time of esculetin treatment). Data represents the mean ± SD of three independent experiments. The significance was determined using ANOVA (Bonferroni’s test). Key:*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

Fig. 3

Esculetin induces loss of Mitochondrial membrane potential: a Flow cytometric analysis of PANC-1 cells stained with JC-1 dye after 100 μM esculetin treatment for indicated time showed a temporal decrease in ratio of red fluorescence (JC-1 aggregates) to green florescence (JC-1 monomers). b Percentage of cells exhibiting monomers and aggregates of JC-1 after treatment of cells with esculetin for different time intervals. c Western blot analysis showing temporal increase in cytosolic cytochrome C in PANC-1 cells. (VC stands for vehicle control, E stands for esculetin treatment sample for indicated time, CCCP stands for positive control i.e., carbonyl cyanide 3-chlorophenylhydrazone treated cells, numerals represent time of esculetin treatment). Data represents the mean ± SD of three independent experiments. The significance was determined using ANOVA (Bonferroni’s test). Key:*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

Fig. 4

Esculetin lowers ROS levels: a Fluorescence intensity of DCFDA, an indicator of ROS level was determined in PANC-1 cells and it was found to decrease upon 100 μM esculetin treatment. b Flow cytometric analysis based histogram showing increase in percentage of PANC-1 cells negative for ROS activity as a function of time of ecsuletin treatment. c Western blot analysis showing temporal decrease in protein levels of ROS dependent transcription factor NK-κB and uniform levels of its inhibitor I-κB in PANC-1 cells. (VC stands for vehicle control, E stands for esculetin treated sample for indicated time, and numerals represent time of esculetin treatment). Data represents the mean ± SD of three independent experiments. The significance was determined using ANOVA (Bonferroni’s test). Key:*p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001)

Fig. 5

Esculetin disrupts Nrf2-KEAP1 interaction: a and b Western Blot analysis of PANC-1 protein extract immuno-precipitated using (a) Nrf2 and (b) KEAP1 antibody and probed with the other, showing loss of their interaction in esculetin treated cells. Input lane represents western blot analysis of 10 % of total protein extract used in CoIP indicating endogenous level of probed protein. c Western Blot analysis of total protein and nuclear extract from esculetin treated PANC-1 cells showing increase in phosphorylated form of Nrf2 and its nuclear accumulation. d Confocal microscopy view of esculetin treated PANC-1 cells incubated with Nrf2 antibody and probed with FITC (green) labeled secondary antibody showing an increase in nuclear accumulation of Nrf2. Nuclear staining was done with DAPI (blue). e Change in expression of NQO1, a target of Nrf2, measured using qPCR, showed about 5 fold increase upon 100 μM esculetin treatment. (C stands for control, VC stands for vehicle control, Iso stands for isotype Ab control, E stands for esculetin treated sample for indicated time). Data represents the mean ± SD of three independent experiments. The significance was determined using t test. Key:*p < 0.05; **p < 0.01; ***p < 0.001)

Fig. 6

Esculetin binds to KEAP1 directly: a 3D Docking model showing esculetin docked to the active site of KEAP1. b 2D interaction diagram of esculetin with KEAP1. Residues involved in hydrogen-bonding, charge or polar interactions are represented by magenta-colored circles. Residues involved in van der Waals interactions are represented by green circles. The solvent accessible surface of a residue is represented by a blue halo around the atom. Hydrogen-bond interactions with amino acid side chain and main chain are represented by a blue and green dashed line, respectively with an arrow head directed toward the electron donor. π-π and π-cationic interactions are represented by an orange line with symbols indicating the interaction. c Western blot analysis of competitive pull down assay carried out with esculetin conjugated beads showing a direct interaction between esculetin and KEAP1. (I stands for input (10 %), B represents protein pulled using non conjugated beads, EB represent protein pulled using esculetin conjugated sepharose beads)